Discovery Of Novel ALK And C-Met Inhibitors For Advanced Lung Cancer Therapy

Lung cancer is a leading cause of cancer-related death in the world, and non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Molecularly targeted therapy has been successfully developed for many malignancies. This strategy is also promising in treating some patients with NSCLC.The tyrosine kinase encoded by anaplastic lymphoma kinase (ALK) gene is normally expressed at high levels in the developmental nervous system. During the past few years, genetic alteration of ALK has been found in many cancers, and is a key component in the development of malignant tumors.In 2007, the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion protein was first reported in Japanese patients with NSCLC and was subsequently indentified as an oncogenic driver in NSCLC. The EML4-ALK fusion rate is detected in approximately 6% of the western world patients with NSCLC, but it can occur at a higher rate of 9.3% in the Chinese non-smoker NSCLC patients. Because ALK’s tyrosine kinase activity is necessary for its transforming activity in tumorigenesis, targeting of ALK kinase activity via a kinase inhibitor represents a promising therapeutic approach for treating EML4-ALK positive NSCLC.c-Met is the cell surface receptor for hepatocyte growth factor (HGF). Genetic alteration and deregulation of c-Met have been linked to a variety of cancers and cancer metastasis.In NSCLC, approximately 4-5% non-smoker patients may harbor oncogenic Met driver mutation. In addition, c-Met gene amplification and protein over-expression in NSCLC are among the important mechanisms leading to clinical resistance to anti-EGFR therapy. In NSCLC patients with EGFR-TKI treatment, c-Met over expression or gene amplification occurs in up to 20%of patients’ tumor tissue. Hence, there is an intense interest in developing c-Met kinase inhibitor therapy for treating NSCLC and other c-Met-driven malignancies.In view of the importance of pharmacological targeting ALK and c-Met in NSCLC, in this thesis project, we have established and validated inhibitor screening platforms on both the biochemical and cellular levels. Employing these assay systems, we performed screening on several series of small molecule chemicals and have indentified a few novel small molecule inhibitors with potent activity against ALK and c-Met. As a major focus of this research, we studied preclinical pharmacology and antitumor activity of CM-118, a novel, potent and selective inhibitor dually targeting the oncogenic ALK and c-Met. Furthermore, we cloned and constructed several disease-related ALK constructs and introduced them into HEK-293 cells. Analysis of ALK-expressing cells versus vector-expressing cells resulted in a better understanding of signaling cascades that may contribute to the ALK-driven tumorigenesis.Part-I. Establishing the biochemical and cellular platforms for screening of novel ALK/c-Met inhibitorsObjective:To establish biochemical and cellular screening platforms for novel ALK/c-Met inhibitors.Methods:1) Biochemical enzymatic screen:We chose Homogeneous Time-Resolved Fluorescence (HTRF) as the routine kinase assay platform. First, the titration was carried out on the enzyme concentration and the enzyme reaction time of ALK and of c-Met, obtaining an optimal enzyme concentration (for which the signal reaches 80% of the maximum) and linear part of the kinase reaction time course (correlation coefficient R2> 0.99). We then employed the optimal enzyme concentration and reaction time, and performed ATP matrix titration to determine ATP Km values.2) Cellular platforms:We chose H2228 and H1993 NSCLC cell lines based on literature, we then performed Western analysis to confirm the ALK/c-Met target status, and determined the growth inhibition sensitivity using positive compound Crizotinib/PF-02341066.Results:1) ALK-HTRF kinase assay conditions:using 0.05 ng/μl ALK,50μM ATP and kinase reaction time of 30 minutes; c-Met-HTRF the Kinase Assay conditions: using 0.03 ng/μl c-Met,50μM ATP and kinase reaction time of 30 minutes.27 compounds