ENDOCRINE SOCIETY
OXFORD
Update on Biology and Genomics of Adrenocortical Carcinomas: Rationale for Emerging Therapies
Antonio Marcondes Lerario, 1.0D Dipika R. Mohan,2,[D and Gary D. Hammer1,3,4,5, [D
“Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, Ann Arbor, Michigan 48109- 2200, USA
2Medical Scientist Training Program, University of Michigan, Ann Arbor, Michigan 48109-2200, USA
3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109-2200, USA
4Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan 48109-2200, USA; and
5Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-2200, USA
Correspondence: Antonio Marcondes Lerario, MD, PHD, Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, 1860 Taubman Biomedical Science Research Bldg, 109 Zina Pitcher PI, Ann Arbor, MI 48109-2200, USA. Email: alerario@umich.edu; or Gary
D. Hammer, MD, PHD, Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, 1528 Taubman Biomedical Science Research Bldg, 109 Zina Pitcher PI, Ann Arbor, MI 48109-2200, USA. Email: ghammer@umich.edu.
Abstract
The adrenal glands are paired endocrine organs that produce steroid hormones and catecholamines required for life. Adrenocortical carcinoma (ACC) is a rare and often fatal cancer of the peripheral domain of the gland, the adrenal cortex. Recent research in adrenal development, homeo- stasis, and disease have refined our understanding of the cellular and molecular programs controlling cortical growth and renewal, uncovering crucial clues into how physiologic programs are hijacked in early and late stages of malignant neoplasia. Alongside these studies, genome-wide approaches to examine adrenocortical tumors have transformed our understanding of ACC biology, and revealed that ACC is composed of dis- tinct molecular subtypes associated with favorable, intermediate, and dismal clinical outcomes. The homogeneous transcriptional and epigen- etic programs prevailing in each ACC subtype suggest likely susceptibility to any of a plethora of existing and novel targeted agents, with the caveat that therapeutic response may ultimately be limited by cancer cell plasticity. Despite enormous biomedical research advances in the last decade, the only potentially curative therapy for ACC to date is primary surgical resection, and up to 75% of patients will develop metastatic disease refractory to standard-of-care adjuvant mitotane and cytotoxic chemotherapy. A comprehensive, integrated, and current bench-to- bedside understanding of our field’s investigations into adrenocortical physiology and neoplasia is crucial to developing novel clinical tools and approaches to equip the one-in-a-million patient fighting this devastating disease.
Graphical Abstract
Capsule
Corticocapsular unit
ZG
Physiologic programs facilitate malignant neoplasia
O
O
O
O
O
O
O .
.
·
O
ZF
O
?
Adrenal gland
ZR
Biomedical advances yield insights into novel therapies
SF1
Adrenal differentiation
Epigenetics
Genomic instability
Immune cells
Plasticity
Wnt/ ß-cat
Steroid
Tyrosine Kinase
Metabolism
Cell cycle
IGF
@ 2022 Endocrine Society
Key Words: adrenocortical carcinoma, targeted therapies, molecular biomarkers, genomics, adrenocortical development and homeostasis
Abbreviations: ACA, adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, adrenocortical tumor; ACTH, adrenocorticotropin; ADS, adrenal differen- tiation score; AGP, adrenogonadal primordium; ATII, angiotensin II; ATP, adenosine 5’-triphosphate; cAMP, cyclic adenosine 5’-monophosphate; CDK, cyclin- dependent kinase; DNMT, DNA methyltransferase; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; FAdE, fetal adrenal-specific enhancer; FDA, US Food and Drug Administration; Fzd, frizzled; IGF1R, insulin-like growth factor receptor 1; LGR, leucine-rich repeat containing G-protein coupled receptor; LOF, loss of function; LOH, loss of heterozygosity; MC2R, melanocortin receptor type 2; PD1, programmed cell death protein 1; PKA, protein kinase A; PRC2, polycomb repressive complex 2; RSPO, R-spondins; SCNA, somatic copy number alteration; SF1, steroidogenesis factor 1; SHH, sonic hedgehog; SNP, single nucleotide polymorphism; SNV, single nucleotide variant; TCGA, The Cancer Genome Atlas project; zF, zona fasciculata; zG, zona glomerulosa.
Adrenocortical carcinoma (ACC) is a rare cancer with an overall dismal prognosis. Despite that approximately half of patients present with metastatic disease at diagnosis (American Joint Committee on Cancer/European Network for the Study of Adrenal Tumors stage IV), surgery remains the only therapy with the potential to cure and is limited to patients with locoregional disease (American Joint Committee on Cancer/European Network for the Study of Adrenal Tumors stage I-III). Furthermore, up to 50% of R0-resected patients recur, indicating an urgent need for improved adjuvant man- agement (1). Limited evidence suggests that adjuvant therapy with the DDT-derived adrenolytic agent mitotane may offer a survival benefit (2). Current clinical guidelines recommend adjuvant mitotane to nearly all patients with ACC (3); how- ever, clinical responses on this regimen are highly heteroge- neous. While a subgroup of patients exhibits rapid recurrence despite standard-of-care adjuvant mitotane (4, 5), it has been recognized that patients with low-risk disease (ie, localized,
ESSENTIAL POINTS
· Adrenocortical carcinoma (ACC) is a rare and aggres- sive cancer, with no curative medical therapies to date
· Recent advances in mouse modeling of adrenocortical development, homeostasis, and disease highlight a crucial role for maintenance of physiologic paracrine (Wnt/ß-catenin) and endocrine (adrenocorticotropin/ protein kinase A [ACTH/PKA]) signaling throughout neoplastic evolution
· Recent integrated pangenomic molecular profiling studies reveal that ACC is composed of 3 molecular subtypes, COC1, COC2, and COC3, associated with good, intermediate, and uniformly dismal prognosis, respectively
· COC1 ACC possesses the highest degree of immune infiltration, and no recurrent somatic alterations ex- cept loss of imprinting of the IGF2 locus (present in 90% of ACC)
· COC2-COC3 ACC possess minimal immune infil- tration, with recurrent somatic alterations leading to constitutive Wnt/ß-catenin signaling, increased ad- renal differentiation, and cortisol production in the setting of epigenetic reprogramming
· COC3 ACC are distinguished by profound disruption of epigenetic programming (genome-wide CpG island hypermethylation, CIMP-high), recurrent somatic al- terations leading to constitutive cell cycle activation, and genomic instability
· These studies illuminate important therapeutic tar- gets for ACC that include adrenal differentiation, steroidogenesis, immune checkpoint activation, cell cycle and genomic instability, Wnt/ß-catenin signaling, epigenetics, metabolism, and cellular plasticity
with low mitotic activity) do not benefit (6). Furthermore, expert opinion suggests that platinum-based adjuvant cyto- toxic chemotherapy should be offered to all high-risk pa- tients (3); this recommendation has been recently supported by a small retrospective study (7), but definitive and robust evidence is still lacking. In addition, the definition of high risk lacks objectivity, relying on clinical judgment with nu- anced interpretation of standardized prognostic markers-a decision-making strategy likely feasible only in expert centers. A phase 3 clinical trial to assess the efficacy of platinum-based therapies for patients with high mitotic activity measured by the Ki67 proliferation index is ongoing (NCT03583710). However, heterogeneous outcomes are observed in existing proliferation-based low-risk and high-risk strata, limiting the value of this strategy to individualize adjuvant therapies (8).
Standard-of-care systemic agents for metastatic ACC in- clude mitotane either as a single agent or in combination with cytotoxic chemotherapy (3). Though mitotane remains the only agent approved by the US Food and Drug Administration (FDA) specifically for ACC, its antitumor effects as a single agent are modest (9). This partially results from its poor pharmacokinetic properties. Because it is an extremely lipo- philic compound with a highly variable metabolic clear- ance among individuals, only a subset of patients will ever achieve therapeutic levels (14-20 mg/L, established in retro- spective studies) after many months of therapy. Furthermore, its narrow therapeutic window, wide array of toxic effects, and potent induction of CYP3A4 activity and hence catab- olism of other drugs (including adrenal hormone replace- ment) are associated with frequent interruptions in therapy (10-12). Recent work has made advances in characterizing some molecular aspects of mitotane action (13, 14); however, predicting individual responses to mitotane remains elusive, and response is likely a complex function of interindividual differences in drug metabolism and intrinsic tumor-specific vulnerabilities (12, 15, 16). A rigorous understanding the molecular basis of mitotane vulnerability will ultimately be required for optimizing clinical strategies using this agent, and for the development of alternative agents targeting these vulnerabilities. Because of its favorable effect in mitigating morbid endocrine manifestations of ACC, mitotane remains a widely prescribed and useful drug for advanced cases with anecdotal reports of complete response (17, 18). Mitotane has also been reported to have favorable interactions with cytotoxic agents via inhibition of efflux pumps (19), and combinations of mitotane and different chemotherapies have been proposed (20). In fact, a randomized phase 3 trial sup- ports the use of combination of etoposide, doxorubicin, and cisplatin plus mitotane (EDP + M) as first-line therapy for advanced disease, albeit with minimal survival benefit (3). Second-line and salvage therapies with other agents, such as gemcitabine, capecitabine, trofosfamide, and streptozotocin have been proposed by a few studies with limited benefit (re- viewed in [21]). Since the original FDA approval of mitotane
in the 1960s, few novel systemic therapies for ACC have been considered for implementation. This is largely secondary to the rarity of ACC restricting our ability to enroll patients in clinical trials, limited risk stratification with widely available tools, and historic lack of knowledge on molecular mechan- isms of ACC pathogenesis, all of which would seed develop- ment of rational, subtype-directed therapeutic strategies.
Recent advances in genome-wide molecular approaches, such as next-generation sequencing, single-nucleotide poly- morphism [SNP] arrays, and methylation arrays, have en- abled an unbiased characterization of the somatic landscape of human cancers. Furthermore, large-scale and systematic studies using these approaches, such as The Cancer Genome Atlas project (TCGA), have provided the opportunity for a high-resolution multiplatform characterization of large and multi-institutional cohorts of ACC (22-25). These initiatives seeded explosive gains in our understanding of the molecular underpinnings of several different cancers, leading to the discovery of novel recurrent somatic alterations, abnormal pathway activation, and the characterization of previously unappreciated molecular subtypes of virtually all cancers in this study. Indeed, with regard to ACC, the data generated by pangenomic, multiplatform studies (22, 23, 25) provided enormous insights into the molecular pathogenesis of ACC. However, practical translation of this knowledge into clin- ically meaningful concepts and tools remains challenging. In this review, we summarize these most recent advances in our understanding of the molecular pathogenesis of ACC and dis- cuss how they expose targetable therapeutic vulnerabilities.
Overview of Physiologic Signaling Pathways Stabilized in Adrenocortical Neoplasia
Developmental and homeostatic signaling pathways, including cell cycle, Wnt/ß-catenin, and protein kinase A (PKA) path- ways are almost universally dysregulated in ACC through a variety of mechanisms that we will detail here. We postulate that in ACC, as recently demonstrated in other cancers (26), such discordant pathway engagement creates a plastic cell state with the capacity to traverse the full spectrum of differ- entiation without terminal differentiation commitment (27). This allows ACC cells to sample transcriptional programs that confer sustained proliferation potential and intrinsic therapy resistance. To place those programs in context, we will first detail the paracrine and endocrine pathways controlling de- velopment, zonation, and renewal of the adrenal cortex.
Development of the Adrenal Cortex
The adrenal cortex derives from cells of the coelomic epithe- lium, a monolayer of squamous cells that lines the surfaces of viscera and the internal body wall. Around E9.5 in mice (fourth to sixth gestational weeks in humans), the coelomic epithelium condenses within the intermediate mesoderm be- tween the urogenital ridge and the dorsal mesentery to form the adrenogonadal primordium (AGP). By E10.5 in mice (eighth gestational week in humans), the AGP has matured with discrete dorsomedial and ventrolateral segments, which give rise to the adrenal and gonadal primordia, respectively. At E13.5 in mice (gestational weeks 8-9 in humans), migrating cells derived from the neural crest invade the adrenal primor- dium and accumulate centrally to nucleate the adrenal me- dulla (28, 29). Concomitantly, mesenchymal cells envelope the adrenal primordium and coalesce as a multilayered fibrous
structure, the adrenal capsule (30). This process is termed en- capsulation. The capsule accompanies all subsequent steps of adrenal development and persists into adulthood. In addition to serving as a physical barrier that defines the organ limits and a scaffold that maintains tissue architecture, the capsule is also required for zonation and renewal of steroidogenic cells throughout life (31-36).
At encapsulation, different histological compartments can be distinguished in the cortex: a central area of large polyhe- dral eosinophilic cells, and a peripheral zone of small baso- philic cells, enveloped by the fibrous capsule. These regions are termed the fetal zone and definitive zone, respectively. By the end of human gestation, the fetal zone comprises approxi- mately 80% of the adrenal mass; however, it completely re- gresses by apoptosis in the first few weeks of life (37-39).
Adrenal formation is dependent on the master transcription factor steroidogenesis factor 1 (SF1, encoded by NR5A1). SF1 expression can be detected early in fetal life during AGP for- mation, and persists throughout adult life in all steroidogenic cells of adrenal and gonadal lineages (33, 40). Genetic models of SF1 deficiency (either targeting Nr5a1 itself or alterna- tive upstream regulators Pbx1, Wt1, and Cited2) exhibit a spectrum of phenotypes characterized by adrenal hypoplasia, underlying the critical role for Nr5a1 in adrenal organogen- esis (28, 30, 39, 41-45). Intact SF1 expression is a hallmark of steroidogenic lineages throughout all stages of life; how- ever, epigenetic mechanisms are responsible for initiation and maintenance of gene expression at different stages of murine fetal development, suggesting the engagement of alternative and context-dependent distal regulators. In mice, Nr5a1 ex- pression is initially maintained by the fetal adrenal-specific enhancer (FAdE), which becomes inactive when the definitive cortex forms. Interestingly, lineage tracing experiments have demonstrated that the definitive cortex originates from FAdE- active cells in the fetal cortex that transiently shut down Nr5a1 expression and get incorporated into the capsule as Gli1-expressing cells (39, 46). These capsular cells ultimately reactivate Nr5a1 expression in a FAdE-independent manner, and give rise to virtually all steroidogenic cells in the defini- tive adrenal cortex. In the postnatal period and into adult- hood, Gli1-positive/SF1-negative cells from the capsule serve as an alternative progenitor cell pool that differentiates into steroidogenic cells in response to homeostatic demands, re- capitulating the cascade that originates the definitive cortex during development (32, 36). In addition to its essential role in organogenesis, differentiation, and steroidogenesis, SF1 controls an array of cellular processes, including glucose me- tabolism, angiogenesis, cell motility, and cell proliferation, that are critical for cell survival (47, 48).
Paracrine Control of Cell Identity in Adrenocortical Development and Homeostasis
Fine transcriptional regulation of signaling programs is a defining feature of adrenocortical homeostasis (49, 50). In addition to SF1, several other transcription factor families and coactivators play a critical role in modulating these programs. Through transduction of paracrine and endocrine signaling cues, these transcriptional modules establish the steroidogenic and differentiation states of adrenocortical cells. For example, progenitor adrenocortical cells, in addition to expressing SF1, express sonic hedgehog (SHH) (33, 51). SHH is a paracrine signaling molecule that centrifugally signals to the capsule and initiates Gli1 expression in the capsular cells that ultimately
serve en masse as a signaling center to initiate centripetal Wnt signaling to the underlying cortex, thereby establishing a closed-loop SHH-Wnt relay system critical for adrenocortical homeostasis. SHH/GLI signaling is rarely targeted for som- atic alteration in adrenocortical neoplasia, and the nuances of this pathway are beyond the scope of this review. However, a classic paracrine signaling pathway that is frequently targeted for activation by driver somatic alterations in adrenocortical neoplasia is the Wnt signaling pathway (22, 23, 52) (Fig. 1).
