Holly Yin

RNAi screen of the druggable genome identifies modulators of proteasome inhibitor sensitivity in myeloma including CDK5

The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters.

Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERTs role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.

Structural Basis and Targeting of the Interaction between Fibroblast Growth Factor-inducible 14 and Tumor Necrosis Factor-like Weak Inducer of Apoptosis

Background: Aberrant TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling is observed in inflammation, autoimmune diseases, and cancers. Results: An integrated computational and experimental study identified small molecule inhibitors of TWEAK-Fn14 interaction. Conclusion: The TWEAK-Fn14 interaction is tractable and can be inhibited by small molecules. Significance: This is the first evidence of small molecules targeting TWEAK-Fn14 signaling. Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr176, but not Trp231, resulted in the loss of TWEAK binding to Fn14 substantiating Tyr176 as the anchoring residue. Importantly, mutation of TWEAK at Tyr176 did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.

Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

Abstract PR-10: RNAi‐directed identification of chemosensitizers of GSK923295 response

Centromere‐associated protein E (CENP‐E) is a kinesin‐like motor protein that plays a central role in chromosome segregation and control of cell division. GSK923295 (GSK295) is a potent and selective small‐molecule inhibitor of human CENP‐E ATPase activity, and has demonstrated broadspectrum activity in vivo against human tumor xenografts. GSK295 is currently being evaluated as a novel anticancer drug in Phase I clinical trials. RNAi phenotype profiling in the Druggable Genome was applied to discover specific genes that regulate GSK295 sensitivity. This study focused on nonsmall cell lung carcinoma (NSCLC) with three cell lines evaluated for HT‐RNAi drug sensitivity analysis. A robust HT‐RNAi cell‐screening assay for GSK295 sensitivity was successfully developed for all cell lines. The sensitization assay was developed as following: cells were reversely transfected with individual siRNA for 24hr followed by the treatment of various concentrations of GSK295 for 72hr when cell viability was measured. Among 3 optimized cell lines, the A427 cell line, which appeared to be relatively more resistant to GSK295, was selected for a primary RNAi screen against 14,000 siRNA sequences covering 7,000 gene targets (Druggable Genome). Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, Citation Information: Mol Cancer Ther 2009;8(12 Suppl):PR-10.

Abstract NTOC-090: TARGETING AND EFFICACY OF THE RECEPTOR TYROSINE KINASE INHIBITOR PONATINIB IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE, WORKS THROUGH INHIBITION OF PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR)

The work that will be presented strongly suggests the identification of a unique vulnerability of Small Cell Carcinoma of the Ovary-Hypercalcemic Type (SCCOHT) to PDGFR inhibition. SCCOHT is an aggressive subtype of ovarian cancer afflicting young women and children with fewer than 35% of patients surviving two years after diagnosis. Currently, these patients must endure high dose chemotherapy because no effective targeted therapies have been identified. We previously demonstrated that these tumors are driven by inactivating mutations in SMARCA4, one of two ATPases in the SWI/SNF chromatin-remodeling complex. In addition, we have also shown that SCCOHT lacks expression of the alternative SWI/SNF ATPase, SMARCA2. SWI/SNF is a key tumor suppressor complex in diverse cancers and its members are mutationally inactivated in up to 50% of other ovarian cancer subtypes. However, no therapeutic approaches selective for SWI/SNF-dysregulation in cancers are routinely used. We have now found through multiomic (DNA/RNA), pathway analysis, immunohistochemistry and functional studies in SCCOHT tumors and cell lines that SMARCA4 loss correlates with increased expression of receptor tyrosine kinases (RTKs) including the platelet derived growth factor receptors (PDGRs). Through integration of high-throughput RNA interference and drug screens in SCCOHT cell lines we have also identified sensitivity to RTK knockdown and inhibitors such as ponatinib. These data corroborate prior studies showing RTK dependence in rhabdoid tumors, rare cancers that are also driven by mutations in the SWI/SNF complex. As ponatinib is approved for cancer treatment and showed great potency, we tested this agent in two patient-derived xenograft (PDX) models of SCCOHT. Ponatinib significantly reduced tumor growth in both models. Of the known ponatinib targets, PDGFR and FGFR1 were highly expressed in SCCOHT tumors, suggesting that SCCOHTs are sensitive to ponatinib through the expression of these RTKs. Further, PDGFR-alpha phosphorylation and downstream signaling are strongly inhibited by ponatinib in SCCOHT. In summary we believe this finding may allow a way to be exploited therapeutically through the FDA-approved inhibitor ponatinib. Citation Format: Jessica D. Lang, William Hendricks, Pilar Ramos, Holly Yin, Chris Sereduk, Jeffrey Kiefer, Yemin Wang, Anthony N. Karnezis, Bernard Weissman, David Huntsman, Jeffrey Trent. TARGETING AND EFFICACY OF THE RECEPTOR TYROSINE KINASE INHIBITOR PONATINIB IN SMALL CELL CARCINOMA OF THE OVARY, HYPERCALCEMIC TYPE, WORKS THROUGH INHIBITION OF PLATELET DERIVED GROWTH FACTOR RECEPTOR (PDGFR) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-090.

