Jaime Reyes

Oncogenic deregulation of EZH2 as an opportunity for targeted therapy in lung cancer

UNLABELLED As a master regulator of chromatin function, the lysine methyltransferase EZH2 orchestrates transcriptional silencing of developmental gene networks. Overexpression of EZH2 is commonly observed in human epithelial cancers, such as non-small cell lung carcinoma (NSCLC), yet definitive demonstration of malignant transformation by deregulated EZH2 remains elusive. Here, we demonstrate the causal role of EZH2 overexpression in NSCLC with new genetically engineered mouse models of lung adenocarcinoma. Deregulated EZH2 silences normal developmental pathways, leading to epigenetic transformation independent of canonical growth factor pathway activation. As such, tumors feature a transcriptional program distinct from KRAS- and EGFR-mutant mouse lung cancers, but shared with human lung adenocarcinomas exhibiting high EZH2 expression. To target EZH2-dependent cancers, we developed a potent open-source EZH2 inhibitor, JQEZ5, that promoted the regression of EZH2-driven tumors in vivo, confirming oncogenic addiction to EZH2 in established tumors and providing the rationale for epigenetic therapy in a subset of lung cancer. SIGNIFICANCE EZH2 overexpression induces murine lung cancers that are similar to human NSCLC with high EZH2 expression and low levels of phosphorylated AKT and ERK, implicating biomarkers for EZH2 inhibitor sensitivity. Our EZH2 inhibitor, JQEZ5, promotes regression of these tumors, revealing a potential role for anti-EZH2 therapy in lung cancer. Cancer Discov; 6(9); 1006-21. ©2016 AACR.See related commentary by Frankel et al., p. 949This article is highlighted in the In This Issue feature, p. 932.

PI3K/AKT Signaling Regulates H3K4 Methylation in Breast Cancer

Post-translational histone H3 modifications regulate transcriptional competence. The mechanisms by which the epigenome is regulated in response to oncogenic signaling remain unclear. Here we show that H3K4me3 is increased in breast tumors driven by an activated PIK3CA allele and that inhibition of PI3K/AKT signaling reduces promoter-associated H3K4me3 in human breast cancer cells. We show that the H3K4 demethylase KDM5A is an AKT target and that phosphorylation of KDM5A regulates its nuclear localization and promoter occupancy. Supporting a role for KDM5A in mediating PI3K/AKT transcriptional effects, the decreased expression in response to AKT inhibition of a subset of cell-cycle genes associated with poor clinical outcome is blunted by KDM5A silencing. Our data identify a mechanism by which PI3K/AKT signaling modulates the cancer epigenome through controlling H3K4 methylation and suggest that KDM5A subcellular localization and genome occupancy may be pharmacodynamic markers of the activity of PI3K/AKT inhibitors currently in clinical development.

Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands.

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.Selective TRIM24 degradation is achieved by co-opting the VHL E3 ubiquitin ligase machinery. TRIM24 degradation outperforms bromodomain inhibition, with an enhanced antiproliferative effect in acute leukemia, a novel context of TRIM24 dependency.

