Aditi Kulkarni

Differential compensation mechanisms define resistance to PI3K inhibitors in PIK3CA amplified HNSCC

OBJECTIVEnRecent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating genomic alterations in PIK3CA (the gene encoding the catalytic subunit of PI3K, targeted inhibitors of PI3K have not shown clinical efficacy as monotherapies. We hypothesized that co-dependent pathways, including the Ras-MEK-ERK pathway, may still be functional in the presence of PI3K inhibitors and might serve as mediators of this resistance.nnnMETHODSnWe assessed the hypothesis using resazurin cell viability and trypan blue exclusion assays. We also used Western blot to characterize Ras-MEK-ERK pathway activity.nnnSTUDY DESIGNnWe evaluated this hypothesis in six PIK3CA-amplified, PI3K inhibitor-resistant HNSCC cell lines following treatment with pan and alpha-isoform selective PI3K inhibitors (BKM120 and HS-173 respectively). We also tested the effect of combination treatment with PI3K inhibitor HS-173 and MEK inhibitor trametinib or EGFR inhibitor gefitinib.nnnRESULTSnOur results displayed maintenance of Ras-MEK-ERK pathway activity in 4 of 6 HNSCC cell lines after PI3K inhibitor treatment. We also found that UM-SCC-69 and UM-SCC-108 cells display synergistic responses to dual therapy.nnnCONCLUSIONnThis study suggests that inhibition of the PI3K and Ras-MEK-ERK pathways might be effective in some HNSCC patients; however, it also prompts the study of additional resistance mechanisms to identify synergistic combination therapies for tumors resistant to these di-therapies.

Whole-Exome Sequencing of Sinonasal Small Cell Carcinoma Arising within a Papillary Schneiderian Carcinoma In Situ

Objective The pathogenetic underpinnings of extrapulmonary small cell carcinomas (EPSCCs) of the head and neck are poorly understood. We sought to describe the clinical case and whole-exome DNA sequencing data of a patient with sinonasal Schneiderian carcinoma in situ whose tumor progressed to small cell carcinoma (SCC). Study Design Case report and whole-exome sequencing of tumor DNA. Setting Academic medical center. Subjects and Methods A 52-year-old man with sinonasal Schneiderian carcinoma in situ whose tumor progressed to small cell carcinoma. We performed whole-exome genetic sequencing and copy-number variation (CNV) analysis of tumor and normal DNA extracted from flash-frozen, paraffin-embedded (FFPE) samples. Results A total of 93 high-confidence, nonsynonymous somatic mutation events were identified in sinonasal SCC, including loss-of-function mutations in TP53, MAML3, a transcriptional coactivator of the Notch pathway, and GAS6, an activating ligand of the TAM family of tyrosine kinase receptors. Focal amplifications of chromosomal regions 6p25-11.1, containing SOX4 and VEGFA, and 14q32.1-32.3, containing AKT1 and the Notch inhibitory ligand DLK1, were also seen. Further CNV analysis revealed deletions in the critical cell cycle regulators CDKN2A, RB1, RBL1, and RBL2 and the chromatin modifier EP300. Conclusions Small cell carcinoma may rarely arise from sinonasal Schneiderian carcinoma in situ and exhibits similar genomic aberrations (eg, SOX amplification, Notch pathway inactivation) to pulmonary small cell carcinoma.

Abstract 54: Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer

Oral squamous cell carcinoma (OSCC) is a disease with poor survival with few novel treatment options in development. While EGFR is commonly over-expressed, targeting EGFR has had limited success possibly because of compensatory pathways rescuing cell survival. Inhibiting compensatory pathways in combination with EGFR signaling may result in greater efficacy. To identify compensatory pathways, we used a Genome-wide CRISPR-Cas9 Knock-Out (GeCKO) library to identify genetic knockouts that sensitized OSCC cell lines to EGFR inhibition. This CRISPR library established a pool of knockouts targeting over 18,000 genes, and upon selection of the GeCKO pool we identified gene knockouts in the FGFR pathway increased sensitivity to the EGFR inhibitor gefitinib. We further profiled this combination with viability assays and found that 6/14 (43%) cell lines responded to the combination of EGFR and FGFR inhibition, suggesting that the FGFR pathway could be a common compensatory mechanism to loss of EGFR signaling. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. In total, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition, with follow-up indicating that the FGFR pathway could be a common compensatory mechanism to EGFR inhibition. Targeting compensatory mechanisms in combination with EGFR-targeted therapy could be clinically beneficial, including the combination of EGFR and FGFR inhibition. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, J. Chad Brenner. Genome-wide CRISPR screen identifies potential therapeutic combination of EGFR and FGFR inhibitors in oral cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 54.

