Chiara Vardabasso

The histone variant macroH2A suppresses melanoma progression through regulation of CDK8

Cancer is a disease consisting of both genetic and epigenetic changes. Although increasing evidence demonstrates that tumour progression entails chromatin-mediated changes such as DNA methylation, the role of histone variants in cancer initiation and progression currently remains unclear. Histone variants replace conventional histones within the nucleosome and confer unique biological functions to chromatin. Here we report that the histone variant macroH2A (mH2A) suppresses tumour progression of malignant melanoma. Loss of mH2A isoforms, histone variants generally associated with condensed chromatin and fine-tuning of developmental gene expression programs, is positively correlated with increasing malignant phenotype of melanoma cells in culture and human tissue samples. Knockdown of mH2A isoforms in melanoma cells of low malignancy results in significantly increased proliferation and migration in vitro and growth and metastasis in vivo. Restored expression of mH2A isoforms rescues these malignant phenotypes in vitro and in vivo. We demonstrate that the tumour-promoting function of mH2A loss is mediated, at least in part, through direct transcriptional upregulation of CDK8. Suppression of CDK8, a colorectal cancer oncogene, inhibits proliferation of melanoma cells, and knockdown of CDK8 in cells depleted of mH2A suppresses the proliferative advantage induced by mH2A loss. Moreover, a significant inverse correlation between mH2A and CDK8 expression levels exists in melanoma patient samples. Taken together, our results demonstrate that mH2A is a critical component of chromatin that suppresses the development of malignant melanoma, a highly intractable cutaneous neoplasm.

Histone variants: emerging players in cancer biology

Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology.

ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globin expression

The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (α-thalassemia/MR, X-linked) as a novel macroH2A-interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2As chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of α-globin expression. Collectively, our results implicate deregulation of macroH2As distribution as a contributing factor to the α-thalassemia phenotype of ATRX syndrome.

Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a transmembrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

Decreased Expression of the Chromatin Remodeler ATRX Associates with Melanoma Progression

To the Editor ATRX is a member of the SWI/SNF family of chromatin remodelers, originally identified as mutated in patients with Alpha Thalassemia/Mental Retardation, X-linked syndrome. The protein product contains several highly conserved domains, including an ADD (ATRX-DNMT3-DNMT3L) domain that binds methylated histone H3 at lysine 9 and an ATPase domain responsible for its remodeling activities (Ratnakumar & Bernstein, 2013). Recently, whole genome sequencing studies identified ATRX mutations in multiple tumors, including those of neural crest cell origin: neuroblastoma, low-grade glioma and glioblastoma (Cheung et al., 2012; Heaphy et al., 2011a; Jiao et al., 2011; Kannan et al., 2012; Schwartzentruber et al., 2012). ATRX alterations encompass point mutations throughout the coding region as well as large N terminal deletions. While mechanistically unclear, ATRX mutations result in loss of protein as assessed by immunohistochemistry (IHC) and often correlate with alternative lengthening of telomeres (ALT) (Cheung et al., 2012; Heaphy et al., 2011a; Kannan et al., 2012; Schwartzentruber et al., 2012). To our knowledge, an investigation of ATRX in cutaneous melanoma is currently lacking. Our previous studies have demonstrated that decreased expression of histone variant macroH2A drives melanoma cell proliferation and metastasis (Kapoor et al., 2010), and that ATRX interacts with macroH2A to negatively regulate its association with chromatin (Ratnakumar et al., 2012). Taken together with recent reports of decreased ATRX protein in neural crest cell-derived tumors, we hypothesized that ATRX function might be compromised in melanoma. In order to test this hypothesis, we performed IHC on a panel of 23 benign nevi, 33 primary melanoma (≥1.0 mm deep) and 25 metastatic melanoma specimens that were formalin fixed paraffin embedded (FFPE) (Figure 1a, c). Slides were evaluated by two dermatopathologists in a blinded fashion using a scoring system based on number of positive nuclei and staining intensity (inter-rater correlation r=0.693, p<0.0001; see Supplemental Methods for details). As depicted in Figure 1a, ATRX protein expression is appreciably reduced with increased malignancy. Benign nevi showed a higher proportion and intensity of nuclear staining when compared to metastatic lesions (Figure 1a, b; p<0.0001). Furthermore, ATRX protein expression was reduced between benign nevi and primary melanoma, with heterogeneous staining observed in the latter (p=0.0026; Figure 1a, b), and between primary and metastatic melanoma (p=0.0113; Figure 1b). This suggests a potential step-wise loss of ATRX expression during melanoma progression. Figure 1 Loss of ATRX protein expression is associated with melanoma progression We further examined whether ATRX levels in primary melanoma correlated with clinicopathologic predictors of prognosis. ATRX staining did not correlate with depth of the lesion (data not shown), however the primary melanomas examined in our cohort were of Breslow thickness greater than 1.0mm (average depth 5.6 mm), and thus quite aggressive. We did however find an inverse correlation with the presence of ulceration, a poor prognostic factor (Figure 1d). Because our study is retrospective with a small sample size, we note that any correlations, or lack thereof, are preliminary. Because structural variations of ATRX exist in neuroblastoma and osteosarcoma (Cheung et al., 2012; Lovejoy et al., 2012), we determined whether such alterations are present in metastatic melanoma. Using a technique to detect structural variations of ATRX, we performed qualitative reverse transcriptase (RT)-PCR of cDNA derived from a cohort of fresh frozen metastatic melanoma samples (n=7). Due to the large ATRX coding region, we amplified the cDNA into five fragments ranging from 1.5-2 kilobase pairs. Because ATRX is located on the X chromosome, we analyzed both male and female patients for potential effects due to gene dosage. Our analysis shows that the ATRX gene product is intact in all metastatic melanomas assayed, as evidenced by appropriately sized bands within each sample (Figure 2a). The cell line WM266-4 derived from a melanoma metastasis served as a positive control for PCR amplicons, as it is devoid of ATRX mutations (Cancer Cell Line Encyclopedia at http://cbioportal.org). The osteosarcoma cell line U2OS, which has large deletions of the ATRX locus (Lovejoy et al., 2012) was used to ensure our assay worked effectively. This analysis suggested that decreased ATRX protein level in metastatic melanoma is unlikely the result of large genomic alterations. Figure 2 ATRX mRNA levels are decreased in metastatic melanoma We next queried whether diminished ATRX protein in metastatic disease was due to transcriptional regulation. We performed qPCR analysis on a cohort of 18 fresh frozen benign nevi and 20 metastatic melanoma tumors, including those samples analyzed for deletions (Figure 2a; indicated in red in Figure 2b). Using both N- and C- terminal primers for ATRX, we found a statistically significant loss of ATRX mRNA levels in metastatic melanoma as compared to benign tissue (p<0.0001; Figure 2b). We next performed IHC on a subset of these tumors, for which FFPE tissue was available (indicated in blue in Figure 2b). The level of ATRX protein indeed corroborated our qPCR findings (Figure 2c). Collectively, these results indicate that ATRX loss occurs, at least in part, by transcriptional repression resulting in loss of protein expression in late stage disease. Collectively, we demonstrate that ATRX loss correlates with melanoma progression. Using two independent cohorts (FFPE and fresh frozen; total of 119 tissues), we found a significant decrease of both mRNA and protein levels of ATRX in metastatic melanoma. While it remains to be tested in a prospective study, ATRX may serve as a biomarker to predict prognosis of disease. Though we did not find evidence of large genomic alterations in a subset of melanoma patients, we do not exclude the possibility of ATRX mutations in melanoma. In fact, a 4-7.5% rate of mutation in cutaneous melanoma is reported by TCGA, Broad and Yale studies (http://www.cbioportal.org). Interestingly, these mutations are distributed throughout the ATRX coding region and do not correlate with decreased mRNA levels (Supplemental Figure S1). This suggests that multiple mechanisms underlie ATRX dysregulation in melanoma – transcriptional regulation as described here and point mutations that may result in loss of protein expression, as reported for other tumor types (Cheung et al., 2012; Kannan et al., 2012; Schwartzentruber et al., 2012). While ATRX staining did not anti-correlate with macroH2A levels (data not shown), we previously showed that macroH2A is transcriptionally silenced by DNA methylation in malignant melanoma and thus might not be regulated at the level of chromatin deposition (Kapoor et al., 2010). The mechanism by which ATRX transcription is suppressed in melanoma may also be through epigenetic silencing (e.g. DNA methylation or histone modifications), or by microRNA mediated regulation (Pacurari et al., 2013). Finally, we posit that investigating the chromatin landscape of tumors that have lost ATRX expression should provide insight into the mechanism(s) by which ATRX loss drives melanoma progression.

