Drug Combination Screening Research Articles

Abstract B021: PARP16 is a novel target of talazoparib which contributes to synergy with adavosertib in SCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly aggressive type of neuroendocrine tumor with a low survival rate, signifying an unmet medical need. Although PARP inhibitors (PARPis) display single-agent anticancer activity in SCLC, the underlying biology is poorly understood. In the present study, we determined the differential efficacy of multiple clinically relevant PARPis and identified a novel non-canonical and actionable PARPi target in SCLC. Methods: A panel of SCLC cell lines was analyzed for sensitivity to PARPis using the Cell Titer Glow (CTG) viability assay. Chemical proteomics and western blotting were performed to investigate the targets of PARPis. The effect of PARP16 on cell viability was analyzed using RNA interference. Drug combination screening for inhibition of viability was performed, network-wide signaling changes were determined using phosphoproteomics, and efficacy of drug combinations was determined in ex vivo PDX clonogenic assays. Results: Compared to other clinically relevant PARPis (rucaparib, olaparib, niraparib) talazoparib displayed the highest potency across a panel of SCLC cell lines, including cells negative for SLFN11, which has been suggested to indicate PARPi sensitivity. Subsequent chemical proteomics with SLFN11-negative SCLC cells identified the non-canonical PARP family member PARP16 as a unique talazoparib target, along with the eminent PARP1. Transient knockdown of PARP16 significantly reduced cell survival in certain SCLC cell lines, particularly in combination with olaparib. Furthermore, drug combination screens revealed synergistic activity of talazoparib when combined with the WEE1 inhibitor adavosertib in SCLC cells, which was more pronounced than the combination of adavosertib with olaparib, which has no activity against PARP16. Silencing of PARP16, in combination with olaparib and adavosertib, with enhanced cell killing, and global phosphoproteomics identified network-wide signaling crosstalk inhibition by the talazoparib/adavosertib drug combination. Conclusion: Our data suggest that in addition to the well-studied PARP1 targeting mechanism, PARP16, a non-canonical member of the PARP family, is likely involved in talazoparib’s overall mechanism of action and may constitute a novel actionable target in SCLC. Citation Format: Vinayak Palve, Clare Knezevic, Yunting Luo, Xueli Li, Slivia Novakova, Eric Welsh, Bin Fang, Fumi Kinose, Eric B Haura, Alvaro N Monteiro, John M Koomen, Harshani R Lawrence, Uwe Rix. PARP16 is a novel target of talazoparib which contributes to synergy with adavosertib in SCLC [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 B021. doi:10.1158/1535-7163.TARG-19-B021

In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells

BackgroundAcute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be “undrugable” because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription.MethodsThis study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt’s lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents.ResultsGQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin.ConclusionThese results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

Abstract A142: Drug combination screen to identify novel synergy with Minnelide

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 C007: A high-throughput screen to identify effective and translationally-relevant drug combinations for breast, colorectal, and pancreatic cancers

Abstract Drug combinations offer a solution to two major issues faced by oncologists, drug resistance and tumour heterogeneity, but identifying the optimum combination of drugs for distinct molecular contexts remains challenging. This is particularly true for cancers where limited targeted therapies are available, for example pancreatic or KRAS-mutant colorectal cancers. We have established a high-throughput, systematic platform for screening large numbers of drug combinations in cell line panels. This includes 650 combinations in 48 colorectal cell lines, over 1,300 combinations in 52 breast cancer cell lines, covering all subtypes of the disease, and 650 combinations in 30 pancreatic cell lines. Combinations are screened in an anchor-library format, with an anchor drug used at two fixed concentrations, combined with the library drug at seven discontinuous concentrations. Drugs were chosen on a per-tissue basis, considering existing standards of care, known genomic features and core tissue pathways. We have prioritised drugs that are currently FDA-approved or are in late-stage clinical trials to ensure maximum clinical relevance of our data. Our screen presents response data for over 100,000 drug combination-cell line pairs, plus single agent drug sensitivities for over 4,800 single agent-cell line pairs. Combinations are classified as effective based on their resultant change in cell viability (ΔEMax) and change in drug sensitivity (ΔIC50). Drug combinations are ranked and prioritised based on potency, number of sensitive cell lines, and clinical relevance, i.e. use of FDA-approved drug(s). Validation of synergistic drug combinations, including using alternative inhibitors of promising target combinations, is ongoing. By screening combinations at this scale, we are able to perform statistically-powerful biomarker analysis, including integration with genomic, transcriptomic and proteomic data, and also clinical classifications such as PAM50 status. This study represents one of the largest drug combination screens performed to date, and focuses on two tissues for which targeted drug therapies are currently limited: pancreatic cancer and colorectal cancer. The third tissue studied, breast, represents a highly heterogenous cancer, meaning that selecting appropriate drug combinations is particularly challenging. We have ranked and prioritised effective drug combinations and identified biomarkers for the molecular contexts in which certain combinations are effective. We are currently validating combinations both in-house and with collaborators. Citation Format: Elizabeth A Coker, Patricia Jaaks, Daniel J Vis, Nanne Aben, Syd Barthorpe, Dieudonne van der Meer, Howard Lightfoot, Lodewyk Wessels, Mathew Garnett, GDSC Screening Team. A high-throughput screen to identify effective and translationally-relevant drug combinations for breast, colorectal, and pancreatic cancers [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 C007. doi:10.1158/1535-7163.TARG-19-C007

Prediction of drug combination effects with a minimal set of experiments.

High-throughput drug combination screening provides a systematic strategy to discover unexpected combinatorial synergies in pre-clinical cell models. However, phenotypic combinatorial screening with multi-dose matrix assays is experimentally expensive, especially when the aim is to identify selective combination synergies across a large panel of cell lines or patient samples. Here we implemented DECREASE, an efficient machine learning model that requires only a limited set of pairwise dose-response measurements for accurate prediction of drug combination synergy and antagonism. Using a compendium of 23,595 drug combination matrices tested in various cancer cell lines, and malaria and Ebola infection models, we demonstrate how cost-effective experimental designs with DECREASE capture almost the same degree of information for synergy and antagonism detection as the fully-measured dose-response matrices. Measuring only the diagonal of the matrix provides an accurate and practical option for combinatorial screening. The open-source web-implementation enables applications of DECREASE to both pre-clinical and translational studies.

Abstract C008: Tolerability and antitumor activity of paclitaxel is improved by the addition of nilotinib in patients with refractory solid tumors

Abstract Background: We initiated the systematic NCI-ALMANAC in vitro combination drug screen of FDA-approved anticancer drugs in the NCI-60 tumor cell line panel to explore novel and effective drug combinations (Holbeck et al., Cancer Res 2017). In this screen and in preclinical xenograft models, the BCR-ABL kinase inhibitor nilotinib demonstrated greater-than-additive activity in combination with the anti-tubulin agent paclitaxel. Additionally, recent preclinical findings suggest a reduced incidence of peripheral neuropathy for this combination relative to paclitaxel monotherapy (Leblanc et al., J Clin Invest 2018). We are performing a phase 1 study to establish the safety, tolerability, and maximum tolerated dose (MTD) of this combination in patients (pts) with advanced solid tumors, and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to better understand the mechanism of synergy. Materials and Methods: Nilotinib was given orally twice-daily (BID), while paclitaxel was administered intravenously on days 1, 8, and 15 of each 28-day cycle. A 1- or 2-day paclitaxel-only run-in during the first cycle enabled comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Dose-limiting toxicities (DLTs) were assessed during cycle 1, and response was evaluated by CT per RECIST 1.1. Results: Thirty pts have been enrolled to date. The MTD was 300 mg nilotinib BID and 80 mg/m2 paclitaxel given on days 1, 8, and 15; DLTs were grade 4 rash and grade 3 myelosuppression. Only 2 pts (7%) have experienced grade 2 peripheral neuropathy, no grade ≥3 neuropathy events occurred. Of the 24 pts assessable for response to date, there are 4 confirmed partial responses (PRs; 17%, 1 pt with endometrial cancer, 1pt with anal cancer and 2 pts with granulosa cell of the ovary) and 12 pts with a best response of stable disease (SD; 50%), including 6 pts with SD for ≥ 8 cycles. Mean time on study to date is 8.7 months (range: 2 – 41 months). Three of 4 responding pts had undergone prior paclitaxel-based therapy and experienced a best response of only SD (1 pt) or progressive disease (2 pts) to carboplatin/paclitaxel or paclitaxel monotherapy, respectively. PK data demonstrate dose-dependent plasma paclitaxel concentrations (for 60 vs. 80 mg/m2 paclitaxel), and no substantial effects of nilotinib on paclitaxel plasma concentrations. Conclusions: The combination of nilotinib and paclitaxel is well-tolerated. Consistent with recent preclinical findings, no grade ≥ 3 peripheral neuropathy has been observed, as compared to rates of 8-30% in prior studies of 80-100 mg/m2 weekly paclitaxel monotherapy in pts with metastatic breast cancer (Rivera and Cianfrocca, Cancer Chemother Pharmacol 2015). PK data indicate dose-dependent plasma paclitaxel concentrations, with no substantial nilotinib-induced changes in plasma paclitaxel levels. Preliminary response data are promising, especially considering the PRs observed in pts who had previously been unresponsive to paclitaxel therapy. Accrual to the expansion cohort and PD analyses of circulating tumor cells and tumor biopsy specimens are ongoing. Citation Format: Geraldine O'Sullivan Coyne, Shivaani Kummar, Jennifer Zlott, Lamin Juwara, Naoko Takebe, Murielle Hogu, Larry Anderson, Jerry Collins, Richard Piekarz, Howard Streicher, Elad Sharon, Arjun Mittra, Sabrina Khan, Brandon Miller, Tony Navas, Apurva Srivastava, Ralph Parchment, Laurence Rubinstein, James H Doroshow, Alice P Chen. Tolerability and antitumor activity of paclitaxel is improved by the addition of nilotinib in patients with refractory solid tumors [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 C008. doi:10.1158/1535-7163.TARG-19-C008

Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

Combination of Venetoclax and Ibrutinib Increases bcl2-Dependent Apoptotic Priming, Reduces ITK-Phosphorylation and Is Clinically Promising in Relapsed/Refractory T-Prolymphocytic Leukemia

Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare and aggressive T-lymphoid malignancy with poor response to current treatment strategies. We recently demonstrated single agent activity of venetoclax in relapsed/refractory (r/r) T-PLL, however resistance developed on mono-therapy. We here set out to identify partners for an effective combinatorial treatment concept. Methods To overcome bcl-2 inhibitor resistance we utilized primary T-PLL patient samples and applied a combinatorial next-generation functional drug screen for venetoclax and 25 additional therapeutic agents (Fig.1a). Molecular mechanisms of drug combinations were evaluated by BH3-family member profiling and mass spectrometry. Protein expression was assessed by Western Blot and viability by AnnexinV/Hoechst staining. The best scoring combination was evaluated in two late stage r/r T-PLL patients. Results Pairwise combinations screen of venetoclax with candidate small molecule inhibitors and chemotherapeutic drugs on primary T-PLL cells revealed synergistic action of venetoclax with ibrutinib, idelalisib, and 5-azacytidine, and to lower extents Olaparib, Temsirolimus, Ruxolitinib and Belinostat whereas cisplatin antagonized the effect of venetoclax across all patient samples tested (Fig 1b). The combination of venetoclax and ibrutinib resulted in substantial reduction of viability in primary T-PLL cells (Fig 1c). BH3-profiling on primary T-PLL samples with and without ibrutinib treatment demonstrated enhanced overall priming with predominant increase of bcl-2 dependency upon ibrutinib treatment (Fig 1d). Addition of ibrutinib to venetoclax led to decreased phosphorylation of ITK in vivo (Fig 1e). Two patients suffering from r/r T-PLL after failing at least two treatment lines including alemtuzumab were treated with the combination of venetoclax and ibrutinib resulting in significant clinical responses with substantial drops in leukocytosis and LDH as well as substantial clinical improvement (Fig 2a, b). The dynamic BH3 profiling in samples taken from these patients confirmed that the addition of ibrutinib is indeed increasing overall priming and Bcl-2 dependency (Fig 2c, d). Conclusion Our findings suggest efficacy of combinatorial treatment of venetoclax with ibrutinib in T-PLL. Mechanistically, ibrutinib dephosphorylated ITK in T-PLL cells and, furthermore, enhanced BCL2 dependency, both, in-vivo and in-vitro. Patients treated with the combination venetoclax and ibrutinib experienced profound clinical responses which needs further evaluation in an prospective clinical study on a larger cohort of r/r T-PLL patients. Disclosures Herbaux: Abbvie: Honoraria; Janssen: Honoraria; Takeda: Honoraria; BMS: Honoraria; Gilead: Honoraria. Mayerhoefer:Siemens: Research Funding, Speakers Bureau; BMS: Speakers Bureau. Jaeger:Novartis, Roche, Sandoz: Consultancy; AbbVie, Celgene, Gilead, Novartis, Roche, Takeda Millennium: Research Funding; Amgen, AbbVie, Celgene, Eisai, Gilead, Janssen, Novartis, Roche, Takeda Millennium, MSD, BMS, Sanofi: Honoraria; Celgene, Roche, Janssen, Gilead, Novartis, MSD, AbbVie, Sanofi: Membership on an entity's Board of Directors or advisory committees. Davids:AbbVie, Acerta Pharma, Adaptive, Biotechnologies, Astra-Zeneca, Genentech, Gilead Sciences, Janssen, Pharmacyclics, TG therapeutics: Membership on an entity's Board of Directors or advisory committees; AbbVie, Astra-Zeneca, Genentech, Janssen, MEI, Pharmacyclics, Syros Pharmaceuticals, Verastem: Consultancy; Acerta Pharma, Ascentage Pharma, Genentech, MEI pharma, Pharmacyclics, Surface Oncology, TG Therapeutics, Verastem: Research Funding; Research to Practice: Honoraria. Staber:Takeda-Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ibrutinib - BTK inhibitor Venetoclax - bcl2 inhibitor

Mapping MALT1 Signaling Connectivity Unveils Novel B-Cell Feedback Mechanisms Directing Assembly of Potent Anti-Lymphoma Regimens

MALT1 inhibition is a promising strategy against B-cell receptor (BCR)-dependent lymphomas including ABC DLBCL, CLL and MCL. MALT1 is downstream of the most frequently mutated genes in the BCR and Toll-like receptor (TLR) pathways. MALT1 inhibitors are active in Ibrutinib-resistant BTK and PLCγ2 mutant CLL or CARD11 mutant ABC DLBCL. Therefore, MALT1 inhibitors, which recently began first in man clinical testing, have the potential to cover a larger patient population than drugs against more upstream targets in the BCR pathway including BTK inhibitors. However, activity of inhibitors targeting signaling mediators can be limited by feedback mechanisms counteracting and/or bypassing the need for a specific pathway. MALT1 is central to NF-κB activation downstream of the BCR. Therefore, activation of alternative pathways leading to full or partial activation of the BCR program or other pro-survival pathways might enable cell survival and set off MALT1i resistance. In order to map the landscape of resistance/sensitivity to MALT1 inhibition in ABC DLBCL and assist design of combinatorial regimens, we carried out a loss-of-function screen in HBL-1 (MALT1i sensitive) with or without MI-2 (small molecule irreversible inhibitor of MALT1) to identify genes capable of modulating response to MALT1 inhibition. Our analyses showed that loss of BCR and PI3K activators enhanced sensitivity, while loss of negative regulators of these pathways promoted MALT1i resistance. These findings were validated by knockdown of individual genes with two independent hairpins against activators CD79B, CARD11, BTK or the negative regulator TNFAIP3. Next, we carried out a combinatorial drug screen anchored in MALT1 inhibition by MI-2 or C3 (irreversible substrate-mimetic MALT1 inhibitor) and focused on inhibitors against signaling hubs in the BCR/PI3K pathways in 4 MALT1i sensitive cell lines. This combinatorial screen confirmed that concurrent inhibition of MALT1 and other BCR and PI3K pathways' proteins is additive (0.9>CI (combination index) <1.1) or synergistic (CI<0.9). MALT1i combinations with PI3K and MTORC1 inhibitors were the most highly synergistic, mean CI<0.5 for 2 MALT1 inhibitors and 4 cell lines, and were further pursued. In depth analysis of proliferation and cell death by CFSE dilution and Annexin V staining revealed that both MALT1/PI3K-i and MALT1/MTORC1-i combinations significantly enhanced growth inhibition and apoptosis in TMD8 and HBL-1 compared to individual agents. Results with MI-2 and C3 were comparable. Short exposure to MI-2 or C3 increased MTORC1 activity as assessed by S6K-Thr389 and S6-Ser235/6 phosphorylation in TMD8 and HBL-1 indicating that MALT1 protease activity modulates MTORC1 activation. MTOR activation is tumorigenic and can mediate chemotherapy resistance. Increased p-S6 following MALT1 inhibition (FC=1.4-2) was blocked by Idelalisib (PI3Ki). However, only Rapamycin, an MTORC1 inhibitor, reduced p-S6 levels relative to vehicle, FC=-5 alone or in combination. In vivo, MALT1/PI3K-i (MI-2/Idelalisib) significantly delayed tumor progression compared to single treatments (p<0.01). In contrast, MALT1/MTORC1-i (MI-2/Rapamycin) promoted tumor regression and significantly improved survival of xenografted mice (median survival 37 days vs 18.5 or 29 days for MI-2 or Rapamycin respectively, p<0.001). Tumors from MI-2/Idelalisib treated mice showed 15-fold increase in p-S6 at 21 days. Short exposure to MI-2 in vivo inhibited MALT1 activity over its targets BCL10 and Roquin-1 and increased MTORC1 activity over p-S6K-Thr389 (FC=1.5-2) and p-S6-Ser235/6 (FC=2-3) in ABC DLBCL xenografts. Rapamycin, but not Idelalisib, effectively blocked p-S6 (p<0.001). MALT1/MTORC1-i regimens were also highly synergistic in 2 patient-derived xenografted (PDX) ABC DLBCL ex vivo. PDXs were cultured in gelatin/silicate nanoparticle hydrogel 3-D organoids and co-cultured with CD40L expressing cells. Compound pairs were assayed for synergy using 4x4 matrices and growth inhibition evaluated by flow cytometry. Synergy ZIP δ-score ranged 7-14 in 2 specimens for 2 MALT1i. Combined, these results suggest that: 1) MTORC1 activation constitutes a survival feedback mechanism activated after MALT1i treatment that might be leveraged by tumoral cells to evade MALT1 inhibition and, 2) that simultaneous targeting of MTORC1 could improve response and prevent resistance to MALT1 inhibitors. Disclosures Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

