Michelle Kassner

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Kinome-wide siRNA screening identifies molecular targets mediating the sensitivity of pancreatic cancer cells to Aurora kinase inhibitors

Aurora kinases are a family of mitotic kinases that play important roles in the tumorigenesis of a variety of cancers including pancreatic cancer. A number of Aurora kinase inhibitors (AKIs) are currently being tested in preclinical and clinical settings as anti-cancer therapies. However, the antitumor activity of AKIs in clinical trials has been modest. In order to improve the antitumor activity of AKIs in pancreatic cancer, we utilized a kinome focused RNAi screen to identify genes that, when silenced, would sensitize pancreatic cancer cells to AKI treatment. A total of 17 kinase genes were identified and confirmed as positive hits. One of the hits was the platelet-derived growth factor receptor, alpha polypeptide (PDGFRA), which has been shown to be overexpressed in pancreatic cancer cells and tumor tissues. Imatinib, a PDGFR inhibitor, significantly enhanced the anti-proliferative effect of ZM447439, an Aurora B specific inhibitor, and PHA-739358, a pan-Aurora kinase inhibitor. Further studies showed that imatinib augmented the induction of G2/M cell cycle arrest and apoptosis by PHA-739358. These findings indicate that PDGFRA is a potential mediator of AKI sensitivity in pancreatic cancer cells.

Functional Genomics Reveals Diverse Cellular Processes That Modulate Tumor Cell Response to Oxaliplatin

Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin. Mol Cancer Res; 9(2); 173–82. ©2010 AACR.

Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

The Application of High-Throughput RNAi in Pancreatic Cancer Target Discovery and Drug Development

Pancreatic cancer is a particularly lethal malignancy and is highly chemoresistant. There is an urgent need for the identification of new therapeutic targets and more effective treatment options. New approaches, such as high-throughput RNAi, enable the functional evaluation of the casual role of numerous genes in regulating cellular processes, such as cell survival and drug response. In the following chapter, we review RNA interference and its application in high-throughput biology. Specifically, an overview is provided highlighting important experimental aspects in transitioning RNAi to a high-throughput platform. In addition, there is a brief review of current applications of high-throughput RNAi for cancer target identification and drug discovery. Lastly, particular applications of genome-scale RNAi to pancreatic cancer target and treatment identification are discussed. In summary, genome-scale RNAi is proving to be a powerful cellular genomics technology that holds great promise for advancing pharmacologically relevant targets and agents in pancreatic cancer research.

In Vitro High-Throughput RNAi Screening to Accelerate the Process of Target Identification and Drug Development.

High-throughput RNA interference (HT-RNAi) is a powerful tool that can be used to knock down gene expression in order to identify novel genes and pathways involved in many cellular processes. It is a systematic, yet unbiased, approach to identify essential or synthetic lethal genes that promote cell survival in diseased cells as well as genes that confer resistance or sensitivity to drug treatment. This information serves as a foundation for enhancing current treatments for cancer and other diseases by identifying new drug targets, uncovering potential combination therapies, and helping clinicians match patients with the most effective treatment based on genetic information. Here, we describe the method of performing an in vitro HT-RNAi screen using chemically synthesized siRNA.

Abstract 2931: Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic alterations responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the resistant clones and RNAi-based systematic synthetic lethal screening revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, we detected increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs refractory patients by immunohistochemistry. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs in a subset of lung cancer patients and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients. Citation Format: Nayoung Kim, Ahye Cho, Hideo Watanabe, Yoon-La Choi, Meraj Aziz, Michelle Kassner, Je-Gun Joung, Angela KJ Park, Joshua Francis, Joon Seol Bae, Soo-min Ahn, Kyoung-Mee Kim, Joon-Oh Park, Woong-Yang Park, Myung-Ju Ahn, Keunchil Park, Hongwei Holly Yin, Jeonghee Cho. Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2931.

Abstract LB-217: CK2 protein kinase promotes resistance to MAPK pathway inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent on the RAS-RAF-MEK-ERK MAPK pathway, yet identification of both de novo/innate/intrinsic and acquired resistance mechanisms will be critical for the successful application of these inhibitors in the clinic. We interrogated the kinome to identify resistance mechanisms towards the novel ERK1/2-selective inhibitor SCH772984. We first utilized a kinome-focused RNAi screen to identify genes that, when silenced, sensitized KRAS-dependent pancreatic cancer cells to SCH772984. In our drug dose-response screen of 711 kinases (QIAGEN library), we used 4 independent siRNA duplexes to knock down each gene, treated at 5 different drug doses, then evaluated viability with a standard CellTiter-Glo assay. Nineteen kinases enhanced sensitivity to SCH772984 (where at least 2 siRNAs for each target decreased IC50) at least 5-fold, indicating that they could drive ERK1/2 inhibitor resistance. Among these were 4 of the 9 protein kinases (Cot, Raf-1, PAK3 and PRKCH) identified in a cDNA expression screen for kinases that caused resistance of BRAF-mutant melanoma to the BRAF inhibitor vemurafenib. We therefore hypothesized that at least some of the kinases that scored positive in this screen might be conserved both across the MAPK pathway and across different cancer types. The tetrameric protein kinase CK2 (formerly casein kinase II) has crucial roles in cell survival, proliferation and differentiation, and its expression and/or activity is dysregulated in cancers including melanoma. In very recent studies, we have now determined that the alpha subunit of CK2 is sufficient to cause resistance to each of the three ERK MAPK pathway inhibitors currently approved for treatment of melanoma: vemurafenib (BRAFi), dabrafenib (BRAFi) and trametinib (MEKi). Co-inhibition of the MAPK pathway and CK2 (e.g., with CX-4945, currently in phase I trials) further enhanced sensitivity. Thus, this combination may offer an effective regimen to forestall or delay tumor relapse. We also observed that CK2alpha maintains ERK phosphorylation in the presence of MAPK inhibitors, and that it posttranslationally reduces abundance of the ERK phosphatase DUSP6/MKP-3, in a kinase-dependent manner. The relationship of these findings to CK2-mediated mechanisms of drug resistance will be discussed. Citation Format: Bingying Zhou, Michelle Kassner, Holly Yin, Channing J. Der, Adrienne D. Cox. CK2 protein kinase promotes resistance to MAPK pathway inhibition. [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-217. doi:10.1158/1538-7445.AM2014-LB-217