Wnt signaling is crucial for embryonic development and morphogenesis, as well as for maintenance of stem/pro- genitor cell pools in virtually all mammalian organs. Wnt signaling through transcriptional coactivator ß-catenin (the canonical Wnt pathway) is essential for adrenal formation, zonation, and renewal. Signaling is initiated by the binding of a Wnt ligand (in the adrenal cortex, this is presumed to be WNT4) to a Frizzled (Fzd) receptor. In the canonical pathway, activation of Fzd neutralizes a major negative regulator of cytoplasmic ß-catenin stability (the destruc- tion complex), enabling rapid cytoplasmic accumulation and nuclear translocation of ß-catenin, where ß-catenin will
WNT4
RSPO3
FZD
LRP5 /6
W
LGR4/5
ZNRF3
ß-catenin PPPP APC
Destruction complex
> Internalization
Degradation
B-catenin
ß-catenin
B-catenin
ß-catenin
TCF/LEF
B-catenin
Stemness
?
Differentiation
classically coactivate a TCF/LEF-dependent transcriptional program. Notably, cytoplasmic ß-catenin stability is regu- lated by phosphorylation; the destruction complex resides in the cytoplasm and phosphorylates key residues on ß-catenin exon 3 to target it for proteasome-mediated degradation. Active Fzd neutralizes the destruction complex by seques- tering it to the cell membrane (53).
While the importance of Wnt/ß-catenin signaling for ad- renal development and homeostasis have been demonstrated by several groups, its precise molecular mechanisms remain incompletely understood. Studies using adrenocortical- specific Cre recombinases to activate or delete ß-catenin in the mouse adrenal have demonstrated that both gain and loss of function (LOF) are associated with a spectrum of pheno- types. While LOF ß-catenin models invariably result in ad- renal agenesis or hypoplasia, gain-of-function models may cause both adrenal agenesis/hypoplasia and hyperplasia with zona glomerulosa (zG) differentiation and increased aldos- terone production, dependent on the timing of the genetic hit and identity of adrenocortical cells affected (54-58). Similar phenotypes are observed in patients with SERKAL syndrome, an autosomal recessive disorder caused by LOF mutations in WNT4. Among several multisystemic malformations, pa- tients with SERKAL syndrome also exhibit adrenal agenesis/ dysgenesis (59). Interestingly, mice engineered to bear adrenocortical-specific Wnt4 deletion have a relatively mild phenotype characterized by zG hypoplasia and aldosterone deficiency, suggesting that other Wnt ligands may explain interspecies differences (60). Collectively, these observations suggest that Wnt/ß-catenin signaling is important not only for adrenal organogenesis and homeostasis, but also for pro- moting functions of the differentiated adrenal cortex such as aldosterone production. These paradoxical actions of Wnt/B- catenin, supporting both stemness and differentiation, may also be mediated by interactions with distinct transcriptional regulators. In fact, it has been demonstrated that ß-catenin binds SF1, suggesting that this complex might control tissue- specific functions such as aldosterone production (61-64).
Wnt/B-catenin activity in the adrenal cortex (measured by intensity of nuclear staining for ß-catenin) is zonally distrib- uted, forming a centripetal gradient where it peaks in the zG, and progressively fades into the inner zones (65). The mo- lecular basis of this compartmentalized expression has been recently elucidated and involves both cell autonomous and nonautonomous mechanisms. The onset of Wnt/ß-catenin signaling in the adrenal cortex overlaps with encapsula- tion and formation of the definitive cortex (28, 29, 55, 66). Indeed, as previously discussed, the entire definitive cortex is derived from capsular precursors, and capsular cells can serve as an alternative progenitor pool throughout life (32, 36, 39). Furthermore, the temporal overlap between encap- sulation and the onset of Wnt signaling suggests that these 2 processes are interconnected. In fact, it has been recently dem- onstrated that the adrenal capsule is a source of R-spondins (RSPO), a family of secreted proteins that potentiate canon- ical Wnt/ß-catenin signaling by interacting with members of the leucine-rich repeat containing G-protein coupled recep- tors (LGR) family. In the presence of RSPO, LGRs form a complex with membrane-bound E3 ubiquitin ligases ZNRF3 and RNF43 (negative Wnt pathway regulators), causing their internalization and degradation. In the absence of RSPO, ZNRF3 and RNF43 inhibit Wnt signaling by promoting in- ternalization and proteasomal degradation of Fzd receptors
(53). Vidal et al (31) have demonstrated that capsular cells expressing Gli1 produce RSPO3 in the mouse. Gli1-driven loss of RSPO3 leads to profound disruption of adrenal zon- ation, with adrenal hypoplasia and an absent zG, obliteration of the SHH + progenitor pool, and downregulation of Wnt/B- catenin target genes. Consistent with the role of RSPO3 in augmenting Wnt signaling, a reduction in Wnt4 expression was also observed, indicating that Wnt4 is likely a Wnt/B- catenin transcriptional target in the adrenal cortex and main- tains Wnt/ß-catenin signaling in an autocrine manner (65).
These observations led to important conceptual advances in the previously proposed model of the corticocapsular homeo- static unit (Fig. 2). According to this model, signaling between SHH-producing cells in the peripheral cortex (SHH+/SF1+) and SHH-responsive cells in the capsule (GLI1+/SF1-) is crit- ical for establishing and maintaining the stem/progenitor cell niche, and the Wnt/B-catenin signaling gradient in the cortex. This is achieved by a bidirectional, interdependent paracrine loop (SHH-Wnt relay), in which cortex-derived SHH induces the expression of GLI targets in the capsule, including RSPO3,
GLISHHRSPO3 WNT4ß-catenin
SF1
Capsule
Corticocapsular unit
zG
zF
ZR
AT2 CAMK ACTH PKA Ki67
which signals back to the cortex, promoting activation of Wnt/ß-catenin signaling. Consistent with this model, mice bearing deletion of either Shh or Gli1 also exhibit reduced Wnt/ß-catenin signaling, adrenal hypoplasia, and disrupted zonation with reduced expression of zG markers (31, 67, 68).
Endocrine Control of Functional Zonation, Transdifferentiation, and Interplay With Paracrine Signaling
Endocrine factors are also major determinants of cortical function and zonation. The most important endocrine fac- tors that regulate steroid production in the adrenal cortex are angiotensin II (ATII) and adrenocorticotropin (ACTH). These hormones are the effectors of 2 independent endo- crine systems predicated on both feed-forward amplification/ activation and feedback inhibition, the renin-angiotensin- aldosterone system and the hypothalamus-pituitary- adrenal axis. The renin-angiotensin-aldosterone system and hypothalamus-pituitary-adrenal axis independently regulate aldosterone and cortisol production, respectively, according to physiologic demands (Fig. 3). Though all the cells in the cortex are derived from the same progenitor pool, different cell populations are deployed to respond to ATII or ACTH with hormone production and secretion. ZG cells respond to ATII and zona fasciculata (zF) cells respond to ACTH (69-73). This is achieved through distinct differentiation states estab- lished in zG and zF cells in response to the Wnt/ß-catenin signaling gradient. Indeed, murine studies have demonstrated that Wnt/B-catenin signaling promotes zG fate and restrains zF differentiation, at least partially through induction of phosphodiesterases, enzymes that convert cyclic adenosine 5’-monophosphate (cAMP) to adenosine 5’-diphosphate terminating cAMP-induced PKA activation (56, 58). As the cells migrate centripetally, progressively lower Wnt/ß-catenin signaling enables maximal zF cellular response to ACTH with initiation of increased expression of the ACTH receptor (melanocortin receptor type 2 [MC2R]) and the essential melanocortin receptor 2 accessory protein (MRAP) (74). Interestingly, despite this antagonistic role, ß-catenin is re- quired for ACTH-dependent zF renewal (32).
MC2R belongs to the G protein-coupled receptor family. On ACTH binding, it signals through the Gas subunit to activate adenylate cyclase, which converts adenosine 5’-tri- phosphate (ATP) to cAMP to activate PKA. In its inactive form, PKA is part of a tetramer formed between two cata- lytic and 2 regulatory subunits. In the presence of cAMP, the tetramer dissociates, releasing the PKA catalytic sub- units, which promote phosphorylation of cAMP response element-binding protein (CREB) transcription factors. pCREB activates the transcription of target genes, including immediate early response genes encoding for AP-1 compo- nents, NR5A1, and several steroidogenic enzymes including HSD3B2, CYP17A1, and CYP11B1 (75-77). In addition, PKA activation strongly represses targets of Wnt/ß-catenin signaling, facilitating the zG to zF transition in cooperation with epigenetic regulators (56, 78). The contribution of PKA activation to zG-zF transdifferentiation is well dem- onstrated in Mc2r and Mrap knockout mouse models (79, 80). Similarly to patients with familial glucocorticoid defi- ciency, these mice exhibit severe corticosterone deficiency with normal aldosterone secretion and die shortly after birth (rescued by in utero corticosterone administration). Histologically, adrenals from these mice possess profoundly
RAAS
HPA
Kidney Renin
Hypothalamus CRH
Liver Angiotensin I
Pituitary ACTH
Lungs Angiotensin II
zG Aldosterone
1BP
zF Cortisol
zG
zF
MN
AT2
CaV
ACTH
AC
ATR
MRAP as
MC2R
as
B
Y
ATP
CAMP
TF
Ca
2+
Ca
2+
CREB
CAMK
Ca
2+
0
PKA
C
C
R
R
Ca
2+
C
C
P
P
TF
CYP11B2
CREB
CYP11B1
disrupted zonation characterized by overall cortical atrophy, thickened capsule, expanded zG, and absent zF. These ab- normalities are accompanied by an increased number of cells positive for Shh, ß-catenin, and Wnt4, with a profound decrease in the expression of Nr5a1 and genes coding for steroidogenic enzymes other than Cyp11b2. Accumulation of Shh-expressing cells with expansion of Wnt/B-catenin gradient is also observed after dexamethasone-induced cor- tical atrophy, which is rapidly restored on dexamethasone withdrawal (32). Collectively, these observations demon- strate the importance of PKA signaling in providing differ- entiation cues that are essential for zG-zF transition, which involves both downregulation of Wnt/ß-catenin signaling at the zG-zF border and increased activation of the PKA- driven zF program.
The ability of the physiologic adrenal cortex to produce mineralocorticoids and glucocorticoids independently, ac- cording to specific demands, relies on anatomic and func- tional compartmentalization. The structural basis of this compartmentalization is the corticocapsular unit, established in embryogenesis shortly after encapsulation (see Fig. 2). The paracrine crosstalk between peripheral cortical progenitors (SHH+/SF1+ cells), and GLI+ capsular cells establishes a zone of high Wnt/B-catenin by releasing capsular RSPO3 into the upper cortex (31). High Wnt/ß-catenin activity is essential to maintain the progenitor cell pool, to promote zG differenti- ation, and to avoid premature zF differentiation by antagon- izing ACTH/PKA (56). In addition, several lines of evidence suggest high Wnt/ß-catenin activity is required for ATII- mediated aldosterone production (31, 60). As Wnt/B-catenin signaling fades centripetally, and cortical cells acquire the ability to respond to ACTH, PKA activity promotes a dramatic transition in the cell state and establishes the zG-zF boundary. In addition to promoting zF differentiation, ACTH also in- duces cell proliferation in the upper zF (32, 81). Interestingly, the mitogenic response to ACTH requires Wnt/ß-catenin signaling, as genetic ablation of Ctnnb1 in Wnt-responsive cells during cortical regeneration after dexamethasone- induced zF atrophy in mice significantly blunts cortical re- growth and zF differentiation (32). In the lower zF, cortical cells ultimately reach terminal differentiation and undergo apoptosis at the inner cortex (82). Cortical cells, therefore, follow a unidirectional trajectory of differentiation that is shaped both by signaling gradients, most important, Wnt/B- catenin signaling, and endocrine signaling (ATII and ACTH). Loss of these key paracrine and endocrine mediators lead to abnormal zonation, differentiation, and organ hypofunction. Interestingly, constitutive activation of paracrine and endo- crine signaling also disrupts the differentiation trajectory by locking cells in specific differentiation states that accumulate and may be prone to malignant transformation (54, 58, 83-86). In fact, benign and malignant adrenocortical neoplasms are characterized by recurrent somatic events targeting these pathways. However, while recurrent driver events provide an opportunity for targeted therapies, considerable challenges for implementing molecular targeted agents in ACC remain. These will be discussed in the following sections.
Unique Clinical Features of Adrenocortical Carcinoma Allude to Mechanisms of Disease
Our current understanding of adrenocortical carcinogenesis is largely informed by the aforementioned murine studies as well as familial genetics and genome-wide investigations that we will discuss in the subsequent sections. However, some interesting and unexpected clinical features of ACC have pro- vided important hints about disease pathogenesis and hetero- geneity, especially when we consider adrenocortical tumors (ACT) as existing along a spectrum of neoplasia, in which both cell identity programs and paracrine/endocrine signaling are uncoupled.
ACT are common human neoplasms affecting approxi- mately 5% to 10% of the population older than 60 years (87). ACT are usually found incidentally during radiological exams for unrelated complaints. Most ACT are benign adrenocortical adenomas (ACA), managed conservatively, with periodic clinical and radiologic follow-up. ACT associ- ated with malignant radiologic features or hormone excess syndromes are managed by surgery (88). In contrast, ACC is a
rare tumor, with a global incidence of 1/million to 1.5/million per year. However, while the prevalence of ACC among ad- renal incidentalomas is low, approximately 10% of all ACC are diagnosed as incidentalomas (89). While ACA increase in frequency with each decade of adulthood, the incidence of ACC has a bimodal distribution peaking around age 5 years and between the fourth and fifth decades of life (1, 90). These observations suggest that molecular events supporting benign tumorigenesis in the adrenal cortex are frequent (50). While there have been anecdotal reports of collision tumors con- taining adenomatous and carcinomatous compartments and carcinomas arising from longstanding incidentalomas (91, 92), these epidemiological data also suggest that a benign to malignant progression in the adrenal cortex is an exquisitely rare event, perhaps unlikely to account for the vast majority of ACC (91, 93, 94). Importantly, observations of malignant adrenocortical phenomena are currently limited by the histo- logical criteria used to diagnose ACC, which necessarily rely on several features that are achieved with sufficiently large tumor size (95, 96). The prevalence of carcinoma in situ that may progress to ACC is unknown.
The male-to-female ratio across all ACC is 1:1.5. Clinical manifestations of ACC are associated with mass effects of the primary tumor or metastasis, and steroid excess syndromes including primary aldosteronism, Cushing syndrome, and vir- ilization/feminization. In contrast to hormonally active ACA, which will routinely secrete one class of hormones (eg, aldos- terone or cortisol), ACC often produce mixed syndromes (most commonly secondary to androgen and cortisol cosecretion). While approximately 40% of ACC do not present with hor- monal excess syndromes, accumulation of steroid precursors (eg, 11-deoxycortisol) can be detected in up to 90% of cases (1, 90, 97). This is also in marked contrast to silent or hormo- nally active ACA, which produce significantly lower levels of steroid precursors. These unique features of ACC illustrate a profound disruption of adrenocortical differentiation specif- ically in malignancy, characterized by discordance between steroidogenesis and endocrine- or paracrine-mediated cell identity programs. Indeed, ACC with activating mutations in the Wnt/ß-catenin program (driving zG fate in physiology) are associated with cortisol rather than aldosterone produc- tion and exhibit features of zF differentiation (23).
Disease stage is the single most important clinical prog- nostic factor, as metastatic disease is refractory to standard therapies. For patients with localized disease, histological grade (assessed by either mitotic counts or the Ki67 score) is a commonly used tool for risk assessment and prognosis prediction. High-grade ACC, characterized by either more than 20 mitoses/50 high-power field or a Ki67 greater than 10% to 20%, bears a significantly higher risk of recurrence after an R0 surgical resection (8). In addition, the presence of hypercortisolism is also associated with an increased risk of recurrence, and a faster progression rate among advanced- disease patients (98, 99). These observations reveal the het- erogeneity that exists across ACC, and suggest that certain differentiation and proliferation cancer cell states support ag- gressive carcinogenesis.
