Single-agent Drug Research Articles

Abstract LB_C06: Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic treatment options and a high rate of metastatic recurrence. TNBC is defined by lack of estrogen and progesterone receptor expression with non-amplified HER2, and accounts for 10-15% of all breast cancer cases. While pembrolizumab, sacituzumab govitecan and PARP inhibitors are beneficial for a subset of TNBC patients, chemotherapy including doxorubicin continues to be the primary treatment. Bocodepsin is a novel, orally bioavailable clinical stage Class 1-targeting HDAC inhibitor with activity in preclinical models of TNBC. The purpose of this study was to investigate OKI-005 (in vitro) and bocodepsin (in vivo) in combination with doxorubicin (DOX) to overcome resistance. Procedures: TNBC cell lines were exposed to a no drug, DOX, OKI-005 or combination for 72 hours and cell viability was assessed via CellTiter-Glo. Synergy scores were calculated from three independent replicates using Synergy Finder+. Apoptosis was assessed following 48 hours of drug exposure by flow cytometry. Immunoblots were performed following 24 hours of drug treatment to evaluate mechanism of action. In vitro senescence-associated beta-galactosidase was performed after 6 days drug treatment. For in vivo studies, 5 million MDA-MB-231 cells were injected subcutaneously in athymic nude mice. Mice were treated with either no drug, 1.5 mg/kg DOX IP QW, 80 mg/kg bocodepsin PO QD, or the combination. Results: Bliss scores calculated from Synergy Finder+ using the CellTiter-Glo data showed synergy for the combination of OKI-005 and DOX in all TNBC cell lines. The combination resulted in statistically significant increased apoptosis measured by Annexin V compared to single agent drug treatment in CAL-51, MDA-MB-231 and CAL-120 cell lines. Immunoblot analysis demonstrated an increase in the pro-apoptotic protein BIM and decrease in anti-apoptotic protein BCL-XL with the combination. The cyclin dependent kinase inhibitor p21 was increased by OKI-005 independently of p53 and doxorubicin for the cell lines MDA-MB-231, Hs 578T, and CAL-120. The combination resulted in increased cleaved caspase-3 for all cell lines evaluated. Senescence-associated beta-galactosidase showed an increase in senescent cells for both DOX and OKI-005, which was eradicated in combination. DOX and bocodepsin were further tested in vivo, where significant tumor growth inhibition was observed in combination compared to DOX (p=0.0016) and bocodepsin (p=0.0068). Conclusion: The addition of OKI-005 or bocodepsin to doxorubicin resulted in improved anti-proliferative and anti-cancer activity with evidence of increased apoptosis and decreased senescence. As a result, bocodepsin is a promising combination partner for doxorubicin to overcome doxorubicin resistance and promote apoptosis in TNBC. Bocodepsin has a favorable toxicity profile compared to other HDAC inhibitors and this work supports the potential clinical investigation of doxorubicin and bocodepsin in patients with metastatic TNBC. Citation Format: Stephen G Smoots, Anna R Schreiber, Marilyn M Jackson, Evan D Dus, Stacey M Bagby, Adrian T A. Dominguez, Cameron A Binns, Jennifer R Diamond, Todd M Pitts. Utilizing a novel HDAC inhibitor bocodepsin (OKI-179) to overcome doxorubicin resistance in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C06.

Abstract A041: Development of novel in-vitro mixed-cell models to capture genomic heterogeneity in prostate tumors

Abstract Prostate cancer (PC), especially castration resistant PC (CRPC), is known to exhibit a high degree of intra- and inter-tumoral (inter-individual) heterogeneity, which have been linked to treatment resistance and disease recurrence. Yet, models employed for PC drug development so far have been derived from cell lines or individual (single) patients and screened independently without consideration of heterogeneity. We hypothesized that the inability to address this critical aspect of tumor biology, has in part led to translational failure in broader patient cohorts. Therefore, we created a number of mixture cell models where multiple PC cell lines, were labeled and co-cultured to capture genomic heterogeneity observed clinically in CRPC patients. Specifically, expression profiles of PC cell lines were clustered with patient-derived single-cell (intra-) or bulk RNA-seq datasets (inter-) to identify cell lines representative of heterogeneous PC tumors, based on their transcriptomic signatures. Cell lines assigned to distinct patient clusters were systematically selected and combined for the mixture model, to reflect the complex in-vivo genomic landscape. To apply these models to drug combination testing, we employed a computational approach to nominate efficacious combinations with Docetaxel (Doc), current Standard of Care. Our mixed-cell model created to recapitulate inter-patient diversity, successfully validated the efficacy of a novel combination of Doc with COL3 (Incyclinide), which is currently in Phase I trial for Refractory Metastatic Cancer. The combination was confirmed to be more potent than individual monotherapies over a range of concentrations. We further tested Doc +Olaparib (clinically approved for patients harboring BRCA mutations), a combination predicted to be inefficacious in the heterogeneous patient cohort as negative control. Indeed, addition of Olaparib did not improve on the inhibitory effect of Doc. To address intra-tumoral heterogeneity, we created a mixture of cell lines which were representative of distinct clonal fractions in single-cell patient datasets. In both models, it was evident that cell lines with similar molecular and phenotypic attributes (such as AR dependency) co-clustered, lending further credibility to our pipeline. We further explored intra-tumoral heterogeneity in the context of acquired phenotypes and advanced disease states, by mixing parent and Doce-resistant clones of DU145 and 22RV1. The binary mixtures were challenged with combinations of Doc and Rigosertib/Selumitinib, drugs that were predicted to selectively target the resistant clone. The combinations produced a higher tumor kill than either single agent drug. Additionally, we observed the effects of altering the relative proportion of constituent clones on drug response. Development of these preclinical models holds enormous promise not only for advancing combinatorial therapies for cancer, but also for providing valuable insights into the mechanisms of resistance and recurrence, by accurately modeling genomic heterogeneity in cancer. Citation Format: Sampreeti Jena, Daniel Kim, Adam M. Lee, Weijie Zhang, R. Stephanie Huang. Development of novel in-vitro mixed-cell models to capture genomic heterogeneity in prostate tumors [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A041.

