Yin, Hongwei

Abstract Triptolide, which was initially extracted from a Chinese medicinal herb, demonstrates potent antitumor, anti-inflammatory, and immunosuppressive properties, yet it is clinically limited due to its poor solubility, bioavailability, and toxicity. Minnelide, a water-soluble analog of triptolide, has potent antiproliferative activity. It is currently in Phase I/II clinical trials for Acute Myeloid Leukemia (AML) and 7 solid tumor indications, including advanced gastrointestinal tumors, pancreatic cancer, and colorectal cancer. To overcome the potential drug resistance and to improve the drug potency as well as to minimize the drug toxicity, we conducted a high throughput screening (HTS) for drug synergy, combining Minnelide with compounds from the LOPAC and/or Prestwick libraries in 3 different cancer cell lines: BxPC3 (pancreatic), HT29 (colon) and SNU5 (gastric). LOPAC and Prestwick libraries consist of drugs approved by FDA and/or drugs that have reached clinical trial stages, representing 2,274 unique compounds in total. After quality control evaluation and data normalization, hits were selected if greater than 20% inhibition was achieved when combined compared with Minnelide alone. A total of 43, 54, and 47 hits were identified for each cell line, representing an average hit rate of 2.1%. We further prioritized the hits by selecting those that were common in two or more cell lines, which resulted in 9 compounds from Prestwick library and 20 compounds from LOPAC library (Harmane is in common between the two libraries). Only 17 compounds were available for purchase for hits confirmation, which was conducted in a similar manner as in primary HTS, except that drugs were tested in a dose-dependent manner instead of a single concentration done in HTS, a duplicated biological run was performed, and a total of 9 cell lines were screened, with 3 cell lines for each tumor type (pancreatic, colon and gastric). IC50 and Combination Index (CI) values for each drug combination were calculated for each cell line. A fold change of 2 or more for the IC50 values and CI value < 1.0 were used for hit selection. In general, colon lines had the most synergistic combinations and gastric had the least. A total of 15 drug candidates were confirmed to have synergistic effect, representing a confirmation rate of 88%, among which 12 drugs demonstrated synergy in >50% of the cell lines tested, with Harmane and Harmine demonstrating synergy in all 9 cell lines tested. In addition, CGS-15943, Amiloride, Naphthalimide, and Idazoxan indicated good synergy with Minnelide in at least 6 out of 9 cell lines. Interestingly, Phenamil, an irreversible inhibitor of amiloride-sensitive Na+ channels, showed synergy in all cell lines tested for pancreatic and colon cancers, but not in gastric cancer cell lines. Overall, our study shed light on potential drug combinations with Minnlide for clinical use. Citation Format: Nanyun Tang, Chris Sereduk, Ashok Saluja, Mohana Velagapudi, Haiyong Han, Daniel D Von Hoff, Hongwei Holly Yin. Drug combination screen to identify novel synergy with Minnelide [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A142. doi:10.1158/1535-7163.TARG-19-A142