Although most ACC is sporadic, inherited forms associ- ated with multiple neoplasia syndromes such as Li-Fraumeni syndrome, Beckwith-Wiedemann syndrome, Lynch syndrome, Birt-Hogg-Dube syndrome, adenomatous polyp- osis coli, and multiple endocrine neoplasia type I comprise
5% to 10% of the cases (100-103). The prevalence of germline TP53 mutations is particularly high among pedi- atric cases, ranging from 50% to 96%. In Southern and Southeastern Brazil, where the overall incidence of ACC is reported to be 10% to 15% higher than elsewhere and up to 4- to 6-fold higher in specific populations (104), more than 90% of pediatric cases and up to 20% of adult cases are associated with the p.R337H TP53 germline variant (103). Early molecular studies on sporadic ACC, based on candi- date gene approaches, were informed by the aforementioned rare inherited syndromes featuring ACC. This approach led to identification of prevalent somatic events in sporadic ACC, including loss of imprinting leading to overexpression of IGF2 (>90%), activating mutations of CTNNB1 (~15%), and inactivating mutations of tumor suppressors TP53 (~20%), APC, and Lynch syndrome-associated genes MSH2 and MSH6 (1, 103). Recent studies performed on larger multi-institutional cohorts using unbiased methods such as next-generation sequencing-based approaches and SNP arrays led to the identification of additional recurrently altered genes, including the Wnt/B-catenin regulator ZNRF3 (~20%); cell cycle regulators RB1, CDKN2A, CDK4, and CCNE1; telomere maintenance genes TERT, TERF2, ATRX, and DAXX, epigenetic regulators including MEN1 and genes encoding MLL and ATP-dependent SWI/SNF chro- matin remodelers; and PKA regulator PRKAR1A (22-24). Together, disruption of homeostatic paracrine (Wnt/ß- catenin), and endocrine (PKA) signaling by somatic events is observed in approximately 30% of ACC, suggesting that dysregulation of these pathways during ACC tumorigenesis is critical for sustained proliferation, recapitulating their im- portance in adrenal development and homeostasis.
Multiplatform Profiling of Adrenocortical Carcinoma Identifies Distinct Molecular Subtypes
High-throughput characterization of ACC using multiomics approaches, including exome sequencing, SNV arrays, Illumina methylation arrays, RNA sequencing, and microRNA sequencing, demonstrated that ACC is composed of distinct molecular subtypes (22, 23). According to ACC-TCGA, the largest and most comprehensive of these studies, 3 molecular subtypes can be distinguished by multiomics clustering, so-called COC1, COC2, and COC3 (Fig. 4) (23). Importantly, these 3 subgroups largely explain the clinical and hormonal heterogeneity that characterizes ACC. Briefly, COC1 ACC are the least aggressive (in terms of disease stage, event-free survival, and high-grade disease), and composed mostly of non-cortisol-producing tumors (including silent tumors and androgen-secreting). COC2 ACC present with intermediate levels of aggressiveness, and a higher prevalence of cortisol- producing tumors. COC3 ACC is the most aggressive and exhibits the highest proportion of cortisol-producing tumors and high-grade disease. Broadly, COC1 and COC2-COC3 overlap with the C1B and C1A transcriptome subgroups, respectively, as first described and correlated with survival outcomes by Assie, de Reynies, Bertherat and colleagues in a series of landmark publications (22, 105). As expected (22), recurrent somatic events are also unevenly distributed among ACC-TCGA molecular subclasses. While few recurrent som- atic SNVs and focal gains and losses are present in COC1, most events targeting Wnt/ß-catenin and cell cycle genes are concentrated in COC2 and COC3 (the latter is particularly
C1B
C1A
Event-free survival
COC1
COC2
COC3
100
IGF2 expression
Immune infiltration
Survival fraction (%)
75
ADS
CpGi methylation
Recurrence
50
Death
Cortisol
IGF2 LOI
Wnt pathway
25
Cell cycle
Chrom / Noisy
0
2
4
6
8
10
12
Years
COC1
COC2
COC3
SF1 low SF1 high ß-catenin PKA Ki67 CpGi methylation int/high
enriched for variants in cell cycle genes). Furthermore, while COC1-COC2 ACC have a somatic copy number alteration (SCNA) profile characterized by whole-chromosome gains and losses (an SCNA signature called “chromosomal”), COC3 ACC possess a high degree of genomic instability with numerous focal gains and losses (an SCNA signature called “noisy”).
Interesting and paradoxical observations emerged from these analyses. Unlike other cancers, in which dedifferenti- ation is associated with aggressive disease, the most aggressive subtype of ACC, COC3, is also the most differentiated from the perspective of the adrenal differentiation score (ADS), a score composed of a combination of several genes exclu- sively/differentially expressed by the normal adrenal cortex (23). As previously alluded to, cortisol-producing tumors are enriched for somatic events targeting genes encoding mem- bers of the Wnt/B-catenin pathway, ZNRF3, CTNNB1, and APC. While in the physiological adrenal cortex, the highest levels of Wnt/ß-catenin activation are associated with zG dif- ferentiation and aldosterone production, in ACC the highest Wnt/ß-catenin activity is associated with zF differentiation and cortisol production. Although these observations seem counterintuitive, they inform us about the potential cell of origin of these particular molecular subtypes of ACC (COC2- COC3). As suggested by observations in human and mouse studies, mitotic activity in the adrenal cortex is concentrated
in the upper zF (81, 82). Furthermore, ACTH-dependent ad- renal regeneration after dexamethasone-induced atrophy re- lies on increased cell proliferation in the zG-zF border in a Wnt/ß-catenin dependent-manner before zF replenishment and differentiation (32). These observations are consistent with a model in which the cell of origin of COC2-COC3 ACC (cortisol-producing; Wnt/ß-catenin-active) are transit- amplifying cells from the zG-zF border. These cells physio- logically exhibit intermediate levels of Wnt/ß-catenin activity (65) but are already committed to zF differentiation and re- spond to ACTH preferentially with proliferation (Fig. 5).
Two additional genetic mouse models that spontaneously develop ACC further support this framework. The first is a model developed Batisse-Lignier et al (106), featuring adrenocortical expression of the SV40 Large T antigen (AdTag), which simultaneously inactivates pRb and p53 to induce sustained cell cycle activation. The second is a model developed by Borges and colleagues (107) possessing gen- etic activation of ß-catenin and inactivation of p53 in all cells of the definitive adrenal cortex that have ever expressed Cyp11b2, termed BPCre. Intriguingly, both models exhibit similar tumor phenotypes with invariable progression to metastatic, glucocorticoid-producing ACC with a preceding dysplasia to carcinoma progression that starts at the zG-zF boundary. Despite genetically encoded differences in the 2 models regarding initial levels of Wnt/B-catenin activation
Recurrent genetic events Epigenetic, private genetic events
B-cat Ki67 PKA
Hyperplasia
Transformation
Metastatic dissemination
Capsule
Corticocapsular unit
zG
zF
ZR
(BPcre is characterized by intrinsic constitutive Wnt/ß-catenin activation, whereas AdTag is not), both models converge to the same phenotype, with selection for cells possessing autono- mous nuclear ß-catenin. We hypothesize that in COC2-COC3 ACC, epigenetic or genetic hits that impair differentiation and lock cells in a Wnt/B-catenin-active/transit amplifying state are selected for and vulnerable to secondary genetic and epi- genetic events that promote rapid cell growth (see Fig. 5).
The maintenance and selection pressure for a differenti- ated state (suggested also by recurrent somatic alterations in PRKAR1A in ACC-TCGA) support an oncogenic role for SF1-mediated transcription in ACC tumorigenesis (Fig. 6). As previously mentioned, SF1 is known to have metabolic and proliferative effects that might be advantageous to tumor cells. In fact, a role for increased SF1 expression and copy number has been demonstrated in pediatric ACC (108, 109). In addition, enforced high expression of NR5A1 in vitro is as- sociated with increased proliferation and invasiveness (110), as well as increased migratory capacity through regulation of cellular cytoskeleton (111). Another interesting observa- tion that supports a tumorigenic role for SF1-mediated zF differentiation is that ACC is among the tumor types with the least immune infiltration in TCGA. Transcriptome ana- lysis of ACC-TCGA data, and from other published micro- array studies, indicates an inverse correlation between ADS (and therefore the ability to synthesize cortisol) and immune cell-associated genes, suggesting that intratumoral cortisol synthesis is a mechanism of immune exclusion in ACC (23). In fact, a negative correlation between immune infiltration and survival was later demonstrated in an independent co- hort (112).
While ACC-TCGA molecular subtypes provide powerful information on mechanisms of tumorigenesis, prognosti- cation, and opportunities for prediction of subtype-specific
response to therapies, translating these information to the clinic is challenging. We and others have proposed the use of targeted molecular biomarkers for risk stratification (4, 105, 113, 114). In fact, a combination of a DNA methylation bio- marker (hypermethylation of the G0S2 locus), and the ex- pression levels of 2 transcripts (BUB1B and PINK1) stratifies ACC into 3 groups according to recurrence risk. Importantly, G0S2 hypermethylation recapitulates a signature of genome- wide CpG island hypermethylation (CIMP-high) that is al- most exclusively observed in COC3 ACC (see Fig. 4) (4). Furthermore, the rapid and homogeneous recurrence kinetics observed in CIMP-high/G0S2 methylated tumors despite ad- juvant mitotane suggest intrinsic resistance to this agent, the subject of our ongoing studies.
Clinical Investigation of Targeted Molecular Agents for Adrenocortical Carcinoma
Targeted molecular agents have emerged as useful therapeutic approaches for several malignancies, including hematological and solid tumors. However, the success of these therapies re- lies on the presence of tumor-specific molecular vulnerabil- ities that can be targeted by such agents, for example, highly expressed fusion transcripts or hotspot-activating mutations of tyrosine kinase receptors, and reliable assays to detect these alterations. Many of these therapeutic agents, therefore, are used in an individualized manner as so-called precision medicine. In addition to increasing therapeutic responses, a major potential advantage of such approaches is to restrict the toxic effects associated with classic cytotoxic agents. In ACC, early molecular studies demonstrating increased ex- pression of IGF2, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor provided the rationale for clinical trials testing tyrosine kinase inhibitors (reviewed in [21]). However, overall, these agents demonstrated overall
Migration & invasion
Differentiation & steroidogenesis
?
ß-cat
metabolism
Glycolytic
SF1
Immune infiltration
9000000 Proliferation
little to no benefit. The most extensively studied targeted agents for ACC are insulin-like growth factor receptor 1 (IGF1R) inhibitors. IGF1R is a tyrosine-kinase coupled re- ceptor by which IGF2 exerts its progrowth effects (115). This receptor appeared to be the ideal molecular target in ACC, since IGF2 is overexpressed in 90% of cases, and preclinical studies using different IGF1R inhibitors demonstrated prom- ising antitumor effects both in vitro and in vivo (116, 117). However, therapeutic responses were limited to 5% to 10% of patients in phase 1 to 3 clinical trials, failing to reach the threshold of uniform efficacy (improved overall survival com- pared to placebo-treated patients) (118-120).
While these clinical trials had disappointing results, further studies illuminated possible reasons for these therapeutic fail- ures. These include inadequate patient selection (all comers), and pharmacological interactions between several of these agents and mitotane (10, 11, 121, 122), later demonstrated to be a potent inducer of CYP3A4 in the liver. Along these lines, newer studies investigating application of tyrosine kinase in- hibitors including cabozantinib in patients with undetectable mitotane levels suggest potential for therapeutic response (123). Importantly, a major barrier to success in prior trials was an incomplete understanding of the landscape of som- atic alterations of ACC before high-throughput molecular profiling studies. Remarkably, these high-throughput studies
revealed that molecular heterogeneity defines key classes of ACC with homogeneous and distinct clinical outcomes, sug- gesting that “one-size-fits-all” approaches are bound to fail.
These data support a possible molecular explanation for intrinsic resistance to IGF1R inhibitors (and other targeted therapies): In addition to IGF2 overexpression, COC2-COC3 tumors exhibit strong activation of other progrowth signaling pathways, including constitutive Wnt/ß-catenin signaling and cell cycle activation due to TP53/RB1 loss. In these cases, IGF1R inhibition would be compensated by these other onco- genic hits. Several lines of evidence support the idea that resist- ance to monotherapy-targeted agents relies on the activation of additional oncogenic signaling pathways, rendering the cells independent from the original oncogenic hit (124). In fact, an in vivo model of ACC supports the synergism between IGF2 and Wnt/ß-catenin signaling for tumorigenesis (83). In other words, these tumors may exhibit prosurvival plastic responses to monotherapy with targeted agents. A recent in vitro study characterizing molecular mechanisms of acquired mitotane resistance supports this hypothesis (125). On treatment with low doses of mitotane, transcriptome analysis shows that the NCI-H295R cells progressively downregulate cholesterol me- tabolism/steroidogenesis genes (including SOAT1, a target of mitotane), and upregulate Wnt/ß-catenin target genes (125). These changes are in parallel with downregulation of genes associated with endoplasmic reticulum (ER) stress, revealing that resistant cells have bypassed the principal mechanism of mitotane toxicity through SOAT1 (13, 126).
More recently, clinical studies using immune checkpoint inhibitors have been conducted in ACC, with heterogeneous responses (127-132). In one of the largest studies, a phase 2 clinical enrolling 39 patients to receive the programmed cell death protein 1 (PD1) inhibitor pembrolizumab, an objective response was observed in 9 patients (23%), with an additional 7 (18%) patients achieving disease stabiliza- tion. In a large phase 1 study in metastatic ACC evaluating checkpoint blockade with avelumab targeting PD-L1, nearly 50% of patients achieved disease stabilization, and 6% ex- hibited an objective response (132). These results clearly in- dicate that pembrolizumab exhibited clinically meaningful antitumoral activity in a considerable subset of patients. However, it remains unclear which patients would benefit most from pembrolizumab since response was not correlated with traditional biomarkers of response such as PD1 expres- sion, mismatch-repair deficiency, and microsatellite instability (127). These promising results were recapitulated by another study using a combination of ipilimumab and nivolumab, anti-CTLA4 and anti-PD1 antibodies, respectively (130). Out of the 6 patients with ACC enrolled in this open-label, multicenter phase 2 trial, 2 exhibited partial response, and 2 exhibited disease stabilization, and tumors from these 4 pa- tients uniformly exhibited MSI-H microsatellite instability. This observation is in striking contrast with the results re- ported by Raj et al (127), in which the MSI-H phenotype was not associated with response to pembrolizumab alone. However, 5 patients exhibited severe (grade 3/4) toxicity, including 4 cases of hepatitis requiring discontinuation of the treatment, adrenalitis, and neutropenia. Collectively, these observations indicate that immunotherapy is a promising sys- temic option for ACC, with a substantial proportion of pa- tients exhibiting durable therapeutic responses-a result that has not yet been observed with any other class of conventional
or targeted systemic agents. Furthermore, immunotherapy may increase antitumorigenic effects of agents targeting other pathways, such as tyrosine kinase inhibitors (133). Additional studies to identify predictive biomarkers, and a better under- standing of the molecular mechanisms of response to treat- ment in the setting of accurate and informative preclinical models (134), are needed to escalate this therapeutic modality to its full potential.
Molecular Subtypes Expose Novel Therapeutic Vulnerabilities in Adrenocortical Carcinoma
Multiplatform studies illuminated that ACC is defined by homogeneous molecular subtypes with distinct clinical out- comes (23) amenable to identification using targeted ap- proaches (4). Importantly, no immediately actionable novel targets, for example, recurrent fusion transcripts or readily targetable hotspot mutations, emerged from these studies. These data suggest that characterization of homogeneous molecular classes and the prominent defining features of each class might provide a rationale for patient selection to specific therapeutic agents. Subtype-defining molecular fea- tures that can potentially be used to guide future therapeutic interventions include the degree of immune cell infiltration, SF1-dependent differentiation (including steroidogenesis capacity), Wnt/ß-catenin activity, constitutive activation of cell cycle genes, genomic/chromosomic instability with as- sociated activation of DNA repair pathways, and epigenetic dysregulation.