Abstract 1238: Targeting the platelet derived growth factor receptor (PDGFR) with the receptor tyrosine kinase inhibitor ponatinib in small cell carcinoma of the ovary, hypercalcemic type

Subunits of the SWI/SNF chromatin-remodeling complex are tumor suppressors that are inactivated in ~20% of all cancers, yet few targeted treatments have shown selective activity in SWI/SNF-mutant cancers. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by SWI/SNF dysregulation. Given that two-year survival following standard high-dose chemotherapy and radiation in SCCOHT is less than 35%, a great need exists for effective targeted therapies to improve outcomes for these women. We previously demonstrated that SCCOHT tumors are driven by inactivating mutations in SMARCA4, one of two mutually exclusive SWI/SNF ATPases. In addition, we have shown that SCCOHT lacks expression of the alternative SWI/SNF ATPase, SMARCA2. We have now found through integrated genomic and functional analyses in SCCOHT tumors and cell lines that SMARCA4 loss correlates with increased expression of receptor tyrosine kinases (RTKs) including the platelet derived growth factor receptors (PDGFRs). Through integration of high-throughput RNA interference and drug screens in SCCOHT cells we have identified sensitivity to RTK knockdown and RTK inhibitors including the FDA-approved oncology drug, ponatinib. These data corroborate prior studies showing RTK dependence in rhabdoid tumors, rare cancers that are also driven by mutations in the SWI/SNF complex. Of the known ponatinib targets, PDGFR-alpha and FGFR1 were highly expressed in SCCOHT tumors, as confirmed in RNA-Seq data (four tumors) and a SCCOHT tissue microarray (TMA; ten tumors). Furthermore, PDGFR-alpha and -beta phosphorylation and downstream signaling are inhibited by ponatinib in SCCOHT cells, suggesting that these tumors are sensitive to ponatinib due to dependence on signaling through these RTKs. Finally, given ponatinib’s potency in vitro and the proposed mechanism of action, we tested this agent in xenograft models of SCCOHT. In addition to confirming efficacy in a SCCOHT cell line xenograft model, superior efficacy was demonstrated in two patient-derived xenograft (PDX) models of SCCOHT with ponatinib. Thus, ponatinib effectively targets SWI/SNF-mutant SCCOHT tumors through inhibition of PDGFR signaling and may have clinical utility for the treatment of these cancers. Citation Format: Jessica Diane Lang, William Hendricks, Pilar Ramos, Holly Yin, Chris Sereduk, Jeffrey Kiefer, Yemin Wang, Anthony N. Karnezis, Bernard Weissman, David Huntsman, Jeffrey Trent. Targeting the platelet derived growth factor receptor (PDGFR) with the receptor tyrosine kinase inhibitor ponatinib in small cell carcinoma of the ovary, hypercalcemic type [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 1238. doi:10.1158/1538-7445.AM2017-1238

Abstract A33: Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcemic type.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a lethal and sometimes familial ovarian tumor of young women and children. We and others recently discovered that over 90% of SCCOHT harbor inactivating mutations in the chromatin remodeling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually-exclusive ATPases of the SWI/SNF chromatin remodeling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry (IHC) in more than 3000 primary gynecologic tumors. Among ovarian tumors, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high-grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumor cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4-negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumors, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase (HDAC) inhibitor trichostatin A. Treatment with HDAC inhibitors or re-expression of either SMARCA4 or SMARCA2 potently inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2 loss, is the first highly sensitive and specific diagnostic immunohistochemical marker of SCCOHT, and that HDAC inhibitors are promising agents to explore for the treatment of SCCOHT. Citation Format: Anthony N. Karnezis, Yemin Wang, Pilar Ramos, William Hendricks, Holly Yin, Esther Oliva, Emanuela D9Angelo, Jaime Prat, Marisa R. Nucci, Torsten O. Nielsen, Bernard E. Weissman, Jeffrey M. Trent, C Blake Gilks, David G. Huntsman. Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcemic type. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A33.

