Michele McTigue

KIT kinase mutants show unique mechanisms of drug resistance to imatinib and sunitinib in gastrointestinal stromal tumor patients.

Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors. Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance.

Acquired Resistance to Crizotinib from a Mutation in CD74–ROS1

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Resensitization to Crizotinib by the Lorlatinib Alk Resistance Mutation L1198F.

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

Multiple Mutations and Bypass Mechanisms Can Contribute to Development of Acquired Resistance to MET Inhibitors

Therapies targeting receptor tyrosine kinases have shown efficacy in molecularly defined subsets of cancers. Unfortunately, cancers invariably develop resistance, and overcoming or preventing resistance will ultimately be key to unleashing their full therapeutic potential. In this study, we examined how cancers become resistant to MET inhibitors, a class of drugs currently under clinical development. We utilized the highly sensitive gastric carcinoma cell line, SNU638, and two related MET inhibitors PHA-665752 and PF-2341066. To our surprise, we observed at least two mechanisms of resistance that arose simultaneously. Both resulted in maintenance of downstream PI3K (phosphoinositide 3-kinase)-AKT and MEK (MAP/ERK kinase)-ERK signaling in the presence of inhibitor. One mechanism, observed by modeling resistance both in vitro and in vivo, involved the acquisition of a mutation in the MET activation loop (Y1230). Structural analysis indicates that this mutation destabilizes the autoinhibitory conformation of MET and abrogates an important aromatic stacking interaction with the inhibitor. The other cause of resistance was activation of the epidermal growth factor receptor (EGFR) pathway due to increased expression of transforming growth factor α. Activation of EGFR bypassed the need for MET signaling to activate downstream signaling in these cells. This resistance could be overcome by combined EGFR and MET inhibition. Thus, therapeutic strategies that combine MET inhibitors capable of inhibiting Y1230 mutant MET in combination with anti-EGFR-based therapies may enhance clinical benefit for patients with MET-addicted cancers. Importantly, these results also underscore the notion that a single cancer can simultaneously develop resistance induced by several mechanisms and highlight the daunting challenges associated with preventing or overcoming resistance.

Axitinib effectively inhibits BCR-ABL1(T315I) with a distinct binding conformation.

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30–50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR–ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR–ABL1-driven leukaemia. Axitinib potently inhibited BCR–ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.

Chapter Twenty-Five – Case History: Xalkori™ (Crizotinib), a Potent and Selective Dual Inhibitor of Mesenchymal Epithelial Transition (MET) and Anaplastic Lymphoma Kinase (ALK) for Cancer Treatment

Abstract Xalkori™ (crizotinib, PF-02341066), a receptor tyrosine kinase (RTK) inhibitor targeting mesenchymal epithelial transition (MET)/anaplastic lymphoma kinase (ALK), was invented using structure-based drug design and medicinal chemistry lead optimization. The U.S. Food and Drug Administration granted fast-track approval of crizotinib on August 26, 2011 based on the marked efficacy of crizotinib in patients with ALK-positive advanced non-small cell lung cancer (NSCLC) and good safety profile in Phases I and II trials. Promising antitumor activity has been observed with crizotinib in patients with abnormal ALK genes of anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, and neuroblastoma; with abnormal ROS1 gene in NSCLC; and with MET gene amplified NSCLC. The broad antitumor activities of crizotinib based on molecular targets across many cancers indicate the importance of understanding tumor biology to identify the oncogenic driver targets for the stratification of the right patient population.

Abstract PR10: Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations.

Oncogenic fusions of anaplastic lymphoma kinase (ALK) define a subset of human lung adenocarcinoma. The 1st generation ALK inhibitor crizotinib demonstrated impressive clinical benefit in ALK-fusion positive lung cancers and was approved by the FDA for the treatment of ALK-fusion positive NSCLC in 2011. However, as seen with most kinase inhibitors, patients treated with crizotinib eventually develop resistance to therapy. Acquired ALK kinase domain mutations and disease progression in the central nervous system (CNS) are reported as main contributors to patient relapse after ALK inhibitor therapy. Preclinically, crizotinib lacks significant brain penetration and does not potently inhibit activity of ALK kinase domain mutants, so a drug discovery program was initiated aimed to develop a second generation ALK inhibitor that is more potent than existing ALK inhibitors, capable of inhibiting the resistant ALK mutants and penetrating the blood-brain-barrier. These objectives present a considerable challenge in kinase inhibitor chemical space. Here we report that PF-06463922, a novel small molecule ATP-competitive inhibitor of ALK/ROS1, showed exquisite potencies against non-mutant ALK (Ki 100 fold kinase selectivity against 95% of the kinases tested in a 207 recombinant kinase panel. Specific design considerations were developed leading to novel ATP-competitive kinase inhibitors with desired low efflux in cell lines over-expressing p-glycoprotein and breast cancer resistance protein, providing excellent blood-brain-barrier and cell penetration properties. Efforts to optimize ligand efficiency and lipophilic efficiency leveraging structure based drug design techniques led to ligands with overlapping broad spectrum potency and low efflux. Single and repeat dose preclinical rat in vivo studies of PF-06463922 demonstrated excellent oral bioavailability and CNS availability with free brain exposure approximately 30% of free plasma levels. In addition, CNS-directed safety studies showed no adverse events at predicted efficacious concentrations. It is anticipated that PF-06463922 with its potent activities on non-mutant ALK, ALK kinase domain mutations and CNS metastases would provide great promise for patients with ALK and ROS1 positive cancers.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):PR10.nnCitation Format: Ted W. Johnson, Simon Bailey, Benjamin J. Burke, Michael R. Collins, J. Jean Cui, Judy Deal, Ya-Li Deng, Martin P. Edwards, Mingying He, Jacqui Hoffman, Robert L. Hoffman, Qinhua Huang, Robert S. Kania, Phuong Le, Michele McTigue, Cynthia L. Palmer, Paul F. Richardson, Neal W. Sach, Graham L. Smith, Lars Engstrom, Wenyue Hu, Hieu Lam, Justine L. Lam, Tod Smeal, Helen Y. Zou. Is CNS availability for oncology a no-brainer? Discovery of PF-06463922, a novel small molecule inhibitor of ALK/ROS1 with preclinical brain availability and broad spectrum potency against ALK-resistant mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr PR10.

