Russell Karp

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

UNLABELLED Patients 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. SIGNIFICANCE We 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.

Selective irreversible inhibition of a protease by targeting a noncatalytic cysteine

Designing selective inhibitors of proteases has proven problematic, in part because pharmacophores that confer potency exploit the conserved catalytic apparatus. We developed a fundamentally different approach by designing irreversible inhibitors that target noncatalytic cysteines that are structurally unique to a target in a protein family. We have successfully applied this approach to the important therapeutic target HCV protease, which has broad implications for the design of other selective protease inhibitors.

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.

Discovery of a potent and isoform-selective targeted covalent inhibitor of the lipid kinase PI3Kα.

PI3Kα has been identified as an oncogene in human tumors. By use of rational drug design, a targeted covalent inhibitor 3 (CNX-1351) was created that potently and specifically inhibits PI3Kα. We demonstrate, using mass spectrometry and X-ray crystallography, that the selective inhibitor covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3Kβ, -γ, and -δ are not covalently modified. 3 is able to potently (EC(50) < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent antiproliferative effect (GI(50) < 100 nM). A covalent probe, 8 (CNX-1220), which selectively bonds to PI3Kα, was used to investigate the duration of occupancy of 3 with PI3Kα in vivo. This is the first report of a PI3Kα-selective inhibitor, and these data demonstrate the biological impact of selectively targeting PI3Kα.

A Novel Methionine Aminopeptidase-2 Inhibitor, PPI-2458, Inhibits Non–Hodgkin's Lymphoma Cell Proliferation In vitro and In vivo

Purpose: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center–derived non–Hodgkins lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center–derived NHL cells. Experimental Design: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center–derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts. Results: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI50 = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice. Conclusions: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.

Abstract C189: CO-1686, an orally available, mutant-selective inhibitor of the epidermal growth factor receptor (EGFR), causes tumor shrinkage in non-small cell lung cancer (NSCLC) with T790M mutations.

Introduction: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond well to EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by the development of resistance. The most common mechanism of resistance is a second site mutation within exon 20 of EGFR (T790M), observed in ∼50% of cases. Our goal was to develop a mutant-selective EGFR inhibitor that potently inhibits activating EGFR mutations as well as the T790M resistance mutation while sparing wild-type EGFR for the treatment of NSCLC patients. Such a drug has the potential to effectively treat first- and second-line NSCLC patients with EGFR mutations without causing the dose limiting toxicities associated with EGFR kinase inhibitors currently in clinical development. Experimental procedures: Using structure-based drug design, we identified CO-1686, a covalent, irreversible small molecule, which selectively inhibits mutant EGFR. We assessed antitumor activity of CO-1686 both in vitro and in vivo in two NSCLC cell lines harboring EGFR mutations: H1975 (EGFR L858R/T790M) and HCC827 (EGFR delE746-A750). We evaluated inhibition of EGFR phosphorylation and downstream signaling by immunoblot analysis in cells and tissue samples. IHC staining on skin samples was performed to address effects on wild-type EGFR. Results: CO-1686 is a potent inhibitor of cell proliferation and EGFR signaling in NSCLC cells harboring the single activating mutation EGFR delE746-A750 as well as the double mutation EGFR L858R/T790M. When administered orally, CO-1686 (3 − 100 mg/kg) significantly suppresses tumor growth of H1975 cells (L858R/T790M) in a dose-dependent manner causing tumor regressions at the highest dose (100 mg/kg) without affecting body weight. Erlotinib at the same dose exhibits no effect against H1975 xenografts. In HCC827 (delE746-A750) xenografts, both agents cause tumor shrinkage. In both NSCLC mouse models, inhibition of EGFR phosphorylation in tumors correlate with the observed anti-tumor activity, while no effect on EGFR signaling is observed in normal lung or skin tissues with CO-1686 treatment, confirming that CO-1686 does not inhibt wild-type EGFR. Conclusions: Our results establish CO-1686 as a mutant-selective, wild-type sparing EGFR inhibitor with in vivo efficacy against tumors with activating EGFR mutations as well as the resistance mutation T790M. These data suggest that treatment with CO-1686 as a single agent can overcome T790M-mediated drug resistance in NSCLC. This hypothesis will be tested clinically. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C189.

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