Abstract A25: Evaluating a role for Rac1-PAK1 signaling in promoting the growth of KRAS-mutant pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer characterized by a high frequency of activating mutations in the KRAS oncogene (95%), which is a well-validated driver of PDAC growth. With limited treatment options for this disease, there is an urgent need to better understand the aberrant signaling that occurs in order to better design therapeutic strategies. However, to date, no successful anti-K-Ras therapies have been developed. Current efforts have focused on inhibition of effectors of K-Ras signaling, in particular the Raf and PI3K effector signaling pathways. However, inhibitors targeting components of these pathways, when used as monotherapy or in combination, have been ineffectual for long-term treatment of KRAS mutant cancers. The lack of success of these inhibitors is due, in part, to the importance of other effectors in K-Ras-dependent cancer growth and the upregulation of compensatory signaling programs that overcome inhibitor activity. Consequently, in order to design effective combinatorial-targeted therapies, there is a pressing need to better understand the role of other effector signaling events that support mutant K-Ras-driven PDAC growth. The small GTPase Rac1 has a known role in driving K-Ras mutant cancers, but the specific effectors through which Rac1 promotes tumor growth have not been defined. We hypothesize that the PAK1 serine/threonine kinase, and related isoforms, PAK2 and PAK3, are key components downstream of Rac1 in mutant K-Ras PDAC. In support of this, we found that PAK1 RNA and protein levels are overexpressed in pancreatic cancer cell lines and in patient tumor samples when compared to normal tissues. Additionally we determined that stable shRNA-mediated suppression of PAK1 protein expression inhibited PDAC anchorage-independent and –dependent growth and Matrigel invasion in vitro. Further, knockdown of K-Ras in PDAC cell lines resulted in reduced phospho-PAK1 (T423) levels, indicating a decrease in PAK1 activity. We also found that a pharmacologic inhibitor of PAK1 and other Group I PAK isoforms (PAK2 and PAK3) inhibited PDAC growth. Additionally, in an RNAi kinome library screen, we identified PAK3 as a driver of resistance to pharmacologic inhibition of ERK. These observations then prompted our studies to evaluate a possible role for PAK2 and PAK3 in PDAC. Finally, our ongoing studies are evaluating the ability of Group I PAK inhibitors in combination with inhibitors of PI3K, mTORC1/2, and MEK1/2 to effectively block KRAS effector signaling and PDAC growth. Citation Format: Nicole M. Baker, Janine LoBello, Haiyong Han, Holly Yin, Jen Jen Yeh, Channing J. Der. Evaluating a role for Rac1-PAK1 signaling in promoting the growth of KRAS-mutant pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A25. doi: 10.1158/1557-3125.RASONC14-A25

Abstract 2709: Identification of Aurintricarboxylic Acid (ATA) as an inhibitor of TWEAK-Fn14 signaling in glioblastoma cells

The long-term survival of patients with glioblastoma (GB) is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. In order to discover a small molecule inhibitors that disrupt Fn14-TWEAK signaling axis and subsequently glioma cell invasion, we developed a cell-based drug screening assay using HEK293 cells that overexpresses Fn14 and harbors NF-κB driven Firefly luciferase protein expression. In preliminary drug screening assay using LOPAC1280 library of 1280 pharmacologically active compound, Aurintricarboxylic Acid (ATA) showed suppression of NF-κB driven Firefly Luciferase activity downstreatm of TWEAK-Fn14 signaling. Conversely, ATA did not show any suppression in NF-κB driven Firefly Luciferase activity downstream of TNFα-TNFR1 signaling, suggesting that ATA is a specific inhibitor of TWEAK-Fn14-NF-κB signaling. In vitro, we demonstrated that ATA suppresses TWEAK induced chemotactic migration of glioma cells (T98G and A172) and display no general cytotoxicity. In summary, this work reports a new small molecule inhibitor of TWEAK-Fn14 signaling that could be useful in enhancing the therapeutic targeting of this deadly disease. Citation Format: Harshil D. Dhruv, Ethan Holiday, Donald Chow, Holly Yin, Michael E. Berens, Nhan L. Tran. Identification of Aurintricarboxylic Acid (ATA) as an inhibitor of TWEAK-Fn14 signaling in glioblastoma cells. [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 2709. doi:10.1158/1538-7445.AM2014-2709