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Fig. S1. Establishment of SB-3123 murine melanoma model. Fig. S2. Schematic of primer design for BRAF c.1799T > A (V600E) mutation analysis in SB-3123p murine melanoma cells. Fig. S3. SB-3123p melanoma is highly sensitive to vemurafenib. Fig. S4. BRAFV600E specific effect of vemurafenib upon SB-3123p established melanomas. Fig. S5. Establishment of SB-3123 murine melanoma cells expressing mouse gp100. Fig. S6. Concurrent vemurafenib therapy is required for sustained tumor growth control. Fig. S7. SB-3123 proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines. Fig. S8. Isobologram analysis from our 8x8 screen for the synergistic (black circle) drug combination of vemurafenib + IFN-γ/TNF-α. Fig. S9. IFN-γ and TNF-α combined with vemurafenib induce growth arrest of SB-3123 melanoma cells. Fig. S10. Melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S11. Human melanoma proliferative capacity is reduced when cultured under combinatorial conditions of vemurafenib and IFN-γ/TNF-α cytokines in a BRAFV600E dependent manner. Fig. S12. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature enriched in genes induced by IFN-γ/TNF-α treatment of SB3123. Supplementary Table 1 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 1. Supplementary Table 2 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 2. Supplementary Table 3 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 3. Supplementary Table 4 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 4. Supplementary Table 5 - All Genes, One Way Anova p FDR < 0.05 - Gene Cluster 5. Table S6. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the vemurafenib only dependent gene expression signature in SB3123. Table S7. Gene set enrichment analysis of vemurafenib + IFN-γ/TNF-α dependent gene expression signature normalized to the IFN- γ/TNF-α only dependent gene expression signature in SB3123. Table S8. Gene set enrichment analysis of vemurafenib dependent gene expression signature normalized to control treated SB3123. Supplementary Table 9 - IFNy/TNFB induced genes vs Control Treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 10 - Vemurafenib induced genes vs control treatment, fold change > 2.0, p-value < 0.01. Supplementary Table 11 - Vem + IFNg/TNFa vs IFNg/TNFa differentially expressed genes, FC > 1.5.

Supplementary Materials and Methods

PDF file - 65KB, Peaks observed for ESI+ mass spectra of Pt complexes dissolved in DMSO, associated with Pt-DMSO adducts.

PDF file - 1000KB, Scheme demonstrating the aquation of cisplatin in water, and prevention when stabilized in high chloride solution.

XLS file - 106KB, Use of solvent for assessing cytotoxicity of platinum complexes in the journal Molecular Cancer Therapeutics (S1). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Public Library of Science (PLoS)(S2). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Journal of Pharmacology and Experimental Therapeutics (JPET) (S3). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Cancer Research (S4). Use of solvent for assessing cytotoxicity of platinum complexes in the journal Molecular Pharmacology (S5). Use of solvent for assessing cytotoxicity of platinum complexes (S6).

Inhibitor of apoptosis (IAP) proteins are frequently upregulated in ovarian cancer, resulting in the evasion of apoptosis and enhanced cellular survival. Birinapant, a synthetic second mitochondrial activator of caspases (SMAC) mimetic, suppresses the functions of IAP proteins in order to enhance apoptotic pathways and facilitate tumor death. Despite on-target activity, however, pre-clinical trials of single-agent birinapant have exhibited minimal activity in the recurrent ovarian cancer setting. To augment the therapeutic potential of birinapant, we utilized a high-throughput screening matrix to identify synergistic drug combinations. Of those combinations identified, birinapant plus docetaxel was selected for further evaluation, given its remarkable synergy both in vitro and in vivo. We showed that this synergy results from multiple convergent pathways to include increased caspase activation, docetaxel-mediated TNF-&alpha, upregulation, alternative NF-kB signaling, and birinapant-induced microtubule stabilization. These findings provide a rationale for the integration of birinapant and docetaxel in a phase 2 clinical trial for recurrent ovarian cancer where treatment options are often limited and minimally effective.