Adrenal Differentiation
In most human cancers, including solid tumors and hemato- logical malignancies, dedifferentiation is associated with ag- gressive disease. As we previously discussed, ACC is unique in that the most aggressive molecular subclass, COC3, bears the highest degree of differentiation with high expression of tissue-specific transcripts (including several SF1 and PKA tar- gets) culminating in increased cortisol production. This sug- gests that SF1-mediated transcription is exploited to confer a proliferative advantage to ACC cells (see Fig. 6); however, it also exposes several vulnerabilities that can be therapeutically targeted, and is the subject of the following sections.
Steroid Production and Immune Exclusion in Adrenocortical Carcinoma
In the last decade, immunotherapy has reemerged as an exciting therapeutic avenue for solid tumors, with several landmark pancancer studies revealing that tumors with mis- match repair deficiency are exquisitely sensitive to inhibition of physiologic checkpoints that restrain autoimmunity (eg, PD1/PD-L1, CTLA4) (135, 136). A widely accepted mech- anism for this sensitivity is that cancer cells upregulate im- mune checkpoints to evade immune detection, and mismatch repair deficiency leads to translation of mutant proteins that may serve as neoantigens with potential for immune recog- nition (137). These studies heralded accelerated pancancer approval of immune checkpoint blockade for mismatch repair-deficient tumors. In fact, limited evidence supports that mismatch repair-deficient ACC might indeed respond to im- mune checkpoint inhibition as discussed in prior sections (128, 130, 138). Contemporaneously and thereafter, a plethora of clinical studies revealed astonishing, long-term remission of previously non-mismatch repair-deficient lethal cancers like
metastatic melanoma and non-small cell lung cancer, leading to clinical practice changes that have significantly prolonged overall survival for patients with advanced forms of these dis- eases (139, 140). In the search for additional predictors of therapeutic response, investigators have also identified clinic- ally significant roles for measurement of immune infiltration and preexisting activation of the checkpoint (141).
As we previously discussed, ACC as a whole have a mixed response to immune checkpoint blockade. Indeed, from a mo- lecular subtype perspective, the vast majority of ACC (COC2- COC3) are immune poor (23, 142). This is consistent with the low level of PD-L1 expression identified in another study, suggesting low activity of this checkpoint in ACC (143). Only COC1 have significant immune infiltration, with non-ACC cells accounting for up to 50% of these tumors (23). This is particularly perplexing in light of the observation that COC3 possesses the highest mutational burden across ACC-TCGA, enriched for the noisy SCNA profile. These data suggest that additional factors that define COC2-COC3 may prevent im- mune infiltration and therefore checkpoint activation, even in the setting of high potential for neoantigen presentation. COC2-COC3 possess frequent somatic alterations leading to constitutive activation of Wnt/ß-catenin activity, known to suppress immune infiltration in other cancer types (144-146) though not clearly associated with resistance to immuno- therapy in ACC (127, 130).
Importantly, COC2-COC3 also possess higher degrees of ad- renal differentiation, with higher expression of steroidogenic enzymes and clinically meaningful hypercortisolism. Cortisol is a well-characterized immune suppressant-patients with Cushing syndrome present with increased risk for infections and disrupted immune cell function including glucocorticoid- induced apoptosis of lymphocytes and defects in myeloid cell migration (147, 148). These observations suggest that gluco- corticoid production may act as a shield to prevent tumor immune infiltration, subverting a requirement for activation of autoimmunity checkpoints. Inhibition of steroidogenesis or glucocorticoid receptor signaling may therefore be a prom- ising therapeutic strategy to sensitize COC2-COC3 tumors and trigger therapeutic response to immune checkpoint blockade. This area is the subject of ongoing research by our group and others (112). Indeed, combined glucocorticoid receptor antagonist relacorilant and pembrolizumab in ad- vanced ACC is the subject of an actively recruiting clinical trial (NCT04373265). Overcoming the intrinsic barriers to immune infiltration in COC2-COC3 ACC will likely be re- quired before evaluating efficacy of engineered cell therapies (eg, CAR T cells or other forms of engineered T cells); how- ever, given the high mutational burden (and likely neoantigen presentation) in COC3, this represents a promising thera- peutic avenue. Conversely, it remains to be seen if androgen production or COC1 status predict intrinsic susceptibility to immunotherapy.
Steroidogenesis as an Intrinsic Vulnerability
Steroid production is the principal and essential physio- logic function of the adrenal gland; early mortality in mice with adrenal agenesis can be prevented with exogenous glucocorticoid supplementation (149). Steroidogenesis in- volves a series of enzymatic oxidative reactions that take place in the mitochondria and ER in which cholesterol is converted into the different classes of steroid hormones. Furthermore, steroidogenesis intrinsically releases a series of
toxic byproducts, including reactive oxygen species (150). Therefore, steroidogenesis is an energetically expensive pro- cess requiring numerous transient and sustained adaptations to facilitate cholesterol transport, scavenging, cellular detoxi- fication, and timely and appropriate expression of synthetic enzymes. This is evidenced by specialized cells in a variety of tissues that metabolize cholesterol and may even engage in steroidogenesis (151-153). In the adrenal cortex, expres- sion of steroidogenic enzymes, lipid transporters, cholesterol scavengers, and other detoxification genes are thought to be directly or indirectly regulated by SF1 (47, 48, 154, 155). The machinery encoded by these genes represent a first-line de- fense mechanism against toxic byproducts of steroidogenesis in an adrenocortical cell.
As we previously described, ACC is characterized by mixed steroid production with the accumulation of steroid pre- cursors even in the setting of overt hypercortisolism, some- times also with concurrent secretion of mineralocorticoids, estrogens, and/or androgens. Patients with ACC and signs/ symptoms of hormone excess often have a higher disease burden (89), suggesting that malignant steroidogenesis is intrinsically inefficient, particularly when compared to the physiologic adrenal cortex and benign tumors (which, when large, may be only < 4 cm in diameter). This cellular program therefore represents a promising therapeutic vulnerability for ACC from multiple standpoints. It offers a high therapeutic index, given the rarity of cells in the body that engage in this program; it would mitigate hormone excess-associated mor- bidity in ACC; and it possesses multiple avenues for thera- peutic targeting.
Clinical utility of targeting steroidogenesis is widely sup- ported by the putative mechanisms of action of mitotane. While the range of molecular targets of mitotane remains poorly characterized, mitotane-induced toxicity is preceded by mitochondria swelling and degeneration, suggesting that this organelle is a major target (156). In fact, later studies demon- strated that mitotane induces a dysfunction in mitochondria- associated membranes, causing impairment of the respiratory chain (and hence steroidogenesis) and inducing caspase 3- and 7-dependent apoptosis (157, 158). Biochemical studies have suggested that mitotane’s cytotoxic actions require an enzymatic activation step, in which the drug is converted to an unstable acyl-chloride intermediate before being metab- olized to o,p’-DDA (159, 160). This unstable acyl-chloride intermediate reacts with unknown mitochondrial proteins, forming adducts that impair mitochondrial function (159, 160). Studies using an I125-labeled analogue of mitotane sug- gest that one such target is the p450-scc enzymes, consistent with inhibitory effects of mitotane on steroidogenesis, and indicating that CYP11A1 might be the enzyme that activates mitotane and therefore explain why adrenal cells are exquis- itely sensitive to this compound (161, 162).
More recently, SOAT1, an enzyme that is essential for cholesterol esterification in the ER, was identified as a mo- lecular target of mitotane. Inhibition of SOAT1 by genetic or pharmacological approaches leads to lipid-dependent toxicity preceded by activation of ER stress signaling (126). While efforts have been made to develop a more toxic form of mitotane (163), a complete characterization of its mo- lecular targets as well as the mechanisms by which it induces cell death would be required to develop alternative and more specific compounds, and to illuminate additional therapeutic targets.
Recent application of this strategy is exemplified by the use of the SOAT1 inhibitor nevanimibe (ATR-101). Initially developed as cholesterol-lowering agents, this class of drugs exhibited unexpected adrenal toxicity secondary to high ad- renal expression of SOAT1 (164, 165). Recently, this agent has been repurposed as an investigational compound to in- hibit steroidogenesis in Cushing syndrome, congenital ad- renal hyperplasia, and ACC (166, 167). Nevanimibe induces cholesterol-dependent apoptotic ACC cell death in vitro and in vivo (126, 168). More recently, a phase 1 clinical trial con- ducted in 63 patients with metastatic ACC has demonstrated disease stabilization in a subset of patients with few toxic ef- fects (167).
Because steroidogenesis is a process that releases substan- tial amounts of reactive oxygen species, defects in clearing these toxic byproducts can induce extensive damage in steroidogenic cells. It has been recently demonstrated that the normal adrenal cortex and ACC express high levels of glutathione peroxidase 4 (GPX4), an enzyme that re- duces hydroperoxides in a glutathione-dependent manner. Inhibition of GPX4 or depletion of glutathione by pharmaco- logical agents in steroidogenic ACC cell lines potently induces ferroptosis, a nonapoptotic iron-dependent form of cell death associated with lipid peroxidation. These observations expose a potential target for future therapies in ACC (169, 170).
Another therapeutic approach that exploits the adrenal differentiation/steroidogenesis program is based on the nonbarbiturate imidazole compound metomidate. Originally designed as an anesthetic, this compound strongly binds to 11ß-hydroxylase (encoded by CYP11B1 and CYP11B2) and inhibits its activity (171, 172). Because of this prop- erty, metomidate has been used as a radiotracer in positron emission tomography and single-photon emission computed tomography imaging techniques (including 11C-methomidate, 123I-methomidate, and 18F-FETO) to distinguish cortical from noncortical adrenal tumors, to identify laterality of aldosterone-producing adenomas, and to identify ACC me- tastasis (173-175). Hahner and colleagues (176, 177) tested the efficacy of 131I-methomidate as a therapeutic agent in a series of 11 patients with advanced ACC. One patient exhib- ited a partial response, with a 51% decrease in the size of target lesions, and 5 patients achieved disease stabilization (including sustained stabilization for > 24 months in some pa- tients). Given the overall good tolerability of this treatment, these results suggest that radiopharmaceuticals are a viable option for advanced ACC and warrant further development and research.
Targeting Genomic Instability and Cell Cycle
ACC is distinguished from ACA by significant upregulation of the cell cycle, measured by mitotic counts, Ki67, and even molecular markers like the BUB1B-PINK1 score (8, 96, 105, 178). Genome-wide multiplatform studies on ACC have re- vealed that even within malignant lesions, cell cycle activation exists along a spectrum, with COC3 tumors possessing the highest expression of proliferation-dependent genes. COC3 ACC is characterized by enrichment for somatic events leading to constitutive cell cycle activation (23). These include amplification of genes encoding cyclins and cyclin-dependent kinases (CDKs), epigenetic silencing or deletions of genes encoding CDK inhibitors (eg, CDKN2A), and recurrent LOF alterations in genes encoding guardians of the G1/S check- point (TP53 and RB1). The high degree of autonomous cell
cycle activation in COC3 ACC is also evidenced by significant enrichment for the noisy SCNA signature in these tumors, characterized by numerous focal gains and losses throughout the genome (23).
COC3 tumors have dismal clinical outcomes with invari- able progression to metastatic disease (4, 23). In light of his- torical observations that a subset of patients with advanced ACC exhibit clinically meaningful responses to cytotoxic chemotherapy known to preferentially target rapidly prolif- erative cells (with cytoreduction culminating in partial re- sponse in some cases) (179), it is possible that patients with COC3 disease may also respond to these traditional agents (4, 180, 181). These observations of course also point to a potentially meaningful role for novel, specific small-molecule inhibitors of CDKs (eg, palbociclib) that have been associated with disease regression for patients with other solid tumors (182, 183). Preliminary in vitro studies have suggested that ACC is susceptible to palbociclib (184, 185). Given the high frequency of LOF TP53 mutations in ACC, it is unlikely that therapeutic strategies targeting intact p53 (eg, MDM2 inhib- ition) will be effective for COC3 tumors as a class, but these agents remain an option for patients with COC3 disease and intact p53 signaling. Other potential cell cycle-associated tar- gets amendable for therapeutic intervention in COC3 tumors include polo-like kinase 1 (PLK1), maternal embryonic leu- cine zipper kinase (MELK), and aurora kinases (186-188).
Given the profound chromosomal instability that prevails in COC3 ACC, it is also possible that patients with ana- tomically accessible metastatic disease may be responsive to attempted cytoreduction with other strategies that induce fur- ther DNA damage, such as targeted radiation. Importantly, while COC3 tumors invariably possess high cell-cycle acti- vation, traditional markers currently implemented in clinical practice to measure proliferation index may be insensitive to capture all tumors that reside in this class (189); using alter- native surrogates to identify patients with COC3 tumors (eg, aberrant DNA methylation) can capture this class even in pa- tients with low-grade disease (4).
Recent pancancer studies incorporating ACC-TCGA sam- ples have also revealed that ACC is characterized by genomic instability signatures that may render them susceptible to ther- apies exploiting DNA damage response machinery. A subset of ACC possesses a genomic signature revealing evidence of defective homologous recombination (190). Tumors with homologous recombination deficiency, classically through inactivating mutations in genes encoding BRCA, FANC, and Rad50 family members, are exquisitely sensitive to PARP in- hibitors (191, 192). ACC do not possess recurrent mutations in these genes. However, the application of this class of ther- apies to tumors with frequent BRCA mutations (eg, ovarian cancer) has significantly prolonged patient survival, and also revealed that genetic events leading to homologous recombin- ation deficiency are not required for therapeutic efficacy (193, 194). Other targets that can be therapeutically exploited in this subgroup of ACC include the heat shock protein 90 (Hsp90) and the Wee1 kinase (195). These proteins are es- sential for an effective DNA damage response, and their in- hibition in different experimental models leads to cell death by mitotic catastrophe. Hsp90 inhibition has demonstrated antitumor activity in different ACC cell lines (196). While molecular predictors of response to these agents have not been fully characterized, dysfunctional p53 has been demon- strated to increase sensitivity to Wee1 inhibitors in different
cancer types, making this target particularly interesting in ACC. Moreover, these agents can be combined with radi- ation therapy and cytotoxic chemotherapy to overcome in- trinsic resistance to any single modality (197-199). In light of new evidence that cytoreduction even by surgical resection of oligometastatic disease is associated with prolonged survival for patients with ACC (200), these strategies hold substantial promise as adjuvant or neoadjuvant approaches.
Targeting Wnt/ß-Catenin Signaling
As previously discussed, one-third of ACC possess activating mutations in the Wnt/ß-catenin pathway, including activating mutations in CTNNB1, and LOF alterations of Wnt/ß- catenin-negative regulators APC and ZNRF3 (22-24). Somatic alterations in ZNRF3, CTNNB1, and APC are en- riched in C1A/COC2-COC3 ACC, suggesting subtype specifi- city of targeting this pathway. While the common denominator for these alterations is the constitutive activation of Wnt/ß- catenin signaling, CTNNB1- and APC-mutated tumors are ligand independent, and ZNRF3-deleted tumors require the presence of an extracellular Wnt ligand for pathway activa- tion (201). Different strategies for targeting Wnt/ß-catenin signaling have been proposed depending on the molecular defect that leads to constitutive pathway activation. These in- clude new monoclonal antibodies or small-molecule agents to promote inhibition of acylation and secretion of Wnt lig- ands (eg, porcupine inhibitors), antagonism of Fzd receptors and/or Wnt ligands, degradation of ß-catenin, or restriction of ß-catenin’s actions as a transcriptional coactivator by inhibiting its interactions with transcription factors like TCF/ LEF family members or epigenetic machinery like CBP (202, 203). Importantly, the high frequency of recurrent deletions in ZNRF3 (ligand-dependent activation) has opened new venues for targeting Wnt/B-catenin in ACC with agents that restrict ligand activity, such as porcupine inhibitors (204). However, despite the wide availability of a plethora of compounds, the clinical utility of pan-Wnt pathway inhibition has yet to be demonstrated. Wnt/ß-catenin is essential for stem/progenitor cell maintenance in a variety of tissues; pathway inhibition has been associated with numerous on-target toxicities (eg, diar- rhea secondary to intestinal mucosa atrophy), preventing any of these agents from advancing to phase 3 clinical trials (205, 206). Indeed, the observation that these agents have a low therapeutic index is actually not surprising, as patients with germline LOF mutations in ligand-dependent Wnt signaling components also exhibit intestinal malabsorption and several additional life-limiting abnormalities (59, 207, 208).