Abstract A02: High-capacity drug screen identification of novel synergistic drug pairs with venetoclax in acute myeloid leukemia

Abstract Advancements in the development of targeted therapies have changed the therapeutic paradigm in acute myeloid leukemia (AML) in recent years. The orally available BCL-2 inhibitor venetoclax is effective and well tolerated in combination with either a hypomethylating agent (HMA) or low-dose cytarabine (LDAC). BCL-2 is a anti-apoptotic protein which binds to and sequesters pro-apoptotic proteins such as BAX, BAK, PUMA and NOXA and prevents pro-apoptotic proteins from initiating mitochondrial apoptosis leading to sustained survival in cancer cells. Venetoclax specifically antagonizes BCL-2 protein and allows unbound pro-apoptotic proteins to initiate apoptosis. Venetoclax antagonizes BCL-2 and allows pro-apoptotic proteins to initiate apoptosis. Venetoclax with HMA or LDAC received FDA approval in 2020 for the frontline treatment of AML patients unfit for standard chemotherapy. However, therapeutic resistance is common and there is an urgent need to improve the efficacy and durability of venetoclax in this setting. Combinatory drug approaches allow for targeting of multiple molecular drivers/pathogenic mutations, lower therapeutic concentrations, increased efficacy, and overcoming resistance. Here, we aimed to develop novel synergistic drug combinations with venetoclax to improve venetoclax safety, efficacy and overcome resistance in cell lines with potential clinical applications in AML. Venetoclax IC50 values in eight AML cell lines range from <0.01 µM to >5 µM highlighting differential response among the panel. THP-1 cells were identified as the most venetoclax resistant cell line with little response observed at any concentration up to 72 hours exposure. Western blot and qPCR analyses demonstrated BCL-2 is ubiquitously expressed across the cell lines. A single agent drug screen of 1,971 FDA approved compounds was performed at two concentrations (100 nM and 1 µM), in THP-1 cells at three timepoints (24, 48, and 72 hours). 364 of 1971 compounds (18.5%) induced >50% cell death and were identified as hits. A secondary screen was performed to investigate the top 180 most effective compounds in combination with venetoclax. Dose response matrices and cell viability data were used to calculate synergy scores. Combinations with synergy scores of >10 were considered synergistic. Many combinations were synergistic at one timepoint (n=117, 65%), others were synergistic at 2 timepoints (n=45, 25%) and few were synergistic at all 3 timepoints (n=5, 2.78%). Validation experiments are currently ongoing to investigate the synergistic relationship of top candidate combinations and identify an optimum dose for further investigations including additional validation experiments, mechanistic studies, and toxicity studies. Citation Format: Ruairidh Harrigan, Evangeline Johnston, Kyle B Matchett, Tony J Bjourson. High-capacity drug screen identification of novel synergistic drug pairs with venetoclax in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A02.

DDRE-28. IDENTIFICATION OF SYNERGISTIC DRUG COMBINATIONS FOR THE THERAPY OF IDHmut GLIOMA

Abstract OBJECTIVE Isocitrate Dehydrogenase (IDH) mutation in glioma results in a multitude of biological differences with consequences for survival and therapy response. Therefore, IDH mutated (IDHmut) and wildtype (IDHwt) tumors are regarded as separate entities with the need for adjusted therapy like the combination of procarbazine, CCNU and vincristine (PCV). However, as vincristine has often severe side effects like neuropathy new effective therapy options are required. Therefore, we searched for combinations of FDA-approved drugs which effectively inhibit the growth of IDHmut cells in vitro. METHODS We tested different drug combinations of a drug library consisting of 146 FDA-approved drugs on two established IDHmut GSC lines. Based on a previous single agent drug screen, six drugs were selected (Idarubicin, Ixazumib, Ponatinib, Neratinib, Romidepsin) to be combined with all 146 drugs of the library. Cell viability was assessed by the CellTiterGlo 3D assay (Promega) in 96 well plates, while Caspase-Glo 3/7 3D assay was used to measure induction of apoptosis. RESULTS Out of 1460 drug combinations tested altogether 21 synergistic drug combinations could be identified and validated. The combination with the highest blood-brain-barrier permeability score was further investigated. Finally, drug-concentrations elucidating the highest synergistic effect on proliferation was further studied in a 8-point dose-response matrix followed by validation in additional four IDHmut GSC lines. CONCLUSION This work can lay the foundation for future improvements of the therapy of patients suffering from LGGs.

The Pan-Clk/Dyrk Inhibitor Cirtuvivint (SM08502) Exposes Mechanistic Underpinnings of Alternative Splicing As a Therapeutic Vulnerability in Heme Malignancies

