1 β-catenin-driven differentiation is a tissue-specific epigenetic vulnerability in adrenal cancer

Dipika R. Mohan1,2, Kleiton S. Borges3,4, Isabella Finco5, Christopher R. LaPensee5, Juilee Rege6, April L. Solon7, Donald W. Little III5, Tobias Else5, Madson Q. Almeida8,9, Derek Dang10,11, James Haggerty-Skeans1,10,11, April A. Apfelbaum2,12, Michelle Vinco10, Alda Wakamatsu13, Beatriz M. P. Mariani8, Larissa Amorim8,9, Ana Claudia Latronico8, Berenice B. Mendonca8, Maria Claudia N. Zerbini13, Elizabeth R. Lawlor12,14, Ryoma Ohi7, Richard J. Auchus5,15,16, William E. Rainey6, Suely K. N. Marie17, Thomas J. Giordano5,10,18, Sriram Venneti6,10,11,19, Maria Candida B. V. Fragoso8,9, David T. Breault3,4,20, Antonio Marcondes Lerario5,21*, and Gary D. Hammer5,6,7,18,21*


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We observed EZH2 binds no DNMTs, with virtually no overlap between EZH2 and DNMT1 353 interactomes ( Figure 2I). DNMT1 binds many chromatin-bound proteins including the HP1 354 family of H3K9me readers, representing a conserved DNMT1 mode (84), and consistent with a 355 model in which H3K9me3, rather than H3K27me3, instructs CIMP-high DNA methylation (16). 356 EZH2 is assembled in PRC2.1, a canonical PRC2 assembly defined by association with PCL 357 accessory proteins that target preferential PRC2 recruitment to unmethylated CpGi ( Figure 2I, 358 (85,86)). 359 360 We next examined EZH2 recruitment genome-wide relative to H3K27me3, active chromatin 361 measured by H3K27 acetylation (H3K27ac), and accessible chromatin. H3K27me3/H3K27ac 362 were mutually exclusive, and most EZH2 peaks co-localized with broad, inaccessible 363 H3K27me3 domains ( Figure 2J). Regions targeted for hypermethylation in CIMP-high ACC 364 (DMR+) and H3K27me3 peaks exhibited minimal overlap, and DNA methylation levels of 365 H3K27me3 peaks were substantially lower than those of DMR+ ( Figure 2K). EZH2 and 366 H3K27me3 were excluded from the hypermethylated G0S2 locus (Supp Fig 3J). In fetal and 367 adult adrenals, we observed strong H3K27me3 deposition at DMR+, and reduced deposition 368 at NCI-H295R H3K27me3 peaks ( Figure 2L). In ACC-TCGA, we observed reduced 369 accessibility of both DMRcate regions and NCI-H295R H3K27me3 in CIMP-high compared to 370 non-CIMP-high ACC (Supp Fig 3K). These observations suggest DNA hypermethylation in 371 CIMP-high ACC is propagated independently of PRC2, leads to epigenetic class switching, 372 PRC2 eviction and recruitment to novel sites for H3K27me3 catalysis.  (Figure 3F-G; Supp Fig 4C). Strikingly, EZH2i disrupted ~70% of genes differentially 406 expressed after forskolin treatment (Figure 3H), and potently downregulated steroidogenic 407 enzymes ( Figure 3I). Suppression of steroidogenic enzymes was dose-dependent and 408 observed with different classes of EZH2i across ACC cell lines ( Figure 3J). Moreover, EZH2i 409 pretreatment followed by forskolin administration diminished both forskolin-induced silencing 410 of canonical Wnt targets and induction of steroidogenic enzymes, ultimately restraining steroid 411 output (Figure 3K-M). 412 413 These observations are consistent with a role for EZH2 in programming the cellular response to 414 ACTH/PKA in CIMP-high ACC, though not solely by dysregulating expression of PKA signaling 415 components (87). Though EZH2i induced few canonical Wnt targets (Figure 3E, K), EZH2i 416 reversed all three core modules of CIMP-high ACC (Figure 3N), while forskolin induced 417 differentiation at the expense of cell cycle and Wnt activation (Figure 3F, K, N). Observing that 418 EZH2i disrupted a spectrum of transcriptional programs (Figure 3C), including those governed 419 by EZH2 in the normal adrenal (Figure 3D), led us to explore if the effects of EZH2i result from 420 an alternative EZH2 role. 421 422 EZH2 binds transcriptional coactivator β-catenin in an off-chromatin complex 423 EZH2 IP-MS revealed several non-PRC2 partners, including nuclear receptors known to 424 regulate adrenocortical biology (90), as well as β-catenin (Figure 4A), constitutively active in 425 NCI-H295R due to the p.S45P mutation (Supp Fig 3A). Given β-catenin's abundance in the 426 EZH2 interactome (Figure 4A), possible role in the chromatin response to EZH2i (Figure 3E) 427 and its well-established role in adrenocortical differentiation and tumorigenesis, we elected to 428 focus our studies on EZH2/β-catenin. 429