COMP-09. A CANCER DRUG ATLAS ENABLES PREDICTION OF PARALLEL DRUG VULNERABILITIES OF GLIOBLASTOMA

Abstract Personalized cancer treatments using synergistic combinations of drugs is attractive but proves to be highly challenging. The combinatorial nature of such problems results in an enormous parameter space that cannot be resolved by empirical research, i.e. testing all combinations for all molecularly defined tumors. In addition, effective drug synergy is hard to predict. Here we present an approach to map data of drug-response encyclopedias and represent these as a drug atlas. This atlas consists of a framework of chemotherapeutic responses that represents a drug vulnerability landscape of cancer. Based on data from the literature we found that many synergistic drug combinations show distinct inter therapy responses and drug sensitivities. We confirmed this by performing a drug combination screen against glioblastoma where we used 270 combination experiments. From the identified dual therapies we were able to predict and validate a triple drug synergy which was validated in vivo. This new and generalizable strategy opens the door to unforeseen personalized multidrug combination approaches.

DrugCombDB: a comprehensive database of drug combinations toward the discovery of combinatorial therapy.

Drug combinations have demonstrated high efficacy and low adverse side effects compared to single drug administration in cancer therapies and thus have drawn intensive attention from researchers and pharmaceutical enterprises. Due to the rapid development of high-throughput screening (HTS), the number of drug combination datasets available has increased tremendously in recent years. Therefore, there is an urgent need for a comprehensive database that is crucial to both experimental and computational screening of synergistic drug combinations. In this paper, we present DrugCombDB, a comprehensive database devoted to the curation of drug combinations from various data sources: (i) HTS assays of drug combinations; (ii) manual curations from the literature; and (iii) FDA Orange Book and external databases. Specifically, DrugCombDB includes 448 555 drug combinations derived from HTS assays, covering 2887 unique drugs and 124 human cancer cell lines. In particular, DrugCombDB has more than 6000 000 quantitative dose responses from which we computed multiple synergy scores to determine the overall synergistic or antagonistic effects of drug combinations. In addition to the combinations extracted from existing databases, we manually curated 457 drug combinations from thousands of PubMed publications. To benefit the further experimental validation and development of computational models, multiple datasets that are ready to train prediction models for classification and regression analysis were constructed and other significant related data were gathered. A website with a user-friendly graphical visualization has been developed for users to access the wealth of data and download prebuilt datasets. Our database is available at http://drugcombdb.denglab.org/.

