Kassner, Michelle

Abstract The lysine deacetylase inhibitor suberoylanilide acid (SAHA) exhibits 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 performed a functional RNA-interference (RNAi) drug modifier screen to identify genes that, when inhibited, potentiate or antagonize the in vitro anti-leukemic activity of SAHA. A total of 901 kinase, phosphatase and closely associated signaling genes were silenced in TF-1, HEL and THP-1 cells, with four different siRNA sequences per gene, both alone and in combination with SAHA treatment. Screen hit lists for each cell line were over-laid on an integrated functional relationship network. A community detection algorithm was then applied to this sub-network and siRNA sensitive modules were identified. Each module represents a highly connected set of genes in the integrated network. To identify pathways represented by each module, enrichment was evaluated using the National Cancer Institute (NCI) Protein Interaction Database (PID) pathways. Both cell line-specific and universal sensitizing targets, grouped into a small number of pathways, emerged from these screens. Seven pharmacologic inhibitors interfering with these pathways have been assessed in a panel of four or five AML cell lines, including SET-2, TF-1, HEL, THP-1, and OCI-AML3. Of the seven tested inhibitors targeting SAHA sensitizing networks, one combination exhibiting universal SAHA sensitization in all AML cell lines tested was further assessed in drug dose response assays using ex vivo cultures of bone marrow mononuclear cell populations derived from patients with various myeloid malignancies. The combination demonstrated “one-sided” SAHA EC50 fold-enhancement, as well as strong synergy by “two-sided” CalcuSyn combination index determination in all ex vivo specimens examined to date (N=6), including polycythemia vera (PV), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndrome, MDS transformed to AML, and de novo AML samples. Thus, this first reported large-scale SAHA RNAi modifier screen in AML has identified a specific pathway/target whose inhibition broadly synergizes with HDAC inhibition in myeloid malignancies. A novel compound putatively targeting this respective pathway/target is currently in early clinical development and has shown limited single-agent activity. With the full dataset presented, we will propose a novel rational combination to enhance the activity of HDAC inhibition in AML and other myeloid malignancies that could be translated into design of a clinical trial. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1851 Background: In previous work we performed RNAi sensitizer screening of epigenetic modulation with 5-Azacytidine and the human kinome. Few kinase hits were identified. Therefore we asked if phosphatases might be involved in modulating epigenetic therapies. Thus in order to identify potentiators of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) we performed high-throughput RNA-interference (RNAi) lethality screening using the myeloid cell lines HEL, THP-1 and TF-1 in combination with SAHA. Methods: A small-interfering RNA (siRNA) library targeting 206 phosphatases with 4 sequences per target gene was used for this study. siRNA were transfected in a single-siRNA-per-well format in 384-well plates using commercial lipid-based transfection reagents. Non-silencing siRNA were included as negative controls to access non-specific siRNA toxicity and universal lethal siRNA were included as positive controls to indirectly assess functional transfection efficiency. After a 96 hour incubation period, cell viability/proliferation was measured with Cell Titer Glo. For analysis of screen data, log2 values of (SAHA + siRNA treatment/siRNA treatment only) were calculated. Potentiation was defined as two standard deviations from the median log2 ratio of all siRNA (excluding positive controls) on a plate-by-plate basis. Hits were defined as ≥ 2/4 siRNA sequences meeting potentiation criteria. Results: Transfection efficiency was 95, 90 and 70% for HEL, THP-1 and TF-1, respectively. All three cell lines were similarly sensitive to SAHA with IC50 values of 0.3, 0.4 and 0.6 μM for HEL, THP-1 and TF-1, respectively. Resulting SAHA concentrations for screens were between the IC5 to IC30. For THP-1, 5 phosphatases qualified according to the hit selection criteria applied indicating strong potentiation to SAHA. Of these 5 hits, 3 did not exhibit strong reductions in viability with siRNA alone, suggesting a SAHA-dependent relationship. For HEL and TF-1, although no phosphatases met the strict selection cutoff, there were several phosphatases exhibiting potentiation, suggesting biological relevance. The primary screening data and preliminary analysis will be validated with rigorous secondary experimentation and top hits for all cell lines will be investigated and advanced. Conclusions: While there are numerous kinase inhibitors in the clinic and in preclinical development, phosphatases are much less explored as therapeutic targets. Validated potentiating targets from this RNAi screen can facilitate the development of rational SAHA combination therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 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 Hepatocellular carcinoma (HCC), the primary malignancy of the liver, is the fourth leading cause of cancer deaths worldwide, with a 5-year survival rate of∼10%. Recently, a new multikinase inhibitor, sorafenib (Nexavar®), which mainly targets the VEGFR and the Raf/Mek/Erk pathways, has shown limited response rate and modest survival benefits in patients with advanced stage HCC. We hypothesize that there exist genetic alterations or contexts of vulnerability (COV) in cancer cells that dictate sensitivity to sorafenib. In order to identify specific COVs and obtain an increased understanding of drug's mechanism of action, we performed a custom chemosensitization high-throughput siRNA screen. We developed and optimized a sorafenib chemosensitization assay in 3 HCC cell lines, one of which, Hep3B, a AFP and HBsAg positive cell line, was selected for primary RNAi screening with a kinase siRNA library, which covers 700 genes (4 siRNA sequences per gene). Primary screen data was rigorously evaluated for multiple quality control metrics (transfection efficiency, CV values, accessing plate-to-plate and set-to-set variation) and found to exceed all expected performance parameters. The top sorefenib sensitizing hits were identified and supplemented with additional genes selected based on related biological concepts. This sensitization effect of the genes was further tested and compared in two additional HCC cell lines (HepG2 and PLC). Additionally, based on the sensitizer list from the RNAi screening, 11 chemical compounds were tested for synergistic combination with sorafenib. Notable synergism with sorafenib was observed in the combination with erlotinib or doxorubicin. More strikingly, we also observed synergy between sorafenib and multiple MEK inhibitors as well as gemcitabine in the selected HCC cell lines. Our data suggest that MEK inhibitors might improve the efficacy of sorafenib in HCC treatment in the clinic. Further mechanistic studies are being conducted for this particular combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-285.