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 wildtype (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 Gquadruplexes, 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 effects cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and inducing senescence by decreasing telomerase activity and telomere length.We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.3

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 IntroductionBortezomib is a potent and selective proteasome inhibitor used in the treatment of multiple myeloma (MM) and low-grade lymphoma patients. Bortezomib produces significant clinical responses in both newly diagnosed and advanced MM, but only 40% of patients respond to the single agent, 1,2 and the majority of these patients become resistant over time. The mechanism of antimyeloma activity of bortezomib is therefore a subject of intense study with inhibition of the proteasome, autophagy, and activation of the unfolded protein stress response pathway apparently critical. A downstream consequence of proteasome inhibition relevant to MM is blockade of nuclear factor B (NF-B) activity, which may partly mediate bortezomib activity in MM because activating mutations in the NF-B pathway were identified in at least 17% of MM patients and 41% of human myeloma cell lines and appear to mediate accelerated growth and survival of malignant plasma cells. [3][4][5] However, the 35% to 50% response rate to bortezomib cannot be totally interpreted by NF-B abnormality, suggesting that other specific molecular targets, resistance mechanisms, or perhaps unique pharmacokinetics are inherent in patients. Furthermore, resistance to bortezomib therapy often develops even in initially sensitive patients; and although certain mechanisms such as mutations in proteasome subunits have been postulated, 6 the underlying mechanism defining this nonresponsiveness is largely unknown. Understanding the cooperating mechanisms of sensitivity to proteasome inhibition will not only allow more targeted use of proteasome inhibitors but should also make it possible to rationally design synergistic drug combinations and predict patient response to therapy.To begin to address these issues, a druggable genome RNAi screen was used to identify modifiers of bortezomib sensitivity in human MM cells. Through this high-throughput screen, we identified a panel of genes whose loss of expression enhances bortezomib sensitivity (sensitizer). Using shRNA expression systems and a small-molecule inhibitor, we have further validated one of the most potent bortezomib sensitizer genes as cyclin-dependent kinase 5 (...

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 D'Angelo, 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.

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.

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.

BackgroundPancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer characterized by complex aberrant genomes. A fundamental goal of current studies is to identify those somatic events arising in the variable landscape of PDA genomes that can be exploited for improved clinical outcomes.MethodsWe used DNA content flow sorting to identify and purify tumor nuclei of PDA samples from 50 patients. The genome of each sorted sample was profiled by oligonucleotide comparative genomic hybridization and targeted resequencing of STAG2. Transposon insertions within STAG2 in a KRASG12D-driven genetically engineered mouse model of PDA were screened by RT-PCR. We then used a tissue microarray to survey STAG2 protein expression levels in 344 human PDA tumor samples and adjacent tissues. Univariate Kaplan Meier analysis and multivariate Cox Regression analysis were used to assess the association of STAG2 expression relative to overall survival and response to adjuvant therapy. Finally, RNAi-based assays with PDA cell lines were used to assess the potential therapeutic consequence of STAG2 expression in response to 18 therapeutic agents.ResultsSTAG2 is targeted by somatic aberrations in a subset (4%) of human PDAs. Transposon-mediated disruption of STAG2 in a KRASG12D genetically engineered mouse model promotes the development of PDA and its progression to metastatic disease. There was a statistically significant loss of STAG2 protein expression in human tumor tissue (Wilcoxon-Rank test) with complete absence of STAG2 staining observed in 15 (4.3%) patients. In univariate Kaplan Meier analysis nearly complete STAG2 positive staining (>95% of nuclei positive) was associated with a median survival benefit of 6.41 months (P = 0.031). The survival benefit of adjuvant chemotherapy was only seen in patients with a STAG2 staining of less than 95% (median survival benefit 7.65 months; P = 0.028). Multivariate Cox Regression analysis showed that STAG2 is an independent prognostic factor for survival in pancreatic cancer patients. Finally, we show that RNAi-mediated knockdown of STAG2 selectively sensitizes human PDA cell lines to platinum-based therapy.ConclusionsBased on these iterative findings we propose that STAG2 is a clinically significant tumor suppressor in PDA.