Mutations in spliceosomal genes are one of the most common classes of somatic alterations in patients with Myelodysplastic Syndrome (MDS) and occur across the entire spectrum of myeloid malignancies, including 10‒25% of patients with acute myeloid leukemia (AML). These mutations occur in higher proportions in AML subjects greater than 60 years of age, or when AML has transformed from an antecedent MDS. Spliceosomal gene mutations are implicated in the production of pathological RNA splicing patterns that block cell differentiation and maintain a myeloid precursor phenotype. This suggests deranged pre-mRNA splicing is a mechanistic determinant of many heme malignancies and, as such, has provoked interest in therapeutic modulation of pre-mRNA splicing as a treatment paradigm. The CLK/DYRK family of protein kinases has been recognized as an integration hub for signal transduction-dependent modulation of alternative pre-mRNA splice junction selection through direct phosphorylation of the serine/arginine-rich splicing factor (SRSF) splice junction enhancer-binding proteins. Thus, these kinases potentially represent a druggable intervention point in alternative splicing-dependent cancers. The isoquinoline SM08502 (cirtuvivint) is a potent ATP-competitive inhibitor of the Cdc2-like kinases (CLK1-4) and the dual specificity tyrosine phosphorylation-regulated kinases (DYRK1-4) with activity against only a minimal number of the remaining members of the CMGC-family kinases and the kinome as a whole. Here the consequence of pan-CLK/DYRK kinase inhibition on cell viability and tumorigenicity was evaluated across a panel of human tumor-derived AML, DLBCL, MCL, myeloma, T-ALL, and CML/CLL models. EC 50s in response to cirtuvivint ranged from 0.014 μM‒0.495 μM in 4-day in vitro cell viability assays, with low-dose responders enriched in subsets of AML, myeloma, DLBCL, MCL and T-ALL. Cell viability EC 50s were associated with induction of programed cell death at drug exposures that inhibited accumulation of phosphorylated SRSF proteins and the anti-apoptotic protein MCL-1. To directly evaluate the contribution of CLK/DYRK kinases to alternative splicing profiles, high-depth RNAseq analysis was performed across 4 cell lines (3 acute myeloid leukemia cell lines and 1 mantle cell lymphoma cell line) +/- a 6-hour exposure to 1µM cirtuvivint. Both baseline and drug-induced changes in alternative splicing events (ASEs) were measured using a multivariate analysis of splicing transcripts (rMATS). The frequency of cirtuvivint-induced ASEs was approximately 20% of total detected ASEs. Concordant drug-induced ASEs among the tested cell lines were in genes enriched in pathways known to drive oncogenesis in hematopoietic lineages, including the MAP kinase and mTOR signaling pathways. Tumor growth inhibition assays in immunocompromised mice showed a range of model-specific responses, including tumor stasis and partial to complete tumor regression at clinically relevant exposures. A cell-based synthetic-lethal screen of cirtuvivint across 36 small molecule inhibitors identified multiple BCL-2 inhibitors among the most prominent synergistic combinations. Consistent with this, combination of the BCL-2 inhibitor venetoclax with cirtuvivint was sufficient to induce tumor regressions in AML xenograft models (KG-1 and HL-60) that were resistant to either single-agent drug at the same concentrations. These observations support further evaluation of CLK/DYRK inhibitors as a therapeutic strategy for heme malignancies dependent upon alternative pre-mRNA splicing. DisclosuresBossard: Biosplice Therapeutics: Current Employment. McMillan: Prizer: Ended employment in the past 24 months. Beaupre: Pfizer: Ended employment in the past 24 months. White: Pfizer: Ended employment in the past 24 months.

Alzheimer's Disease: Efficacy of Mono- and Combination Therapy. A Systematic Review.

Importance:Alzheimer’s disease (AD) is a complex neurodegenerative disorder and the most prevalent cause of dementia. In spite of the urgent need for more effective AD drug therapy strategies, evidence of the efficacy of combination therapy with existing drugs remains unclear.Objective:To assess the efficacy of combined drug therapy on cognition and progress in patients with AD in comparison to single agent drug therapy.Methods:The electronic databases MEDLINE and EMBASE were systematically searched to identify relevant publications. Only randomized controlled clinical trials were included, but no limits were applied to language or time published. Data were extracted from May 27th until December 29th, 2020.Results:Three trials found that a combination of ChEI with additional memantine provides a slight benefit for patients with moderate to severe AD over ChEI monotherapy and placebo. However, a further 4 trials could not replicate this effect. One trial reported benefits of add-on Gingko biloba in donepezil-treated patients with moderate AD (using a formula containing Gingko and other antioxidants) compared to donepezil with placebo. A further trial found no significant effect of combining EGb 761® and donepezil in patients with probable AD over donepezil with placebo. Approaches with idalopirdine, atorvastatin or vitamin supplementation in combination with ChEI have not proven effective and have not been retried since. Fluoxetine and ST101 have shown partial benefits in combination with ChEI over ChEI monotherapy and placebo. However, these effects must be replicated by further research.Conclusion:Additional memantine in combination with ChEI might be of slight benefit in patients with moderate to severe AD, but evidence is ambiguous. Longer trials are needed. No major cognitive benefit is missed, if solely appropriate ChEI monotherapy is initiated.