Reviving B-Factors: Retrospective Normalized B-Factor Analysis of C-ros Oncogene 1 Receptor Tyrosine Kinase and Anaplastic Lymphoma Kinase L1196M with Crizotinib and Lorlatinib

Structure-based drug design (SBDD) is commonly leveraged in rational drug design. Usually, ligand and binding site atomic coordinates from crystallographic data are exploited to optimize potency and selectivity. In addition to traditional, static views of proteins and ligands, we propose using normalized B-factors to study protein dynamics as a part of the drug optimization process. A retrospective case study of crizotinib and lorlatinib bound to both c-ros oncogene 1 kinase (ROS1) and anaplastic lymphoma kinase (ALK) L1196M related normalized B-factors to differences in binding affinity. This analysis showed that ligand binding can have protein-stabilizing effects that start near the ligand but propagate through nearby residues and structural waters to more distal motifs. The potential opportunities for analyzing normalized B-factors in SBDD are also discussed.

Reviving B-Factors: Activating ALK Mutations Increase Protein Dynamics of the Unphosphorylated Kinase

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics. Crystallographic B-factors were converted to normalized B-factors, which allowed analysis of wildtype ALK, ALK-C1156Y, and ALK-L1196M. This data suggests that mobility of the P-loop, αC-helix, and activation loop (A-loop) may be important in catalytic activity increases, with or without phosphorylation. Both molecular dynamics simulations and hydrogen-deuterium exchange experimental data corroborated the normalized B-factors data.

Abstract 4456: Biochemical and structural characteristics of crizotinib-resistant ALK mutants.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnAnaplastic lymphoma kinase (ALK), when aberrantly regulated through gene translocations or mutations, has been implicated as an oncogenic driver in a variety of cancers. XALKORI (crizotinib) is an FDA-approved therapy for locally advanced or metastatic (ALK)-positive non-small cell lung cancer (NSCLC). Mutations of certain residues within the ALK kinase domain have been associated with crizotinib resistance. To understand the molecular basis of mutational resistance, we have kinetically and structurally characterized several clinical mutations of ALK kinase domain. In the basal-state (nonphosphorylated) proteins, mutations C1156Y, F1174L, L1152R and L1196M resulted in 14 to 52-fold increase in catalytic efficiency of phosphorylation of an activation loop peptide. Accordingly, these mutants were more rapidly autophosphorylated, compared to wild-type enzyme. In addition, F1174L, C1156Y and L1156Y had 2 to 6-fold higher catalytic efficiencies when fully activated by autophosphorylation. Conversely, catalytic efficiencies of nonphosphorylated and phosphorylated G1269A variant decreased by 1.2 and 1.6-fold, respectively. Inhibition of L1152R, L1196M and G1269A by crizotinib was reduced by 3-28-fold, compared to wild-type enzyme, from Ki determinations. From direct binding studies of the L1196M “gatekeeper” mutation, the loss of crizotinib binding occurred 6 and 13-fold for phosphorylated and nonphosphorylated proteins, respectively. By crystallographic studies apoenzyme and crizotinib complexes of ALK kinase domain displayed an inactive kinase conformation that is stabilized by an extended hydrophobic network of residues. F1174 and L1196 are part of this hydrophobic core, and mutations of these residues are predicted to destabilize the inactive ALK conformation. Taken together, these results suggest that most of the resistant mutations (e.g. L1196M, F1174L, C1156Y, L1152R) result in a more dynamic protein that increases substrate turnover. In addition, the reduction of crizotinib binding for the mutations in the vicinity of the inhibitor binding site (L1196M, G1269A) is likely a contributing factor for the resistance. This structural and kinetic analysis of mutational resistance may be useful for design of new inhibitors targeting multiple clinical mutations of ALK.nnCitation Format: Sergei Timofeevski, Wei Liu, Ya-Li Deng, Alexei Brooun, Simon Bergqvist, Shannon Karlicek, Brion Murray, Ben Bolanos, Michele McTigue. Biochemical and structural characteristics of crizotinib-resistant ALK mutants. [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 4456. doi:10.1158/1538-7445.AM2013-4456