Abstract 4092: Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous cell carcinoma

Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated, oncogenic pathway in this cancer type. Despite the frequency of activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K), targeted inhibitors of PI3K have not shown clinical efficacy as monotherapies. We tested a panel of more than 20 patient-derived oral cavity squamous cell carcinoma (OCSCC) cell lines, which were profiled by Nimblegen V3 exome sequencing, and observed resistance to PI3K inhibitors despite PIK3CA copy number amplification and/or mutation. Of six inhibitors tested, only alpha-isoform selective and pan-PI3K agents were somewhat effective; these inhibitors, despite on-target and downstream activity, did not cause an appreciable reduction in cell viability when administered at submicromolar concentrations. We hypothesized that other oncogenic pathways might still be functional in the presence of PI3K inhibitors and might serve as mediators of this resistance. For example, our group and others have evaluated co-dependence on the Ras-MEK-ERK pathway in OCSCC, showing that this serves as a compensatory mechanism in some models. In order to more fully characterize other novel mechanisms of resistance, we have also developed a high-throughput screening approach that utilizes a resazurin cell viability assay. This screen serves as an unbiased means of testing ~1400 inhibitors as monotherapies and in combination with alpha-isoform selective PI3K inhibitor HS-173 and pan-PI3K inhibitor BKM120 in several OCSCC models. Further validation of “hits” from this screen, which are drugs that are effective in combination but not as monotherapies, may identify additional resistance mechanisms and lead to combination therapies to improve HNSCC patient prognosis. Citation Format: Nicole L. Michmerhuizen, Elizabeth Leonard, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Hui Jiang, Chad Brenner. Targeting compensatory mechanisms of resistance to phosphatidylinositol 3-kinase inhibitors in head and neck squamous cell carcinoma [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 4092. doi:10.1158/1538-7445.AM2017-4092

Abstract 3198: Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer

Oral squamous cell carcinoma (OSCC) has remained a disease with poor survival for decades with few novel treatment options. We anticipate great potential for personalized targeted therapies, facilitated by recent characterizations of the mutational landscape of OSCCs. Combinations of targeted therapies may have greater efficacy through inhibiting compensatory pathways. For example, targeting EGFR has had limited success. To identify synergistic combinations with EGFR-targeted therapy, we introduced Genome-scale CRISPR-Cas9 Knock-Out (GeCKO) libraries into OSCC cell lines. CRISPR-Cas9 generates individual genetic knockouts through targeted gene editing. We used a pool of CRISPRs targeting over 18,000 genes to perform genome-scale screening for drivers of sensitivity to EGFR-targeted therapy. Upon selection of the OSCC GeCKO pool, we identified gene knockouts in the FGFR pathway that increased sensitivity to the EGFR inhibitor gefitinib. Using resazurin viability assays we tested combinations of EGFR and FGFR inhibitors in 14 OSCC cell lines. Six/14 (43%) of the cell lines were responsive to the combination, indicating that the FGFR pathway is an alternate mechanism of resistance to EGFR-targeted therapy in some tumors. In complement, we again used CRISPR-Cas9 to generate a syngeneic EGFR knockout (KO) OSCC cell line. The parent OSCC cell line is responsive to the EGFR and FGFR dual inhibition, and the EGFR KO derivative retains sensitivity to FGFR-targeted monotherapies. We expect that this EGFR KO cell line will be a useful tool in further evaluating the compensatory mechanism of the FGFR pathway. OSCC remains a common and frequently lethal cancer with great potential for the development of personalized targeted therapies. Here, we describe the use of genome-wide CRISPR-Cas9 library to discover the synergistic combination of EGFR and FGFR inhibition. Further investigation suggests the FGFR pathway is a common compensatory mechanism to EGFR inhibition. We hope to use this approach to identify additional compensatory mechanisms of resistance to targeted therapies with the eventual goal of translating these findings to clinic. Citation Format: Megan Ludwig, Andrew Birkeland, Sai Nimmagadda, Sue Foltin, Aditi Kulkarni, Hui Jiang, Thomas Carey, Chad Brenner. Using a genome-wide CRISPR-Cas9 knockout library to identify therapeutic combinations in oral cancer [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 3198. doi:10.1158/1538-7445.AM2017-3198

Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (>20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors. Citation Format: Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach [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 1667. doi:10.1158/1538-7445.AM2017-1667

Abstract 30: Small molecule profiling uncovers the landscape of combinatorial PI3K inhibitor responses in HNSCC