PIKfyve Inhibitors for the Treatment of Multiple Myeloma

IntroductionUsing a high throughput screening approach employing ~80 FDA approved, late stage clinical trial, or drugs of interest to multiple myeloma (MM), we examined single agent drug sensitivity of 35 MM and lymphoma cell lines and in 65 primary MM samples. We identified APY-0201, a PIKfyve inhibitor (P5I), as lethal at nanomolar concentrations in 48% of human myeloma cell lines (HMCLs) and 28.6% of primary MM samples after 24 hour of drug exposure. In 20% of patient samples, in vitro sensitivity was less than 100nM EC50. PIKfyve is a lipid and protein kinase that controls pleiotropic cellular functions, notably regulation of endomembrane homeostasis; mechanisms of sensitivity and resistance were explored.MethodsApilimod, a key P5I initially developed as an interleukin (IL)-12 and IL-23 inhibitor, has been used as an immunomodulatory agent in the treatment of inflammatory diseases and has been recently suggested for the treatment of neoplastic diseases, including B-cell Non-Hodgkin's Lymphoma (NHL). YM201636 has also been described as a potent P5I. The cellular efficacies of APY-0201, apilimod, and YM201636 in HMCLs were assessed using the CellTiter Glo assay (Promega), in 20-point 2-fold dilutions of drug concentrations at 72 hours; and HMCL P5I sensitivity was correlated with RNA-SEQ data publically available at www.keatslab.org. Cell lysates were extracted for western blot expression analysis of transcription factor EB (TFEB), cathepsin D, caspase 3, poly (ADP-ribose) polymerase (PARP), at different drug exposure time points (3, 6, 12, 24, 48, and 72 hours). Ex vivo sensitivity to APY-0201, the most potent studied P5I, was measured in CD138+ primary cells of 56 primary MM patients at 24 hours. In vivo activity of apilimod at 100-150 mg/kg and APY-0201 at 30-45 mg/kg was evaluated over 2 weeks in the Vk*MYC transgenic mouse model of MM. Tumor burden was measured by weekly evaluation of M-spike serum levels.ResultsAPY-0201 was the most potent P5I, followed by YM201636 and apilimod. However, dose-dependent inhibition of cell viability was observed in all HMCLs. APY-0201 EC50 ranged from 2.8 to 266.7nM (mean 123.4nM), 63.8 to 8425.8nM (mean 1329nM) for YM201636, and 62.5 to 3561.2nM (mean 2026nM) for apilimod. APY-0201 maximum inhibition ranged from 68.2 to 100% (mean 87.2%), 33.2 to 100% (mean 64.6%) for YM201636, and 56.7 to 100% (mean 83.6%) for apilimod. A significant correlation between low RNA-SEQ expression of TFEB, which regulates lysosomal biogenesis and autophagy, and lower sensitivity for APY-0201 was found (Wilcoxon signed-rank test p-value = 0.017). No correlation with baseline OSTM1, CLCN7 or SNX10 levels was seen.APY-0201 dose dependent sensitivities were observed in 37.5% of ex vivo patient samples. Of the 21 patients demonstrating sensitivity, 2 were inhibited at an EC50<10nM (median 8.58nM), 10 samples presented EC50 between 10 and 100nM (median 64.67nM), and 9 samples presented EC50>100nM (median 1000.16nM). Thus, 12 (21.4%) primary MM patient samples exhibited lethality with less than 100nM concentration of APY-0201 after 24 hours. P5I activity profile was not obviously correlated with clinical findings, such as stage of disease or genetic subtype, in either cell lines or primary samples.As demonstrated previously in B-cell NHL, treatment of the most sensitive cell line, KMS26, with apilimod and APY-0201 was associated with activation of TFEB, found in its dephosphorylated state. An accumulation of the inactive precursor procathepsin D was also observed in KMS26 following treatment, demonstrating a decrease in maturation of lysosomal proteases. Furthermore, the cytotoxic activity of both compounds was shown by induction of apoptosis makers, i.e. caspase 3 and PARP. In ongoing studies, in vivo activity was evident in 2 of 6 Vk*MYC transgenic MM mice treated with a P5I; dose finding and longer treatment duration studies are underway.ConclusionsInhibition of PIKfyve surprisingly demonstrated promising anti-myeloma activity in vitro, ex vivo and in vivo, thus providing a rationale for further evaluation in the treatment of MM. As reported by others in NHL, a novel mechanism of action involving disruption of endolysosomal function is suggested in PIKfyve mediated cytotoxicity in MM.AcknowledgmentThis work was supported in part by the Multiple Myeloma Research Foundation. DisclosuresSharik:Phosplatin Therapeutics: Research Funding. Sepetov:Nanosyn Inc.: Employment. Romanov:Nanosyn Inc.: Employment. Bergsagel:Phosplatin Therapeutics: Research Funding. Chesi:Phosplatin Therapeutics: Research Funding. Stewart:Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Immunosuppression in Lung Transplantation.

Immunosuppression in lung transplantation is an area devoid of robust clinical data. This chapter will review the history of immunosuppression in lung transplantation. Additionally, it will evaluate the three classes of induction, maintenance, and rescue immunosuppression in detail. Induction immunosuppression in lung transplantation aims to decrease incidence of lung allograft rejection, however infectious risk must be considered when determining if induction is appropriate and which agent is most favorable. Similar to other solid organ transplant patient populations, a multi-drug approach is commonly prescribed for maintenance immunosuppression to minimize single agent drug toxicities. Emphasis of this review is placed on key medication considerations including dosing, adverse effects, and drug interactions. Clinical considerations will be reviewed per drug class given available literature. Finally, acute cellular, antibody mediated, and chronic rejection are reviewed.

Modeling Adaptive Resistance of KRAS Mutant Colorectal Cancer to MAPK Pathway Inhibitors with a Three-Dimensional Tumor Model.

Single-agent drug treatment of KRASmut colorectal cancers is often ineffective because the activation of compensatory signaling pathways leads to drug resistance. To mimic cyclic chemotherapy treatments of patients, we showed that intermittent treatments of 3D tumor spheroids of KRASmut colorectal cancer cells with inhibitors of mitogen-activated protein kinase (MAPK) signaling pathway temporarily suppressed growth of spheroids. However, the efficacy of successive single-agent treatments was significantly reduced. Molecular analysis showed compensatory activation of PI3K/AKT and STAT kinases and EGFR family proteins. To overcome the adaptation of cancer cells to MAPK pathway inhibitors, we treated tumor spheroids with a combination of MEK and EGFR inhibitors. This approach significantly blocked signaling of MAPK and PI3K/AKT pathways and prevented the growth of spheroids, but it was not effective against STAT signaling. Although the combination treatment blocked the matrix invasion of DLD1 cells, additional treatments with STAT inhibitors were necessary to prevent invasiveness of HCT116 cells. Overall, our drug resistance model elucidated the mechanisms of treatment-induced growth and invasiveness of cancer cells and allowed design-driven testing and identifying of effective treatments to suppress these phenotypes.

Bifunctional HDAC Therapeutics: One Drug to Rule Them All?

Histone deacetylase (HDAC) enzymes play crucial roles in epigenetic gene expression and are an attractive therapeutic target. Five HDAC inhibitors have been approved for cancer treatment to date, however, clinical applications have been limited due to poor single-agent drug efficacy and side effects associated with a lack of HDAC isoform or complex selectivity. An emerging strategy aiming to address these limitations is the development of bifunctional HDAC therapeutics—single molecules comprising a HDAC inhibitor conjugated to another specificity targeting moiety. This review summarises the recent advancements in novel types of dual-targeting HDAC modulators, including proteolysis-targeting chimeras (PROTACs), with a focus on HDAC isoform and complex selectivity, and the future potential of such bifunctional molecules in achieving enhanced drug efficacy and therapeutic benefits in treating disease.