The high toxicity associated with different pan-Wnt pathway inhibition strategies suggest that novel approaches to target tissue-specific Wnt/ß-catenin signaling components are required. Possible strategies are targeting production or signaling through tissue-specific Wnt ligands (eg, WNT4), tissue-specific Fzd receptors, or tissue-specific nuclear part- ners of ß-catenin. More research into such components of this pathway will be required to develop novel agents with a high therapeutic index; ongoing investigations into this area are being led by our group and others. Furthermore, patients bearing tumors with Wnt pathway activation (regardless of ligand dependence of the alteration) also develop metastatic disease, necessitating systemic therapies. However, it remains to be seen if tumors with ligand-dependent alterations possess pathway activation at metastatic sites, for example, through autocrine stimulation by WNT4 or paracrine stimulation
by other Wnt ligands native to the metastatic site. If meta- static seeding away from the Wnt-active adrenal cortex erases evidence of Wnt signaling in ligand-dependent tumors, it is possible that Wnt pathway activation may be required only for early stages of tumorigenesis, limiting the spectrum of ACC for which targeting Wnt signaling would be clinically meaningful.
Targeting Epigenetic Programs
Transcriptional dysregulation is a core hallmark of cancer, and ACC is no exception. Abnormal transcription is sus- tained by alterations in protein-coding regions, encoding transcription factors, upstream signaling components, and regulators of chromatin dynamics; and also through alter- ations in noncoding regions like cis-regulatory elements. The contributions of each class of alterations to transcriptional dysregulation in ACC have been discussed previously and elsewhere (142). A unique type of epigenetic dysregulation in ACC involves the disruption of imprinted genes. The first evidence of dysregulation of imprinting in ACC came from studies exploring the mechanisms of IGF2 overexpression in these tumors. Loss of imprinting within the 11p15 locus could be detected in most cases, usually associated with re- duced expression of the cell-cycle regulator CDKN1C (209-211). Later studies have demonstrated abnormal ac- tivity of another imprinted region, the MEG3-DLK1 locus, characterized by hypermethylation and reduced expres- sion of a cluster of microRNAs located within this region. This abnormal pattern is almost exclusively observed in the C1B/COC1 molecular subclass of ACC (22, 23). In ACC, dysregulation of these imprinted regions is frequently asso- ciated with whole chromosome loss-of-heterozygosity (LOH) events, indicating a selective pressure for the chromosomal SCNA signature (and perhaps explaining why hypodiploidy is so frequently observed in ACC). Furthermore, widespread whole-chromosomal LOH and hypodiploidy may confer vulnerabilities to secondary recurrent events that inactivate tumor-suppressor genes. For example, in ACC-TCGA almost all tumors that exhibit focal deletion of ZNRF3 already lost the first copy by whole-chromosome LOH events involving chr22 (23).
Abnormal DNA methylation is perhaps the next most prom- inent feature of epigenetic dysregulation in ACC. In particular, as previously discussed, widespread DNA hypermethylation targeting CpG islands (CIMP-high) is a feature of COC3 ACC and a marker of aggressive disease, frequently associ- ated with constitutive cell-cycle activation, differentiation, and activation of Wnt/B-catenin signaling (4, 23). However, the role of CIMP-high in transcriptional dysregulation has not been characterized, and it is possible that this is a by- stander phenomenon secondary to rapid proliferation, wide- spread genomic instability (212), or an abnormal metabolic state (213). DNA hypermethylation is written by the DNA methyltransferases DNMT1 and DNMT3A/B, which are also regulated in a cell cycle-dependent manner by transcription factors of the E2F family-explaining the link with a consti- tutively active cell cycle observed in ACC (214, 215). While DNMT inhibitors are clinically available and are standard of care for certain hematological malignancies, their role for solid tumors remains to be demonstrated (216). Our prelim- inary in vitro studies using zebularine, a cytidine analogue that inhibits DNMT activity, demonstrated a mild cytostatic effect in the NCI-H295R (which possesses the CIMP-high
signature) (217) after a prolonged treatment, suggesting that targeting DNA hypermethylation in ACC may not be a vi- able therapeutic option, or the inhibitory effects on DNMTs obtained with conventional cytidine analogues are not potent enough. More recently, a new class of DNMT inhibitors that relies on allosteric inhibition has shown extremely potent inhibitory effects in other models (218) and remains to be tested in ACC. Given the strong association between CIMP- high and dismal outcomes for patients with ACC, a better understanding of the biological processes driving CIMP-high, including a full characterization of metabolic dysregulation in ACC subtypes, and other epigenetic alterations involving histone marks may reveal additional opportunities to target epigenetic dysregulation in ACC (181).
While considerable advances in therapeutic approaches targeting epigenetic dysregulation in solid tumors have been reported, it remains a challenging enterprise and an active area of research. Novel approaches targeting chromatin regulators have recently been described for several solid tumors (219, 220), but they largely rely on tumor-specific vulnerabilities and metabolic profiles. Recently, alterations in several chro- matin remodelers have been described in ACC, adding to early studies that identified ACC as part of the MEN1 spectrum. In addition to recurrent mutations in MEN1 in sporadic ACC, other somatic alterations target genes encoding MLL and SWI/ SNF family members (23, 221), suggesting that dysregulation of chromatin dynamics is a feature of the molecular patho- genesis of ACC. Interestingly, dysregulation of chromatin dynamics seems to converge on activation of an oncogenic SF1-driven transcriptional program, as suggested by a recent pancancer chromatin accessibility study incorporating sam- ples from ACC-TCGA. This study revealed that ACCs possess a unique epigenetic profile, defined by accessibility and ac- tivation of the SF1-dependent transcriptional and epigenetic program characterized by increased accessibility not only in the promoter regions of target genes, but also several de novo putative distal regulatory elements. These observations sug- gest inhibition of pioneer factors unique to ACC and adrenal tissue may offer a high therapeutic index through targeting of a cell identity/differentiation program that is selected for in COC2-COC3 ACC (222).
Advances in the understanding of epigenetic repro- gramming in cancer have illuminated several novel cancer codependencies. Given the variable activating and repressive effects of different epigenetic marks, cancers that possess de- creased activation of one pathway may be vulnerable to inhib- ition of the other pathway. This is best exemplified by tumors possessing LOF alterations in SWI/SNF machinery, which are exquisitely sensitive to inhibition of an antagonistic repres- sive epigenetic complex, the Polycomb repressive complex 2 (PRC2) (223, 224). The catalytically active member of the PRC2 is a histone H3 lysine 27 methyltransferase, EZH2, that has been the subject of extensive investigation in many can- cers including ACC secondary to its high cell-cycle dependence (225-227). SWI/SNF mutations are frequent in human can- cers (228), and the discovery of this therapeutic vulnerability along with the simultaneous discovery of recurrent activating EZH2 mutations in lymphomas (229, 230) has spurred the rapid development of several small molecules targeting PRC2 (231), culminating in the recent FDA approval of tazemetostat for patients with tumors bearing gain-of-function alterations in EZH2. Intriguingly, the physiologic role of EZH2 is to pro- mote stemness and pluripotency (232-234); however, in the
adrenal, EZH2 facilitates zG to zF transdifferentiation and response to ACTH (78). These data suggest that targeting EZH2 may also target the differentiation program that pre- vails COC3 ACC, supported by our ongoing work (217).
Conclusion and Future Directions
Despite substantial advances in our understanding of the molecular pathogenesis of ACC, treatment outcomes remain dismal for the majority of patients. Therapeutic responses to conventional and targeted agents are heterogeneous, indicating that the current “one-size-fits-all” approaches are suboptimal, at best, in all settings. Furthermore, recent
multiomics studies have provided important conceptual ad- vances that can be used to guide the development of new therapeutic strategies using existing and novel agents. Clinical heterogeneity in ACC is mirrored by distinct molecular sub- types that possess unique features, including recurrent gen- omic alterations targeting few core signaling pathways; distinct epigenetic patterning; distinct forms of chromosomal instability; differential engagement of DNA repair programs; and transcriptional modules that capture variable degrees of adrenocortical differentiation, immune cell infiltration, mitotic activity, and Wnt/B-catenin signaling (Fig. 7). Overcoming the limitations of current therapeutic approaches necessitates ac- counting for molecular heterogeneity. Molecular information
Wnt secretion and signaling
2
2
WNT4
RSPO3
WNT4
FZD
LGR4/5
LRP5 /6
m
Ub
Ub
Ub
ZNRF3
Immune checkpoint
B-catenin P
Destruction complex
M
APC
G1
P
P
P
G2
Cellular metabolism
Growth factor signaling (e.g. IGF2/IGF1R)
S
2
WNT4
Steroidogenesis
PORCN
Cell cycle and genomic instability
ß-catenin
CBP
TCF/LEF
SF1
ß-catenin
EZH2
?
Fo
2+
DNMT
Cell identity, differentiation, stemness and epigenetic programming
GPX4
Ferroptosis
can lead to improved risk stratification strategies to guide ad- juvant therapies, and therapeutic interventions for advanced disease. However, while several studies have recently demon- strated the clinical relevance of molecular data, translating these discoveries into clinical practice remains challenging. Minimalistic targeted approaches that capture the most clin- ically relevant molecular information from widely available clinical specimens such as formalin-fixed, paraffin-embedded tissue (235) and plasma (236-239) need to be further devel- oped and validated. These putative biomarkers can be used for guiding mechanism-based therapeutic interventions to specific and well-defined subgroups both in the adjuvant set- ting and in late-stage disease.
Given the rarity of ACC and therefore the challenges of rapidly accruing clinical data on a timeline at pace with sci- entific and mechanistic discoveries, it will be crucial to recon- sider the clinical trial model. For patients with slower-growing disease (eg, COC1 and some COC2), practice-changing clin- ical data through traditional phase 1 to 3 clinical trials may emerge only after a decade or more of enrollment (6). For patients with rapidly growing disease (eg, COC2-COC3), this approach is viable only in the relapsed/recurrent setting and at high risk of trial failure. Given the recent advances in preclinical modeling of ACC (134, 138, 240-242), bench- to-bedside approaches hold considerable promise improving clinical trial outcomes by prioritizing therapeutic agents that have a higher potential to provide therapeutic benefit. If com- prehensive care centers develop pipelines adopting these ap- proaches in combination with rapid molecular subtyping and high-throughput screening platforms (eg, patient-derived organoids), targeted small-molecule based therapies may soon have opportunities for success in ACC.
ACC is widely known for being resistant to several forms of systemic therapy. Understanding the molecular basis of resist- ance is essential to develop new therapeutic strategies, even provided the existence of high-throughput screening plat- forms. Moreover, within a single patient with ACC, different foci of disease (eg, metastatic vs adrenal sites) may exhibit dis- cordant responses to medical agents. These data suggest that cancer-cell heterogeneity plays an important and clinically relevant role. While genetic, genomic, and epigenetic hetero- geneity has been documented among primary and metastatic ACC lesions (4, 243-245), cellular plasticity might ultimately be the culprit of therapeutic resistance (246). Plasticity can be defined as an ability to adopt different identities along the phenotypic spectra that resemble distinct developmental lin- eages, cell identities, and metabolic states. This is achieved by dynamic and reversible epigenetic reprogramming of different transcriptional modules. Plasticity is thought to be an adaptive response to stressors, including hypoxia, fuel deprivation, im- mune system activity, and systemic therapies, enabling adap- tation and survival (247, 248). Examples of plasticity include epithelial-to-mesenchymal transition, a process by which cells from carcinomas assume mesenchymal characteristics ren- dering the migratory and invasive capacity essential for meta- static spread (249). Recently, plasticity has been documented as a mechanism of resistance to systemic targeted therapies. Prostate cancer and EGFR-mutated non-small cell lung cancer that initially respond to agents targeting the androgen receptor and EGFR, respectively, assume a neuroendocrine- like identity as therapeutic resistance emerges (250, 251, 252). This new cellular identity, characterized by the expression of
several neuroendocrine markers, renders cells independent of the initial oncogenic hits, and therefore, resistant to agents targeting these programs (124, 254). Plasticity may emerge as a result of therapeutic interventions (125); however, recent studies using lineage-tracing techniques in murine models of prostate and lung cancers have demonstrated the existence of plastic cell states within a tumor even before therapeutic intervention. Changes in cell identity in these systems are as- sociated with certain genetic hits, including RB1 and TP53 loss, and the APOBEC signature (26, 253, 254). These data support a model that certain cell types within a tissue with intrinsic lineage infidelity may be selected for in early stages of carcinogenesis.
While plasticity has been recently recognized as a major driver of therapeutic resistance, there are currently no effica- cious therapeutic interventions that restrict or mitigate plas- ticity. Preclinical studies suggest that inhibition of epigenetic programs may restrict plasticity, and clinical trials evaluating combination therapy with agents targeting the original onco- genic hit and emerging plasticity programs are ongoing (124, 248). Little is known about cellular states that ACC can as- sume spontaneously or as a result of systemic therapies. A comprehensive characterization of cell heterogeneity in ACC using single-cell technologies both in human samples and mouse models may provide useful biological insights. Indeed, these studies in the physiologic adrenal gland are cur- rently underway (255). Understanding the diversity of states a single ACC cell can adopt might be the most critical in- sight to enable the development of new strategies to overcome therapy resistance for patients facing this devastating disease.
Financial Support
This work was supported by the National Institutes of Health (grant Nos. R01 DK043140 to A.M.L. and R01 DK062027 to A.M.L. and G.D.H.), the United States Department of Defense (grant Nos. CA180750 and CA18751 A.M.L., D.R.M., and G.D.H.), the Cissell-Roell Innovation Fund (to A.M.L., D.R.M., and G.D.H.), the University of Michigan Medical Scientist Training Program (No. T32 GM7863 to D.R.M.), and the Drew O’Donoghue Fund (to D.R.M. and G.D.H.).
Disclosures
A.M.L., D.R.M., and G.D.H. are coinventors on 3 pending patent applications owned by the Regents of the University of Michigan on methods for characterizing and treating adrenocortical carcinoma. G.D.H. is founder and stock owner of Vasaragen, Inc (private).
References
1. Else T, Kim A, Sabolch A, et al. Adrenocortical carcinoma. Endocr Rev. 2014;35(2):2372-2380.
2. Terzolo M, Angeli A, Fassnacht M, et al. Adjuvant mitotane treatment for adrenocortical carcinoma. N Engl J Med. 2007;356(23):2372-2380.
3. Fassnacht M, Dekkers OM, Else T, et al. European Society of Endocrinology Clinical Practice Guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol. 2018;179(4):G1-G46.
4. Mohan DR, Lerario AM, Else T, et al. Targeted assessment of G0S2 methylation identifies a rapidly recurrent, routinely fatal molecular subtype of adrenocortical carcinoma. Clin Cancer Res. 2019;25(11):3276-3288.
5. Glenn JA, Else T, Hughes DT, et al. Longitudinal patterns of re- currence in patients with adrenocortical carcinoma. Surgery. 2019;165(1):186-195.
6. Terzolo M, Fassnacht M, Perotti P, et al. Results of the ADIUVO Study, the first randomized trial on adjuvant mitotane in adrenocortical carcinoma patients. J Endocr Soc. 2021;5(Suppl 1):A166-A167.
7. Kimpel O, Bedrose S, Megerle F, et al. Adjuvant platinum-based chemotherapy in radically resected adrenocortical carcinoma: a co- hort study. Br J Cancer. 2021;125(9):1233-1238.
8. Beuschlein F, Weigel J, Saeger W, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resec- tion. J Clin Endocrinol Metab. 2015;100(3):841-849.
9. Megerle F, Herrmann W, Schloetelburg W, et al. German ACC Study Group. Mitotane monotherapy in patients with ad- vanced adrenocortical carcinoma. J Clin Endocrinol Metab. 2018;103(4):1686-1695.
