Sekulić, Aleksandar

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

Abstract Despite remarkable therapeutic advances in malignant melanoma, an incomplete understanding of melanoma's extraordinary genetic diversity, complex disease course and ability to resist treatment continues to impede rational, curative clinical intervention. This is particularly true for the understudied “dark matter” of the melanoma genome, i.e. cases that lack mutations in the three most commonly-mutated melanoma genes (BRAF, NRAS, NF1) and that display extensive mutational heterogeneity. Clearly a pressing need exists for transformative approaches to melanoma medicine. We have therefore broadened our traditional focus on melanoma in human patients to include canine melanoma as a model of human disease. Given canine melanoma's clinicopathological resemblance to human non-cutaneous triple-negative subtypes, study of this disease in each species may inform the other. Further, the accelerated clinical course of canine melanoma and flexibility in veterinary clinical trial design may allow for evaluation of novel treatment designs that would not be possible in human cancer trials. Thus, we have undertaken a first-in-dog genomic characterization study of canine melanomas from a cohort of 37 dogs to further explore the landscape of this comparative model. This analysis combines comprehensive genomics (whole genome, whole genome long-insert, RNA sequencing, and array comparative genomic hybridization) in 7 matched canine melanoma/normal tissues with targeted analysis in an additional 27 tumors and 3 cell lines. These canine tumors display striking patterns of mutation and subtype heterogeneity resembling that of their non-cutaneous human counterparts with the majority being BRAF, NRAS, and NF1 wild type and lacking recurrent single nucleotide variants. Despite heterogeneity at the single nucleotide level, shared patterns of structural variation and gene expression exist and these patterns suggest core pathways that may define therapeutic vulnerabilities in these disease subtypes. These data provide the genomic framework for further clinical pursuit of melanoma in the canine model as a paradigm for drug development. Citation Format: William P. Hendricks, Victoria Zismann, Berry Button, Christophe Legendre, Waibhav Tembe, Holly Yin, Michael Bittner, Donald Berry, Matthew Huentelman, Chand Khanna, Patricia LoRusso, Matthew Breen, Aleksandar Sekulic, Jeffrey Trent. The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation. [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 3417. doi:10.1158/1538-7445.AM2014-3417

AbstractMatching molecularly targeted therapies with cancer subtype‐specific gene mutations is revolutionizing oncology care. However, for rare cancers this approach is problematic due to the often poor understanding of the disease's natural history and phenotypic heterogeneity, making treatment of these cancers a particularly unmet medical need in clinical oncology. Advanced Sézary syndrome (SS), an aggressive, exceedingly rare variant of cutaneous T‐cell lymphoma (CTCL) is a prototypical example of a rare cancer. Through whole genome and RNA sequencing (RNA‐seq) of a SS patient's tumor we discovered a highly expressed gene fusion between CTLA4 (cytotoxic T lymphocyte antigen 4) and CD28 (cluster of differentiation 28), predicting a novel stimulatory molecule on the surface of tumor T cells. Treatment with the CTLA4 inhibitor ipilimumab resulted in a rapid clinical response. Our findings suggest a novel driver mechanism for SS, and cancer in general, and exemplify an emerging model of cancer treatment using exploratory genomic analysis to identify a personally targeted treatment option when conventional therapies are exhausted.

