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Supplementary Figure S1. This supplementary figure shows that SNF5 KD in normal human fibroblasts does not alter mRNA levels of SWI/SNF components.

Table shows genes with significantly altered expression by RNA-seq after BRG1 or BRM knockout.

Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP–Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs.Implications: SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation. Mol Cancer Res; 12(11); 1574–85. ©2014 AACR.

Supplementary Figure 1 shows BRG1 deficiency activates transcription of a subset of NRF2 target genes in second H358 BRG1 knockdown cell line; Supplementary Figure 2 shows western blot of BRG1 and BRM knockout samples used for RNA-seq.

Purpose:Most aggressive thyroid cancers are commonly associated with a BRAFV600E mutation. Preclinical and clinical data in BRAFV600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAFV600E thyroid cancer than in single-agent or BRAF and MEK inhibitors.Experimental Design:The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs.Results:The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAFV600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAFV600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control.Conclusions:Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAFV600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Supplementary method and data

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)‐associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.

The NF-E2–related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. Implications: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.

Purpose: Most aggressive thyroid cancers are commonly associated with a BRAFV600E mutation. Preclinical and clinical data in BRAFV600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAFV600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. Experimental Design: The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. Results: The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAFV600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAFV600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Conclusions: Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAFV600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Renal medullary carcinoma (RMC) is a rare, aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses with a median overall survival of 13 months. Patients are typically young (median age—22) and male (male:female ratio of 2:1) and tumors are characterized by complete loss of expression of the SMARCB1 tumor suppressor protein. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to better understand the pathogenesis of this disease and to identify effective forms of therapy. Two novel cell line models, UOK353 and UOK360, were derived from primary RMCs that both demonstrated the characteristic SMARCB1 loss. Both cell lines overexpressed EZH2 and other members of the polycomb repressive complex and EZH2 inhibition in RMC tumor spheroids resulted in decreased viability. High throughput drug screening of both cell lines revealed several additional candidate compounds, including bortezomib that had both in vitro and in vivo antitumor activity. The activity of bortezomib was shown to be partially dependent on increased oxidative stress as addition of the N‐acetyl cysteine antioxidant reduced the effect on cell proliferation. Combining bortezomib and cisplatin further decreased cell viability both in vitro and in vivo that single agent bortezomib treatment. The UOK353 and UOK360 cell lines represent novel preclinical models for the development of effective forms of therapy for RMC patients.

Kidney cancer is not a single disease but represents several distinct types of cancer that have defining histologies and genetic alterations and that follow different clinical courses and have different responses to therapy. Mutation of genes associated with kidney cancer, such as VHL, FLCN, TFE3, FH, or SDHB, dysregulates the tumor's responses to changes in oxygen, iron, nutrient, or energy levels. The identification of these varying genetic bases of kidney cancer has increased our understanding of the biology of this cancer, allowing the development of targeted therapies and the appreciation that it is a cancer driven by metabolic alterations. Significance: Kidney cancer is a complex disease composed of different types of cancer that present with different histologies, clinical courses, genetic changes, and responses to therapy. This review describes the known genetic changes within kidney cancer, how they alter tumor metabolism, and how these metabolic changes can be therapeutically targeted.

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel a FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Renal medullary carcinoma (RMC) is one of the most aggressive renal cell carcinomas. It predominantly afflicts young adults and adolescents with sickle cell trait and other sickle hemoglobinopathies, and is refractory to targeted and antiangiogenic therapies used in patients with clear-cell renal cell carcinoma. Platinum-based cytotoxic chemotherapy is the mainstay for RMC treatment. On the basis of recent advances in the diagnosis, management, and clinical trial development for RMC, a panel of experts met in October 2017 and developed updated consensus recommendations to inform clinicians, researchers, and patients. Because RMC often aggressively recurs while patients are still recovering from nephrectomy, upfront chemotherapy should be considered for most patients, including those with localized disease. After safety and dosing information has been established in adults, phase II and III trials enrolling patients with RMC should allow patients aged 12 years and older to be accrued. Patients with the very rare unclassified renal cell carcinoma with medullary phenotype variant should be included in RMC trials. Medical providers should be aware that RMC can afflict subjects of all races, and not only those of African descent, and that the presence of sickle cell trait, or of other sickle hemoglobinopathies, can affect drug responses and toxicity.

