Azorsa, David O.

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

Abstract Abstract 3513 To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures: Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding.

Abstract Abstract 229 Introduction: Optimal rational combination targets with Cytarabine (AraC) have not been identified. After the development of a high-throughput, small-interfering RNA (siRNA) platform for suspension cells, we performed the first RNAi sensitizer screen of the human kinome with AraC in myeloid cell lines. Herein we present the final data of this first siRNA-sensitizer screen in leukemias in combination with AraC. Our results include full validation of the selected targets as well as ex vivo data assessing the first in class clinically tested WEE1 inhibitor MK1775 with AraC providing a pre-clinical rationale for using the combination of AraC with WEE1 inhibitors in leukemias. RNAi Screens and siRNA Validation: In siRNA sensitizer screens, we silenced 572 kinases of the human kinome in combination with AraC. Few kinases strongly sensitized to AraC and the targets CHEK1 and the WEE1 related kinase PKMYT1 emerged as the most potent genes whose silencing sensitized leukemia cells to AraC. In secondary siRNA validation experiments, silencing of WEE1 kinase was found to be more potent than silencing of CHEK1, PKMYT1 or other kinases (i.e. ATR, BRCA, AURKB) in combination with AraC treatment. Importantly, the sensitizing activity observed with WEE1 inhibition was universal across all AML cell lines examined, whereas it was not for CHEK1 inhibition. siRNA assays were robust, exhibiting little (<10%) non-specific toxicity and siRNA silencing was specific for individual genes (i.e. WEE1 or CHEK1) as demonstrated by selective reduction of mRNA and protein level for the respective genes; confirming the validity of the siRNA assay. Pharmacological WEE1 Inhibitor Experiments In vitro and Ex vivo: In vitro treatment of eight AML, ALL and CML cell lines with commercial and the first clinically developed WEE1 kinase inhibitor MK1775, demonstrated strong sensitization to AraC up to 97-fold in individual cell lines. Ex vivo, MK1775 substantially reduced viability in 13 of 14 AML, MDS and CML patient samples compared to AraC alone. The optimal dose to achieve maximum sensitization was observed for AraC between ∼10–120 nM and for MK1775 between 200–400 nM. Synergy at these optimal concentrations was confirmed in all ex vivo samples tested for which sufficient data for analysis with CalcuSyn Software was available (n=7): with Combination Indices (CI) ranging from 0.21–0.45. At higher AraC and MK1775 concentrations antagonism was observed both ex vivo and in vitro. Thus, we have also established optimal suggested concentration dose ranges for the currently ongoing design of a clinical trial. Clinically relevant, WEE1 is expressed in primary AML, ALL and advanced CML specimens with a trend towards higher expression in relapsed samples. However, there was no direct correlation with WEE1 mRNA expression or TP53 status. Our initial in vitro experiments further suggest that inhibition of WEE1 with MK1775 in combination with AraC at optimal concentrations was associated with higher degree of caspase 3 cleavage, DNA damage induction as measured by gamma-H2AX, and greatest inhibition of proliferation. Thereby establishing a context of optimal drug concentration ranges with molecular correlates and anti-leukemic activity directly informing design of the clinical trial and correlative biomarkers. In conclusion, data from this first siRNA sensitizer screen of the kinome in leukemias identifies inhibition of WEE1 kinase as the most potent sensitizing target to AraC. Inhibiting WEE1 with pharmacological inhibitors in combination with AraC is a rational treatment strategy for myeloid and lymphoid leukemias that warrants clinical investigation. Furthermore, our RNAi loss-of-function genomics approach has high potential to rapidly identify and validate crucial genes whose inhibition modulate the response to cytotoxic agents. Establishment of the siRNA sensitizer platform allows accelerating the development of new rational combinations for leukemia therapy. Disclosures: Off Label Use: AraC in combination with the experimental Wee1 inhibitor MK1775. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding. Demuth:Merck and CO: Employment.