Abstract 2674: High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring

Introduction: The mechanisms underlying the association between high dietary fat intake and prostate cancer (PCa) are unknown. Using a MYC-driven PCa mouse model, we sought to identify metabolic and epigenomic alterations driven by high fat diet (HFD) that facilitate PCa progression. Additionally, we investigated whether these alterations were relevant to PCa progression and lethality in humans. Material and Methods: Wild-type (WT) and transgenic Hi-MYC (MYC) mice were assigned either a HFD or control diet and were sacrificed at 12, 24, and 36 weeks of age for histologic and phenotypic characterization. Metabolic and epigenomic analyses were carried on the ventral prostates of 12-week old mice. Human PCa gene expression profiling data were obtained from 319 men with PCa and well-annotated post-diagnostic saturated fat intake (SFI) data from the Physicians’ Health Study and Health Professionals Follow-up Study prospective cohorts. Results: HFD does not affect the incidence of MYC-induced murine prostate intraepithelial neoplasia (mPIN) at 12 weeks, but increases mPIN proliferative index (Ki-67) at 24 weeks and tumor burden at 36 weeks. MYC overexpression, as expected, induces a significant metabolic reprogramming and HFD further enhances this rewiring to provide additional anabolic metabolites to sustain the increased proliferation of MYC prostate while having little effect on the WT prostate. Moreover, MYC altered key metabolites of the methionine cycle in a direction suggestive of a global hypomethylation, again amplified by HFD. Targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature, including a global demethylation of H3K27 and H4K20 marks, the latter enhanced by HFD. Moreover, ChIP-seq revealed an intricate crosstalk between MYC and the H4K20me1 demethylase PHF8, resulting in enhanced genomic instability in the context of HFD. Finally, RNA-seq and ATAC-seq analyses showed that HFD rewires MYC-driven PCa through the alteration of genes implicated in chromatin function and remodeling. In humans, SFI was associated with enrichment in genes associated with increased MYC transcriptional activity in the prostate. Furthermore, this MYC transcriptional signature was associated with PCa lethality overall (OR = 3.21; 95% CI = 1.47, 7.35 comparing extreme score tertiles), and the association was stronger among men with high post-diagnostic SFI (OR = 1.32; 95%CI = 1.11, 1.66) than those with low SFI (OR = 1.05; 95%CI = 0.98, 1.12). Conclusions: HFD supports a coordinated metabolomic and epigenomic rewiring to increase epigenomic plasticity and MYC transcriptional activity prior to the appearance of phenotypic alterations in the prostate. Importantly, HFD requires MYC-mediated transformation to trigger its deleterious effects. In humans, SFI also enhances MYC transcriptional activity, which is associated with increased PCa lethality. Citation Format: David P. Labbe’, Giorgia Zadra, Meng Yang, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Ericka M. Ebot, Maura B. Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Jorge E. Chavarro, Massimo Loda, Myles Brown. High fat diet accelerates MYC-driven prostate cancer through metabolic and epigenomic rewiring. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2674.

Abstract 981: The design and characterization of a selective TRIM24 degrader

A fundamental biological understanding of the individual contributions of functional domains within multidomain proteins is critical to inform therapeutic approaches to targeting mechanisms driving human disease. TRIM24 is a multi-domain protein that has been broadly characterized as a co-regulator of transcription. It is therapeutically relevant as it is implicated as a dependency in many human cancers, however, the potent and selective inhibitors of the TRIM24 bromodomain do not have well-characterized phenotypic consequences. Where inhibition of one activity of a protein does not appear to be efficacious, chemical knockdown tools allow for the acute depletion of the entire protein, therefore eliminating all protein activities. In this study, we have used chemical degradation as one strategy to target the entire TRIM24 protein, where we have shown that a bifunctional degrader molecule hijacks the ubiquitin ligase machinery for targeted TRIM24 degradation. We have shown that TRIM24 degradation is required to perturb the oncogenic state in leukemia. In this context, TRIM24 degradation rather than bromodomain inhibition alone is required to displace TRIM24 from chromatin. Using this probe, a further understanding of the contribution of TRIM24 domains to its transcriptional activation function will provide mechanistic insight as to how TRIM24 promotes a gene expression program permissive of the oncogenic state, as well as inform a therapeutic approach to target multidomain proteins, such as TRIM24, that are tightly linked to disease. Citation Format: Lara N. Gechijian, Dennis Buckley, Matthew Lawlor, Thomas Scott, Joshiawa Paulk, Jaime Reyes, Georg Winter, Michael Erb, Chris Ott, Sirano Dhe-Paganon, James Bradner. The design and characterization of a selective TRIM24 degrader [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 981. doi:10.1158/1538-7445.AM2017-981

Abstract A10: High-fat diet enhances MYC-driven prostate cancer through epigenomic and metabolomic rewiring

Diet is hypothesized to be a critical environmental risk factor for prostate cancer (PCa) development, and progression; however, the mechanisms underlying these associations remain elusive. In a MYC-driven PCa mouse model we find that a high fat diet significantly alters the transcription of genes implicated in chromatin function and remodeling in prostatic tumor tissues but not in the normal prostate. Importantly, this chromatin associated gene expression signature was observed well before the appearance of a high fat diet-driven phenotype that was characterized by greater cell proliferation and increased tumor burden. Consistent with this finding, high-throughput targeted quantitative histone mass spectrometry revealed a robust MYC-driven signature affecting more than half of the 68 histone marks profiled. Surprisingly, high fat diet further enhanced the MYC-induced epigenetic signature while it was unable to affect the normal murine prostate. Epigenetic remodeling relies on substrates and cofactors that are obtained from the diet. Untargeted metabolomic analyses revealed that MYC overexpression, as expected, impacted glutamine uptake. In addition, high fat diet leads to additional carbohydrates, amino acids, lipids and nucleotides necessary to sustain an increased cellular proliferation in MYC-driven cancers while it had little influence on the normal prostate. Moreover, the pool of metabolites altered by high fat diet in the context of MYC overexpression is highly suggestive of a global methylation defect. Finally, using the genome-wide mRNA profiles of tumor (N=402) and adjacent normal (N=200) prostate tissues from the Health Professionals Follow-up Study and the Physicians9 Health Study cohorts, we have discovered an enrichment in genes implicated in chromatin function and remodeling in tumor tissues from overweight/obese men, but not in normal adjacent tissues, consistent with the high fat diet signature observed in mice. Strikingly, men whose tumors had high expression of this chromatin signature had worse clinical characteristics and were more likely to die from prostate cancer (OR = 5.01; 95% CI = 2.31, 11.38 comparing extreme score quartiles). Taken together, these results demonstrate that a high fat diet does not drive significant epigenomic and metabolomic alterations in the normal prostate while it leads to important alterations in MYC-driven PCa that results in increased aggressiveness. Our results suggest that the impact of diet on PCa risk may be to augment the growth of already established subclinical disease. In addition, as MYC is one of the most commonly amplified genes in PCa, the ability of a high fat diet to augment MYC-driven cancers in this pre-clinical model suggest that a healthy diet may slow the progression of the disease. Citation Format: David P. Labbe, Giorgia Zadra, Ericka M. Ebot, Charles Y. Lin, Jaime M. Reyes, Stefano Cacciatore, Maura Cotter, Amanda L. Creech, Jacob D. Jaffe, Philip W. Kantoff, James E. Bradner, Lorelei A. Mucci, Massimo Loda, Myles Brown. High-fat diet enhances MYC-driven prostate cancer through epigenomic and metabolomic rewiring. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A10.

Abstract 2209: Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape

Unrestrained activation of receptor tyrosine kinase (RTK) pathways drives tumorigenesis through the persistent activation of critical signaling pathways, including the Ras-ERK axis. This aberrant signaling is unleashed by mutations in signaling effectors such as Ras, as well as through disruption of feedback control by regulatory proteins such as the Sprouty family. Mutations in epigenetic regulators also commonly occur during cancer progression, modulating chromatin architecture and gene expression. While unrestrained signaling and epigenetic deregulation are root causes of tumorigenesis, the relationship between aberrant RTK signaling and chromatin modifications at cis-regulatory elements remains to be fully elucidated. We establish linkage between these processes by examining the effects of oncogenic HRasG12V or loss of feedback regulation by Sprouty on global changes in gene expression and enhancer-associated chromatin modifications. Using RNA-sequencing and ChIP-sequencing, we demonstrate that aberrant RTK signaling unleashed by oncogenic HRasG12V or Sprouty gene deletion disrupts gene expression programs and remodels histone modifications associated with active enhancers, including histone 3 lysine 27 acetylation (H3K27ac). Abolishing persistent Ras-Erk signaling through chemical inhibition of MEK activity reverses the aberrant transcriptional program and H3K27ac remodeling at deregulated enhancers. While both lesions disrupt the Ras-Erk axis, the specific target genes and enhancers modulated upon HRasG12V-transformation or Sprouty deletion are largely distinct. Specifically, oncogenic HRasG12V significantly elevates the expression and H3K27ac levels near key target genes encoding the transcription factor Gata4 and the kinase Prkcb. Gata4 is necessary for the HRasG12V expression program and coordinates H3K27ac marking at enhancers of deregulated target genes. We further show that HRasG12V-driven cells are sensitive to chemical inhibition of Prkcb, which reduces the viability and clonogenicity of HRasG12V-transformed cells. These oncogenic effects upon HRasG12V-transformation are also reduced by chemical inhibition of the chromatin regulators BET bromodomain proteins and p300/CBP, which recognize and deposit H3K27ac, respectively. Taken together, our data support a model in which dynamic reprogramming of the cellular enhancer landscape is a key effect of oncogenic RTK signaling. Furthermore, our study shows that identification of enhancers regulated by oncogenic Ras yields insight into targeting key epigenetic regulators and downstream factors that promote Ras-driven malignancies. Citation Format: Behnam Nabet, Pilib O Broin, Jaime Reyes, Kevin Shieh, Charles Y. Lin, Christine M. Will, Relja Popovic, Teresa Ezponda, James E. Bradner, Aaron A. Golden, Jonathan D. Licht. Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2015-2209