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To understand the genomic architecture and physiologic relevance of these SE, we examined 468 promoter capture Hi-C, single-cell RNA-seq, and single-cell ATAC-seq from normal adrenals 469 (58,(65)(66)(67), and integrated these studies with our epigenomics data. We observed SF1/β-470 catenin SE and enhancers contact promoters of many genes critical for zone-specific and 471 adrenocortical steroidogenic identity, including HSD3B2 and NR5A1 itself (Figure 4K-M). 472 These enhancers are present in normal adrenal (Figure 4M, Supp Fig 4I), and more accessible 473 in outer cortex, consistent with nuclear localization of β-catenin in these regions (Figure 1A, 474

in vivo 545
To determine if differentiation programs targeted by EZH2i represent a viable therapeutic 546 strategy in zF-differentiated ACC bearing SF1/β-catenin and EZH2/β-catenin complexes, we 547 developed a subcutaneous allograft model using a cell line (BCH-ACC3A) derived from 548 BPCre AS/+ ACC with metastatic potential, Figure 7A. Recipient mice were treated with vehicle 549 or EZH2i, which was well tolerated (Supp Fig 5G). EZH2i-treated mice exhibited diminished 550 tumor growth (Figure 7B) with decreased H3K27me3 deposition ( Figure 7C) and proliferation 551 ( Figure 7D, Supp Fig 5H). Tumors from EZH2i-treated mice also exhibited dedifferentiation 552 ( Figure 7E-F) with a reduction in SF1/β-catenin complexes ( Figure 7G) and retention of 553 EZH2/β-catenin complexes (Figure 7H), recapitulating the molecular features we observed in 554 vitro (Figures 3-4, 6; Supp Fig 4F, 6). Taken together, these studies point to zF differentiation 555 as a targetable epigenetic vulnerability selected for in CIMP-high ACC and provide proof-of-556 principle support for efficacy of dedifferentiating therapies in ACC treatment. 557 558 DISCUSSION 559 ACC is exquisitely rare and outcomes remain dismal. CIMP-high ACC is prevalent, invariably 560 metastatic, and lethal (8,9). Molecularly, CIMP-high ACC is defined by abnormal DNA 561 methylation and paradoxical activation of cell cycle, Wnt/β-catenin signaling, and zF 562 differentiation. We discovered that abnormal CpGi hypermethylation, in addition to serving as a 563 pathognomonic marker of aggressive disease, displaces EZH2/PRC2 to novel sites. CIMP-high 564 DNA hypermethylation is uniform, with many targets possessing binary and complete 565 methylation (8). These data, consistent with literature examining etiology and emergence of 566 CIMP-high (14,15), suggest acquisition of this signature is an early selection event in 567 adrenocortical carcinogenesis. 568 We reconcile the convergence of zF differentiation and Wnt/β-catenin activation in ACC by 570 discovering uniform SF1/β-catenin control of CIMP-high SE, including an SE that regulates 571 expression of SF1 itself. Selection for zF differentiation is counterintuitive given the many 572 events other cancers acquire to harness proliferation potential at the expense of differentiation 573 (106,107). Our data suggest that the cell originating CIMP-high ACC is one which relies on β-574 catenin and zF differentiation for sustained proliferation, perhaps a transit-amplifying ACTH-575 responsive cell of the upper zF which fails to proliferate in the setting of congenital 576 adrenocortical EZH2 deficiency or β-catenin ablation (87,108). This is further supported by the 577 demonstrations that combined β-catenin/cell cycle GOF generates zF-differentiated 578 glucocorticoid producing ACC (22), and adrenocortical deficiency of negative Wnt pathway 579 regulator ZNRF3 induces zF hyperplasia with eventual malignant transformation (109)(110)(111). 580