10. Chortis V, Taylor AE, Schneider P, et al. Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5a-reductase, explaining the need for personalized glucocorticoid and androgen replacement. J Clin Endocrinol Metab. 2013;98(1):161-171.
11. Kroiss M, Quinkler M, Lutz WK, Allolio B, Fassnacht M. Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma. Clin Endocrinol (Oxf). 2011;75(5):585-591.
12. Puglisi S, Calabrese A, Basile V, et al. New perspectives for mitotane treatment of adrenocortical carcinoma. Best Pract Res Clin Endocrinol Metab. 2020;34(3):101415.
13. Sbiera S, Leich E, Liebisch G, et al. Mitotane inhibits sterol-O-Acyl transferase 1 triggering lipid-mediated endoplasmic reticulum stress and apoptosis in adrenocortical carcinoma cells. Endocrinology. 2015;156(11):3895-3908.
14. Warde KM, Schoenmakers E, Ribes Martinez E, et al. Liver X re- ceptor inhibition potentiates mitotane-induced adrenotoxicity in ACC. Endocr Relat Cancer. 2020;27(6):361-373.
15. Altieri B, Sbiera S, Herterich S, et al. Effects of germline CYP2W1*6 and CYP2B6*6 single nucleotide polymorphisms on mitotane treatment in adrenocortical carcinoma: a multicenter ENSAT study. Cancers (Basel). 2020;12(2):359.
16. D’Avolio A, De Francia S, Basile V, et al. Influence of the CYP2B6 polymorphism on the pharmacokinetics of mitotane. Pharmacogenet Genomics. 2013;23(6):293-300.
17. Reidy-Lagunes DL, Lung B, Untch BR, et al. Complete responses to mitotane in metastatic adrenocortical carcinoma-a new look at an old drug. Oncologist. 2017;22(9):1102-1106.
18. El Ghorayeb N, Rondeau G, Latour M, et al. Rapid and complete re- mission of metastatic adrenocortical carcinoma persisting 10 years after treatment with mitotane monotherapy: case report and re- view of the literature. Medicine (Baltimore). 2016;95(13):e3180.
19. Bates SE, Shieh CY, Mickley LA, et al. Mitotane enhances cyto- toxicity of chemotherapy in cell lines expressing a multidrug re- sistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas. J Clin Endocrinol Metab. 1991;73(1):18-29.
20. Dogliotti L, Berruti A, Pia A, Paccotti P, Alì A, Angeli A. Cytotoxic chemotherapy for adrenocortical carcinoma. Minerva Endocrinol. 1995;20(1):105-109.
21. Megerle F, Kroiss M, Hahner S, Fassnacht M. Advanced adrenocortical carcinoma-what to do when first-line therapy fails? Exp Clin Endocrinol Diabetes. 2019;127(2-03):109-116.
22. Assié G, Letouzé E, Fassnacht M, et al. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet. 2014;46(6):607-612.
23. Zheng S, Cherniack AD, Dewal N, et al. Cancer Genome Atlas Research Network. Comprehensive pan-genomic characterization of adrenocortical carcinoma. Cancer Cell. 2016;29(5):723-736.
24. Juhlin CC, Goh G, Healy JM, et al. Whole-exome sequencing characterizes the landscape of somatic mutations and copy number alterations in adrenocortical carcinoma. J Clin Endocrinol Metab. 2015;100(3):E493-E502.
25. Pinto EM, Chen X, Easton J, et al. Genomic landscape of paediatric adrenocortical tumours. Nat Commun. 2015;6:6302.
26. Marjanovic ND, Hofree M, Chan JE, et al. Emergence of a high- plasticity cell state during lung cancer evolution. Cancer Cell. 2020;38(2):229-246.e13.
27. Chang HH, Hemberg M, Barahona M, Ingber DE, Huang S. Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature. 2008;453(7194):544-547.
28. Val P, Martinez-Barbera JP, Swain A. Adrenal development is initiated by Cited2 and Wt1 through modulation of Sf-1 dosage. Development. 2007;134(12):2349-2358.
29. Xing Y, Lerario AM, Rainey W, Hammer GD. Development of adrenal cortex zonation. Endocrinol Metab Clin North Am. 2015;44(2):243-274.
30. Keegan CE, Hammer GD. Recent insights into organogenesis of the adrenal cortex. Trends Endocrinol Metab. 2002;13(5):200-208.
31. Vidal V, Sacco S, Rocha AS, et al. The adrenal capsule is a signaling center controlling cell renewal and zonation through Rspo3. Genes Dev. 2016;30(12):1389-1394.
32. Finco I, Lerario AM, Hammer GD. Sonic hedgehog and WNT signaling promote adrenal gland regeneration in male mice. Endocrinology. 2018;159(2):579-596.
33. King P, Paul A, Laufer E. Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages. Proc Natl Acad Sci U S A. 2009;106(50):21185-21190.
34. Revest JM, Spencer-Dene B, Kerr K, De Moerlooze L, Rosewell I, Dickson C. Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4. Dev Biol. 2001;231(1):47-62.
35. Guasti L, Cavlan D, Cogger K, et al. Dlk1 up-regulates Gli1 expres- sion in male rat adrenal capsule cells through the activation of beta 1 integrin and ERK1/2. Endocrinology. 2013;154(12):4675-4684.
36. Grabek A, Dolfi B, Klein B, Jian-Motamedi F, Chaboissier MC, Schedl A. The adult adrenal cortex undergoes rapid tissue renewal in a sex-specific manner. Cell Stem Cell. 2019;25(2):290-296.e2.
37. Mesiano S, Jaffe RB. Developmental and functional biology of the primate fetal adrenal cortex. Endocr Rev. 1997;18(3):378-403.
38. Narasaka T, Suzuki T, Moriya T, Sasano H. Temporal and spatial distribution of corticosteroidogenic enzymes immunoreactivity in developing human adrenal. Mol Cell Endocrinol. 2001;174(1-2):111-120.
39. Zubair M, Parker KL, Morohashi K. Developmental links between the fetal and adult zones of the adrenal cortex revealed by lineage tracing. Mol Cell Biol. 2008;28(23):7030-7040.
40. Honda S, Morohashi K, Nomura M, Takeya H, Kitajima M, Omura T. Ad4BP regulating steroidogenic P-450 gene is a member of steroid hormone receptor superfamily. J Biol Chem. 1993;268(10):7494-7502.
41. Bamforth SD, Bragança J, Eloranta JJ, et al. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat Genet. 2001;29(4):469-474.
42. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC complementation shows a requirement for Wt1 in the develop- ment of epicardium, adrenal gland and throughout nephrogenesis. Development. 1999;126(9):1845-1857.
43. Schnabel CA, Selleri L, Cleary ML. Pbx1 is essential for ad- renal development and urogenital differentiation. Genesis. 2003;37(3):123-130.
44. Luo X, Ikeda Y, Parker KL. A cell-specific nuclear receptor is essen- tial for adrenal and gonadal development and sexual differentia- tion. Cell. 1994;77(4):481-490.
45. Achermann JC, Ito M, Hindmarsh PC, Jameson JL. A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet. 1999;22(2):125-126.
46. Wood MA, Acharya A, Finco I, et al. Fetal adrenal capsular cells serve as progenitor cells for steroidogenic and stromal adrenocortical cell lineages in M. musculus. Development. 2013;140(22):4522-4532.
47. Baba T, Otake H, Sato T, et al. Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1. Nat Commun. 2014;5:3634.
48. Lalli E, Doghman M, Latre de Late P, El Wakil A, Mus-Veteau I. Beyond steroidogenesis: novel target genes for SF-1 discovered by genomics. Mol Cell Endocrinol. 2013;371(1-2):154-159.
49. Nishimoto K, Rigsby CS, Wang T, et al. Transcriptome anal- ysis reveals differentially expressed transcripts in rat ad- renal zona glomerulosa and zona fasciculata. Endocrinology. 2012;153(4):1755-1763.
50. Nishimoto K, Tomlins SA, Kuick R, et al. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands. Proc Natl Acad Sci U S A. 2015;112(33):E4591-E4599.
51. Laufer E, Kesper D, Vortkamp A, King P. Sonic hedgehog signaling during adrenal development. Mol Cell Endocrinol. 2012;351(1):19-27.
52. Tissier F, Cavard C, Groussin L, et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res. 2005;65(17):7622-7627.
53. Nusse R, Clevers H. Wnt/ß-catenin signaling, disease, and emerging therapeutic modalities. Cell. 2017;169(6):985-999.
54. Berthon A, Sahut-Barnola I, Lambert-Langlais S, et al. Constitutive beta-catenin activation induces adrenal hyperplasia and promotes adrenal cancer development. Hum Mol Genet. 2010;19(8):1561-1576.
55. Kim AC, Reuter AL, Zubair M, et al. Targeted disruption of beta- catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex. Development. 2008;135(15):2593-2602.
56. Drelon C, Berthon A, Sahut-Barnola I, et al. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development. Nat Commun. 2016;7:12751.
57. Huang CC, Liu C, Yao HH. Investigating the role of adrenal cortex in organization and differentiation of the adrenal medulla in mice. Mol Cell Endocrinol. 2012;361(1-2):165-171.
58. Pignatti E, Leng S, Yuchi Y, et al. Beta-catenin causes adrenal hyperplasia by blocking zonal transdifferentiation. Cell Rep. 2020;31(3):107524.
59. Mandel H, Shemer R, Borochowitz ZU, et al. SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function muta- tion in WNT4. Am J Hum Genet. 2008;82(1):39-47.
60. Heikkilä M, Peltoketo H, Leppäluoto J, Ilves M, Vuolteenaho O, Vainio S. Wnt-4 deficiency alters mouse adrenal cortex func- tion, reducing aldosterone production. Endocrinology. 2002;143(11):4358-4365.
61. Gummow BM, Winnay JN, Hammer GD. Convergence of Wnt signaling and steroidogenic factor-1 (SF-1) on transcription of the rat inhibin alpha gene. J Biol Chem. 2003;278(29):26572-26579.
62. Mizusaki H, Kawabe K, Mukai T, et al. Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) gene transcription is regulated by Wnt4 in the female developing gonad. Mol Endocrinol. 2003;17(4):507-519.
63. Hossain A, Saunders GF. Synergistic cooperation between the ß-catenin signaling pathway and steroidogenic factor 1 in the acti- vation of the mullerian inhibiting substance type II receptor. J Biol Chem. 2003;278(29):26511-26516.
64. Kennell JA, O’Leary EE, Gummow BM, Hammer GD, MacDougald OA. T-cell factor 4N (TCF-4N), a novel isoform of mouse TCF-4, synergizes with beta-catenin to coactivate C/ EBPalpha and steroidogenic factor 1 transcription factors. Mol Cell Biol. 2003;23(15):5366-5375.
65. Basham KJ, Rodriguez S, Turcu AF, et al. A ZNRF3-dependent Wnt/ß-catenin signaling gradient is required for adrenal homeo- stasis. Genes Dev. 2019;33(3-4):209-220.
66. Lerario AM, Finco I, LaPensee C, Hammer GD. Molecular mechanisms of stem/progenitor cell maintenance in the adrenal cortex. Front Endocrinol (Lausanne). 2017;8:52.
67. Ching S, Vilain E. Targeted disruption of Sonic Hedgehog in the mouse adrenal leads to adrenocortical hypoplasia. Genesis. 2009;47(9):628-637.
68. Huang CC, Miyagawa S, Matsumaru D, Parker KL, Yao HH. Progenitor cell expansion and organ size of mouse adrenal is regulated by sonic hedgehog. Endocrinology. 2010;151(3):1119-1128.
69. Yates R, Katugampola H, Cavlan D, et al. Adrenocortical de- velopment, maintenance, and disease. Curr Top Dev Biol. 2013;106:239-312.
70. Chan LF, Metherell LA, Clark AJL. Effects of melanocortins on ad- renal gland physiology. Eur J Pharmacol. 2011;660(1):171-180.
71. Kramer RE, Gallant S, Brownie AC. Actions of angiotensin II on aldosterone biosynthesis in the rat adrenal cortex. Effects on cyto- chrome P-450 enzymes of the early and late pathway. J Biol Chem. 1980;255(8):3442-3447.
72. Fujita K, Aguilera G, Catt KJ. The role of cyclic AMP in aldoste- rone production by isolated zona glomerulosa cells. J Biol Chem. 1979;254(17):8567-8574.
73. Aguilera G, Capponi A, Baukal A, Fujita K, Hauger R, Catt KJ. Metabolism and biological activities of angiotensin II and des-Asp1- angiotensin II in isolated adrenal glomerulosa cells. Endocrinology. 1979;104(5):1279-1285.
74. Chan LF, Webb TR, Chung TT, et al. MRAP and MRAP2 are bidi- rectional regulators of the melanocortin receptor family. Proc Natl Acad Sci U S A. 2009;106(15):6146-6151.
75. Baccaro RB, Mendonça PO, Torres TE, Lotfi CF. Immunohistochemical Jun/Fos protein localization and DNA syn- thesis in rat adrenal cortex after treatment with ACTH or FGF2. Cell Tissue Res. 2007;328(1):7-18.
76. Rosenberg D, Groussin L, Jullian E, Perlemoine K, Bertagna X, Bertherat J. Role of the PKA-regulated transcription factor CREB in development and tumorigenesis of endocrine tissues. Ann N Y Acad Sci. 2002;968:65-74.
77. Aesøy R, Mellgren G, Morohashi K, Lund J. Activation of cAMP- dependent protein kinase increases the protein level of steroidogenic factor-1. Endocrinology. 2002;143(1):295-303.
78. Mathieu M, Drelon C, Rodriguez S, et al. Steroidogenic dif- ferentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex. Proc Natl Acad Sci USA. 2018;115(52):E12265-E12274.
79. Chida D, Sato T, Sato Y, et al. Characterization of mice deficient in melanocortin 2 receptor on a B6/Balbc mix background. Mol Cell Endocrinol. 2009;300(1-2):32-36.
80. Novoselova TV, Hussain M, King PJ, et al. MRAP deficiency impairs adrenal progenitor cell differentiation and gland zonation. FASEB J. 2018;32(11):fj201701274RR.
81. Chang SP, Morrison HD, Nilsson F, Kenyon CJ, West JD, Morley SD. Cell proliferation, movement and differentiation during maintenance of the adult mouse adrenal cortex. PLoS One. 2013;8(12):e81865.
82. Sasano H, Imatani A, Shizawa S, Suzuki T, Nagura H. Cell prolifer- ation and apoptosis in normal and pathologic human adrenal. Mod Pathol. 1995;8(1):11-17.
83. Heaton JH, Wood MA, Kim AC, et al. Progression to adrenocortical tumorigenesis in mice and humans through insulin- like growth factor 2 and ß-catenin. Am J Pathol. 2012;181(3): 1017-1033.
84. Sahut-Barnola I, de Joussineau C, Val P, et al. Cushing’s syndrome and fetal features resurgence in adrenal cortex-specific Prkar1a knockout mice. PLoS Genet. 2010;6(6):e1000980.
85. Morin E, Mete O, Wasserman JD, Joshua AM, Asa SL, Ezzat S. Carney complex with adrenal cortical carcinoma. J Clin Endocrinol Metab. 2012;97(2):E202-E206.
86. Taylor MJ, Ullenbruch MR, Frucci EC, et al. Chemogenetic acti- vation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation. J Clin Invest. 2020;130(1): 83-93.
87. Arnaldi G, Boscaro M. Adrenal incidentaloma. Best Pract Res Clin Endocrinol Metab. 2012;26(4):405-419.
88. Fassnacht M, Arlt W, Bancos I, et al. Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol. 2016;175(2):G1-G34.
89. Abiven G, Coste J, Groussin L, et al. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J Clin Endocrinol Metab. 2006;91(7):2650-2655.
90. Wajchenberg BL, Albergaria Pereira MA, Medonca BB, et al. Adrenocortical carcinoma: clinical and laboratory observations. Cancer. 2000;88(4):711-736.
91. Kohli HS, Manthri S, Jain S, et al. An adrenocortical carci- noma evolving after nine years of latency from a small adrenal incidentaloma. Cureus. 2021;13(8):e16851.