Disease recurrence is the major cause of morbidity and mortality of ovarian cancer (OC). In terms of maintenance therapies after platinum-based chemotherapy, PARP inhibitors significantly improve the overall survival of patients with BRCA mutations but is of little benefit to patients without homologous recombination deficiency (HRD). The stem-like tumor-initiating cell (TIC) population within OC tumors are thought to contribute to disease recurrence and chemoresistance. Therefore, there is a need to identify drugs that target TICs to prevent relapse in OC without HRD. RNA sequencing analysis of OC cells grown in TIC conditions revealed a strong enrichment of genes involved in drug metabolism, oxidative phosphorylation and reactive oxygen species (ROS) pathways. Concurrently, a high-throughput drug screen identified drugs that showed efficacy against OC cells grown as TICs compared to adherent cells. Four drugs were chosen that affected drug metabolism and ROS response: disulfiram, bardoxolone methyl, elesclomol and salinomycin. The drugs were tested in vitro for effects on viability, sphere formation and markers of stemness CD133 and ALDH in TICs compared to adherent cells. The compounds promoted ROS accumulation and oxidative stress and disulfiram, elesclomol and salinomycin increased cell death following carboplatin treatment compared to carboplatin alone. Disulfiram and salinomycin were effective in a post-surgery, post-chemotherapy OC relapse model in vivo, demonstrating that enhancing oxidative stress in TICs can prevent OC recurrence.

// Marianne K. Kim 1 , Dong J. Min 2 , George Wright 3 , Ian Goldlust 4 , Christina M. Annunziata 1 1 Women’s Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 2 Transgenic Oncogenic and Genomics Section, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 3 Biometrics Research Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892 4 NIH Chemical Genomics Center, Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Bethesda, MD 20892 Correspondence to: Christina M. Annunziata, e-mail: annunzic@mail.nih.gov Keywords: shRNAs, therapeutic targets, IKKe, CHEK1, p21, ovarian cancer Received: October 02, 2014 Accepted: October 27, 2014 Published: November 15, 2014 ABSTRACT Ovarian cancer (OC) is extremely heterogeneous, implying that therapeutic strategies should be specifically designed based on molecular characteristics of an individual’s tumor. Previously, we showed that IKKe promotes invasion and metastasis in a subset of OCs. Here, we identified CHEK1 as an IKKe-dependent lethal gene from shRNA kinome library screen. In subsequent pharmacological intervention studies, the co-inhibition of IKKe and CHEK1 was more effective in killing OC cells than single treatment. At the molecular level, co-inhibition dramatically decreased pro-survival proteins, but increased proteins involved in DNA damage and apoptosis. IKKe-knockdown increased p21 levels, while overexpression of wild-type IKKe, but not a kinase dead IKKe mutant decreased p21 levels. We further demonstrated that the depletion of p21 rendered OC cells more resistant to cell death induced by co-inhibition of IKKe and CHEK1. In conclusion, we revealed a novel interplay between IKKe, CHEK1 and p21 signaling in survival of OC. Our study provides a rationale for the clinical development of specific IKKe inhibitor and for usage of IKKe as an exploratory marker for resistance to CHEK1 inhibitors in the clinic. The interplay provides one potential explanation as to why very few clinical responses were achieved in patients treated with single-agent CHEK1 inhibitors.

The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence are extensively studied in the laboratory setting. In this study, we examined the literature and found a significant number of studies (11%–34%) in prominent cancer journals utilizing cisplatin dissolved in DMSO. However, dissolving cisplatin in DMSO for laboratory-based studies results in ligand displacement and changes to the structure of the complex. We examined the effect of DMSO on platinum complexes, including cisplatin, carboplatin, and oxaliplatin, finding that DMSO reacted with the complexes, inhibited their cytotoxicity and their ability to initiate cell death. These results render a substantial portion of the literature on cisplatin uninterpretable. Raising awareness of this significant issue in the cancer biology community is critical, and we make recommendations on appropriate solvation of platinum drugs for research. Cancer Res; 74(14); 3913–22. ©2014 AACR.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