Abstract Abstract 180 Background: Proliferative signals in myeloid malignancies/leukemias are often driven by kinase activating events. Phosphatases often counteract these effects, though some phosphatases are oncogenes as well. To characterize these opposing signaling networks and build a loss of function intervention map, we performed the first phosphatome RNAi screen in conjunction with a kinome RNAi screen in myeloid leukemia cells. Methods: Four myeloid cell lines, TF-1, THP-1, HEL and MDS-L were incubated with siRNA against 206 phosphatases and 711 kinases with 4 different siRNA sequences (4x) per gene. After 96 hours, viability/proliferation was assessed by luminescence with Cell Titer Glo. Tranfection efficacy was determined by the ratio of the median of an universal lethal control vs. all siRNA. Normalization was done calculating the percent of sample median according to % proliferation = [siRNA signal × 100] / [median all siRNA signals]. Hits were defined using the median absolute deviation (MAD) of > 2 standard deviation above or below the individual siRNA signal (Birmingham et al, Nature Methods 2009) for at least 2 of 4 siRNA per gene. Results: Transfection efficacy was around 70, 95, 95 and 80 percent for TF-1, THP-1, HEL and MDS-L cells respectively. In TF-1, five kinases met hit selection criteria reducing proliferation whereas surprisingly no phosphatase was a hit. In THP-1, five kinases each reduced or increased proliferation respectively and only one phosphatase was a hit altogether increasing proliferation upon siRNA silencing. A different trend was seen in HEL cells: siRNA silencing of 16 kinases decreased and silencing of one kinase increased proliferation. Interestingly, inhibition of six phosphatases reduced proliferation in HEL whereas none increased proliferation. In MDS-L cells, siRNA inhibition of 9 kinases reduced and of 1 kinases increased viability respectively; inhibition of two phosphatases increased and of one decreased viability. The most common universal hit was PLK1 across 3 of 4 cell lines. No phosphatase was found to be a common hit across several lines. Conclusion: RNAi kinome-phosphatome profiling of 917 phosphatases and kinases reveals distinct signaling patterns among representative myeloid leukemia cell lines. Some cell lines, like HEL, which harbors a Jak2 mutation, are possibly more driven by kinase activating events but also show putative phosphatases as therapeutic targets. Candidate kinases whose siRNA silencing reduces viability/proliferation may uncover synthetic lethal interactions between known mutational and genomic abnormalities. The identified siRNA hits are potential novel targets for drug development. This combined functional genomics approach also highlights the complexity of signaling and that inhibition of some kinases can accelerate growth at least in vitro, e.g. inhibition of PIM3 in THP-1 cells. Phosphatases are an emerging network of cellular regulators with occasional oncogenic potential. A “loss-of-phosphatome/ase” RNAi approach is a fast way to gain new insights into the complex and little researched field. Disclosures: No relevant conflicts of interest to declare.

Abstract Objective The prevalence and mortality for head and neck cancer (HNC) has not improved significantly in the last several decades. It is the sixth most common cancer with an estimated 600,000 new cases diagnosed worldwide every year and has ∼50% 5-year survival rate. Of these, 47000 new cases are diagnosed in the US annually. The most common risk factors for HNC are tobacco and alcohol use but a growing subset is also attributed to human papillomavirus (HPV) infection. Late stage HNCs are treated with a combination of surgery, radiation, and chemotherapy (often platinum-based), but the cancers treated with platinum-based chemotherapy often develop drug resistance. Identification of target genes using high-throughput RNAi (HT-RNAi) gene-silencing technology has become an increasingly popular method in the field of cancer research. The objective of our study is to use this functional genomics based approach to identify genes that, when silenced, increase the sensitivity of the platinum drugs. This would possibly help us in identifying potentiators of platinum-based drugs to more effectively treat HNC patients. Methods We performed drug dose response (DDR) experiments on nine HNC cell lines; including two are HPV-positive lines, to identify cisplatin-resistant and -sensitive cancer cell lines. To identify sensitizers of cisplatin response, we used a siRNA library targeting 572 kinases. Each kinase is represented by two different siRNA sequences in the library. Based on the data mined from published literature on head and neck cancer as well as genes thought to confer a functional relationship between HPV infection and HNC, a more focused siRNA library was also designed and included in the screening. Cell lines were reverse transfected with the siRNAs in a 384 well format, dosed with cisplatin after 24 hours and the plates were read 72 hours later. Results We calculated the inhibitory concentrations using DDR experiments for all the nine HNC cell lines and identified cisplatin-resistant (UMSCC47 and SCC9) and -sensitive (Cal27, UMSCC14C, and SCC090) HNC cell lines. HT-RNAi experiments have been performed on the cisplatin-resistant cell lines, one of which is HPV-positive. After data analysis, confirmation of silencing and validation of drug modulation will be done on a subset of candidate genes. Conclusions HNC patients with surgically non-resectable tumors could have improved treatment with chemotherapeutic drugs; however, acquired drug resistance often inhibits a successful treatment. Identification of sensitizing targets may provide hope for a better outcome, but may also allow for use of smaller doses of more toxic drugs, such as cisplatin. In addition, this study may also identify alternate genes that can be targeted in the subset of HPV-positive HNC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5136. doi:1538-7445.AM2012-5136

AbstractTo identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

Abstract In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. 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-128.

AbstractA paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma.