Juswinder Singh

Discovery of a Mutant-Selective Covalent Inhibitor of EGFR that Overcomes T790M-Mediated Resistance in NSCLC

UNLABELLEDnPatients with non-small cell lung cancer (NSCLC) with activating EGF receptor (EGFR) mutations initially respond to first-generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR(T790M) mutation. CO-1686 is a novel, irreversible, and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR-mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition and demonstrated increased sensitivity to AKT inhibitors. These results suggest that CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC.nnnSIGNIFICANCEnWe report the preclinical development of a novel covalent inhibitor, CO-1686, that irreversibly and selectively inhibits mutant EGFR, in particular the T790M drug-resistance mutation, in NSCLC models. CO-1686 is the fi rst drug of its class in clinical development for the treatment of T790M-positive NSCLC, potentially offering potent inhibition of mutant EGFR while avoiding the on-target toxicity observed with inhibition of the WT EGFR.

Inhibition of Btk with CC-292 Provides Early Pharmacodynamic Assessment of Activity in Mice and Humans

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton’s tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro and in the collagen-induced arthritis model of autoimmune disease. Recently, CC-292 has entered human clinical trials with a trial design that has provided rapid insight into safety, pharmacokinetics, and pharmacodynamics. This first-in-human healthy volunteer trial has demonstrated that a single oral dose of 2 mg/kg CC-292 consistently engaged all circulating Btk protein and provides the basis for rational dose selection in future clinical trials. This targeted covalent drug design approach has enabled the discovery and early clinical development of CC-292 and has provided support for Btk as a valuable drug target for B-cell mediated disorders.

In vitro and In vivo Characterization of Irreversible Mutant-Selective EGFR Inhibitors that are Wild-type Sparing

Patients with non–small cell lung carcinoma (NSCLC) with activating mutations in epidermal growth factor receptor (EGFR) initially respond well to the EGFR inhibitors erlotinib and gefitinib. However, all patients relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR (EGFRWT). These inhibitors have not provided compelling clinical benefit in T790M-positive patients, apparently because of dose-limiting toxicities associated with inhibition of EGFRWT. Thus, there is an urgent clinical need for therapeutics that overcome T790M drug resistance while sparing EGFRWT. Here, we describe a lead optimization program that led to the discovery of four potent irreversible 2,4-diaminopyrimidine compounds that are EGFR mutant (EGFRmut) selective and have been designed to have low affinity for EGFRWT. Pharmacokinetic and pharmacodynamic studies in H1975 tumor–bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. Importantly, evaluation of normal lung tissue from the same animals showed no inhibition of EGFRWT. Of all the compounds tested, compound 3 displayed the best efficacy in EGFRL858R/T790M-driven tumors. Compound 3, now renamed CO-1686, is currently in a phase I/II clinical trial in patients with EGFRmut-advanced NSCLC that have received prior EGFR-directed therapy. Mol Cancer Ther; 13(6); 1468–79. ©2014 AACR.

Superiority of a novel EGFR targeted covalent inhibitor over its reversible counterpart in overcoming drug resistance

Recently, the importance of targeted covalent inhibitors in addressing potency, selectivity and drug resistance has become of great interest, especially in the area of non-small cell lung cancer (NSCLC). Although several covalent EGFR TKIs that are advancing in NSCLC clinical development are active against mutations which are refractory to the reversible TKI drugs Tarceva and Iressa, limited chemical diversity has been explored; all of the irreversible and reversible clinical compounds share the same quinazoline scaffold. We describe the design of a novel pyrimidine-based irreversible inhibitor of EGFR (CNX17) which is active against both the WT EGFR as well as the resistance mutation L858R/T790M in biochemical assays. The inhibitor is also a potent inhibitor of EGFR signaling, including the L858R/T790M resistance mutation in cells (H1975 cell line, EC50 441 nM). Importantly, it also potently inhibits proliferation in both HCC827 (EGFRΔ746–750 EC50 < 5 nM) and H1975 (EC50 134 nM). This novel chemical scaffold may be an important addition to the armamentarium in overcoming drug resistance to current EGFR therapies.

