Trent, Jeffrey M.

10572 Background: Adrenocortical carcinoma (ACC) is a rare and lethal cancer with an average incidence of 1 in 1.4 million. Response to current standard therapy including mitotane is approximately 22%. Herein we use gene expression profiling to identify novel targets for therapeutic intervention. Methods: The transcriptomes of 29 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix Human Genome and Agilent 22K element expression arrays in order to identify genes that could represent potential therapeutic targets. Agents known to inhibit the selected targets were tested in comparison with mitotane in two ACC cell lines (H295R and SW-13) in vitro and are currently being tested in a Scid mouse xenograft system. Results: Expression of numerous kinesin family members is dysregulated within the ACC tumor population. Of interest in the ACC cells EG5 (KIF11, KSP), a kinesin important for mitotic spindle formation, is up-regulated 5.81+ 4.42 (μ+SD) and 6.48 + 0.99 fold on the Agilent and Affymetrix platforms respectively. In addition, SPARC, an albumin binding matrix associated protein is up-regulated in ACC tumors by 3.69 + 2.37 (μ+SD) and 1.56 + 0.44 on the two platforms. The anti-microtubular drugs paclitaxel and albumin bound paclitaxel (abraxane) exhibited in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 μM and 0.0078 μM for paclitaxel and 0.35 μM and 0.0087 μM for Abraxane compared to mitotane concentrations of 15.9 μM and 46.4 μM. Treatment effects of monastrol, paclitaxel, and Abraxane are currently being tested in xenograft murine models. Testing of the EG5 inhibitor S-trityl-L- cysteine is ongoing in the in vitro system. Conclusions: KIF11 inhibitors have been proposed in the treatment of cancer. These compounds may offer better anti-mitotic activity than other tubulin interacting agents with less neurotoxic side effects. Based on biological insight garnered through expression profiling of ACC tumors, the current ongoing research is investigating the use of abraxane and EG5 inhibitors in the in vitro and in vivo treatment of ACC. No significant financial relationships to disclose.

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

Abstract Purpose: 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. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4-mutant 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. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Abstract Purpose: Glioblastoma is an aggressive and molecularly heterogeneous cancer with few effective treatment options. We hypothesized that next-generation sequencing can be used to guide treatment recommendations within a clinically acceptable time frame following surgery for patients with recurrent glioblastoma. Experimental Design: We conducted a prospective genomics-informed feasibility trial in adults with recurrent and progressive glioblastoma. Following surgical resection, genome-wide tumor/normal exome sequencing and tumor RNA sequencing were performed to identify molecular targets for potential matched therapy. A multidisciplinary molecular tumor board issued treatment recommendations based on the genomic results, blood–brain barrier penetration of the indicated therapies, drug–drug interactions, and drug safety profiles. Feasibility of generating genomics-informed treatment recommendations within 35 days of surgery was assessed. Results: Of the 20 patients enrolled in the study, 16 patients had sufficient tumor tissue for analysis. Exome sequencing was completed for all patients, and RNA sequencing was completed for 14 patients. Treatment recommendations were provided within the study's feasibility time frame for 15 of 16 (94%) patients. Seven patients received treatment based on the tumor board recommendations. Two patients reached 12-month progression-free survival, both adhering to treatments based on the molecular profiling results. One patient remained on treatment and progression free 21 months after surgery, 3 times longer than the patient's previous time to progression. Analysis of matched nonenhancing tissue from 12 patients revealed overlapping as well as novel putatively actionable genomic alterations. Conclusions: Use of genome-wide molecular profiling is feasible and can be informative for guiding real-time, central nervous system–penetrant, genomics-informed treatment recommendations for patients with recurrent glioblastoma. Clin Cancer Res; 24(2); 295–305. ©2017 AACR. See related commentary by Wick and Kessler, p. 256

Abstract Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. Contrasting with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis, and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM. Citation Format: Winnie S. Liang, William Hendricks, Jeffrey Kiefer, Jessica Schmidt, Shobana Sekar, John Carpten, David W. Craig, Jonathan Adkins, Lori Cuyugan, Zarko Manojlovic, Rebecca F. Halperin, Adrienne Helland, Sara Nasser, Christophe Legendre, Laurence H. Hurley, Karthigayini Sivaprakasam, Douglas B. Johnson, Holly Crandall, Klaus J. Busam, Victoria Zismann, Valerie De Luca, Jeeyun Lee, Aleksandar Sekulic, Charlotte E. Ariyan, Jeffrey Sosman, Jeffrey Trent. Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma [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 3389. doi:10.1158/1538-7445.AM2017-3389