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 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 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 2801 Poster Board II-777 To provide a rationale basis for targeted drug development for multiple myeloma patients, we have conducted high-throughput genome-scale RNA interference (RNAi) synthetic lethality studies in human myeloma cell lines (HMCL) to generate a comprehensive map of critical genes and molecular vulnerabilities in human myeloma tumor cells. KMS11 human myeloma cells were screened with a 13,982-oligo library targeting the ‘druggable' genome (6,991 genes) using optimized conditions that resulted in >95% transfection efficiency. Each gene was screened with 2 or more distinct oligos, in duplicate, using a single-siRNA-per-well format, testing >34,000 wells. Replicate high throughput experiments yielded highly reproducible results (R2=0.82). Viability was measured at 96h by ATP-dependent luminescence. Universally lethal siRNA and non-targeted siRNA were employed as controls. The specificity (FDR) of lethal RNAi was evaluated by custom-developed statistical methods based on RNAi result concordancy. From screening, 5.8% of druggable genome siRNA caused statistically relevant reductions in HMCL viability (greater than three standard deviations from control samples treated with non-silencing siRNA, compared with an anticipated rate due to chance of only 0.135%). Of these, two hundred and nineteen genes, targeted by the most lethal siRNA, were forwarded to validation studies. Validation high-throughput RNAi studies, using 4 oligos per gene, were conducted in order to verify target gene vulnerability. Ultimately, seventy two genes were validated as highly critical for myeloma cell survival (each with multiple concordant siRNA hits plus high reproducibility of R2 =0.94). Among top-ranked lethal molecular vulnerabilities in KMS11 myeloma cells we recurrently identified the proteasome (8 PSM subunits were independently identified as highly vulnerable); and BCL2 family member MCL1; both known therapeutic targets, validating a functional genomics approach to target discovery. In addition, a number of novel, equally lethal, molecular vulnerabilities were identified in KMS11 including WEE1, kinetochore complex component KNTC2, the aurora kinases, polo-like kinase 1, a previously uncharacterized DNA methyl transferase, ribonucleotide reductase, ribosomal protein L38, leptin receptor overlapping transcript LEPROT and SLC25A23 (a carrier responsible for mitochondrial adenine nucleotide flux). Various ubiquitous cellular proteins involved in RNA or protein processing (e.g. SF3a, SNW1, SNRPA1, EIF3s8, amongst others) were also identified as non redundant and critical for cellular viability. Thirty nine top-ranked molecular vulnerabilities in KMS11 were evaluated for comparative vulnerability in a second myeloma cell line, JJN3, and in A549 and 293 epithelial cells. A majority were vulnerable in JJN3. While 25 genes proved equally lethal on silencing in A549 and 293 epithelial cells, several, notably including MCL1 and three proteasome subunits, appear differentially susceptible in myeloma cells versus epithelial cells. From this genome scale study, MCL1 ranks third as a target capable of inducing cell death in myeloma cells, highlighting the potent vulnerability of myeloma cell viability to alterations in this anti-apoptosis regulator. Moreover, from active comparative studies of molecular vulnerability in myeloma versus epithelial cell lines, MCL1 as an RNAi target demonstrates perhaps the greatest cytotoxic selectivity between myeloma and epithelial cell lines of all targets examined. Gene expression studies indicate that MCL1 is highly expressed in myeloma tumor cells but is absent or only weakly expressed in human primary somatic tissues. Overall, therefore, from an unsupervised genome-scale screening approach, our data support MCL1 as an optimal Achilles heel molecular target in multiple myeloma, potentially offering greater specificity and deeper cytotoxic effect than proteasome inhibition. Disclosures: No relevant conflicts of interest to declare.

Abstract Purpose: Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive ovarian cancer in young women that is universally driven by loss of the SWI/SNF ATPase subunits SMARCA4 and SMARCA2. A great need exists for effective targeted therapies for SCCOHT. Experimental Design: To identify underlying therapeutic vulnerabilities in SCCOHT, we conducted high-throughput siRNA and drug screens. Complementary proteomics approaches profiled kinases inhibited by ponatinib. Ponatinib was tested for efficacy in two patient-derived xenograft (PDX) models and one cell-line xenograft model of SCCOHT. Results: The receptor tyrosine kinase (RTK) family was enriched in siRNA screen hits, with FGFRs and PDGFRs being overlapping hits between drug and siRNA screens. Of multiple potent drug classes in SCCOHT cell lines, RTK inhibitors were only one of two classes with selectivity in SCCOHT relative to three SWI/SNF wild-type ovarian cancer cell lines. We further identified ponatinib as the most effective clinically approved RTK inhibitor. Reexpression of SMARCA4 was shown to confer a 1.7-fold increase in resistance to ponatinib. Subsequent proteomic assessment of ponatinib target modulation in SCCOHT cell models confirmed inhibition of nine known ponatinib target kinases alongside 77 noncanonical ponatinib targets in SCCOHT. Finally, ponatinib delayed tumor doubling time 4-fold in SCCOHT-1 xenografts while reducing final tumor volumes in SCCOHT PDX models by 58.6% and 42.5%. Conclusions: Ponatinib is an effective agent for SMARCA4-mutant SCCOHT in both in vitro and in vivo preclinical models through its inhibition of multiple kinases. Clinical investigation of this FDA-approved oncology drug in SCCOHT is warranted. Clin Cancer Res; 24(8); 1932–43. ©2018 AACR.

Key Points High-throughput RNAi screening identified lenalidomide sensitizer genes, including RSK2, RAB, peroxisome, and potassium channel family members. Knockdown or inhibition of RSK2 synergized with lenalidomide to induce myeloma cytotoxicity and downregulation of interferon regulatory factor 4 and MYC.

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 Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non–multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma. Cancer Res; 72(3); 757–68. ©2011 AACR.

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.