Abstract 3681: Selective vulnerability of GBM PDX to a panel of EGFR tyrosine kinase inhibitors

Abstract Glioblastoma (GBM), a particularly aggressive form of primary brain tumor, has poor survival due to a lack of effective treatments and high recurrence. Epidermal growth factor receptor (EGFR) (mapped to 7p 11.2), is frequently amplified and mutated (&amp;gt;50%) in GBM, and is suggested as a potential therapeutic target. To correlate chemo-vulnerability with genomic aberrations and further determine biomarkers for targeted therapies, a panel of seven EGFR tyrosine kinase inhibitors were tested in a selected nine human GBM patient-derived xenograft (PDX) panel; these preclinical models harbor different mutation combinations of CDKN2A deletion (C), PTEN deletion (P), wild-type EGFR (E) and/or EGFRvIII (Ev3) overexpression. A single agent drug dose response assay (DDR) was performed on GBM PDX neurospheres using drug concentrations ranging from 100uM to 0.5nM with a 12-point serial 3-fold dilution scheme. The cytotoxic efficacy of EGFR inhibition was dependent on both the PDX line and the EFGR inhibitor applied. GBM59 and GBM76 – both triple CEv3P mutants – were the most responsive to EGFR inhibition relative to the other GBM models tested. For GBM59, the IC50 values of afatinib, canertinib and neratinib, were sub-micromolar, and low micromolar (&amp;lt;3uM) for erlotinib, gefitinib and lapatinib. GBM76 had IC50 values of 1-8uM with all seven EGFR inhibitors including AZD3759. However, GBM155 and GBM126 with CEv3 but wildtype PTEN only responded to afatinib, canertinib and neratinib at low micromolar IC50 values (&amp;lt;3uM) but not to erlotinib, gefitinib and lapatinib. A deletion in PTEN may sensitize GBM cells with CEv3 mutations to erlotinib, gefitinib and lapatinib in vitro. The other group of GBM PDX lines (C only) represented by GBM122, GBM150 and GBM182 responded well to afatinib, canertinib and neratinib with IC50 values of 2- 9uM. GBM156 with CE had a similar profile to the group of C only or CEv3. GBM56 with CP showed the most resistance with mild responses to afatinib, canertinib and neratinib, as observed in its high micromolar IC50 values (6-9uM). Overall, neratinib is the most effective compound among the tested EGFR inhibitors based on IC50 values. Canertinib, the triple tyrosine kinase inhibitor against EGFR/HER2/ErbB-4, is the second most effective agent. Afatinib has similar drug efficacy to canertinib. All three compounds moderately inhibit all GBM PDX lines. The drug response mechanism and its correlation with the mutation status of CDKN2A, EGFR, and PTEN will be further investigated cross a broad array of GBM PDX models. Supported by NIH NCI R01 CA204136 Citation Format: Nanyun Tang, George Reid, C Ryan Miller, Michael E. Berens. Selective vulnerability of GBM PDX to a panel of EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3681.

Abstract A45: Targeted drug therapies for osteosarcoma

Abstract Osteosarcoma (OS) is a highly aggressive cancer that has had no new treatments options in over 30 years. OS is a heterogeneous disease that is characterized by widespread and recurrent somatic copy-number alterations (SCNAs) with relatively few recurrent point mutations. Our laboratory has recently published that SCNAs contain key oncogenic drivers that can be used to identify patient-specific candidates for targeted therapies (Sayles, Breese, et al., Cancer Discovery Jan. 2019). Using patient-derived tumor xenografts (PDX), we demonstrated that targeting of patient-specific oncogenes within SCNAs leads to significant decrease in tumor burden. However, no single-agent therapy was able to regress tumors completely, suggesting that combination therapies would be required for disease management. In order to assess the applicability of our PDX models to the patient tumor and the stability of the oncogenes within SCNAs, we performed whole-genome sequencing (WGS). We observed that SCNAs are highly stable across samples from the same patient in addition to multiple PDX passages and PDX-derived cell lines, highlighting the equivalence between the PDX models and their derived cell lines to the human disease. This allows us to use the PDX cell lines as a surrogate for the identification of combination drug therapies that may be of benefit in OS. Currently, we have generated 6 PDX cell lines that encompass the most common SCNAs observed in patients, including MYC, CCNE1, and CDK4 gain and alterations in the PI3K/PTEN pathway. We have performed a single-agent drug screen with 38 agents and have identified several efficacious compounds, including HDAC inhibitors and cell cycle and Wee1 inhibitors. We also observed a differential response between PDX cell lines to various chemotherapeutics, including gemcitabine, paclitaxel, and SN-38. We are currently testing combination drug therapies in vitro and will validate using our PDX xenografts in vivo. While these experiments are still ongoing, we can report that MYC-driven OS PDX xenografts show a striking resensitization to cisplatin after AURKB inhibitor (barasertib) pretreatment. We have 2 aggressive PDX models with high MYC amplification generated from metastatic lesions. These PDX models are resistant to cisplatin, which is part of the standard of care. We treated these PDX with barasertib and then with cisplatin for two cycles. This resulted in a decrease in tumor volume for the combination therapy compared to vehicle or either single agent alone. Additionally, we observed retention of the platinum adduct 24 hrs after cisplatin dosing only when tumors were pretreated with barasertib. Further work is needed to assess the mechanism of this synergy and whether it can be of use in other SCNA-driven OS and to identify other possible combination therapies that could be of importance in this disease. Citation Format: Leanne C. Sayles, Marcus R. Breese, Henry Martell, Alex G. Lee, Stanley Leung, Avanthi T. Shah, E. Alejandro Sweet-Cordero. Targeted drug therapies for osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A45.