92. Nogueira TM, Lirov R, Caoili EM, et al. Radiographic character- istics of adrenal masses preceding the diagnosis of adrenocortical cancer. Horm Cancer. 2015;6(4):176-181.
93. Belmihoub I, Silvera S, Sibony M, et al. From benign adrenal incidentaloma to adrenocortical carcinoma: an exceptional random event. Eur J Endocrinol. 2017;176(6):K15-K19.
94. Rebielak ME, Wolf MR, Jordan R, Oxenberg JC. Adrenocortical carcinoma arising from an adrenal adenoma in a young adult fe- male. J Surg Case Rep. 2019;2019(7):rjz200.
95. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol. 1984;8(3):163-169.
96. Weiss LM, Medeiros LJ, Vickery AL. Pathologic features of prog- nostic significance in adrenocortical carcinoma. Am J Surg Pathol. 1989;13(3):202-206.
97. Arlt W, Biehl M, Taylor AE, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab. 2011;96(12):3775-3784.
98. Libé R, Borget I, Ronchi CL, et al; ENSAT Network. Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study. Ann Oncol. 2015;26(10):2119-2125.
99. Berruti A, Fassnacht M, Haak H, et al. Prognostic role of overt hypercortisolism in completely operated patients with adrenocortical cancer. Eur Urol. 2014;65(4):832-838.
100. Raymond VM, Everett JN, Furtado LV, et al. Adrenocortical car- cinoma is a Lynch syndrome-associated cancer. J Clin Oncol. 2013;31(24):3012-3018.
101. Raymond VM, Long JM, Everett JN, et al. An oncocytic ad- renal tumor in a patient with Birt-Hogg-Dubé syndrome. Clin Endocrinol (Oxf). 2014;80(6):925-927.
102. Raymond VM, Else T, Everett JN, Long JM, Gruber SB, Hammer GD. Prevalence of germline TP53 mutations in a pro- spective series of unselected patients with adrenocortical carci- noma. J Clin Endocrinol Metab. 2013;98(1):E119-E125.
103. Lerario AM, Moraitis A, Hammer GD. Genetics and epigenetics of adrenocortical tumors. Mol Cell Endocrinol. 2014;386(1-2):67-84.
104. Costa TEJ, Gerber VKQ, Ibañez HC, et al. Penetrance of the TP53 R337H mutation and pediatric adrenocortical carcinoma incidence associated with environmental influences in a 12-year observational cohort in Southern Brazil. Cancers (Basel). 2019;11(11):1804.
105. de Reyniès A, Assié G, Rickman DS, et al. Gene expression pro- filing reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol. 2009;27(7):1108-1115.
106. Batisse-Lignier M, Sahut-Barnola I, Tissier F, et al. P53/Rb inhibi- tion induces metastatic adrenocortical carcinomas in a preclinical transgenic model. Oncogene. 2017;36(31):4445-4456.
107. Borges KS, Pignatti E, Leng S, et al. Wnt/ß-catenin activation cooperates with loss of p53 to cause adrenocortical carcinoma in mice. Oncogene. 2020;39(30):5282-5291.
108. Figueiredo BC, Cavalli LR, Pianovski MAD, et al. Amplification of the steroidogenic factor 1 gene in childhood adrenocortical tumors. J Clin Endocrinol Metab. 2005;90(2):615-619.
109. Almeida MQ, Soares IC, Ribeiro TC, et al. Steroidogenic factor 1 overexpression and gene amplification are more frequent in adrenocortical tumors from children than from adults. J Clin Endocrinol Metab. 2010;95(3):1458-1462.
110. Doghman M, Karpova T, Rodrigues GA, et al. Increased steroidogenic factor-1 dosage triggers adrenocortical cell prolifer- ation and cancer. Mol Endocrinol. 2007;21(12):2968-2987.
111. Ruggiero C, Doghman-Bouguerra M, Sbiera S, et al. Dosage- dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion. Sci Signal. 2017;10(469):eaal2464.
112. Landwehr LS, Altieri B, Schreiner J, et al. Interplay between glucocorticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma. J Immunother Cancer. 2020;8(1):e000469.
113. Jouinot A, Assié G, Libé R, et al. DNA methylation is an inde- pendent prognostic marker of survival in adrenocortical cancer. J Clin Endocrinol Metab. 2017;102(3):923-932.
114. Assié G, Jouinot A, Fassnacht M, et al. Value of molecular clas- sification for prognostic assessment of adrenocortical carcinoma. JAMA Oncol. 2019;5(10):1440-1447.
115. Fürstenberger G, Senn HJ. Insulin-like growth factors and cancer. Lancet Oncol. 2002;3(5):298-302.
116. Almeida MQ, Fragoso MCBV, Lotfi CFP, et al. Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors. J Clin Endocrinol Metab. 2008;93(9):3524-3531.
117. Barlaskar FM, Spalding AC, Heaton JH, et al. Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma. J Clin Endocrinol Metab. 2009;94(1):204-212.
118. Haluska P, Worden F, Olmos D, et al. Safety, tolerability, and pharmacokinetics of the anti-IGF-1R monoclonal antibody figitumumab in patients with refractory adrenocortical carci- noma. Cancer Chemother Pharmacol. 2010;65(4):765-773.
119. Lerario AM, Worden FP, Ramm CA, et al. The combina- tion of insulin-like growth factor receptor 1 (IGF1R) antibody cixutumumab and mitotane as a first-line therapy for patients with recurrent/metastatic adrenocortical carcinoma: a multi-institutional NCI-sponsored trial. Horm Cancer. 2014;5(4):232-239.
120. Fassnacht M, Berruti A, Baudin E, et al. Linsitinib (OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study. Lancet Oncol. 2015;16(4):426-435.
121. van Erp NP, Guchelaar HJ, Ploeger BA, Romijn JA, den Hartigh J, Gelderblom H. Mitotane has a strong and a durable inducing ef- fect on CYP3A4 activity. Eur J Endocrinol. 2011;164(4):621-626.
122. Kroiss M, Quinkler M, Johanssen S, et al. Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, open-label trial. J Clin Endocrinol Metab. 2012;97(10):3495-3503.
123. Kroiss M, Megerle F, Kurlbaum M, et al. Objective response and prolonged disease control of advanced adrenocortical carcinoma with cabozantinib. J Clin Endocrinol Metab. 2020;105(5):1461-1468.
124. Boumahdi S, de Sauvage FJ. The great escape: tumour cell plas- ticity in resistance to targeted therapy. Nat Rev Drug Discov. 2020;19(1):39-56.
125. Seidel E, Walenda G, Messerschmidt C, et al. Generation and characterization of a mitotane-resistant adrenocortical cell line. Endocr Connect. 2020;9(2):122-134.
126. LaPensee CR, Mann JE, Rainey WE, Crudo V, Hunt SW III, Hammer GD. ATR-101, a selective and potent inhibitor of Acyl- CoA acyltransferase 1, induces apoptosis in H295R adrenocortical cells and in the adrenal cortex of dogs. Endocrinology. 2016;157(5):1775-1788.
127. Raj N, Zheng Y, Kelly V, et al. PD-1 blockade in advanced adrenocortical carcinoma. J Clin Oncol. 2020;38(1):71-80.
128. Head L, Kiseljak-Vassiliades K, Clark TJ, et al. Response to immu- notherapy in combination with mitotane in patients with meta- static adrenocortical cancer. J Endocr Soc. 2019;3(12):2295-2304.
129. Habra MA, Stephen B, Campbell M, et al. Phase II clinical trial of pembrolizumab efficacy and safety in advanced adrenocortical carcinoma. J ImmunoTher Cancer. 2019;7(1):253.
130. Klein O, Senko C, Carlino MS, et al. Combination immunotherapy with ipilimumab and nivolumab in patients with advanced adrenocortical carcinoma: a subgroup analysis of CA209-538. Oncoimmunology. 2021;10(1):1908771.
131. Mota JM, Sousa LG, Braghiroli MI, et al. Pembrolizumab for metastatic adrenocortical carcinoma with high mu- tational burden: two case reports. Medicine (Baltimore). 2018;97(52):e13517.
132. Le Tourneau C, Hoimes C, Zarwan C, et al. Avelumab in patients with previously treated metastatic adrenocortical carci- noma: phase 1b results from the JAVELIN solid tumor trial. J ImmunoTher Cancer. 2018;6(1):111.
133. Bedrose S, Miller KC, Altameemi L, et al. Combined lenvatinib and pembrolizumab as salvage therapy in advanced adrenal cor- tical carcinoma. J ImmunoTher Cancer. 2020;8(2):e001009.
134. Lang J, Capasso A, Jordan KR, et al. Development of an adrenocortical cancer humanized mouse model to characterize anti-PD1 effects on tumor microenvironment. J Clin Endocrinol Metab. 2020;105(1):26-42.
135. Marabelle A, Le DT, Ascierto PA, et al. Efficacy of pembrolizumab in patients with noncolorectal high microsatellite instability/ mismatch repair-deficient cancer: results from the phase II KEYNOTE-158 study. J Clin Oncol. 2020;38(1):1-10.
136. Azad NS, Gray RJ, Overman MJ, et al. Nivolumab is effective in mismatch repair-deficient noncolorectal cancers: results from arm Z1D-A subprotocol of the NCI-MATCH (EAY131) study. J Clin Oncol. 2020;38(3):214-222.
137. Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357(6349):409-413.
138. Kiseljak-Vassiliades K, Zhang Y, Bagby SM, et al. Development of new preclinical models to advance adrenocortical carcinoma research. Endocr Relat Cancer. 2018;25(4):437-451.
139. Wolchok JD, Chiarion-Sileni V, Gonzalez R, et al. Overall sur- vival with combined nivolumab and ipilimumab in advanced mel- anoma. N Engl J Med. 2017;377(14):1345-1356.
140. Borghaei H, Gettinger S, Vokes EE, et al. Five-year outcomes from the randomized, phase III trials CheckMate 017 and 057: nivolumab versus docetaxel in previously treated non-small-cell lung cancer. J Clin Oncol. 2021;39(7):723-733.
141. Taube JM, Klein A, Brahmer JR, et al. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvi- ronment with response to anti-PD-1 therapy. Clin Cancer Res. 2014;20(19):5064-5074.
142. Crona J, Beuschlein F. Adrenocortical carcinoma-towards genomics guided clinical care. Nat Rev Endocrinol. 2019;15(9):548-560.
143. Fay AP, Signoretti S, Callea M, et al. Programmed death ligand-1 expression in adrenocortical carcinoma: an exploratory bio- marker study. J Immunother Cancer. 2015;3:3.
144. Trujillo JA, Luke JJ, Zha Y, et al. Secondary resistance to im- munotherapy associated with ß-catenin pathway activation or PTEN loss in metastatic melanoma. J Immunother Cancer. 2019;7(1):295.
145. Ruiz de Galarreta M, Bresnahan E, Molina-Sánchez P, et al. ß-Catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma. Cancer Discov. 2019;9(8):1124-1141.
146. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic B-catenin signalling prevents anti-tumour immunity. Nature. 2015;523(7559):231-235.
147. Savino W, Mendes-da-Cruz DA, Lepletier A, Dardenne M. Hormonal control of T-cell development in health and disease. Nat Rev Endocrinol. 2016;12(2):77-89.
148. Ince LM, Weber J, Scheiermann C. Control of leukocyte trafficking by stress-associated hormones. Front Immunol. 2018;9:3143.
149. Majdic G, Young M, Gomez-Sanchez E, et al. Knockout mice lacking steroidogenic factor 1 are a novel genetic model of hypo- thalamic obesity. Endocrinology. 2002;143(2):607-614.
150. Prasad R, Kowalczyk JC, Meimaridou E, Storr HL, Metherell LA. Oxidative stress and adrenocortical insufficiency. J Endocrinol. 2014;221(3):R63-R73.
151. Pezzi V, Mathis JM, Rainey WE, Carr BR. Profiling transcript levels for steroidogenic enzymes in fetal tissues. J Steroid Biochem Mol Biol. 2003;87(2-3):181-189.
152. Sirianni R, Seely JB, Attia G, et al. Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates tran- scription of genes encoding steroidogenic enzymes. J Endocrinol. 2002;174(3):R13-R17.
153. Acharya N, Madi A, Zhang H, et al. Endogenous glucocorti- coid signaling regulates CD8+ T cell differentiation and develop- ment of dysfunction in the tumor microenvironment. Immunity. 2020;53(3):658-671.e6.
154. Ferraz-de-Souza B, Hudson-Davies RE, Lin L, et al. Sterol O-acyltransferase 1 (SOAT1, ACAT) is a novel target of steroidogenic factor-1 (SF-1, NR5A1, Ad4BP) in the human ad- renal. J Clin Endocrinol Metab. 2011;96(4):E663-E668.
155. Lala DS, Rice DA, Parker KL. Steroidogenic factor I, a key regu- lator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol. 1992;6(8):1249-1258.
156. Hart MM, Reagan RL, Adamson RH. The effect of isomers of DDD on the ACTH-induced steroid output, histology and ul- trastructure of the dog adrenal cortex. Toxicol Appl Pharmacol. 1973;24(1):101-113.
157. Hescot S, Slama A, Lombès A, et al. Mitotane alters mitochon- drial respiratory chain activity by inducing cytochrome c oxi- dase defect in human adrenocortical cells. Endocr Relat Cancer. 2013;20(3):371-381.
158. Lehmann TP, Wrzesiński T, Jagodziński PP. The effect of mitotane on viability, steroidogenesis and gene expression in NCI-H295R adrenocortical cells. Mol Med Rep. 2013;7(3):893-900.
159. Cai W, Counsell RE, Djanegara T, Schteingart DE, Sinsheimer JE, Wotring LL. Metabolic activation and binding of mitotane in ad- renal cortex homogenates. J Pharm Sci. 1995;84(2):134-138.
160. Martz F, Straw JA. The in vitro metabolism of 1-(o-chlorophenyl)- 1-(p-chlorophenyl)-2,2-dichloroethane (o,p’-DDD) by dog adrenal mitochondria and metabolite covalent binding to mitochondrial macromolecules: a possible mechanism for the adrenocorticolytic effect. Drug Metab Dispos. 1977;5(5):482-486.
161. Cai W, Counsell RE, Schteingart DE, Sinsheimer JE, Vaz AD, Wotring LL. Adrenal proteins bound by a reactive intermediate of mitotane. Cancer Chemother Pharmacol. 1997;39(6):537-540.
162. Waszut U, Szyszka P, Dworakowska D. Understanding mitotane mode of action. J Physiol Pharmacol. 2017;68(1):13-26.
163. Schteingart DE, Sinsheimer JE, Benitez RS, Homan DF, Johnson TD, Counsell RE. Structural requirements for mitotane activity: development of analogs for treatment of adrenal cancer. Anticancer Res. 2012;32(7):2711-2720.
164. Dominick MA, McGuire EJ, Reindel JF, Bobrowski WF, Bocan TM, Gough AW. Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs. Fundam Appl Toxicol. 1993;20(2):217-224.
165. Reindel JF, Dominick MA, Bocan TM, Gough AW, McGuire EJ. Toxicologic effects of a novel acyl-CoA:cholesterol acyltransferase inhibitor in cynomolgus monkeys. Toxicol Pathol. 1994;22(5):510-518.
166. El-Maouche D, Merke DP, Vogiatzi MG, et al. A phase 2, multicenter study of nevanimibe for the treatment of congenital adrenal hy- perplasia. J Clin Endocrinol Metab. 2020;105(8):2771-2778.
167. Smith DC, Kroiss M, Kebebew E, et al. A phase 1 study of nevanimibe HCl, a novel adrenal-specific sterol O-acyltransferase 1 (SOAT1) inhibitor, in adrenocortical carcinoma. Invest New Drugs. 2020;38(5):1421-1429.
168. Burns VE, Kerppola TK. ATR-101 inhibits cholesterol efflux and cortisol secretion by ATP-binding cassette transporters, causing
cytotoxic cholesterol accumulation in adrenocortical carcinoma cells. Br J Pharmacol. 2017;174(19):3315-3332.