were screened, of which two compounds (X6, X7) showed potent inhibition against ALK and c-Met kinase activity.2) H2228 is a model for EML4-ALK-driven NSCLC, H1993 is a model for c-Met driven NSCLC. These two NSCLC lines are highly sensitive to the positive control drug PF-02341066.Conclusions:1) We have validated the HTRF format biochemical assays for ALK and c-Met kinase inhibitors.2) We have validated NCSLC H2228 and HI993 cancer cell lines as the respective ALK- and c-Met-driven disease models for inhibitor screening and preclinical pharmacology studies.Part-II. Preclinical in vitro pharmacology studies of a novel ALK/c-Met dual inhibitor CM-118Objective:1) To evaluate CM-118’s potency in inhibiting ALK and c-Met enzyme activity; 2) To determine CM-118’s potency and selectivity profile in growth inhibition assays against a panel of human cancer lines; 3) Analysis of mechanism of CM-118 in targeting ALK, c-Met signaling pathways in NSCLC cell growth, survival and migration; 4) To explore the efficacy and mechanism of combination therapy with CM-118 and additional inhibitors.Methods:We performed HTRF format ALK and c-Met kinase assay with CM-118; 2) We then assessed CM-118 in cell proliferation assays in a cell panel including H2228, H1993 and other NSCLC lines; 3) We also constructed and transfected various ALK expression vectors into HEK-293 cells for determining CM-118’s ALK-targeting in cells; 4) We assayed CM-118 in HGF-stimulated c-Met phosphorylation activation in A549 cells; 5) We studied the effects and mechanisms of CM-118 in targeting c-Met and ALK signaling on NSCLC cell growth, survival and migration by Western blot, flow cytometry and Transwell migration experiments; 6) We combined CM-118 with EGFR inhibitor Afatinib or mTOR inhibitor Rapamycin to observe their impact on NSCLC cell survival.Results:1) CM-118 potently inhibited ALK and c-Met kinase catalytic activity with respective IC50 values of 60±10 nM and 40±5 nM; 2) CM-118 specifically inhibited the proliferation of EML4-ALK-driven H2228 and c-Met-driven H1993 NSCLC cells with respective IC50 values of 1.16±0.43 μM and 0.54±0.06μM, which are significantly lower than the other non-sensitive cell lines; 3) CM-118 can completely suppress the phosphorylation of the transiently over-expressed ALK in HEK-293 cells and HGF-induced c-Met phosphorylation in A549 cells; 4) In H2228 and H1993 NSCLC cells, CM-118 inhibited ALK, c-Met signaling, inactivated downstream signaling cascades of PI3K/mTOR, ERK and Stat3,and mediated growth inhibition via G1 cell cycle and loss of survival. CM-118 was able to significantly inhibit HGF-induced cell migration; 5) Combination treatment of CM-118 with EGFR kinase inhibitor Afatinib or mTOR inhibitor Rapamycin additively and/or synergistically enhanced cancer cell apoptosis.Conclusion:CM-118 is a potent and selective inhibitor dually targeting oncogenic ALK and c-Met activity. In NSCLC disease models, CM-118 blocks the oncogenic signaling of AKL and c-Met, inhibits the proliferation of ALK, c-Met-driven NSCLC H2228 and H1993 cells. There is an additive and/or synergistic antitumor activity when in combination with an EGFR inhibitor or mTOR inhibitor.Part-Ⅲ. Better understanding of ALK signaling mechanismObjective:To investigate the effects of expression of various oncogenic ALK proteins on downstream signaling pathways in HEK-293 cells.Methods:We transfected the full-length ALK and EML4-ALKvl fusion constructs into HEK-293 cells and analyzed ALK and several key signaling pathway molecule proteins by Western blot.Results:Over expression of both the full-length ALK and EML4-ALKvl fusion in HEK-293 can activate the ERK, mTOR and Stat3 signaling pathways. In particular, expression of EML4-ALKvl fusion resulted in a much more dramatic elevation of phosphorylation of Stat3 than that induced by the full-length ALK or ALK F1174L.Conclusion:While various oncogenic ALK constructs all activated signaling cascades of ERK and mTOR, the EML4-ALKvl induced a dramatic activation of Stat3, implicating an important role of Stat3 pathway in tumorigenesis of NSCLC.