Interleukin 2-inducible T cell kinase (ITK) facilitates efficient egress of HIV-1 by coordinating Gag distribution and actin organization

Interleukin 2-inducible T cell kinase (ITK) influences T cell signaling by coordinating actin polymerization and polarization as well as recruitment of kinases and adapter proteins. ITK regulates multiple steps of HIV-1 replication, including virion assembly and release. Fluorescent microscopy was used to examine the functional interactions between ITK and HIV-1 Gag during viral particle release. ITK and Gag colocalized at the plasma membrane and were concentrated at sites of F-actin accumulation and membrane lipid rafts in HIV-1 infected T cells. There was polarized staining of ITK, Gag, and actin towards sites of T cell conjugates. Small molecule inhibitors of ITK disrupted F-actin capping, perturbed Gag-ITK colocalization, inhibited virus like particle release, and reduced HIV replication in primary human CD4+ T cells. These data provide insight as to how ITK influences HIV-1 replication and suggest that targeting host factors that regulate HIV-1 egress provides an innovative strategy for controlling HIV infection.

Abstract 3388: Novel aurora plus Bruton's tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin's lymphoma.

Exploring synthetic lethality in the context of signaling pathways with novel targeted agents may yield effective combination therapies for aggressive B-NHL. Auroras are a family of mitotic oncogenic serine/threonine kinases intimately involved in high fidelity regulation of cell division. We previously demonstrated that aberrant Aurora (A and B) expression portends a poor survival in mantle cell lymphoma (MCL) patients. Aberrant aurora expression leads to genetic instability, polyploidy, and resistance to microtubule targeted agents (MTAs). We demonstrated that combination of an Aurora inhibitor MLN8237 (alisertib) or AT9283 with MTAs (e.g. taxol or vincas) leads to a mechanistic decoupling of the mitotic spindle manifesting as a synergistic apoptotic response in cell culture and in mouse xenograft models of aggressive B-NHL. Gene expression profiling of harvested mouse xenograft tumors at the end of treatment (3 weeks) with an aurora inhibitor + MTA showed over-expression of several markers of resistance of which BTK (Bruton9s tyrosine kinase) was the most prominent. BTK plays a role in uncontrolled activation and proliferation of malignant B-cells via the chronic active B-cell receptor pathway in aggressive B-NHL. We hypothesized that targeting proliferation [BTK] and replication [Aurora] would be a novel therapeutic strategy in aggressive B-NHL. We demonstrate that both aurora (A and B) and BTK are highly over-expressed in B-NHL cell lines (Granta-519, SUDHL4, SUDHL10, OCI-Ly-10, TMD8, U-2932) and harvested mouse xenograft tumour treated with an Aurora inhibitor + vincristine and in patient samples. IC 50 values for AT9283 is 5-10nM, and CNX652 is 0.07-5μM. The combination index (CI) indicated significant synergism for AT9283 plus CNX-652 or vice versa. Combination therapy showed increased apoptosis (flow cytometry, PARP-cleavage) in a dose-dependent manner. Both AT9283 and AVL-292 (CNX-652 is a structural analog with similar in vitro and in vivo properties to AVL-292) are in early phase clinical trials in hematologic malignancies. We utilized clinically relevant doses to evaluate the efficacy of single agent and combination therapy of AT9283 and CNX652 in mouse xenograft models (Granta-519) of MCL. The first mouse model evaluated single agent dose-response and target modulation by Western blotting (pHisH3, pBTK) in tumors harvested at the end of 3 weeks of treatment (CNX-652 daily oral for 3 weeks or AT9283 daily IV for 3 weeks, n=12 mice per cohort). The second study evaluated the combination of AT9283 and CNX-652 for endpoints of response, overall survival and target modulation. Gene expression and protein analysis of harvested tumors at the end of treatment (3 weeks) have been interrogated to ascertain the mechanistic role of Aurora plus BTK inhibition. The combination of AT9283 + CNX652 represents a novel therapeutic strategy for aggressive B-NHL [Funded by the Hope Foundation]. Citation Format: Daruka Mahadevan, Carla Morales, Russell Karp, Laurence Cooke, John Lyons, Juswinder Singh, Wenqing Qi. Novel aurora plus Bruton9s tyrosine kinase-targeted therapies for aggressive B-cell non-Hodgkin9s lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2013-3388