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA‐based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z‐nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate‐specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z‐specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome‐wide mapping reveals that PWWP2A binds selectively to H2A.Z‐containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C‐terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z‐specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

Histone variant H2A.Z.2: A novel driver of melanoma progression

ABSTRACT Histone variants are attracting attention in the field of cancer epigenetics. Our study has established a novel role for the uncharacterized histone variant H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 promotes cellular proliferation by recruiting BRD2 and E2F1 to E2F target genes and facilitating their transcription. High H2A.Z.2 expression correlates with poor survival in patients, and its depletion sensitizes cells to chemotherapy and targeted therapies.

The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

Abstract A12: Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma

Malignant melanoma is the most lethal form of skin cancer with rising incidence. Once metastasis occurs, patients have a dismal prognosis, largely due to limited systemic treatment with chemotherapy and resistance to targeted therapies. Thus, effective therapies with long-term responses are currently lacking. Although much effort has focused on characterizing and targeting the genetic alterations in melanoma, the identification of epigenetic players remains poorly understood. Chromatin dynamics have recently been shown to exert a critical function in a number of cancers, including melanoma, and emerging evidence points towards a role of histone variants as key regulatory molecules in cancer. H2A.Z is a highly conserved H2A variant, harboring two different isoforms in vertebrates, H2A.Z.1 and H2A.Z.2. High levels of H2A.Z promote cell proliferation in breast, prostate and bladder cancers, however studies so far have focused primarily on H2A.Z.1 or did not clearly distinguish between the two isoforms. Here, we report a role for the unappreciated isoform H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma. To our knowledge, this is the first evidence to implicate a distinct role for this H2A.Z isoform in any tumor type. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation, which occurs through a G1 to S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, which are highly expressed and acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic cells. We further identified the BET (bromodomain and extraterminal domain) family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and our data suggest that H2A.Z.2 exerts its oncogenic function by maintaining the global levels of BRD2 and histone H4 acetylation. Furthermore, H2A.Z.2 depletion sensitizes melanoma cells to targeted therapies and chemotherapy. Collectively, our findings implicate H2A.Z.2 as a driver of melanoma pathogenesis. Owing to the fact that histone modification is a reversible process, H2A.Z.2 and BRD2 hold translational potential for novel therapeutic strategies. Citation Format: Chiara Vardabasso, Alexandre Gaspar-Maia, Sebastian Punzeler, David Valle-Garcia, Dan Hasson, Tobias Straub, Eva C. Keilhauer, Thomas Strub, Taniya Panda, Miguel F. Segura, Chi-Yeh Chung, Amit K. Verma, Matthias Mann, Eva Hernando, Sandra B. Hake, Emily Bernstein. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A12.