A high-throughput drug combination screen of targeted small molecule inhibitors in cancer cell lines

While there is a high interest in drug combinations in cancer therapy, openly accessible datasets for drug combination responses are sparse. Here we present a dataset comprising 171 pairwise combinations of 19 individual drugs targeting signal transduction mechanisms across eight cancer cell lines, where the effect of each drug and drug combination is reported as cell viability assessed by metabolic activity. Drugs are chosen by their capacity to specifically interfere with well-known signal transduction mechanisms. Signalling processes targeted by the drugs include PI3K/AKT, NFkB, JAK/STAT, CTNNB1/TCF, and MAPK pathways. Drug combinations are classified as synergistic based on the Bliss independence synergy metrics. The data identifies combinations that synergistically reduce cancer cell viability and that can be of interest for further pre-clinical investigations.

Imatinib revives the therapeutic potential of metformin on ewing sarcoma by attenuating tumor hypoxic response and inhibiting convergent signaling pathways.

Ewing sarcoma (EwS) is an aggressive pediatric tumor treated with intensive cytotoxic chemotherapies. Overall survival for metastatic or relapsed disease is only 20-30%. Metformin has long been an attractive therapeutic option for EwS, but hypoxia limits its efficacy. Through a systematic integration of drug combination screening, bioinformatics analyses, functional and in vivo studies, and correlation with clinical outcome, we identified another known drug, imatinib that could augment the in vivo anti-tumor capacity of metformin by attenuating tumor hypoxic response. This drug combination regimen widely suppressed multiple dominant mechanisms in EwS genesis, growth, and metastasis, including key EWS-FLI1 downstream targets that converge into the PI3K/AKT/mTOR signaling pathway. In addition, the combination significantly enhanced inhibition on tumor cell proliferation by standard EwS chemotherapy drugs, including cyclophosphamide and ifosfamide. This suggests a potential clinical benefit of the metformin/imatinib combination by allowing the reduction in dose intensity of standard chemotherapy without compromising survival outcome and represents a potential faster track application for EwS patients.

Dendronized fluorosurfactant for highly stable water-in-fluorinated oil emulsions with minimal inter-droplet transfer of small molecules

Fluorosurfactant-stabilized microfluidic droplets are widely used as pico- to nanoliter volume reactors in chemistry and biology. However, current surfactants cannot completely prevent inter-droplet transfer of small organic molecules encapsulated or produced inside the droplets. In addition, the microdroplets typically coalesce at temperatures higher than 80 °C. Therefore, the use of droplet-based platforms for ultrahigh-throughput combination drug screening and polymerase chain reaction (PCR)-based rare mutation detection has been limited. Here, we provide insights into designing surfactants that form robust microdroplets with improved stability and resistance to inter-droplet transfer. We used a panel of dendritic oligo-glycerol-based surfactants to demonstrate that a high degree of inter- and intramolecular hydrogen bonding, as well as the dendritic architecture, contribute to high droplet stability in PCR thermal cycling and minimize inter-droplet transfer of the water-soluble fluorescent dye sodium fluorescein salt and the drug doxycycline.

Mask-free generation of multicellular 3D heterospheroids array for high-throughput combinatorial anti-cancer drug screening

Since cancer incidence and mortality are rapidly growing worldwide, it is imperative to develop the effective tumor models in vitro for anti-cancer drug screening. Cellular spheroid as an excellent simple 3D model can bridge the gap between cell culture and live tissue. Although numerous approaches for generating cellular spheroids emerges, the long cultivation time and the heterospheroids formation with multiple cell types are still challenging. In the present study, a mask-free method of multicellular heterospheroids arrays formation using digital micromirror device (DMD)-based hydrogel fabricating system and microfluidic system is proposed. Microwell arrays were designed and manufactured online based on shadowed light which shows high flexible. Besides, heterospheroids with multiple cells controlled in different ratios can be generated and cultured for a long time in microfluidic system. Furthermore, the drug screening shows that heterospheroids have high rate of drug resistance compared with homospheroids and combinatorial drugs works better than the single drugs. These studies provide a 3D heterospheroids generating process for high-throughput combinatorial anti-cancer drug screening with potential cancer therapeutic application.