Recent sequencing studies of head and neck squamous cell carcinomas (HNSCCs) have identified the phosphatidylinositol 3-kinase (PI3K) pathway as the most frequently mutated oncogenic pathway in this cancer type. Although activating mutations or amplification in PIK3CA (the gene encoding the catalytic subunit of PI3K) are frequently observed in HNSCC, monotherapies targeting PI3K have shown limited clinical efficacy. We tested a panel of more than 20 patient-derived oral cavity squamous cell carcinoma (OCSCC) cell lines, which were profiled by Nimblegen V3 exome sequencing. Despite PIK3CA copy number amplification and/or mutations, we observed resistance to PI3K inhibitors in these models. Of six inhibitors tested, the only agents with marginal effects were alpha-isoform selective and pan-PI3K agents; these inhibitors, despite on-target and downstream activity, did not cause an appreciable reduction in cell viability when administered at submicromolar concentrations. We hypothesized that other oncogenic pathways, such as the Ras-MEK-ERK pathway, might still be functional in the presence of PI3K inhibitors and serve as compensatory mechanisms of resistance to these drugs. In order to more fully characterize other novel co-dependent pathway in PI3K inhibitor resistant HNSCC models, we have developed a high-throughput screening approach that utilizes a resazurin cell viability assay. This approach is an unbiased means of testing ~1400 inhibitors as monotherapies and in combination with alpha-isoform selective PI3K inhibitor HS-173 and pan-PI3K inhibitor BKM120 and has been utilized in several in vitro models of OCSCC to date. Testing of additional cellular models and validation of “hits” from this screen, which are drugs that are effective in combination but not as monotherapies, may identify additional resistance mechanisms and are currently ongoing. Developing a better understanding of these combination therapies and the patients in which they might be most effective may lead to improved prognosis for this disease. Citation Format: Nicole L. Michmerhuizen, Elizabeth Leonard, Micah Harris, Susan K. Foltin, Aditi Kulkarni, Thomas E. Carey, Carol R. Bradford, Jiang Hui, J. Chad Brenner. Small molecule profiling uncovers the landscape of combinatorial PI3K inhibitor responses in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 30.

Abstract 28: Identifying mechanisms of PD-L1 regulation in HNSCC

Evidence of immunosuppression is frequently reported in head and neck squamous cell carcinoma (HNSCC). Thus, there has been growing interest in advancing therapies for HNSCC that relieving immunosuppression to permit anti-tumor immunity. The immune checkpoint inhibitor Pembrolizumab targets the coinhibitory PD-1/PD-L1 pathway, which serves to shield tumor cells from attack by cytotoxic T lymphocytes. While Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, the initial trial reported overall response rates of only 18%. A more comprehensive understanding of the intrinsic and extrinsic mechanisms inducing PD-L1 expression on tumor cells may reveal therapeutic targets that improve response to immune modulation. We have employed a high-throughput genetic screen to identify targetable signaling pathways that may regulate PD-L1 expression in patients with specific genetic lesions. The Genome-scale CRISPR Knock-Out (GeCKO) library, established in multiple HNSCC cell lines, consists of a pool of stable knockouts targeting >20,000 genes with representation of approximately 300 gRNAs per target gene. Sub-populations with enhanced or diminished expression of PD-L1 were sequenced to identify gRNA targets whose knockout causes a change in PD-L1 expression. We expect that by performing these screens across multiple cell lines with diverse genetic lesions, pathways consistently regulating PD-L1 expression will be revealed, and that targeting such pathways will provide novel avenues for modulating this immunosuppressive mechanism. Citation Format: Jacqueline E. Mann, Megan Ludwig, Aditi Kulkarni, Rebecca Hoesli, Judy Kafelghazal, Alexey Nesvizhskii, J. Chad Brenner. Identifying mechanisms of PD-L1 regulation in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 28.

Abstract 19: Establishment of an upstream head and neck precision oncology tumor board

Background: Precision medicine is rapidly becoming incorporated into the management of head and neck cancers. Institutional precision tumor boards are emerging, mostly in frameworks that are not specific to head and neck cancers, and are focused on recurrent and metastatic (R/M) disease. There are significant benefits to a subspecialized focus on head and neck cancers without restricting enrollment to R/M cases. Herein, we discuss the establishment of a unique, upstream head and neck precision oncology program. Methods: We have established a head and neck precision oncology model that is run by head and neck surgeons and translational scientists, in combination with colleagues from radiation oncology, medical oncology, genetics, and ethics. Inclusion criteria stipulate adult patients with biopsy-proven malignancy of the head and neck (newly diagnosed or previously treated, including all sites, stages and histologies) who have pathologic samples available for analysis (fresh frozen or paraffin embedded). Results: We have begun enrolling patients prospectively (n=47 to date; 39 primary tumors and 8 R/M tumors). A custom Illumina 250 gene sequencing panel is utilized for targeted sequencing compared to matched blood. At the time of subject enrollment, we have incorporated a novel informed consent process involving ethicists and genetics counselors, with prospective collection to validate our informed consent process, discussion of disclosure of incidental findings, and clarifying patient expectations. Sequencing data are then presented formally at our head and neck precision oncology tumor board. Discussion: A primary benefit of a head and neck precision oncology tumor board is the integration of multidisciplinary expertise both from a clinical and cancer biology perspective. Our model of counseling, in which we employ a flexible default model of information disclosure, provides unique and valuable prospective information on patient expectations and understanding in disclosure of genetic results, clinical translation, and enrollment in targeted therapy trials. Unique to this model is a focus on enrolling patients up-front, at their initial presentation to our institution. This platform allows for early identification of potential targetable genetic aberrations (in adjuvant and neoadjuvant settings), and allows for evaluation of tumor genomic evolution/mutational selection if the patient develops R/M disease (which may allow for more specific targeted therapy to the mutation that appears to be driving the aggressive tumor biology). Citation Format: Andrew Birkeland, Aditi Kulkarni, Collin Brummel, Susan Foltin, Mark Prince, Carol Bradford, Andrew Shuman, J. Chad Brenner. Establishment of an upstream head and neck precision oncology tumor board [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 19.