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

MCL-1 Inhibitor-Induced Killing of Multiple Myeloma Is Modulated By the Relative Level of Expression of Pro-Survival Members of the BCL-2 Family and the Proto-Oncogene MYC

Background: The anti-apoptotic protein Myeloid cell leukemia sequence 1 (MCL-1) is involved in the regulation of programmed cell death and is highly expressed in multiple myeloma (MM) playing an important role in promoting the survival of MM cells. Recent data suggests that the efficacy against MM of the selective BCL-2 inhibitor Venetoclax (V) may be correlated with the relative level of expression of the pro-survival members of the BCL-2 protein family. Against this background we have evaluated the activity of the MCL-1 inhibitor (MCL1i) S63845 (S) against human myeloma cell lines (HMCL) and primary MM tumours (1ºMM) both as a single agent and in combination with other anti-MM agents. Moreover, we have used RNASeq to identify biomarkers of responsiveness to S in both HMCL and 1ºMM. Methods: A panel of 16 HMCL and 46 1ºMM were treated for 72 hours with S (100nM and 500nM) either alone or in combination with V (500nM), the BCLxL inhibitor A1331852 (A) (500nM), bortezomib (B) (10nM) or panobinostat (P) (10nM). Apoptosis was measured by flow-cytometric enumeration of either propidium iodide positivity (HMCL) or Apo2.7 positivity following cell permeabilisation (1ºMM). Drug synergy was calculated based on the net effect of cell death by Drug A plus Drug B divided by cell death caused by drugs A and B singly, with a Synergy Quotient &gt;1 indicating a synergistic effect. Paired samples from 30 of the 1ºMM were subject to CD138 enrichment utilizing CD138 micro-beads. Subsequently total RNA was extracted from both HMCL (n = 16) and CD138 enriched 1ºMM (n = 30). mRNAs were sequenced on a HiSeq 4000 platform with a paired-end 150 bp strategy. Raw data were evaluated and quality controlled by FASTQC (v0.11.5) and SOAPnuke. Hisat2 and StringTie were used to align clean reads to the reference genome (GRCH38) and profile gene expression, respectively. HTSeq-count was used to count the reads aligned to each gene then differentially expressed genes (DEGs) (S sensitive vs resistant) were defined by edgeR (fold change &gt; 2, p &lt; 0.05 and false discovery rate &lt; 0.05). DAVID Bioinformatics Resources was used to analyse the Gene Ontology and KEGG pathway enrichment by the DEGs. The co-expression in both HMCL and 1ºMM of apoptosis-related genes (ARG) (BIM, BCL2, BAD, BCL2A1, NOXA, BID, BAK1, MCL1, BAX, BCLw, TP53, PUMA, BCLxL, BMF) and MYC was specifically evaluated using Pearson correlation. The Spearman correlation coefficient was used to define any correlation between S sensitivity and ARG/MYC expression. The correlation scatter plots and p-values were calculated using 'gg_scatter' function (from ggpubr package) in R. Results: HMCL killing with S ranged from 0.9-91.6% (100nM) and 1.0-93.3% (500nM) with 70%, 80%, 37% and 61% demonstrating synergy when S was combined with V, A, B or P, respectively. S killing of HMCL was modestly but statistically significantly correlated with MCL-1 expression but strongly correlated with both BCL-2 expression and the BCLxL:BCL-2, MCL-1:BCL-2 and BCLxL:MYC expression ratios (Table 1). 1ºMM, cells were classified as sensitive when &gt;20% cell death was induced and resistant with ≤20% cell death. The proportion of sensitive 1ºMM with single agent drug treatments was S100nM 37%, S500nM 66%, V50%, A77%, B47% and P45%. Synergy with S was seen with 70%, 80%, 37% and 61% of the 1ºMM with V, A, B or P, respectively. 1ºMM demonstrated a statistically significant, albeit modest, correlation with the BCLxL:MCL-1 ratio (reduced 1ºMM death with high BCLxL coupled with low MCL-1, R=-0.43, p=0.019) (Figure A) and the MCL-1:MYC ratio (increased 1ºMM death with high MCL-1 coupled with low MYC, R=0.43, p=0.018) (Figure B), the former consistent with the high frequency of synergistic killing of 1ºMM when S was combined with BCLxL inhibition. Compared to 1ºMM sensitive to the MCL1-inhibitor, we identified 985 genes (440 up-regulated and 545 down-regulated) differentially expressed in the 1ºMM resistant to the drug. DEGs were annotated demonstrating that the top 2 KEGG pathways involved by the DEGs were "hsa03010:Ribosome" (p&lt;0.0001) and "hsa04010:MAPK signaling pathway" (p=0.04). Conclusions: The efficiency of MCL1i-induced killing of both HMCL and 1ºMM is modulated by the co-expression of pro-survival members of the BCL-2 family and MYC. These data provide a framework for future MCL1i MM treatment by potentially enabling rational patient selection and providing insights into strategies to optimize MCL1i-based MM therapy. Disclosures Spencer: Takeda: Other: Consulting/advisory role, Research Funding; Janssen Oncology: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Amgen: Other: Consulting/advisory role, Research Funding; AbbVie: Other: Consulting/advisory role, Research Funding; Servier: Other: Consulting/advisory role; Secura Bio: Other: Consulting/advisory role; Haemalogix: Other: Consulting/advisory role; Celgene: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Sanofi: Other: Consulting/advisory role; Specialised Therapeutics Australia: Consultancy, Honoraria. Khong:Novartis Oncology: Research Funding.

Assessing the effectiveness of empiric antimicrobial regimens in cases of septic/infected abortions

IntroductionInfected abortion is a life-threatening condition that requires immediate surgical and medical interventions. We aimed to assess the common pathogens associated with infected abortion and to test the microbial coverage of various empiric antimicrobial regimens based on the bacteriological susceptibility results in women with infected abortions. MethodsA retrospective study in a single university-affiliated tertiary hospital. Electronic records were searched for clinical course, microbial characteristics, and antibiotic susceptibility of all patients diagnosed with an infected abortion. The effectiveness of five antibiotic regimens was analyzed according to bacteriological susceptibility results. ResultsOverall, 84 patients were included in the study. The mean age of patients was 32.3(SD ± 5.8) years, and the median gestational age was 15 (IQR 8–19) weeks. Risk factors for infection were identified in 23 patients (27.3%), and included lack of medical insurance (n = 12), recent amniocentesis/chorionic villus sampling or fetal reduction due to multifetal pregnancies (n = 10). The most common pathogens isolated were Enterobacteriaceae (35%), Streptococci (31%), Staphylococci (9%) and Enterococci (9%). The combination of intravenous ampicillin, gentamicin and metronidazole showed significant superiority over all the other tested regimens according to the susceptibility test results. Piperacillin-tazobactam as an empiric single-agent drug of choice and provided a superior microbial coverage, with a coverage rate of 93.3%. ConclusionsA combination of ampicillin, gentamicin, and metronidazole had a better spectrum of coverage as a first-line empiric choice for patients with infected abortion.