Abstract Introduction: Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is arguably the most aggressive ovarian cancer. Most patients are diagnosed at an advanced stage, do not respond to chemotherapy, and die of disease within 1-2 years. It affects children and young women and is reported to occur in families. The cause of the disease is poorly understood. Therefore, we used next generation sequencing technology to identify the genetic basis of the disease. Method: We performed whole genome, whole exome sequencing or targeted sequencing on tumours and germline samples from 17 SCCOHT patients and on the SCCOHT cell line BIN-67 and SCCOHT-1. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumors from 23 patients and on a tissue microarray of 485 primary ovarian tumours of other subtypes. BIN-67 cells harbouring biallelic inactivation of SMARCA4 were transduced with a lentivirus expressing wild type SMARCA4. Result: We identified inactivating germline and somatic mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (11/14) of SCCOHT patients, 2 of whom bore germline mutations, and in both cell lines. SMARCA4 protein was lost in 87% (20/23) of SCCOHT tumours but in only 0.4% (2/485) of other primary ovarian tumours. Reintroduction of wild-type SMARCA4 into BIN-67 cells resulted in altered cell morphology and growth arrest. Conclusions: The mutation spectrum, IHC profile and cell culture phenotype implicate SMARCA4 as a critical tumour suppressor in SCCOHT pathogenesis. Citation Format: Pilar Ramos, Anthony N. Karnezis, David W. Craig, Aleksandar Sekulic, Megan L. Russell, William P.D. Hendricks, Jason J. Corneveaux, Michael T. Barrett, Karey Shumansky, Yidong Yang, Sohrab P. Shah, Leah M. Prentice, Marco A. Marra, Jeffrey Kiefer, Victoria L. Zismann, Troy A. McEachron, Bodour Salhia, Jaime Prat, Blaise A. Clarke, Joseph G. Pressey, John H. Farley, Stephen P. Anthony, Richard B.S. Roden, Heather E. Cunliffe, David G. Huntsman, Jeffrey M. Trent. Small cell carcinoma of the ovary, hypercalcemic type displays frequent inactivating germline and somatic mutations in SMARCA4 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1327.

Abstract Targeted therapies and immunotherapies have led to significant improvements in the treatment of advanced cancers, including metastatic melanoma. However, new strategies are desperately needed to overcome therapeutic resistance to these agents, as well as to identify effective treatment approaches for cancer patients that fall outside major targetable mutational subtypes (e.g., non–V600 BRAF melanoma). One such strategy is to extend the paradigm of individually tailored, molecularly targeted therapy into a broader spectrum of melanoma patients, particularly those bearing tumors without commonly recognized therapeutic targets, as well as having failed or were ineligible for immunotherapy. In this nontreatment pilot study, next-generation sequencing (NGS) technologies were utilized, including whole genome and whole transcriptome sequencing, to identify molecular aberrations in patients with non–V600 BRAF metastatic melanoma. This information was then rationally matched to an appropriate clinical treatment from a defined pharmacopeia. Five patients with advanced non–V600 BRAF metastatic melanoma were enrolled. We demonstrated successful performance of the following during a clinically relevant time period: patient tumor biopsy, quality DNA/RNA extraction, DNA/RNA-based sequencing for gene expression analysis, analysis utilizing a series of data integration methodologies, report generation, and tumor board review with formulated treatment plan. Streamlining measures were conducted based on the experiences of enrolling, collecting specimens, and analyzing the molecular signatures of patients. We demonstrated the feasibility of using NGS to identify molecular aberrations and generate an individualized treatment plan in this patient population. A randomized treatment study utilizing lessons learned from the conduct of this pilot study is currently underway. Mol Cancer Ther; 14(8); 1962–71. ©2015 AACR.

