Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP‐site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP‐competitive inhibitors and myristoyl‐site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP‐competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP‐competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation‐selective ATP‐site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP‐competitive inhibitors in a series of biochemical and cellular assays.

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP‐site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP‐competitive inhibitors and myristoyl‐site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP‐competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP‐competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation‐selective ATP‐site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP‐competitive inhibitors in a series of biochemical and cellular assays.

Purpose: There is a need for biomarkers of drug efficacy for targeted therapies in triple-negative breast cancer (TNBC). As a step toward this, we identify multi-omic molecular determinants of anti-TNBC efficacy in cell lines for a panel of oncology drugs. Methods:Using 23 TNBC cell lines, drug sensitivity scores (DSS 3 ) were determined using a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS 3 values were correlated with molecular readouts using an FDRcorrected significance cutoff of p* < 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy.Results: Six molecular determinant panels were obtained from 12 drugs we prioritized based on their efficacy. Determinant panels were largely devoid of DNA mutations of the targeted pathway. Molecular determinants were obtained by correlating DSS 3 with molecular readouts. We found that co-inhibiting molecular correlate pathways leads to robust synergy across many cell lines. Conclusions:These findings demonstrate an integrated method to identify biomarkers of drug efficacy in TNBC, where DNA predictions correlate poorly with drug response. Our work outlines a framework for the identification of novel molecular determinants and optimal companion drugs for combination therapy based on these correlates.Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. https://www.springer.com/aamterms-v1

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

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Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 μM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (C max = 1.6-3.7 μM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 μM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (C max = 1.6-3.7 μM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.

There are diverse resources available for data relevant to the drug screening of bladder cancer cell lines in 2D culture, such as the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) databases; however, 3D culture is more representative of in vivo response in patients. Moreover, drug screens in 3D reveal differential drug response and expression of genes and proteins that indicate the potential of more effective selection of drugs for specific patients: the basic tenet of personalized oncology. Therefore, we screened bladder cancer cell lines in 3D culture using a library of curated small-molecule inhibitors to identify compounds and classes of compounds with efficacy in bladder cancer. Genomic and transcriptomic data for these bladder cancer cell lines were used to correlate genomic molecular parameters with drug response with the aim of discerning biomarkers of response or to suggest plausible drug combinations. This allowed us to identify groups of tumors that are vulnerable to specific drugs or classes of drugs. We found that MEK inhibitors were a promising target among basal bladder cancers, an RNA-based molecular subtype of bladder cancer, especially aggressive and exhibiting poor prognosis. Furthermore, trametinib was found to be a “very active” MEK inhibitor in basal cancers, classified using drug sensitivity 3 (DSS3) scoring. PI3K/AKT/MTOR inhibitors target a parallel signaling pathway to MEK inhibition; therefore, we tested PI3K/AKT/MTOR combinations with trametinib in basal bladder cancer to query for synergistic properties. Additionally, we tested PI3K/AKT/MTOR combinations with trametinib using ex vivo organoids of bladder cancer patient samples collected directly at operation. Taken together, MEK inhibition is a promising therapeutic strategy in the basal subtype of bladder cancer, particularly in combination with parallel pathway inhibition. Moving forward, we plan to test PI3K/AKT/MTOR combinations with trametinib in trametinib-resistant basal bladder cancer cell lines to investigate how this affects signaling and re-sensitization to trametinib. Citation Format: Athena M. Apfel, Nathan M. Merrill, Nathalie M. Vandecan, Liwei Bao, Xu Cheng, Lluis A. Lopez-Barcons, Kathleen C. Day, Phillip L. Palmbos, Mark L. Day, Aaron M. Udager, Matthew B. Soellner, Sofia D. Merajver. MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1194.