Purpose Specificity protein 1 (SP1) is involved in the transcription of several genes implicated in tumor maintenance. We investigated the effects of mithramycin A (MTA), an inhibitor of SP1 DNA binding, on radiation response. Methods and Materials Clonogenic survival after irradiation was assessed in 2 tumor cell lines (A549, UM-UC-3) and 1 human fibroblast line (BJ) after SP1 knockdown or MTA treatment. DNA damage repair was evaluated using γH2AX foci formation, and mitotic catastrophe was assessed using nuclear morphology. Gene expression was evaluated using polymerase chain reaction arrays. In vivo tumor growth delay was used to evaluate the effects of MTA on radiosensitivity. Results Targeting of SP1 with small interfering RNA or MTA sensitized A549 and UM-UC-3 to irradiation, with no effect on the BJ radiation response. MTA did not alter γH2AX foci formation after irradiation in tumor cells but did enhance mitotic catastrophe. Treatment with MTA suppressed transcription of genes involved in cell death. MTA administration to mice bearing A549 and UM-UC-3 xenografts enhanced radiation-induced tumor growth delay. Conclusions These results support SP1 as a target for radiation sensitization and confirm MTA as a radiation sensitizer in human tumor models.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Chromophobe renal cell carcinoma (ChRCC) represents 5% of all RCC cases and frequently demonstrates multiple chromosomal losses and an indolent pattern of local growth, but can demonstrate aggressive features and resistance to treatment in a metastatic setting. Cell line models are an important tool for the investigation of tumor biology and therapeutic drug efficacy. Currently, there are few ChRCC-derived cell lines and none is well characterized. This study characterizes a novel ChRCC-derived cell line model, UOK276. A large ChRCC tumor with regions of sarcomatoid differentiation was used to establish a spontaneously immortal cell line, UOK276. UOK276 was evaluated for chromosomal, mutational, and metabolic aberrations. The UOK276 cell line is hyperdiploid with a modal number of 49 chromosomes per cell, and evidence of copy-neutral loss of heterozygosity, as opposed to the classic pattern of ChRCC chromosomal losses. UOK276 demonstrated a TP53 missense mutation, expressed mutant TP53 protein, and responded to treatment with a small-molecule therapeutic agent, NSC319726, designed to reactivate mutated TP53. Xenograft tumors grew in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The xenograft pathology and genetic analysis suggested that UOK276 was derived from the sarcomatoid region of the original tumor. In summary, UOK276 represents a novel in vitro and in vivo cell line model for aggressive, sarcomatoid-differentiated, TP53 mutant ChRCC. This preclinical model system could be used to investigate the novel biology of aggressive, sarcomatoid ChRCC and evaluate the new therapeutic regimes.