Abstract A28: Identification of HDAC inhibitor potentiating targets in acute myeloid leukemia cells by large-scale RNA-interference

The lysine deacetylase inhibitor suberoylanilide acid (SAHA) has shown promising but limited activity in the treatment of acute myeloid leukemia (AML). To identify potential targets for rational combination therapies that increase the efficacy of SAHA in AML, we used a functional RNA-interference (RNAi) screening approach to identify genes, that when inhibited, potentiate the in vitro anti-leukemic activity of SAHA. A total of 901 kinase, phosphatase and associated signaling genes were silenced, with four different siRNA sequences per gene, in combination with SAHA treatment. Log2 values of the ratio [(siRNA + SAHA)/(siRNA alone)] were calculated, with median and standard deviation determined on a per-plate basis. Hits were defined as ≥ 2 standard deviations from the log2 ratio median. Hit lists for each cell line were over-laid on an integrated functional relationship network. We applied a community detection algorithm to this sub-network and identified siRNA sensitive modules. Each module represents a highly connected set of genes in the integrated network. To identify the pathways represented by each module, we evaluated enrichment using the National Cancer Institute (NCI) Protein Interaction Database (PID) pathways. Several novel sensitizing targets, grouped into a small number of pathways, emerged from these screens. Some hits exhibit little to no anti-leukemic activity when silenced alone, indicative of synthetic lethal interaction with SAHA treatment. Initial validation experiments with siRNA and novel small molecule inhibitors confirm RNAi screen results and pharmacological sensitization is observed. The first reported large-scale HDAC inhibitor RNAi screen in leukemias has identified a novel rational combination that can be translated into design of a clinical trial. Citation Format: James M. Bogenberger, James E. Rudd, Donald Chow, Michelle Kassner, Holly Yin, Casey S. Greene, Raoul Tibes. Identification of HDAC inhibitor potentiating targets in acute myeloid leukemia cells by large-scale RNA-interference. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A28.

Abstract B09: High-throughput RNAi sensitization screening identifies a novel drug combination for metastatic melanoma

Metastatic malignant melanoma is an incurable disease with a poor 5-year survival rates. Despite the recent development of several novel therapeutic agents, there remains an urgent need to improve the effectiveness of conventional chemotherapeutics such as temozolomide (TMZ). In this study, we developed RNA-interference-based (RNAi) TMZ chemosensitization assays in melanoma cell lines in order to better understand how to pharmacologically augment TMZ response. Initially 65 melanoma cell lines were surveyed for siRNA optimization, with 18 selected to carry out extensive assay optimization across a multitude of assay conditions. Then we applied these optimized RNAi screening parameters and performed high-throughput RNAi screens with a 7,000 gene siRNA library targeting the druggable genome. Five melanoma cell lines demonstrating various response in the presence of TMZ were selected for the primary screens: two primary screens were performed using two siRNAs per target, followed by three screens using four siRNAs per target. The sensitizer screen was conducted with a drug dose-response methodology using five different concentrations of TMZ (targeting IC10 to IC80), and cell viability was measured 120 hour post-treatment. The IC50 was calculated for each siRNA and individual siRNA hits were selected when their IC50 values were significantly different from that of the scrambled siRNA control. Genes that had two or more siRNAs demonstrating TMZ sensitization were selected for confirmation and further validation in a panel of 15 total melanoma cell lines. 137 genes were validated as TMZ sensitizers in two or more melanoma cell lines. Because multiple members of the CHEK1/CHEK2 pathway were among these validated sensitizers, we chose to focus on this pathway and evaluate checkpoint kinase inhibition as a potential means of augmenting TMZ response. We assessed four CHEK1 inhibitors for synergistic effects when combined with TMZ. Notably, we observed measurably synergistic effects for four CHEK1 inhibitors in each of the most TMZ-resistant cell lines, with only moderate/marginal effects in the most TMZ-sensitive lines. These synergistic effects on cell viability were accompanied by an increase in apoptotic events, and the synergy between TMZ and CHEK1 inhibition was further validated in a mouse xenograft model. Our findings suggest that targeting the CHEK1/CHEK2 pathway may augment TMZ response in melanoma, and lay the foundation for the testing of novel combination therapies for melanoma patients who are vulnerable to conventional treatment. Citation Format: Hongwei Holly Yin, Donald Chow, Chris Sereduk, Meraj Aziz, Jeff Kiefer, Mai Xu, Michael Garside, Nanyun Tang, Chao Sima, Jianping Hua, Michelle Kassner, Mike Bittner, Nick Hayward, Kevin Brown, Trent Jeff. High-throughput RNAi sensitization screening identifies a novel drug combination for metastatic melanoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B09.