Abstract 2880: Targeted drug therapies for osteosarcoma

Abstract Osteosarcoma (OS) is a highly aggressive cancer that has had no new treatments options in over 30 years. OS is a heterogeneous disease which is characterized by widespread and recurrent somatic copy-number alterations (SCNAs) with relatively few recurrent point mutations. We have previously reported that SCNAs contain key oncogenic drivers that can be used to identify patient-specific candidates for targeted therapies (Sayles and Breese, et al. Cancer Discovery 2018). Using patient-derived tumor xenografts (PDX), we demonstrated that targeting of patient-specific oncogenes within SCNAs leads to significant decrease in tumor burden. However, no single agent therapy was able to regress tumors completely suggesting that combination therapies would be required for disease management. In order to assess the applicability of OS PDX models to the patient tumor and the stability of the oncogenes within SCNAs, we preformed whole genome sequencing (WGS). We observed that SCNAs are highly stable across samples from the same patient in addition to multiple PDX passages and PDX derived cells lines, highlighting the equivalence between the PDX models and their derived cell lines to the human disease. This allows us to use the PDX cell lines as a surrogate for the identification of combination drug therapies that may be of benefit in OS. Currently, we have generated six PDX cell lines that encompass the most common SCNAs observed in patients including MYC, CCNE1 and CDK4 gain and alterations in the PI3K/PTEN pathway. We have performed single agent drug screens in these cell lines and have identified heterogeneous responses as would be anticipated by the genomic diversity observed in OS. We are currently testing combination drug therapies and will validate using the PDX xenografts in vivo. We have found that MYC driven OS PDX cell lines and PDX xenografts show a striking re-sensitization to cisplatin after AURKB inhibitor (barasertib) pretreatment. We have two PDX and derived cell lines that were generated from highly aggressive metastatic disease. Both of these PDX have high MYC amplification and were resistant to cisplatin, which is part of the standard of care. We pretreated the PDX cell lines for 24hrs with barasertib then added cisplatin and saw a synergistic effect in vitro. Further studies in the PDX model demonstrated a decrease in tumor volume for the combination therapy compared to vehicle or either single agent alone. Additionally, we observed retention of the platinum adduct 24 hrs after cisplatin dosing only when tumors were pretreated with barasertib. Further work is needed to assess the mechanism of this synergy and whether it can be of use in other SCNA driven OS and to identify other possible combination therapies that could be of importance in this disease. Citation Format: Leanne C. Sayles, Amanda Koehne, Kieren Marini, Alex G. Lee, Stanley G. Leung, Avanthi T. Shah, Marcus R. Breese, E. Alejandro Sweet-Cordero. Targeted drug therapies for osteosarcoma [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 2880.

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma

BackgroundCancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments.MethodsTissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination.Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient’s epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient’s primary tumor.Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay.ResultsOur computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model).ConclusionsThese proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

Dual silencing of EGFR and HER2 enhances the sensitivity of gastric cancer cells to gefitinib.

Gefitinib exhibits very limited efficacy in gastric cancer (GC). Indeed, the limited clinical results obtained with gefitinib alone justify investigation of additional therapeutic strategies. Here, we demonstrate the importance of EGFR and HER2 in GC malignancy using RNA interference (RNAi). Additionally, we explored the ability of RNAi targeting EGFR and HER2 to enhance the sensitivity of GC cells to gefitinib. Specific small interfering RNAs (siRNAs) significantly inhibited mRNA and protein expression of target genes. EGFR-specific siRNA, EGFR/HER2 siRNAs, and gefitinib inhibited growth and induced apoptosis in GC cell lines in a dose-dependent manner. In contrast, resistance to HER2-siRNA-induced growth inhibition and apoptosis was linked to compensatory activation of EGFR. Moreover, gefitinib dramatically reduced p-EGFR and p-HER2 levels in the cell lines tested, and sensitivity to gefitinib was enhanced through dual silencing of EGFR and HER2 via suppression of AKT and ERK activation. These findings are in agreement with the profound inhibitory effect of gefitinib on activation of both EGFR and HER2. Overall, EGFR/HER2 knockdown by siRNAs further decreased the growth of GC cells treated with gefitinib alone, confirming that single-agent drug targeting does not achieve a maximal biological effect. The combination of gefitinib with EGFR/HER2 siRNAs should be further investigated as a new strategy for the treatment of GC and other EGFR/HER2-dependent cancers.