BackgroundRenal cell carcinomas (RCC) harboring aTFE3gene fusion (TfRCC) represent an aggressive subset of kidney tumors. Key signaling pathways of TfRCC are unknown and preclinical in vivo data are lacking. We investigated Akt/mTOR pathway activation and the preclinical efficacy of dual mTORC1/2 versus selective mTORC1 inhibition in TfRCC.MethodsLevels of phosphorylated Akt/mTOR pathway proteins were compared by immunoblot in TfRCC and clear cell RCC (ccRCC) cell lines. Effects of the mTORC1 inhibitor, sirolimus, and the dual mTORC1/2 inhibitor, AZD8055, on Akt/mTOR activation, cell cycle progression, cell viability and cytotoxicity were compared in TfRCC cells. TfRCC xenograft tumor growth in mice was evaluated after 3-week treatment with oral AZD8055, intraperitoneal sirolimus and respective vehicle controls.ResultsThe Akt/mTOR pathway was activated to a similar or greater degree in TfRCC than ccRCC cell lines and persisted partly during growth factor starvation, suggesting constitutive activation. Dual mTORC1/2 inhibition with AZD8055 potently inhibited TfRCC viability (IC50 = 20-50 nM) due at least in part to cell cycle arrest, while benign renal epithelial cells were relatively resistant (IC50 = 400 nM). Maximal viability reduction was greater with AZD8055 than sirolimus (80–90% versus 30–50%), as was the extent of Akt/mTOR pathway inhibition, based on significantly greater suppression of P-Akt (Ser473), P-4EBP1, P-mTOR and HIF1α. In mouse xenograft models, AZD8055 achieved significantly better tumor growth inhibition and prolonged mouse survival compared to sirolimus or vehicle controls.ConclusionsAkt/mTOR activation is common in TfRCC and a promising therapeutic target. Dual mTORC1/2 inhibition suppresses Akt/mTOR signaling more effectively than selective mTORC1 inhibition and demonstrates in vivo preclinical efficacy against TFE3-fusion renal cell carcinoma.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is characterized by germline mutations of the FH gene that encodes for the TCA cycle enzyme, fumarate hydratase. HLRCC patients are at risk for the development of an aggressive form of type 2 papillary renal cell carcinoma. By studying the mechanism of action of marizomib, a proteasome inhibitor able to cross the blood-brain barrier, we found that it modulates the metabolism of HLRCC cells. Marizomib decreased glycolysis in vitro and in vivo by downregulating p62 and c-Myc. C-Myc downregulation decreased the expression of lactate dehydrogenase A, the enzyme catalyzing the conversion of pyruvate to lactate. In addition, proteasomal inhibition lowered the expression of the glutaminases GLS and GLS2, which support glutamine metabolism and the maintenance of the redox balance. Thus, in HLRCC cells, proteasome inhibition disrupts glucose and glutamine metabolism, restricting nutrients and lowering the cells’ anti-oxidant response capacity. Although the cytotoxicity induced by proteasome inhibitors is complex, the understanding of their metabolic effects in HLRCC may lead to the development of effective therapeutic strategies or to the development of markers of efficacy.

Figure S1. Flow cytometry representing suppression of S-phase of cell cycle in TfRCC cells using mTOR inhibitors Cell cycle profile of mTOR inhibitor-treated UOK120 and UOK146 cells measured by flow cytometry and displayed as time-course experiment showing percentage of cells in G2/M-phase, S-phase and G0/G1 phase of the cell cycle at 12 h, 24 h, 48 h and 72 h following drug treatment with 50 nM and 500 nM of Sirolimus and AZD8055 (a and b). Representative scatter plots of total DNA content versus newly synthesized DNA content are shown in c and d. Dose-dependent reduction in the proportion of cells in S-phase is apparent in both cell lines at all time points, with a greater reduction observed using dual mTORC1/2 inhibition (AZD8055) than selective mTORC1 inhibition (sirolimus). An accumulation over time of cells arrested in G0/G1 phase of the cell cycle can be observed. Figure S2. Dual mTORC1/2 inhibitor and selective mTORC1 inhibitor treatments achieve on-target effects in TfRCC xenograft models. A quantitative analysis of the changes of phosphorylated protein levels of mTOR pathway proteins in UOK120 and UOK146 xenograft tumors 6 h after treatment with a selective mTORC1 inhibitor (sirolimus), a dual mTORC1/2 inhibitor (AZD8055) or respective vehicle controls (see Fig. 5) is shown as normalized intensity based on β-actin protein levels. (PDF 670 kb)