169. Belavgeni A, Bornstein SR, von Mässenhausen A, et al. Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis. Proc Natl Acad Sci U S A. 2019;116(44):22269-22274.
170. Weigand I, Schreiner J, Röhrig F, et al. Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis. 2020;11(3):192.
171. Bergström M, Bonasera TA, Lu L, et al. In vitro and in vivo primate evaluation of carbon-11-etomidate and carbon-11-metomidate as potential tracers for PET imaging of the adrenal cortex and its tumors. J Nucl Med. 1998;39(6):982-989.
172. Weber MM, Lang J, Abedinpour F, Zeilberger K, Adelmann B, Engelhardt D. Different inhibitory effect of etomidate and ketoconazole on the human adrenal steroid biosynthesis. Clin Investig. 1993;71(11):933-938.
173. Hahner S, Stuermer A, Kreissl M, et al. [123 I]Iodometomidate for molecular imaging of adrenocortical cytochrome P450 family 11B enzymes. J Clin Endocrinol Metab. 2008;93(6):2358-2365.
174. Hahner S, Kreissl MC, Fassnacht M, et al. Functional character- ization of adrenal lesions using [123IJIMTO-SPECT/CT. J Clin Endocrinol Metab. 2013;98(4):1508-1518.
175. Kreissl MC, Schirbel A, Fassnacht M, et al. [123I]Iodometomidate imaging in adrenocortical carcinoma. J Clin Endocrinol Metab. 2013;98(7):2755-2764.
176. Hahner S, Kreissl MC, Fassnacht M, et al. [131I]Iodometomidate for targeted radionuclide therapy of advanced adrenocortical car- cinoma. J Clin Endocrinol Metab. 2012;97(3):914-922.
177. Hahner S, Hartrampf PE, Mihatsch PW, et al. Targeting 11-beta hydroxylase with [131I]IMAZA: a novel approach for the treat- ment of advanced adrenocortical carcinoma. J Clin Endocrinol Metab. 2022;107(4):e1348-e1355.
178. Giordano TJ, Kuick R, Else T, et al. Molecular classification and prognostication of adrenocortical tumors by transcriptome pro- filing. Clin Cancer Res. 2009;15(2):668-676.
179. Fassnacht M, Terzolo M, Allolio B, et al. FIRM-ACT Study Group. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med. 2012;366(23):2189-2197.
180. Mohan DR, Lerario AM, Hammer GD. Therapeutic targets for adrenocortical carcinoma in the genomics era. J Endocr Soc. 2018;2(11):1259-1274.
181. Mohan DR, Lerario AM, Finco I, Hammer GD. New strategies for applying targeted therapies to adrenocortical carcinoma. Curr Opin Endocr Metab Res. 2019;8:72-79.
182. Turner NC, Ro J, André F, et al. PALOMA3 Study Group. Palbociclib in hormone-receptor-positive advanced breast cancer. N Engl J Med. 2015;373(3):209-219.
183. O’Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4/6 inhibitors. Nat Rev Clin Oncol. 2016;13(7):417-430.
184. Liang R, Weigand I, Lippert J, et al. Targeted gene expression profile reveals CDK4 as therapeutic target for selected patients with adrenocortical carcinoma. Front Endocrinol (Lausanne). 2020;11:219.
185. Fiorentini C, Fragni M, Tiberio GAM, et al. Palbociclib inhibits proliferation of human adrenocortical tumor cells. Endocrine. 2018;59(1):213-217.
186. Bussey KJ, Bapat A, Linnehan C, et al. Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carci- noma. Clin Transl Med. 2016;5(1):1.
187. Kiseljak-Vassiliades K, Zhang Y, Kar A, et al. Elucidating the role of the maternal embryonic leucine zipper kinase in adrenocortical carcinoma. Endocrinology. 2018;159(7):2532-2544.
188. Borges KS, Andrade AF, Silveira VS, et al. The aurora kinase inhib- itor AMG 900 increases apoptosis and induces chemosensitivity to anticancer drugs in the NCI-H295 adrenocortical carcinoma cell line. Anticancer Drugs. 2017;28(6):634-644.
189. Papathomas TG, Pucci E, Giordano TJ, et al. An international Ki67 reproducibility study in adrenal cortical carcinoma. Am J Surg Pathol. 2016;40(4):569-576.
190. Knijnenburg TA, Wang L, Zimmermann MT, et al. Genomic and molecular landscape of DNA damage repair deficiency across The Cancer Genome Atlas. Cell Rep. 2018;23(1):239-254.e6.
191. Mateo J, Carreira S, Sandhu S, et al. DNA-repair defects and olaparib in metastatic prostate cancer. N Engl J Med. 2015;373(18):1697-1708.
192. Moore K, Colombo N, Scambia G, et al. Maintenance olaparib in patients with newly diagnosed advanced ovarian cancer. N Engl J Med. 2018;379(26):2495-2505.
193. Takaya H, Nakai H, Takamatsu S, Mandai M, Matsumura N. Homologous recombination deficiency status-based classi- fication of high-grade serous ovarian carcinoma. Sci Rep. 2020;10(1):2757.
194. Mirza MR, Monk BJ, Herrstedt J, et al. ENGOT-OV16/ NOVA Investigators. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer. N Engl J Med. 2016;375(22):2154-2164.
195. Pennisi R, Ascenzi P, di Masi A. Hsp90: a new player in DNA re- pair? Biomolecules. 2015;5(4):2589-2618.
196. Siebert C, Ciato D, Murakami M, et al. Heat shock protein 90 as a prognostic marker and therapeutic target for adrenocortical carcinoma. Front Endocrinol (Lausanne). 2019;10:487.
197. Bridges KA, Hirai H, Buser CA, et al. MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells. Clin Cancer Res. 2011;17(17):5638-5648.
198. De Witt Hamer PC, Mir SE, Noske D, Van Noorden CJF, Würdinger T. WEE1 kinase targeting combined with DNA- damaging cancer therapy catalyzes mitotic catastrophe. Clin Cancer Res. 2011;17(13):4200-4207.
199. Rajeshkumar NV, De Oliveira E, Ottenhof N, et al. MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. Clin Cancer Res. 2011;17(9):2799-2806.
200. Srougi V, Bancos I, Daher M, et al. Cytoreductive surgery of the primary tumor in metastatic adrenocortical carcinoma: impact on patients’ survival. J Clin Endocrinol Metab. 2022;107(4):964-971.
201. Kleeman SO, Leedham SJ. Not all Wnt activation is equal: ligand- dependent versus ligand-independent Wnt activation in colorectal cancer. Cancers (Basel). 2020;12(11):3355.
202. Katoh M. Canonical and non-canonical WNT signaling in cancer stem cells and their niches: cellular heterogeneity, omics reprogramming, targeted therapy and tumor plasticity (Review). Int J Oncol. 2017;51(5):1357-1369.
203. Kahn M. Can we safely target the WNT pathway? Nat Rev Drug Discov. 2014;13(7):513-532.
204. Koo BK, van Es JH, van den Born M, Clevers H. Porcupine inhibitor suppresses paracrine Wnt-driven growth of Rnf43;Znrf3-mutant neoplasia. Proc Natl Acad Sci U S A. 2015;112(24):7548-7550.
205. Chatterjee A, Paul S, Bisht B, Bhattacharya S, Sivasubramaniam S, Paul MK. Advances in targeting the WNT/ß-catenin signaling pathway in cancer. Drug Discov Today. 2022;27(1):82-101.
206. Krishnamurthy N, Kurzrock R. Targeting the Wnt/beta-catenin pathway in cancer: update on effectors and inhibitors. Cancer Treat Rev. 2018;62:50-60.
207. O’Connell AE, Zhou F, Shah MS, et al. Neonatal-onset chronic di- arrhea caused by homozygous nonsense WNT2B mutations. Am J Hum Genet. 2018;103(1):131-137.
208. Chai G, Szenker-Ravi E, Chung C, et al. A human pleiotropic multiorgan condition caused by deficient Wnt secretion. N Engl J Med. 2021;385(14):1292-1301.
209. Gicquel C, Bertagna X, Schneid H, et al. Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors. J Clin Endocrinol Metab. 1994;78(6):1444-1453.
210. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab. 1997;82(8):2559-2565.
211. Liu J, Kahri AI, Heikkilä P, Voutilainen R. Ribonucleic acid ex- pression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab. 1997;82(6):1766-1771.
212. Vaz M, Hwang SY, Kagiampakis I, et al. Chronic cigarette smoke- induced epigenomic changes precede sensitization of bronchial epithelial cells to single-step transformation by KRAS mutations. Cancer Cell. 2017;32(3):360-376.e6.
213. Venneti S, Thompson CB. Metabolic modulation of epigenetics in gliomas. Brain Pathol. 2013;23(2):217-221.
214. Petryk N, Bultmann S, Bartke T, Defossez PA. Staying true to your- self: mechanisms of DNA methylation maintenance in mammals. Nucleic Acids Res. 2021;49(6):3020-3032.
215. McCabe MT, Davis JN, Day ML. Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway. Cancer Res. 2005;65(9):3624-3632.
216. Mehdipour P, Murphy T, De Carvalho DD. The role of DNA- demethylating agents in cancer therapy. Pharmacol Ther. 2020;205:107416.
217. Mohan DR, Finco I, LaPensee CR, et al. SAT-LB34 repressive epigenetic programs reinforce steroidogenic differentiation and Wnt/ß-catenin signaling in aggressive adrenocortical carcinoma. J Endocr Soc. 2020;4(Suppl 1):SAT-LB34.
218. Pappalardi MB, Keenan K, Cockerill M, et al. Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia. Nat Cancer. 2021;2(10):1002-1017.
219. Chung C, Sweha SR, Pratt D, et al. Integrated metabolic and epigenomic reprograming by H3K27M mutations in diffuse in- trinsic pontine gliomas. Cancer Cell. 2020;38(3):334-349.e9.
220. Sweha SR, Chung C, Natarajan SK, et al. Epigenetically defined therapeutic targeting in H3.3G34R/V high-grade gliomas. Sci Transl Med. 2021;13(615):eabf7860.
221. Pozdeyev N, Fishbein L, Gay LM, et al. Targeted genomic anal- ysis of 364 adrenocortical carcinomas. Endocr Relat Cancer. 2021;28(10):671-681.
222. Corces MR, Granja JM, Shams S, et al. Cancer Genome Atlas Analysis Network. The chromatin accessibility landscape of pri- mary human cancers. Science. 2018;362(6413):eaav1898.
223. Kim KH, Kim W, Howard TP, et al. SWI/SNF-mutant cancers de- pend on catalytic and non-catalytic activity of EZH2. Nat Med. 2015;21(12):1491-1496.
224. Kadoch C, Copeland RA, Keilhack H. PRC2 and SWI/SNF chro- matin remodeling complexes in health and disease. Biochemistry. 2016;55(11):1600-1614.
225. Drelon C, Berthon A, Mathieu M, et al. EZH2 is overexpressed in adrenocortical carcinoma and is associated with disease progres- sion. Hum Mol Genet. 2016;25(13):2789-2800.
226. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for prolif- eration and amplified in cancer. EMBO J. 2003;22(20):5323-5335.
227. Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419(6907):624-629.
228. Kadoch C, Hargreaves DC, Hodges C, et al. Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy. Nat Genet. 2013;45(6):592-601.
229. Bödör C, Grossmann V, Popov N, et al. EZH2 mutations are fre- quent and represent an early event in follicular lymphoma. Blood. 2013;122(18):3165-3168.
230. Morin RD, Johnson NA, Severson TM, et al. Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet. 2010;42(2):181-185.
231. Knutson SK, Kawano S, Minoshima Y, et al. Selective inhibi- tion of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Cancer Ther. 2014;13(4):842-854.
232. Kamminga LM, Bystrykh LV, de Boer A, et al. The polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood. 2006;107(5):2170-2179.
233. Ezhkova E, Pasolli HA, Parker JS, et al. Ezh2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell. 2009;136(6):1122-1135.
234. O’Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T. The polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol. 2001;21(13):4330-4336.
235. Lippert J, Appenzeller S, Liang R, et al. Targeted molecular anal- ysis in adrenocortical carcinomas: a strategy toward improved personalized prognostication. J Clin Endocrinol Metab. 2018;103(12):4511-4523.
236. Garinet S, Nectoux J, Neou M, et al. Detection and monitoring of circulating tumor DNA in adrenocortical carcinoma. Endocr Relat Cancer. 2018;25(3):L13-L17.
237. Creemers SG, Korpershoek E, Atmodimedjo PN, et al. Identification of mutations in cell-free circulating tumor DNA in adrenocortical carcinoma: a case series. J Clin Endocrinol Metab. 2017;102(10):3611-3615.
238. Szabó DR, Luconi M, Szabó PM, et al. Analysis of circu- lating microRNAs in adrenocortical tumors. Lab Invest. 2014;94(3):331-339.
239. Perge P, Butz H, Pezzani R, et al. Evaluation and diagnostic poten- tial of circulating extracellular vesicle-associated microRNAs in adrenocortical tumors. Sci Rep. 2017;7(1):5474.
240. Beuschlein F, Jakoby J, Mentz S, et al. IGF1-R inhibition and liposomal doxorubicin: progress in preclinical evaluation for the treatment of adrenocortical carcinoma. Mol Cell Endocrinol. 2016;428:82-88.
241. Hantel C, Shapiro I, Poli G, et al. Targeting heterogeneity of adrenocortical carcinoma: evaluation and extension of preclin- ical tumor models to improve clinical translation. Oncotarget. 2016;7(48):79292-79304.
242. Pinto EM, Morton C, Rodriguez-Galindo C, et al. Establishment and characterization of the first pediatric adrenocortical car- cinoma xenograft model identifies topotecan as a potential chemotherapeutic agent. Clin Cancer Res. 2013;19(7):1740-1747.
243. Jouinot A, Lippert J, Fassnacht M, et al. Intratumor heterogeneity of prognostic DNA-based molecular markers in adrenocortical carcinoma. Endocr Connect. 2020;9(7):705-714.
244. Gara SK, Lack J, Zhang L, Harris E, Cam M, Kebebew E. Metastatic adrenocortical carcinoma displays higher mutation rate and tumor heterogeneity than primary tumors. Nat Commun. 2018;9(1):4172.
245. Fojo T, Huff L, Litman T, et al. Metastatic and recurrent adrenocortical cancer is not defined by its genomic landscape. BMC Med Genomics. 2020;13(1):165.
246. Meacham CE, Morrison SJ. Tumour heterogeneity and cancer cell plasticity. Nature. 2013;501(7467):328-337.
247. Yuan S, Norgard RJ, Stanger BZ. Cellular plasticity in cancer. Cancer Discov. 2019;9(7):837-851.
248. Quintanal-Villalonga Á, Chan JM, Yu HA, et al. Lineage plasticity in cancer: a shared pathway of therapeutic resistance. Nat Rev Clin Oncol. 2020;17(6):360-371.
249. Nieto MA, Huang RY, Jackson RA, Thiery JP. EMT: 2016. Cell. 2016;166(1):21-45.
250. Sequist LV, Waltman BA, Dias-Santagata D, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 2011;3(75):75ra26.
251. Davies AH, Beltran H, Zoubeidi A. Cellular plasticity and the neuroendocrine phenotype in prostate cancer. Nat Rev Urol. 2018;15(5):271-286.
252. Davies A, Nouruzi S, Ganguli D, et al. An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer. Nat Cell Biol. 2021;23(9):1023-1034.
253. Lee JK, Lee J, Kim S, et al. Clonal history and genetic predictors of transformation into small-cell carcinomas from lung adenocarcinomas. J Clin Oncol. 2017;35(26):3065-3074.
254. Aggarwal R, Huang J, Alumkal JJ, et al. Clinical and genomic characterization of treatment-emergent small-cell neuroendocrine prostate cancer: a multi-institutional prospective study. J Clin Oncol. 2018;36(24):2492-2503.
255. Lopez JP, Brivio E, Santambrogio A, et al. Single-cell molec- ular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation. Sci Adv. 2021;7(5):eabe4497.
Downloaded from https://academic.oup.com/edrv/article/43/6/1051/6585478 by National Library of Medicine user on 04 April 2026