Darmood Wei

SNF5 reexpression in malignant rhabdoid tumors regulates transcription of target genes by recruitment of SWI/SNF complexes and RNAPII to the transcription start site of their promoters.

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. Visual Overview

SNF5/INI1 Deficiency Redefines Chromatin Remodeling Complex Composition During Tumor Development

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.

A chemoproteomic portrait of the oncometabolite fumarate

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.

Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers

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.

Updated Recommendations on the Diagnosis, Management, and Clinical Trial Eligibility Criteria for Patients With Renal Medullary Carcinoma

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.

MP98-10 UTILITY OF HIGH THROUGHPUT SCREENING IN IDENTIFYING AND REPURPOSING SMALL MOLECULE INHIBITORS FOR UROTHELIAL CARCINOMA

INTRODUCTION AND OBJECTIVES: We developed a novel treatment for localized and metastatic bladder cancer comprised of gold nanoparticle-based photothermal therapy and immunotherapy (SYMPHONY). We demonstrate that it effectively ablates primary tumors, destroys metastases abscopally, and induces potent anti-tumor immunity. METHODS: MB49 murine bladder cancer cells were injected into the bilateral flanks of C57BL/6 mice and grown until 100 mm3 in size. PEG-functionalized gold nanostars, developed and manufactured by our team, were administered intravenously. A 808-nm laser (0.6 W/ cm2) was used to trigger plasmonic heat production from the gold nanostars in the left flank 24 hours after injection, while the contralateral flank was left untreated. Anti-PD-L1 antibody immunotherapy was coadministered intraperitoneally and repeated q3days. Mice were assessed for ipsilateral and contralateral tumor response and survival. Flow cytometry, multiplex cytokine profiling, and T cell receptor sequencing were used to characterize the immune response. Mice achieving a complete response were rechallenged with an additional injection of MB49 tumor cells 90 days later. RESULTS: Gold nanostar-mediated phototherapy alone completely ablated ipsilateral tumors in 4/5 of mice (pT0 at necropsy) but contralateral tumors grew and all 5 mice required sacrifice within 14 days. Anti-PD-L1 therapy alone slowed tumor growth in 3/5 mice, but tumors rapidly began growing again and 5/5 mice required sacrifice by 45 days. Combined treatment (i.e. SYMPHONY) ablated 5/5 ipsilateral tumors and resulted in partial (3/5) and complete responses (2/5) of untreated contralateral tumors, demonstrating a strongabscopal effect. After 90days of follow-up, the two mice achieving a complete response with SYMPHONYwere rechallengedwithMB49and neither developed a tumor over the ensuing 4weeks indicating strong and effective immunememory. Flow cytometry showed CD4 and CD8 T cell proliferation, decreased myeloid derived suppressor cells, and increased IL2 with SYMPHONY. CONCLUSIONS: SYMPHONY treatment resulted not only in effective ablation of primary tumors but also in immune-mediated abscopal destruction of untreated distant tumors. Strong and permanent anti-tumor immunity developed in some mice, indicating that with further optimization, SYMPHONY may be able to cure more advanced bladder cancers.

Abstract 1400: Using tumor spheroids to evaluate the efficacy of EZH2 inhibitors in clear cell renal cell carcinoma (ccRCC)

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

Abstract A19: A novel cell line model for chromophobe renal cell carcinoma, UOK276, derived from an aggressive sarcomatoid differentiated tumor

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.

Abstract 2727: LOX is a novel mitotic spindle-associated protein essential for mitosis

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.

Abstract 4707: Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC)

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.