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth and survival. Understanding these mechanisms and developing biomarkers to identify their presence thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of the covalent targets of fumarate, an oncometabolite whose accumulation marks the genetic cancer predisposition syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). First, we validate the ability of known and novel chemoproteomic probes to report on fumarate reactivity in vitro. Next, we apply these probes in concert with LC-MS/MS to identify cysteine residues sensitive to either fumarate treatment or fumarate hydratase (FH) mutation in untransformed and human HLRCC cell models, respectively. Mining this data to understand the structural determinants of fumarate reactivity reveals an unexpected anti-correlation with nucleophilicity, and the discovery of a novel influence of pH on fumarate-cysteine interactions. Finally, we show that many fumarate-sensitive and FH-regulated cysteines are found in functional protein domains, and perform mechanistic studies of a fumarate-sensitive cysteine in SMARCC1 that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the influence of fumarate on reactive cysteine residues, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15–20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Malignant rhabdoid tumor (MRT), a highly aggressive cancer of young children, displays inactivation or loss of the hSNF5/INI1/SMARCB1 gene, a core subunit of the SWI/SNF chromatin-remodeling complex, in primary tumors and cell lines. We have previously reported that reexpression of hSNF5 in some MRT cell lines causes a G1 arrest via p21CIP1/WAF1 (p21) mRNA induction in a p53-independent manner. However, the mechanism(s) by which hSNF5 reexpression activates gene transcription remains unclear. We initially searched for other hSNF5 target genes by asking whether hSNF5 loss altered regulation of other consensus p53 target genes. Our studies show that hSNF5 regulates only a subset of p53 target genes, including p21 and NOXA, in MRT cell lines. We also show that hSNF5 reexpression modulates SWI/SNF complex levels at the transcription start site (TSS) at both loci and leads to activation of transcription initiation through recruitment of RNA polymerase II (RNAPII) accompanied by H3K4 and H3K36 modifications. Furthermore, our results show lower NOXA expression in MRT cell lines compared with other human tumor cell lines, suggesting that hSNF5 loss may alter the expression of this important apoptotic gene. Thus, one mechanism for MRT development after hSNF5 loss may rely on reduced chromatin-remodeling activity of the SWI/SNF complex at the TSS of critical gene promoters. Furthermore, because we observe growth inhibition after NOXA expression in MRT cells, the NOXA pathway may provide a novel target with clinical relevancy for treatment of this aggressive disease. Visual Overview: http://mcr.aacrjournals.org/content/11/3/251/F1.large.jpg. Mol Cancer Res; 11(3); 251–60. ©2013 AACR.

Renal medullary carcinoma (RMC) is a rare and aggressive disease that predominantly afflicts individuals of African or Mediterranean descent with sickle cell trait. RMC comprises 1% of all renal cell carcinoma diagnoses, and the median overall survival for RMC patients is only 13 months. Patients are typically young (median age-22yo) and male (male:female ratio of 2:1). RMC was first characterized in 1995, and its hallmark SMARCB1 loss was identified in 2008. Due to the low incidence of RMC and the disease's aggressiveness, treatment decisions are often based on case reports. Thus, it is critical to develop preclinical models of RMC to both better understand the pathogenesis of this disease and identify an effective therapy. These RMC cell lines overexpress EZH2, relative to normal renal proximal tubule epithelium, thus making it an attractive druggable target. EZH2 inhibition in RMC tumor spheroids did not result in decreased viability. We have evaluated a potential clinical treatment (bortezomib) found in the literature and validate bortezomib's potency in vitro. Intriguingly, these cells lines not only lack NQO1, an antioxidant enzyme, but also the addition of N-acetyl cysteine, an antioxidant, has a protective effect against bortezomib. Together the data suggest that RMC are highly sensitive to oxidative stress. This evidence is consistent with case reports in the literature that found bortezomib treatment of RMC resulted in positive outcomes. Citation Format: Darmood Wei, Youfeng Yang, Christopher J. Ricketts, Cathy D. Vocke, Mark W. Ball, Carole Sourbier, Darawalee Wangsa, Danny Wangsa, Rajarshi Guha, Xiaohu Zhang, Paul Meltzer, Thomas Reid, Craig J. Thomas, Maria J. Merino, W. Marston Linehan. Novel renal medullary carcinoma cell lines, UOK353 and UOK360, provide preclinical tools to identify new therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1169.