A combination of oligonucleotide and single-nucleotide polymorphism probes on the same array platform can detect copy-number abnormalities and copy-neutral aberrations such as uniparental disomy and long stretches of homozygosity. The single-nucleotide polymorphism probe density in commercially available platforms varies widely, which may affect the detection of copy-neutral abnormalities. We evaluated the ability of array platforms with low (Oxford Gene Technology CytoSure ISCA uniparental disomy), mid-range (Agilent custom array), and high (Affymetrix CytoScan HD) single-nucleotide polymorphism probe density to detect copy-number variation, mosaicism, uniparental isodisomy, and absence of heterozygosity in 50 clinical samples. All platforms reliably detected copy-number variation, mosaicism, and uniparental isodisomy; however, absence-of-heterozygosity detection varied significantly. The low-density array called absence-of-heterozygosity regions not confirmed by the other platforms and also overestimated the length of true absence-of-heterozygosity regions. Furthermore, the low- and mid-density platforms failed to detect some small absence-of-heterozygosity regions that were identified by the high-density platform. Variation in single-nucleotide polymorphism density can lead to major discrepancies in the detection of and confidence in copy-neutral abnormalities. Although suitable for uniparental disomy detection, copy-number plus single-nucleotide polymorphism arrays with 30,000 or fewer unique single-nucleotide polymorphism probes miscall absence-of-heterozygosity regions due to identity by descent. Genet Med 15 9, 706–712.

Quality control (QC) metrics are critical in high throughput screening (HTS) platforms to ensure reliability and confidence in assay data and downstream analyses. Most reported HTS QC metrics are designed for plate level or single well level analysis. With the advent of high throughput combination screening there is a need for QC metrics that quantify the quality of combination response matrices. We introduce a predictive, interpretable, matrix-level QC metric, mQC, based on a mix of data-derived and heuristic features. mQC accurately reproduces the expert assessment of combination response quality and correctly identifies unreliable response matrices that can lead to erroneous or misleading characterization of synergy. When combined with the plate-level QC metric, Z’, mQC provides a more appropriate determination of the quality of a drug combination screen. Retrospective analysis on a number of completed combination screens further shows that mQC is able to identify problematic screens whereas plate-level QC was not able to. In conclusion, our data indicates that mQC is a reliable QC filter that can be used to identify problematic drug combinations matrices and prevent further analysis on erroneously active combinations as well as for troubleshooting failed screens. The R source code of mQC is available at http://matrix.ncats.nih.gov/mQC.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

The discovery of chemotherapeutic agents for the treatment of cancer commonly uses cell proliferation assays in which cells grow as two-dimensional (2D) monolayers. Compounds identified using 2D monolayer assays often fail to advance during clinical development, most likely because these assays do not reproduce the cellular complexity of tumors and their microenvironment in vivo. The use of three-dimensional (3D) cellular systems have been explored as enabling more predictive in vitro tumor models for drug discovery. To date, small-scale screens have demonstrated that pharmacological responses tend to differ between 2D and 3D cancer cell growth models. However, the limited scope of screens using 3D models has not provided a clear delineation of the cellular pathways and processes that differentially regulate cell survival and death in the different in vitro tumor models. Here we sought to further understand the differences in pharmacological responses between cancer tumor cells grown in different conditions by profiling a large collection of 1912 chemotherapeutic agents. We compared pharmacological responses obtained from cells cultured in traditional 2D monolayer conditions with those responses obtained from cells forming spheres versus cells already in 3D spheres. The target annotation of the compound library screened enabled the identification of those key cellular pathways and processes that when modulated by drugs induced cell death in all growth conditions or selectively in the different cell growth models. In addition, we also show that many of the compounds targeting these key cellular functions can be combined to produce synergistic cytotoxic effects, which in many cases differ in the magnitude of their synergism depending on the cellular model and cell type. The results from this work provide a high-throughput screening framework to profile the responses of drugs both as single agents and in pairwise combinations in 3D sphere models of cancer cells.

Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer, but responses can be either short lived or incompletely effective. Oncogene inhibition can augment the efficacy of immune-based therapy, but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAFV600E-mutant murine melanoma model (SB-3123), we explored potential mechanisms of synergy between the selective BRAFV600E inhibitor vemurafenib and adoptive cell transfer (ACT)–based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8+ T cells, as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells in vitro. This combinatorial effect was recapitulated in human melanoma–derived cell lines and was restricted to cancers bearing a BRAFV600E mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the antiproliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest has major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer. Cancer Immunol Res; 3(1); 37–47. ©2014 AACR. See related commentary by Riddell, p. 23

Obesity is a highly heritable condition and a risk factor for other diseases, including type 2 diabetes, cardiovascular disease, hypertension, and cancer. Recently, genomic copy number variation (CNV) has been implicated in cases of early onset obesity that may be comorbid with intellectual disability. Here, we describe a recurrent CNV that causes a syndrome associated with intellectual disability, seizures, macrocephaly, and obesity. This unbalanced chromosome translocation leads to duplication of over 100 genes on chromosome 12, including the obesity candidate gene G protein β3 (GNB3). We generated a transgenic mouse model that carries an extra copy of GNB3, weighs significantly more than its wild-type littermates, and has excess intraabdominal fat accumulation. GNB3 is highly expressed in the brain, consistent with G-protein signaling involved in satiety and/or metabolism. These functional data connect GNB3 duplication and overexpression to elevated body mass index and provide evidence for a genetic syndrome caused by a recurrent CNV.

Sphere forming cells persist in the ascitic fluid of patients with high-grade serous ovarian cancer after first-line therapy and likely contribute to relapse and metastasis. These residual tumor spheres, which are enriched for cancer cells by up to 95% based on detectable TP53 mutations, are slow growing, resistant to platinum-based chemotherapy, and remain a large obstacle towards durable remission. Screening established, rapidly dividing monolayer cell lines in proliferation assays has failed to produce chemotherapeutics capable of eradicating this slow growing population. To identify a consolidation therapy that targets these residual tumor cells we screened nearly 2000 mechanistically annotated, approved and investigational drugs in three cell lines (PEO1, PANC1, A375M) cultured in spheroid and conventional monolayer. To elucidate targetable genes and pathways responsible for spheroid maintenance we performed a whole-genome RNAi screen against PEO1 in both culture conditions. Our pharmacological and genetic profiling provided mechanistic insight into the baseline changes that occur when cells are grown in three dimensional, anchorage independent conditions and identified susceptibilities specific to spheroid populations. Consistent with residual disease, cultured spheres were resistant to many common chemotherapeutics, most notably proteasome inhibitors, but were highly sensitive to the pro-oxidant elesclomol. We verified elesclomol's activity in ex vivo cancer spheroids extracted from the ascitic fluid of patients with advanced high-grade serous ovarian cancer using a simple, culture-free technique. Expression profiling of cultured and ex vivo spheres revealed broad downregulation of genes involved in cell cycle signaling (AURKA, AURKB, PLK1, CDK1, CCNA2, CCNB1, CCNB2, GMNN, CHEK1). Treatment with elesclomol induced expression of chaperone proteins (HSP6, HSP7, HSPA1A, HSPA1B, DNAJA4, DNAJB1), metallothionein proteins (MT1F, MT1M, MT1P2, MT1X, MT2A), and genes involved with the oxidative stress response (HMOX1, ABCB1, SLC7A11) consistent with increased reactive oxygen species caused by high intracellular Cu2+. To pursue this drug as a consolidation therapy in vivo, we evaluated elesclomol's toxicity, pharmacokinetic properties, and efficacy in a murine model for recurrent high-grade serous ovarian cancer. By targeting residual tumor cells with elesclomol after successful treatment with platinum-based therapeutics we hope to prevent recurrence. Citation Format: Ian S. Goldlust, Kelli Wilson, Ludmila Szabova, Xiaohu Zhang, Lesley Mathews-Griner, Maria Vias, Anna Piskorz, Rory Stark, Lee Mendil, Monica Kasbekar, John Braisted, Rajarshi Guha, Crystal McKnight, Paul Shinn, Donna Michelle-Smith, Zoe Weaver Ohler, Mindy Davis, Udo Rudloff, Sam Michael, Madhu Lal-Nag, Scott Martin, Christina Annunziata, Marc Ferrer, James D. Brenton, Craig Thomas. No cell left behind: Residual ovarian spheroids drive recurrence and are sensitive to the pro-oxidant elesclomol. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 278.

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hESCs, we find that iPSCs tend to form large-scale (100 kb-1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (~80%) with aberrant iPSC-ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC-hESC differences.