Abstract Metastatic malignant melanoma is an incurable disease with average 5-year survival rates for patients with disseminated disease ranging from 5-18%. Despite the recent development of promising targeted and immunological approaches for treatment of melanoma patients, there remains an urgent need to improve the effectiveness of conventional chemotherapeutics such as TMZ treatment for those patients ineligible or unresponsive to these approaches. We hypothesized that genetic alterations or contexts of vulnerability (COVs) exist in melanoma cells that mediate sensitivity to TMZ, and thus developed RNA-interference-based (RNAi) TMZ chemosensitization assays in melanoma cell lines in order to better understand how to pharmacologically augment TMZ response. First, we extensively developed RNAi sensitization assay in 17 melanoma cell lines followed by screening siRNA kinome library in these cell lines. This screening result, combined with our previous melanoma genomic data (gene expression, aCGH, SNP, and gene mutations), would prioritize the critical essential genes in melanoma. In addition, we profiled 17 melanoma cell lines for TMZ sensitivity and subsequently optimized RNAi screening parameters for detection of TMZ sensitization in one sensitive (UACC903) and four resistant (UACC647, UACC1308, UACC91 and UACC1940) cell lines. We then performed high-throughput RNAi screens with a 7,000 gene siRNA library targeting the druggable genome in these five cell lines. The data from the screens were rigorously evaluated and top TMZ-sensitizing hits were identified and confirmed. Based on these data, we prioritized the ATR/ATM/Chk1 pathway for further evaluation. We tested two Chk1 inhibitors for synergistic effects when combined with TMZ. Notably, both Chk1 inhibitors demonstrated good synergistic effects in the TMZ-resistant cell lines, with moderate/marginal effects in the sensitive line UACC903. These data are consistent with previous reports that inhibition of Chk1 can sensitize TMZ-induced cytotoxicity in glioma cells, and suggest that targeting this pathway in melanoma may augment TMZ response in melanoma. We are presently following up these results across a larger panel of melanoma cell lines, as well as evaluating pharmacological inhibitors of additional TMZ-sensitizing siRNA hits and pathways with the hope that these results may serve as a starting point for the development of novel combination therapies for melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2011-5457

Abstract Cutaneous squamous cell carcinoma (SCC) occurs commonly and can metastasize. Identification of specific molecular aberrations and mechanisms underlying the development and progression of cutaneous SCC may lead to better prognostic and therapeutic approaches and more effective chemoprevention strategies. To identify genetic changes associated with early stages of cutaneous SCC development, we analyzed a series of 40 archived skin tissues ranging from normal skin to invasive SCC. Using high-resolution array-based comparative genomic hybridization, we identified deletions of a region on chromosome 10q harboring the INPP5A gene in 24% of examined SCC tumors. Subsequent validation by immunohistochemistry on an independent sample set of 71 SCC tissues showed reduced INPP5A protein levels in 72% of primary SCC tumors. Decrease in INPP5A protein levels seems to be an early event in SCC development, as it also is observed in 9 of 26 (35%) examined actinic keratoses, the earliest stage in SCC development. Importantly, further reduction of INPP5A levels is seen in a subset of SCC patients as the tumor progresses from primary to metastatic stage. The observed frequency and pattern of loss indicate that INPP5A, a negative regulator of inositol signaling, may play a role in development and progression of cutaneous SCC tumors. Cancer Prev Res; 3(10); 1277–83. ©2010 AACR.

Abstract Purpose. Rash occurs in >50% of patients prescribed epidermal growth factor receptor (EGFR) inhibitors. This study was undertaken to determine whether sunscreen prevents or mitigates these rashes. Methods. This placebo-controlled, double-blinded trial enrolled rash-free patients starting an EGFR inhibitor. Patients were randomly assigned to sunscreen with a sun protection factor of 60 applied twice a day for 28 days versus placebo. They were then monitored for rash and quality of life (Skindex-16) during the 4-week intervention and for an additional 4 weeks. Results. Fifty-four patients received sunscreen, and 56 received placebo; the arms were balanced at baseline. During the 4-week intervention, physician-reported rash occurred in 38 (78%) and 39 (80%) sunscreen-treated and placebo-exposed patients, respectively (p = 1.00); no significant differences in rash rates emerged over the additional 4 weeks. There were no significant differences in rash severity, and patient-reported outcomes of rash yielded similar conclusions. Adjustment for sun intensity by geographical zone, season, and use of photosensitivity medications did not yield a significant difference in rash across study arms (p = .20). Quality of life scores declined but remained comparable between arms. Conclusions. Sunscreen, as prescribed in this trial, did not prevent or attenuate EGFR inhibitor–induced rash.