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects of two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC tumor spheroids. Our preliminary data suggests EZH2 inhibition results in reduced growth of PBRM1 mutant cell lines grown as tumor spheroids. Citation Format: Darmood Wei, Youfeng Yang, Christopher J. Ricketts, Carole Sourbier, Laura S. Schmidt, William M. Linehan. Using tumor spheroids to evaluate the efficacy of EZH2 inhibitors in clear cell renal cell carcinoma (ccRCC) [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 1400. doi:10.1158/1538-7445.AM2017-1400

Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP–Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs. Implications: SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation. Mol Cancer Res; 12(11); 1574–85. ©2014 AACR . This article is featured in Highlights of This Issue, [p. 1533][1] [1]: /lookup/volpage/12/1533?iss=11

Malignant rhabdoid tumours (MRTs) represent an aggressive paediatric cancer with limited treatment options. Although MRTs mainly arise in the kidney and brain of patients under the age of 8 years, they may appear in other major organs as well as in soft tissues. Remarkably, these tumours possess few mutations other than the ones that inactivate the SNF5/INI1 gene, the smallest member of the SWI/SNF chromatin-remodelling complex. In addition, these cancers lack other hallmarks of adult malignancies including genomic instability, aberrant karyotypes and abnormal regulation of cellular stress-response pathways. Their major defect appears to arise from epigenetic instability, presumably through changes in nucleosome positioning due to defective chromatin-remodelling activity. Recent reports have shown that SNF5 loss affects key signalling pathways such as cell cycle regulation, deoxyribonucleic acid (DNA) damage repair and gene transcription. Thus, MRTs offer a unique model for studying the role of epigenetics in driving tumourigenesis and for the development of novel treatment approaches. Key Concepts: Malignant rhabdoid tumours are rare and aggressive paediatric cancers. MRT was the first tumour in which mutations in the SWI/SNF chromatin-remodelling complex were identified. Inactivation of the SNF5/INI1 tumour suppressor gene drives MRT development. MRTs often show no changes in copy number or karyotype. The key mechanisms by which SNF5/INI1 loss induces MRT development remain unclear. MRTs offer a unique model for epigenetic-driven tumourigenesis. Keywords: malignant rhabdoid tumour; SWI/SNF complex; SNF5; INI1; chromatin remodeling; epigenetics

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices. LOX is highly expressed in aggressive cancers; has been associated with a shorter survival and regulates cancer progression in a variety of human malignancies. Here, we report a new function of LOX in mitosis in anaplastic thyroid cancer cell line (THJ-16T), breast cancer cell line (MDA-MB231) and HeLa cells. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression suggesting that LOX binds to stabilized microtubules only. Functional studies of LOX depletion resulted in decreased cellular proliferation, G2/M cell cycle arrest, and formation of gigantic nuclei. Further, LOX knockdown led to reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B expression. Lastly, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules and this effect was synergistic. Our results show identify a novel function of LOX in cancer cell mitosis, and that loss of LOX expression in cancer cells can enhanced effects of anti-microtubules agents used in cancer therapy. Citation Format: Myriem Boufraqech, Darmood Wei, Urbain Weyemi, Lisa Zhang, Martha Quezado, Petr Kalab, Electron Kebebew. LOX is a novel mitotic spindle-associated protein essential for mitosis. [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 2727.

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors such as CDKN2A/p16. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects on two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC cell lines both in vitro and in vivo. Our preliminary data suggests EZH2 inhibition results in reduced growth of ccRCC cell lines with PBRM1 mutations. Citation Format: Darmood Wei, Christopher J. Ricketts, Laura S. Schmidt, Youfeng Yang, Cathy D. Vocke, William M. Linehan. Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC). [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 4707.