Abstract IA13: Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors represent one of the most exciting recent developments in cancer therapy. While substantial efficacy has been shown with clinically available PARP inhibitors (PARPis), to date, in treatment of hereditary deletions of BRCA1/2 in breast and ovarian cancers, the high promise of these drugs has not yet been realized in sporadic cancers. We present here strong preclinical data for a novel, mechanistically based, combinatorial approach to using DNA methyltransferase inhibitors (DNMTis), such as decitabine (DAC) and 5-Azacytidine (5-AZA), with PARP inhibitors (PARPis) as a treatment strategy for acute myelogenous leukemias (AML) and triple negative breast cancer (TNBC). We have previously demonstrated that low doses of 5-AZA and DAC alone show efficacy in AML and TNBC, and propose treatment with PARPis to enhance sensitivity of cancer cells to DNMTis. The mechanistic rationale for our approach is based upon: 1) data from our group and others showing DNMT1 and PARP1 associate in a complex, and this association increases with DNA damage; 2) the fact that 5-AZA and DAC trap DNMTs led us to hypothesize that these drugs might also increase PARP trapping at DNA damage sites; and 3) the cytotoxicity of the most potent PARPis (e.g. BMN 673) appears to correlate with the degree of trapping of PARP1 in chromatin. We first find that in cultured human AML and TNBC cells, the DNMTis (5 to 20 nM DAC or 100 to 200nM 5-AZA) and PARPis (1 to 10 nM BMN 673) alone trap PARP into chromatin, and this effect is enhanced when the drugs are combined. In addition, the PARPi-DNMTi combination treatment of TNBC cell line MDA-MB-231 resulted in significantly enhanced retention of PARP1 and DNMT1 at sites of double strand breaks (DSBs) induced by laser microirradiation. Concomitant with this, the combined doses resulted in significant increases in cytotoxic DSBs, observed 4-24 hours after DSB induction, when compared to single-drug treatments. Homologous recombination (HR) DSB repair activity also appears decreased, as measured by GFP reporter assays. In keeping with these findings, colony survival assays demonstrated that the combination treatment, compared to either drug alone, strongly inhibited colony formation of TNBC cell lines (N=4). Notably non-tumorigenic MCF10A cells showed no significant differences in colony numbers with single or combination drug treatments. Similar to TNBCs, AML cell lines (N=3) as well as primary AML cells (N=8) showed dramatic decreases in colonies in combination vs single agent drug treatments. In the most important translational implications of the preliminary studies, in in vivo therapy TNBC and AML models in immune-deficient mice, our low dose combinations of DNMTis and PARPis provide for potent anti-tumor responses. Mouse xenograft experiments using BRCA mutant TNBC cell line SUM149PT demonstrated that the combination treatment has a significant (p&amp;lt;0.05) survival advantage compared to control (vehicle), AZA (0.5mg/kg) or BMN (0.3 mg/kg) alone. Importantly, mouse MDA-MB-231 xenografts with intact BRCA1 showed significant survival with the combined drug treatment. Likewise in AML xenografts of MOLM14 and MV411 cell lines treated with the drug combination show significantly decrease leukemia burden, as measured by luciferase imaging. Our data suggest a novel use of both DNMTis and PARPis in a compelling therapeutic strategy for TNBCs independent of BRCA mutations and poor prognosis AML; the latter will be investigated in a clinical trial to be based at the University of Maryland and funded by Van Andel-SU2C. Citation Format: Nidal Muvarak, Khadiza Chowdhury, Carine Robert, Xia Limin, Eun Yong Choi, Yi Cai, Marina Bellani, Michael Seidman, Maria R. Baer, Rena Lapidus, Stephen B. Baylin, Feyruz V. Rassool. Combination of DNA methyltransferase and PARP inhibitors as a novel therapy strategy for multiple cancers: Key data in AML and triple negative breast cancer [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA13.

Abstract 1778: A clinical genomic biomarker study of the CHK1 inhibitor prexasertib in advanced head and neck squamous cancer and squamous cell carcinoma of the anus

Abstract CHK1 plays a critical role in DNA damage repair (DDR) pathways as well as in coordinating DNA replication. Selective CHK1/CHK2 compounds are being tested in clinical trials but predictive biomarkers of patient response are lacking. A phase 1b expansion cohort study (I4D-MC-JTJA, NCT01115790) with the CHK 1 inhibitor, prexasertib, included patients with advanced, metastatic head and neck squamous cell carcinoma (HNSCC) or squamous cell carcinoma of the anus (SCCA). To identify genomic biomarkers associated with single-agent drug response, pre-treatment tissues (archived or biopsy) from 71 consented patients (HNSCC=47, SCCA=24) were subjected to next-generation sequencing (NGS) using the FoundationOne gene panel. In this subset of patients, the disease control rate (DCR) (Complete Response (CR) + Partial Response (PR) + Stable Disease (SD) based on RECIST Criteria (v 1.1)) was 60% (28/47) and 75% (18/24), respectively. We present here the observed genetic alterations corresponding to three pathways, Cell Cycle, DNA Damage Repair (DDR) and PI3K. In addition, patients’ human papillomavirus (HPV) carrier status was inferred from DNA sequencing using HPV-specific capture probes. HPV+ was 47% for HNSCC and 87% for SCCA. HPV+ and TP53 mutations were mutually exclusive across the two patient cohorts. In HNSCC patients with evaluable progression-free survival (PFS) data, greater clinical efficacy was observed in the HPV+ cohort (median PFS: 4.5 vs 1.4 months, log-rank p = 0.0008). Known or likely loss-of-function (LOF) mutations in FBXW7 and PARK2, two genes implicated in Cyclin E1 proteolysis, were noted in patients with favorable response in both tumor types. Across both HNSCC and SCCA cohorts, mutations and/or germline variants in the DDR genes BRCA1, BRCA2, MRE11A and ATR but not in Fanconi (FANC) pathway genes, were found in patients with treatment benefit. Whereas PIK3CA mutations were infrequent in the HNSCC cohort, in SCCA, mutations occurred in 5/8 (63%) patients with disease control vs 1/6 (17%) with PD. All 7 PI3KCA mutations observed in HPV+ HNSCC and SCCA patients mapped to the helical domain suggestive of Apobec-induced mutagenesis as their source of origin. The enhanced clinical benefit to prexasertib associated with HPV+ in HNSCC may reflect a prognostic effect. Alternatively, the clinical biomarker findings may support the hypothesis that oncogene-induced replication stress (RS) (i.e. arising from HPV E6/E7 and/or FBXW7 loss-dependent Cyclin E1 dysregulation) in the context of attenuated DDR (i.e. BRCA1/2, MRE11A mutations) may sensitize patients to prexasertib monotherapy. Citation Format: Ricardo Martinez, Sameera R. Wijayawardana, David Hong, Johanna Bendell, Anna Maria Russell, Richard P. Beckmann, Aimee Bence Lin. A clinical genomic biomarker study of the CHK1 inhibitor prexasertib in advanced head and neck squamous cancer and squamous cell carcinoma of the anus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2017-1778