Combinatorial drug screening of mammary cells with induced mesenchymal transformation to identify drug combinations for triple-negative breast cancer.

Mesenchymal stem-like (MSL) breast cancers are enriched for cells with tumor reconstituting and mesenchymal characteristics. These cancers are often triple-negative and have a poor prognosis. Few effective targeted treatment options exist for patients with these cancers, and even when targeted therapies exist, resistance often arises and tumors recur, due in part to drug-tolerant persisting tumor cells with self-renewal capability. Effective treatment strategies will combine agents that target the bulk-tumor and reconstituting cells. In order to identify such a combination therapy, we conducted an inhibitor screen using 40 targeted agents at three different doses in all pairwise combinations. Checkpoint Kinase 1 (CHK1) inhibitors were identified as potent inhibitors of MSL breast cancers. When combined with a pro-apoptotic agent/B Cell Lymphoma 2 (BCL2) inhibitor, the effectiveness of the combination regimen was super-additive compared to either treatment alone and was selective for MSL cancers. Treatment of MSL breast cancer cells results in DNA damage, cell-cycle defects characterized by a prolonged S-phase, increased apoptosis and decreased colony forming abilities compared to untreated cells. These data suggest that a combination of a CHK1 and BCL2 inhibitor could be an effective treatment for patients with MSL breast cancer. Several other effective drug combinations were also identified.

Multi-targeted kinase inhibition alleviates mTOR inhibitor resistance in triple-negative breast cancer

PurposeOwing to its genetic heterogeneity and acquired resistance, triple-negative breast cancer (TNBC) is not responsive to single-targeted therapy, causing disproportional cancer-related death worldwide. Combined targeted therapy strategies to block interactive oncogenic signaling networks are being explored for effective treatment of the refractory TNBC subtype.MethodsA broad kinase inhibitor screen was applied to profile the proliferative responses of TNBC cells, revealing resistance of TNBC cells to inhibition of the mammalian target of rapamycin (mTOR). A systematic drug combination screen was subsequently performed to identify that AEE788, an inhibitor targeting multiple receptor tyrosine kinases (RTKs) EGFR/HER2 and VEGFR, synergizes with selective mTOR inhibitor rapamycin as well as its analogs (rapalogs) temsirolimus and everolimus to inhibit TNBC cell proliferation.ResultsThe combination treatment with AEE788 and rapalog effectively inhibits phosphorylation of mTOR and 4EBP1, relieves mTOR inhibition-mediated upregulation of cyclin D1, and maintains suppression of AKT and ERK signaling, thereby sensitizing TNBC cells to the rapalogs. siRNA validation of cheminformatics-based predicted AEE788 targets has further revealed the mTOR interactive RPS6K members (RPS6KA3, RPS6KA6, RPS6KB1, and RPS6KL1) as synthetic lethal targets for rapalog combination treatment.ConclusionsmTOR signaling is highly activated in TNBC tumors. As single rapalog treatment is insufficient to block mTOR signaling in rapalog-resistant TNBC cells, our results thus provide a potential multi-kinase inhibitor combinatorial strategy to overcome mTOR-targeted therapy resistance in TNBC cells.

A nanodroplet cell processing platform facilitating drug synergy evaluations for anti-cancer treatments

Therapeutic drug synergism intervened in cancer treatments has been demonstrated to be more effective than using a single effector. However, it remains inherently challenging, with a limited cell count from tumor samples, to achieve potent personalized drug cocktails. To address the issue above, we herein present a nanodroplet cell processing platform. The platform incorporates an automatic nanodroplet dispenser with cell array ParaStamp chips, which were fabricated by a new wax stamping approach derived from laser direct writing. Such approach enables not only the on-demand de-wetting with hydrophobic wax films on substrates but also the mask-less fabrication of non-planar microstructures (i.e. no photolithography process). The ParaStamp chip was pre-occupied with anti-cancer drugs and their associate mixtures, enabling for the spatially addressable screening of optimal drug combinations simultaneously. Each droplet with a critical volume of 200 nl containing with 100 cells was utilized. Results revealed that the optimal combination reduces approximate 28-folds of conducted doses compared with single drugs. Tumor inhibition with the optimally selected drug combination was further confirmed by using PC-3 tumor-bearing mouse models. Together, the nanodroplet cell processing platform could therefore offer new opportunities to power the personalized cancer medicine at early-stage drug screening and discovery.