About 10% of papillary renal cell carcinomas (PRCC) bear mutations in NF2 or SAV1, two members of the Hippo pathway, which controls cell growth by regulating the transcriptional activity of YES1-Associated Protein YAP1. NF2 or SAV1 mutations lead to aberrant YAP1 activation resulting in cell proliferation and anti-apoptotic signals. The src family kinase YES1 is a known regulator of YAP1 transcriptional activity in the context of β-catenin-dependent tumors. Dasatinib and saracatinib are 2 src inhibitors that have shown to also have potent inhibitory effects on YES1. We hypothesized that inhibition of the Hippo pathway might provide a valuable therapeutic approach for patients with PRCC tumors bearing NF2 mutation, and that inhibiting YES1 might inhibit YAP1 in PRCC. Our data demonstrate that inhibition of the Hippo pathway is lethal to NF2-deficient PRCC cell lines, and that inhibiting YES1 with dasatinib or saracatinib effectively inhibits YAP1 and its downstream targets BIRC5 (survivin), CCND1 (cyclin D1) and CTGF (CTGF). Further, dasatinib proves to have an anti-tumor effect in vivo and was cytotoxic in vivo and in vitro, causing G0-G1 cell cycle arrest in NF2-deficient PRCC cell lines. Thus, inhibiting YES1 and the subsequent transcriptional activity of YAP1 with dasatinib or saracatinib might be a viable therapeutic approach for NF2-deficient PRCC tumors. Citation Format: Carole Sourbier, Penny Liao, Christopher J. Ricketts, Darmood Wei, Youfeng Yang, Sarah M. Baranes, Toshiki Kijima, Louis S. Krane, Myriem Boufraqech, Lernik Ohanjanian, Ben Gibbs, Ming-Hui Wei, Len Neckers, Cathy D. Vocke, W. Marston Linehan. Targeting the Hippo pathway in NF-2 deficient papillary kidney cancers. [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 1288.

Background: Renal cell carcinoma (RCC) represents a heterogeneous group of cancers that arise from the nephron and are subtyped by histopathological features. The most common subtypes are clear cell RCC (~75%) and papillary RCC (~15%); whereas chromophobe renal cell carcinoma (ChRCC) represents only 5% of RCC cases. ChRCC typically demonstrate a well-known karyotype of multiple chromosomal losses and a relatively indolent pattern of local growth, but can present with aggressive features and demonstrate resistance to treatment in a metastatic setting. Some ChRCC cases demonstrate regions of sarcomatoid RCC and the exact cause of this differentiation has yet to be elucidated. Cell line models are an important tool for both the investigation of tumor biology and therapeutic drug efficacy. Currently, numerous cell lines models exist that have been derived from sporadic clear cell or papillary RCCs, but there are few cell lines derived from chromophobe RCCs and none are well characterized. This study produced a novel ChRCC-derived cell line model and provides an initial genetic and metabolic characterization. Materials and Methods: A patient presented with a 20 cm ChRCC with regions of sarcomatoid differentiation that was surgically excised and a section of this tumor was used to establish a spontaneously immortal cell line model, UOK276. This line was grown for over 20 passages and cytogenetically assessed by spectral karyotyping (SKY). Mutation analysis was performed using a cancer gene specific chip, OncoVar V3, which analyses 232 genes. Identified mutations were confirmed in both UOK276 and the original tumor tissue and further investigated for their effects of mRNA and protein expression. UOK276 cells were injected into nude mice to assess the production of xenograph tumors. The metabolic, bioenergetic profile was assessed using a Seahorse XF96 Extracellular Flux Analyzer. Results: The chromosomal SKY analysis did not demonstrate the classic pattern of chromophobe chromosomal losses, but demonstrated hyper-aneuploidy, with a modal number of 49 chromosomes per cell, and identified a balanced translocation t(X;8)(q10;q24). The break on chromosome 8q occurred near the MYC gene, but break-apart FISH analysis demonstrated no alterations to MYC although amplification of this derivative chromosome was observed and increased MYC mRNA expression was demonstrated. Mutation analysis identified a missense mutation (p.H193Y) of TP53, commonly mutated in ChRCC, which was only present in the sarcomatoid region of the tumor. Mutation of TP53 has previously been associated with sarcomatoid differentiation. Protein expression analysis demonstrated the presence of the mutant TP53 protein in UOK276. A heterozygous germline mutation in TRAF7 was identified resulting in an in-frame loss of 4 amino acids (del T22-P25) that was homozygous in the sarcomatoid tumor region and UOK276. Xenograph tumors were successfully grown in nude mice and provide an in vivo animal model for the investigation of potential therapeutic regimes. The recent TCGA study of ChRCC demonstrated increased expression of the electron transport chain (ETC) genes suggesting increased oxidative phosphorylation within these tumors. Metabolic analysis of UOK276 demonstrated a relatively low level of oxygen consumption (OCR) in comparison to a normal kidney cell line and this was supported by mRNA expression data showing normal or reduced levels of expression for several ETC-related genes. Conclusions: Our study has produced a novel ChRCC cell line model that exhibits a TP53 mutation, commonly seen in ChRCC, and represents a sarcomatoid differentiated region of the tumor. UOK276 should provide a unique in vitro and in vivo preclinical model system for studying the deregulated pathways and testing therapeutic strategies in sarcomatoid differentiated ChRCC. Citation Format: Youfeng Yang, Cathy D. Vocke, Christopher J. Ricketts, Darmood Wei, Hesed M. Padilla-Nash, Shawna L. Boyle, Robert Worrell, Thomas Ried, Maria J. Merino, W. Marston Linehan. A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A19.