Background: Inhibitors of apoptosis (IAPs) are attractive anti-cancer therapeutic targets as a mechanism to sensitize cancer cells to apoptosis. IAPs are overexpressed in ovarian cancer and portend a poor prognosis. SMAC- mimetics are IAP antagonists that have shown in vitro and in vivo antitumor activity in ovarian cancer, but preclinical and clinical studies suggest SMAC mimetics may be more effective in combination therapies. Methods: Birinapant (B) was initially screened in combination with each of 1,912 compounds using an automated acoustic drug dispensation system in 6×6 matrices. Seventy compounds were chosen from the initial screen, based on synergistic characteristics, and verified in 10×10 matrices. The most clinically rational drug combinations with B were further evaluated in 8 high-grade serous ovarian cancer cell lines (CAOV3, CAOV4, OV-90, OVCAR4, OVCAR5, OVCAR8, PEO1, PEO4), focusing on 3 synergistic (docetaxel, panobinostat, volasertib) and 2 antagonistic combinations (cisplatin, olaparib) for exploration of mechanism. Combination index was determined using Compusyn software. Results: Specific classes of drugs were identified to have synergism with B, including taxanes, HDAC inhibitors, and PLK1 inhibitors, while platinum agents and PARP inhibitors were found to be antagonistic. Four of the 8 cell lines were sensitive to B. Caspase-Glo 3/7 assay was performed in B-sensitive cell lines and confirmed increased caspase-3 activity in the combination treatments. Ongoing studies include genomic analysis, mechanistic interrogation, and in vivo xenograft studies. Conclusions: B is a promising novel therapy for use in combination treatment for ovarian, fallopian tube, or primary peritoneal cancers. Our findings provide insight on optimal therapies to combine with B, and constitute a foundation for further evaluation of predictive and pharmacodynamic biomarkers to select the patient population most likely to respond to this targeted therapeutic approach. Citation Format: Kristen Bunch, Ian Goldlust, Craig Thomas, Lidia Hernandez, Rajarshi Guha, Christina Annunziata. High-throughput drug matrix screen with SMAC (second mitochondria-derived activator of caspases)-mimetic birinapant in high-grade serous ovarian cancer cell lines identifies synergism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-254. doi:10.1158/1538-7445.AM2015-LB-254

Drug combinations are used commonly to treat patients with cancer. Combination studies are conducted in vitro, to support advancement to in vivo and/or clinical research. In order to interpret in vitro data, mathematical models and numerical methods are used to quantify degrees of drug synergism. Several traditional approaches exist but they have been questioned with regard to how resulting synergy or antagonism levels should be interpreted. Our aim was to develop a robust method to analyse in vitro drug combination data, avoiding the issues related to traditional models and methods. To facilitate its implementation, software to enable rigorous and systematic assessment of combinations in vitro was also developed. We have derived a new mathematical model to identify synergy distributions based on drug combination dose response surfaces. Our new model and the classical Bliss, Loewe and HSA models were incorporated into the newly developed Combenefit software. Combenefit enables systematic quantification of high-throughput screening data, incorporating statistical assessment, metrics to describe synergy distributions and advanced graphical visualisation. Using multiple simulations of combination data with expected outcomes, our approach showed that it always provided appropriate evaluation while classical models failed to do so. We showed how our new model is in principle better aligned with in vivo requirements of identifying combinations which provide improvements compared to single agents. We then proceeded to analyse a large high-throughput combination screen. We first demonstrated that dataset assessment is strongly model-sensitive. We then demonstrated that our new model identified the best combinations in terms of dose response, thus confirming our simulated cases. Therefore, we have developed a new mathematical model that solves fundamental issues of model relevance that are encountered with classical models in the context of drug combinations. The newly developed Combenefit software enables easily performing rigorous and systematic quantification of in vitro combinations based on this novel approach. (Combenefit is freely available on our institutional website http://www.cruk.cam.ac.uk/combenefit). The resulting procedure is robust and better aligned with our perception of in vivo requirements. Citation Format: Giovanni Y. Di Veroli, Duncan I. Jodrell, Frances M. Richards, Ian Goldlust. A new mathematical model and software, to enable improved quantification and interpretation of combinatorial drug effects. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2540. doi:10.1158/1538-7445.AM2015-2540