Abstract 1927: High-throughput cell line panel drug screening in organoids and 3D systems

Abstract Cell panel screening of cancer lines grown in standard 2D conditions is a well-established component of the drug discovery toolbox. A sufficiently large cell line panel, reflecting the molecular heterogeneity within a cancer type/sub-type, is required to detect correlations with robust statistical power that aid biomarker identification and patient stratification. However, the heterogenous exposure of tumor cells to nutrients, oxygen and other physical or chemical stresses found in three-dimensional solid tumors also has a role to play in patient response and this is not well mimicked in 2D monolayer cell cultures. To meet the need for high-throughput cell-panel screening in more complex culture systems, we have developed 3D cell-based screens to more closely model the complex physiological environment found in tumors. We have characterised our OncoSignature panel of 300 cell lines for growth as spheroids to produce a 200 strong panel of spheroid forming lines. Using our high-throughput platform to compare drug activity in 2D and 3D cultures, we have identified clear differentials for some compounds in single agent and drug combination screens. For example, MEK and EGFR inhibitors have significantly higher activity in 3D cultures than 2D cultures, with a greater than 10-fold shift in EC50 values. These results were validated in 3D soft agar assays, with similar compound potencies being observed in the HTS spheroid assay and the lower throughput soft-agar assay. We have further extended our 3D screening into organoid cultures. Organoid cultures, with increased complexity in terms of structure and cell heterogeneity compared to spheroids, have historically been challenging to use in high-throughput screening. However, our initial results show encouragingly robust data, with compound activity that is congruent with the genetic characteristics of the organoids. Finally, we used our integrated screening platform to evaluate the power of CRISPR approaches to examine genetic dependencies in KRAS mutant cells in 2D, 3D spheroid and organoid cell cultures. Pooled, next-generation sequencing-linked CRISPRko allows an alternative and complementary genomics approach to arrayed cell panel screening, providing a powerful and robust target identification and validation strategy. Overall, Horizon’s 3D screening platforms will enable large scale interrogation of both drug and gene interactions in more complex cell culture systems. Citation Format: Tim M. Scales, Anett Rada-Kovacs, Helen N. Pemberton, Ceri M. Wiggins, Nicola J. McCarthy, Annette S. Little, David A. Sorrell. High-throughput cell line panel drug screening in organoids and 3D systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1927.

Confirmation of Selected Synergistic Cancer Drug Combinations Identified in an HTS Campaign and Exploration of Drug Efflux Transporter Contributions to the Mode of Synergy

Systematic unbiased high-throughput screening (HTS) of drug combinations (DCs) in well-characterized tumor cell lines is a data-driven strategy to identify novel DCs with potential to be developed into effective therapies. Four DCs from a DC HTS campaign were selected for confirmation; only one appears in clinicaltrials.gov and limited preclinical in vitro data indicates that the drug pairs interact synergistically. Nineteen DC-tumor cell line sets were confirmed to interact synergistically in three pharmacological interaction models. We developed an imaging assay to quantify accumulation of the ABCG2 efflux transporter substrate Hoechst. Gefitinib and raloxifene enhanced Hoechst accumulation in ABCG2 (BCRP)-expressing cells, consistent with inhibition of ABCG2 efflux. Both drugs also inhibit ABCB1 efflux. Mitoxantrone, daunorubicin, and vinorelbine are substrates of one or more of the ABCG2, ABCB1, or ABCC1 efflux transporters expressed to varying extents in the selected cell lines. Interactions between ABC drug efflux transporter inhibitors and substrates may have contributed to the observed synergy; however, other mechanisms may be involved. Novel synergistic DCs identified by HTS were confirmed in vitro, and plausible mechanisms of action studied. Similar approaches may justify the testing of novel HTS-derived DCs in mouse xenograft human cancer models and support the clinical evaluation of effective in vivo DCs in patients.