Malignant Rhabdoid Tumor (MRT) is a rare and aggressive pediatric cancer that most frequently appears in the kidney (RTK) and brain (AT/RT). They are generally characterized by the lack of SNF5 (SMARCB1/INI1) expression, a subunit of the SWI/SNF chromatin remodeling complex. Previous studies have shown that reintroducing SNF5 into MRT cell lines reconstitutes the SWI/SNF complex and results in an inhibition of growth through induction of p21WAF1/CIP1, p16INK4A and p57KIP2 expression. However, the mechanisms behind this growth arrest remain incompletely characterized. In this current report, we used a RT-PCR Cell Cycle SuperArray to identify six candidate genes that showed increased expression after SNF5 reexpression but not after expression of a constitutively active RB gene. One of these genes, Cyclin G2 (CCNG2), a member of the non-canonical Cyclin G family, is thought to act as a negative regulator of the cell cycle. Using a panel of RTK and AT/RT cell lines, we confirmed that SNF5 reexpression leads to induction of CCGN2 in additional MRT cell lines. To determine if CCNG2 is a direct target of SNF5, we used chromatin immunoprecipitation analyses (ChIP) to verify that SNF5 binds to the CCNG2 promoter with peak binding at the transcription start site (TSS) after its reexpression in a MRT cell line. Importantly, primary MRT samples display reduced CCNG2 expression when compared to normal brain tissue or other types of pediatric brain cancers. Therefore, CCNG2 represents a new SNF5 target gene whose downregulation may play a role during MRT development. Citation Format: Darmood Wei, Aubri Charboneau, Ho-yoon Chung, Donastas Sakellariou-Thompson, Brian Davies, Dennis A. Simpson, Yasumichi Kuwahara, William K. Kaufmann, Charles W. M. Roberts, Bernard E. Weissman. Cyclin G2, a novel target of the SNF5/BAF47 tumor suppressor gene. [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 4787. doi:10.1158/1538-7445.AM2015-4787