581
We identify a series of protein complexes, EZH2/β-catenin and SF1/β-catenin, that shuttle β-582 catenin off and on chromatin. These complexes are zonally distributed and exist in the 583 presence of active Wnt signaling, stabilized either by genetic alteration or physiologic Wnt 584 ligands ( Figure 5). They are conserved across murine and human adrenocortical 585 carcinogenesis, and selected for throughout ACC evolution. EZH2/β-catenin formation is a 586 necessary but costly consequence of Wnt and cell cycle activation in CIMP-high, destabilizing 587 the SF1/β-catenin program. CIMP-high ACC may thus require high PRC2 catalytic activity to 588 restrain EZH2/β-catenin in the setting of CpGi hypermethylation, in contrast to other tissues 589 that use CIMP-high to select for PRC2 loss of function with malignancy (23). The EZH2/β-590 catenin/SF1 triangulation therefore creates a dependence of zF differentiation on PRC2, 591 illuminating an intrinsic tissue-specific vulnerability in CIMP-high ACC with therapeutic 592 significance in vitro and in vivo ( Figure 7I). 593 594 Repressive epigenetic modifiers are often modeled as complexes that maintain stemness. 595 PRC2 is critical for embryonic pluripotency and gastrulation (4), and in many cancers restrains 596 differentiation for sustained proliferation potential (5), in apparent contrast to our work. A 597 perhaps more nuanced interpretation is that PRC2/H3K27me3 deposition facilitates cell state 598 transitions required for accurate differentiation. Indeed, our studies reveal that catalytically 599 active PRC2 stabilizes a pro-proliferative differentiation state in CIMP-high ACC, by limiting 600 EZH2's interaction with a transcriptional coactivator core to adrenocortical cell type 601 specification, β-catenin. 602 The Wnt/β-catenin pathway is recurrently activated by genetic alteration in ~20% of cancer 604 and >50% of CIMP-high ACC (cBioPortal (112,113), (8)(9)(10)). Efforts to target this pathway 605 clinically have failed, due to life-limiting on-target toxicities in Wnt-dependent organs with rapid 606 turnover (103). Here, we discover that selection for active β-catenin in ACC is dependent on 607 maintenance of a tissue specific program: SF1-dependent zF differentiation. Given the paucity 608 of organs that require both programs for homeostatic renewal, this opens a large therapeutic 609 window for targeting oncogenic β-catenin. Do alternative context-specific nuclear receptors 610 bind β-catenin in other cancers? A paradigm geared towards tissue-specific disruption of 611 oncogenic programs will be essential to combat cancers that rely on differentiation for growth 612 and dissemination, like ACC. 613

614
This study has several important limitations. We rely heavily on small molecules, in part 615 because we seek to demonstrate how pharmacologic tools unveil vulnerabilities in disease. 616 While EZH2i induces a reduction in H3K27me3 at low doses, the doses of EZH2i that induce 617 loss of viability in vitro exceed the effective doses of EZH2i in EZH2-mutant lymphomas for 618 which EZH2i has been FDA approved (114). At such high doses, it is possible that EZH2i also 619 targets EZH1 (115), though this appears unlikely in our system for multiple reasons (Supp Fig  620   3I, 6), including our demonstration of concordant findings in vivo, where our dosing regimen 621 yields steady state plasma levels in the low micromolar range (114  A. Corticocapsular unit of adrenocortical homeostasis depicting peripheral mesenchymal cells (capsule) and human cortical populations zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), that produce mineralocorticoids, glucocorticoids, and androgens, respectively. Differentiation in the cortex is centripetal, and zG, zF, and zR cells are supplied by peripheral capsular and cortical progenitors (arrow). The entire cortex is SF1 positive. ZG cells possess active Wnt/β-catenin signaling and lower zF/zR cells possess active ACTH signaling through protein kinase A (PKA). Wnt/β-catenin signaling fades in the upper zF, and these cells respond to ACTH/PKA with proliferation (Ki67). Mice do not have a zR.
B. GSVA was used on ACC-TCGA RNA-seq to calculate the expression score of genes that define zF differentiation or are regulated in a cell-cycle-or Wnt-dependent manner across ACC-TCGA. Score validation detailed in Methods and Supp Fig 1. Radar plot depicts average score for each ACC CIMP class, with values mapped onto an arbitrary scale along each axis. Heatmap below depicts p-value for comparisons. C. 10 most significant gene sets from curated GSEA (53,54) on genes with promoters targeted for hypermethylation in CIMP-high ACC.
H. Disease-free (after R0/RX resection in patients without metastatic disease at diagnosis) and overall survival (all patients) stratified by ACC EZH2 expression.