Abstract Targeted BRAF inhibition in melanoma yields frequent and dramatic responses, which are unfortunately followed by almost universal development of therapeutic resistance. In contrast to MAP kinase pathway activation, (e.g., differential BRAF splicing, MEK1 mutations), direct gatekeeper mutations, seen with other targeted therapies, have not yet been reported in association with resistance to targeted BRAF inhibition. Through a combination of genomic profiling of clonal diversity and molecular modeling, we documented a new molecular mechanism of vemurafenib resistance in a melanoma patient, occurring by selection of a pre-existing clonal subpopulation harboring a novel mutation in the vemurafenib-binding site of BRAF. We assessed melanoma clonal diversity using a novel approach for studying tumor heterogeneity in solid tumors, based on separating cellular subpopulations by nuclear flow sorting coupled to next generation sequencing. This approach identified distinct, coexisting melanoma subpopulations prior to initiation of vemurafenib. While vemurafenib treatment suppressed multiple clonal populations, a distinct population was noted at the time of relapse, harboring both the common activating V600E and a novel L567V mutation in the vemurafenib-binding site. Molecular modeling predicted that L567V would interfere with drug binding leading to decreased therapeutic response. Using a lentiviral expression system we assessed the functional effects of the L567V mutation both alone, and in combination with V600E. Assessment of the kinase activity in an in vitro kinase assay using MEK as a substrate indicates that the L567V mutant retains kinase activity. However, expression in a panel of melanoma cell lines demonstrates that while both the V600E single mutant and the L567V single mutant respond to BRAF inhibition by decreasing the levels of phospho-Erk, the double mutant actually responds to treatment by increasing phospho-Erk levels. This is also reflected in the increased viability of cells expressing double mutant in presence of increasing drug levels (PLX4720), compared to either of the single mutants. Taken together, the data suggest that the BRAF L567V/V600E double mutant confers insensitivity to vemurafenib, and that patients harboring both mutations may demonstrate clinically significant resistance to vemurafenib. Further, this data illustrates the role of clonal heterogeneity in mediating key clinical events including response to therapy and development of resistance, a major challenge of targeted BRAF therapies. Citation Format: Aleksandar Sekulic, Ashani T. Weeraratna, Jefferey L. MacKeigan, Amanpreet Kaur, Michael O'Connell, Megan L. Goodall, Michael Barrett, Elizabeth Lenkiewitz, Tara Holley, Victoria Zismann, Michael Gordon, Gideon Bollag, Chao Zhang, Jeffrey M. Trent. Discovery and molecular modeling of a novel mutation in the vemurafenib binding site of BRAF associated with therapeutic resistance in a patient with metastatic melanoma. [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 LB-213. doi:10.1158/1538-7445.AM2014-LB-213

Abstract Small Cell Carcinoma of the Ovary Hypercalcemic Type (SCCOHT) is a rare and highly aggressive malignancy that affects children and young women at a mean age of 24 (range 14 months - 58 years). SCCOHT is refractory to standard of care therapy for ovarian cancer, with ∼75% mortality within 18 months of diagnosis. The early age of onset of SCCOHT and reports of familial occurrence in some cases, strongly suggest an underlying hereditary etiology. To understand the molecular pathogenesis of SCCOHT, we performed next-generation genomic sequencing on a series of tumor and germline samples from SCCOHT patients. This analysis revealed germline and somatic inactivating mutations in SMARCA4, a subunit of the SWI/SNF chromatin-remodeling complex, in 75% (9/12) of SCCOHT patients. Moreover, immunohistochemical (IHC) analysis of 15 tumors revealed that 87% (13/15) of tumors lacked SMARCA4 protein. The high prevalence of SMARCA4 mutations in SCCOHT has not been previously reported in other, more common ovarian carcinomas. We therefore examined the expression of SMARCA4 protein in 300 ovarian carcinomas of different histologies by IHC and found SMARCA4 protein loss in only 6 tumors. In addition, the BIN-67 SCCOHT cell line, which harbors 2 splice site mutations in SMARCA4, showed complete absence of SMARCA4 protein by Western blot while representative cell lines from 4 other ovarian carcinoma subtypes as well as immortalized granulosa cells (SVOG) and adult granulosa tumor cells (KGN) all maintained SMARCA4 expression. The prevalence of germline and sporadic SMARCA4 mutations as well as frequent SMARCA4 protein loss in SCCOHTs implicates this gene as a tumor suppressor in this cancer and more broadly suggests a role for the SWI/SNF complex in its pathogenesis. In addition to providing evidence to the pathogenesis of SCCOHT, this finding provides the opportunity to develop treatment approaches for SCCOHT based on targeting vulnerabilities of SMARCA4-deficient cells. Citation Format: Pilar Ramos, Anthony Karnezis, David Craig, Aleksandar Sekulic, Megan Russell, William Hendricks, Michael Barrett, Karey Shumansky, Yidong Yang, Sohrab Shah, Leah Prentice, Marco Marra, Jeffrey Kiefer, Victoria Zismann, Troy McEachron, Bodour Salhia, Joseph Pressey, John Farley, Stephen Anthony, Richard Roden, Heather Cunliffe, David Huntsman, Jeffrey Trent. The rare, highly malignant small cell carcinoma of the ovary displays common inactivating germline and somatic mutations in the tumor suppressor SMARCA4. [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 LB-202. doi:10.1158/1538-7445.AM2014-LB-202