Cellular senescence was initially described as the phenomenon by which normal diploid cells cease to divide after reaching the Hayflick limit. However, with the identification of replicative senescence, other inducers of this process have appeared in the literature including oncogene induced senescence (OIS). Of interest, different oncogenes can initiate OIS through alternative pathways e.g. HRAS (G12V) and BRAF (V600E) that effect senescence through p16 dependent and p16 independent pathways, respectively. Alternatively, loss of tumor suppressor gene function can also induce senescence. In malignant rhabdoid tumors (MRTs), SNF5 behaves as a tumor suppressor and its re-expression in MRT cell lines results in growth arrest via the RB pathway. Paradoxically, when we knocked out SNF5 in normal human fibroblasts (NHF-1), we also observed senescence. We therefore hypothesize that perturbation of the SWI/SNF complex (e.g. knocking down SNF5) results in cellular senescence in most normal tissues. This suggests an epigenetic mechanism of senescence occurring through a p16 independent pathway, as previous publications have demonstrated a critical role for SNF5 in p16 expression. Additionally, in BRAF induced senescence, changes in levels of CD44, phospho-MEK1/2, and p16 are observed. However, we do not find these changes during SNF5 loss. Our findings suggest a relationship between SWI/SNF's chromatin remodeling activity and the regulation of cellular senescence. Importantly, our studies will identify the key signaling pathways that allow MRTs to escape cellular senescence after SNF5 loss. These results will further elucidate the role of SNF5 loss in MRT development and provide new targets for clinical intervention. Acknowledgement: We thank the members of the Weissman lab for their help, advice, and support and R01CA91048 (B.E.W.) , T32ES007126 (D.W.), UNC Dissertation Completion Fellowship (D.W.) for financial support. Citation Format: Darmood Wei, HoYoon Chung, Dennis A. Simpson, William K. Kaufmann, Bernard E. Weissman. Mechanism of replicative senescence induced by SNF5 loss. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2245. doi:10.1158/1538-7445.AM2014-2245

Malignant Rhabdoid Tumors (MRTs), a pediatric renal cancer, lack SNF5, a subunit of the SWI/SNF chromatin remodeling complex, a regulator of nucleosome positioning and gene expression. Recent studies showed that PBRM1, another subunit of SWI/SNF, was mutated in adult clear cell renal carcinoma (ccRCC). Intriguingly, both SNF5 and PBRM1 regulate the cell cycle through control of expression of p21WAF1/CIP1. We hypothesized that SWI/SNF complex mutations provide a link between adult and pediatric renal cancers. To test this notion, we re-expressed SNF5 in MRT cell lines and followed PBRM1 and p21 expression and cell growth. We also examined SWI/SNF complex composition and recruitment to the p21 promoter. Finally, we investigated PBRM1’s role in SNF5-induced G1 cell cycle arrest using PBRM1-deficient MRT cell lines. We utilized adenoviral vectors to express human SNF5 (hSNF5) in several different MRT cell lines. The expression of hSNF5 and other complex members was quantified through immunoblotting and QT-PCR. Immunoprecipitation (IP) was utilized to determine the composition of the SWI/SNF complex. Cell cycle analysis was conducted using flow cytometry with BrdU and PI staining. PBRM1 knockdown cell lines were generated using lentiviral-RNAis (Open Biosystems). Reexpression of SNF5 in MRT cell lines led to increased PBRM1 protein levels without concomitant increases in mRNA levels. Immunoprecipitation experiments showed that SNF5 re-expression caused PBRM1 levels in the complex to increase, as compared to the BRG1 ATPase levels that remained constant after SNF5 reexpression. These results were consistent across 4 MRT cell lines examined. Interestingly, levels of other complex proteins, such as BAF155, also increased in a similar fashion to PBRM1. After reexpression of SNF5, we also observed an increase of both SNF5 and PBRM1 at the p21 promoter, with a peak at the transcription start site. In contrast, other complex members showed only a modest increase across the entire promoter region with no apparent peak of binding. To further investigate PBRM1’s role in SNF5-induced G1 cell cycle arrest, we generated PBRM1-deficient MRT cell lines using RNAi technology. We are currently testing these knockdown cell lines for cell cycle arrest and p21 induction after hSNF5 reexpression. Our data show that SNF5 expression increases PBRM1 protein levels, presumably through stabilization within the SWI/SNF complex, suggesting a high degree of interplay between PBRM1 and SNF5 during chromatin remodeling activity. Therefore, identifying genes regulated by both proteins will prove critical in further understanding their contribution to MRT development. Ultimately, our studies will identify key signaling pathways that contribute to the initiation and/or progression of renal cancers including MRTs and ccRCCs. Citation Format: Darmood Wei, Bernard E. Weissman, Yasumichi Kuwahara. Elucidating the role of PBRM1 in SNF5 regulated gene expression in malignant rhadboid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3821. doi:10.1158/1538-7445.AM2013-3821