Structured AbstractPurpose: Subunits of the SWI/SNF chromatin-remodeling complex are tumor suppressors inactivated in ∼20% of all cancers. Yet, few targeted treatments for SWI/SNF-mutant cancers exist. Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits, SMARCA4 and SMARCA2. Given poor two-year survival rates for these women, a great need exists for effective targeted therapies.Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches comprehensively profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two PDX models and one cell line xenograft model of SCCOHT.Results: FGFRs and PDGFRs were overlapping hits between screens and the receptor tyrosine kinase (RTK) family was enriched in the siRNA screen hits. Evaluation of eleven RTK inhibitors in three SCCOHT cell lines identified ponatinib, an inhibitor of multiple RTKs, as the most effective clinically approved agent. Proteomics approaches confirmed inhibition of known targets of ponatinib and more than 20 non-canonical ponatinib targets. Ponatinib also delayed tumor doubling time 4-fold in SCCOHT-1 xenografts and reducing final tumor volumes in two SCCOHT patient-derived xenograft (PDX) models by 58.6% and 42.5%.Conclusion: Ponatinib is an effective agent for SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted.Additional InformationThis work was supported by research funds from the Canadian Cancer Society Research Institute 34 (#703458, D.G.H.), the National Institutes of Health (R01 CA195670-01, B.E.W., D.G.H., and 35 J.M.T., and T32 HL007106-39 to E.M.C), the Terry Fox Research Institute Initiative New Frontiers Program in Cancer (#1021, D.G.H.), the British Columbia Cancer Foundation (D.G.H.), the VGH & UBC Foundation (D.G.H.), the Anne Rita Monahan Foundation (P.R.), the Marsha Rivkin Center for Ovarian Cancer Research (J.M.T.), the Ovarian Cancer Alliance of Arizona (J.M.T.), the Small Cell Ovarian Cancer Foundation (P.R., J.D.L., B.V., and J.M.T.), and philanthropic support to the TGen Foundation (J.M.T.).COI disclosure statement: The authors declare no potential conflicts of interest.

e18579 Background: Patients with anaplastic thyroid cancer (ATC) have poor outcomes due to treatment resistance with an estimated 5-year survival rate of 7%. Although “classic” ATC can arise de novo from follicular thyroid cells, it can also arise in association with papillary thyroid carcinoma (PTC), as a “mixed” histology. The immunologic and molecular differences between these histological subtypes have not been well-characterized. We aimed to investigate the differences in immune signatures in these histological subtypes to assess immune pathway differences in the classic ATC and mixed ATC/PTC tumor microenvironment. Methods: Classic ATC and mixed ATC/PTC cases (N = 12) from 1998 to 2018 with viable formalin-fixed paraffin-embedded (FFPE) tissue were identified and selected by a board-certified pathologist. 4 samples exhibited both PTC and ATC components while 8 were classic ATC cases, as confirmed by a pathologist. In the 4 mixed ATC/PTC histology specimens, 3 were identified to contain greater than 80% ATC involvement while the fourth was characterized as 40% ATC and 60% PTC. The NanoString Immune Profiling panel contains more than 770 genes. It was performed with extracted RNA to evaluate and compare the gene expression of classic ATC versus mixed ATC/PTC. Results: Majority of patients (N = 11) developed metastatic disease, a common feature of ATC, and are currently deceased (N = 10). The NanoString panel identified several differentially expressed immune pathways in the mixed ATC/PTC in comparison to the classic ATC group. There is upregulation of CD3 (p < 0.01) and CD8 (p < 0.01) transcripts in the mixed ATC/PTC group, but not CD4 transcripts (p > 0.05). Additionally, we found that genes associated with B cell and T cell function were significantly upregulated in the mixed ATC group, including CD27 (p < 0.01), CD28 (p < 0.05), BTLA (p < 0.01), CTLA4 (p < 0.01), LAG3 (p < 0.05), and TIGIT(p < 0.01). We did not observe any significant differences in the expression of PD1 (p > 0.05) or MR1 (p > 0.05) between classic ATC and mixed ATC variants. Conclusions: Our study demonstrates a significant difference in the immune landscape between classic and mixed variants of ATC. Our results indicate there may be an increase in immune cell infiltration and tumor inflammation in the mixed ATC variant, which suggests patients with the mixed ATC variant may show a positive response to immunotherapy.