G1202R Mutation Research Articles

Abstract 2132: The novel, rationally-designed, ALK/SRC inhibitor TPX-0005 overcomes multiple acquired resistance mechanisms to current ALK inhibitors

Abstract Non-small cell lung cancers harboring rearrangements involving the ALK gene are sensitive to treatment with the ALK inhibitor crizotinib. However, the emergence of drug resistance is universal and limits clinical applicability. The mechanisms of resistance include ALK gene amplification, acquired ALK missense mutations, bypass activation, and epithelial-mesenchymal transition (EMT). More than ten acquired ALK mutations have been identified from patients and preclinical cell lines resistant to crizotinib. Although the second generation of ALK inhibitors (ceritinib and alectinib) overcome some of the crizotinib resistant mutations, they are plagued by a different spectrum of ALK resistant mutations. For example, the ALK G1202R mutation confers resistance to crizotinib, ceritinib, and alectinib. None of the current ALK inhibitors approved or in clinical trials can overcome bypass and EMT resistance mechanisms. SRC kinase has been identified to contribute broadly to primary intrinsic resistance to ALK inhibitor treatment, the development of bypass resistance and EMT during ALK inhibitor treatment. Saracatinib, a selective SRC inhibitor, can re-sensitize ALK inhibitor-resistant cell lines, suggesting a therapeutic role. There are currently no available ALK/SRC dual inhibitors, and we designed a novel ALK and SRC inhibitor TPX-0005 to address the need to overcome resistance. TPX-0005 is a novel three-dimensional macrocycle with a much smaller size (MW <370) than current ALK inhibitors designed to efficiently target the center of the ATP binding site and circumvent the steric interference from mutations outside the ATP binding boundary. As expected, TPX-0005 potently inhibited WT ALK (IC50 1.01 nM) and mutant ALKs including ALK G1202R (1.26 nM) and ALK L1196M (1.08 nM). Moreover, TPX-0005 effectively inhibited tumor growth in vivo in ALK WT and ALK G1202R xenografts. TPX-0005 is also a potent SRC inhibitor (IC50 5.3 nM). The elevated SRC kinase activity in H2228 lung cancer cell line confers resistance to crizotinib (IC50 1200 nM) and ceritinib (IC50 1000 nM) in cell proliferation assays. TPX-0005 effectively overcame this primary resistance (IC50 100 nM in cell proliferation assay) with strong inhibition of the phosphorylation of EML4-ALK (IC50 13 nM) and the SRC substrate paxillin (IC50 107 nM), along with other downstream signaling targets. TPX-0005 inhibited H2228 cell migration in a wound healing assay with similar activity to saracatinib. Taken together, TPX-0005 was able to not only inhibit the wild-type and a broad spectrum of mutant ALKs, but also overcome primary resistance and suppress metastatic features by inhibiting SRC. Overall, TPX-0005 has a highly favorable profile with ability to overcome multiple ALK resistance mechanisms including secondary mutations, bypass signaling activation, EMT, and warrants clinical investigation. Citation Format: Dayong Zhai, Wei Deng, Zhongdong Huang, Evan Rogers, J. Jean Cui. The novel, rationally-designed, ALK/SRC inhibitor TPX-0005 overcomes multiple acquired resistance mechanisms to current ALK inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2132.

Anaplastic Lymphoma Kinase (ALK) Kinase Domain Mutation Following ALK Inhibitor(s) Failure in Advanced ALK Positive Non–Small-Cell Lung Cancer: Analysis and Literature Review

BackgroundSecondary anaplastic lymphoma kinase (ALK) mutation may occur in patients with advanced ALK-positive non–small cell lung cancer treated with ALK inhibitors, but its nature is not well-known. Patients and MethodsWe analyzed tumor specimens after the failure of treatment with ALK inhibitor(s) (crizotinib, alectinib, and ceritinib) for secondary ALK kinase domain mutation, EGFR, K-ras, and PIK3CA mutations. The literature regarding acquired ALK-inhibitor(s) resistance was also reviewed. ResultsAmong 59 patients who received ALK inhibitor(s) during the period of December 2010 to April 2015, 7 had re-biopsied tumor specimens for analyses following ALK inhibitor(s) failure. One had G1202R after crizotinib and alectinib failure, and 6 were wild type. No EGFR, K-ras, or PIK3CA mutations were found. In our review of the literature and taken together with our patients, 25 of the 88 (28%) patients with crizotinib failure had secondary ALK mutation; L1196M mutation was most common (n = 11). Patients with secondary ALK mutation other than L1196M had a longer progression-free survival after crizotinib than patients with L1196M (median, 12.0 vs. 7.0 months; P = .04). Of the 9 patients with alectinib failure, 5 had I1171 mutation and 2 had G1202R. Of the 11 patients with ceritinib failure, 2 had G1202R, 1 had F1174C, and 1 had both G1202R and F1174V. I1171 mutation, G1202R, and F1174 mutations were also found in crizotinib-failed patients. ConclusionsSome acquired ALK mutations may cause co-resistance to other ALK inhibitors. Re-biopsy for ALK mutation analysis might be suggested prior to choosing a second-line ALK inhibitor treatment.

Molecular Changes Associated with Acquired Resistance to Crizotinib in ROS1-Rearranged Non-Small Cell Lung Cancer.

Although ROS1-rearranged non-small cell lung cancer (NSCLC) is sensitive to crizotinib, development of resistance is inevitable. Here, we identified molecular alterations in crizotinib-resistant tumors from two NSCLC patients with the CD74-ROS1 rearrangement, and in HCC78 cells harboring SLC34A2-ROS1 that showed resistance to crizotinib (HCC78CR cells). ROS1 kinase domain mutations were examined in fresh tumor tissues from two NSCLC patients and HCC78CR1-3 cells by direct sequencing. Ba/F3 cells expressing ROS1 secondary mutations were constructed to evaluate resistance to crizotinib. An upregulated pathway was identified using phospho-receptor tyrosine kinase array, EGFR signaling antibody array, and RNA sequencing (RNA-seq). Cell proliferation and ROS1 downstream signaling pathways were compared between HCC78 and HCC78CR1-3 cells. The ROS1 G2032R mutation was identified in crizotinib-resistant tumors from one patient. Furthermore, HCC78CR1 and CR2 cells harbored a novel ROS1 L2155S mutation (73.3% and 76.2%, respectively). ROS1 G2032R and L2155S mutations conferred resistance to crizotinib in Ba/F3 cells. Evidence of epithelial-to-mesenchymal transition with downregulated E-cadherin and upregulated vimentin was observed in HCC78CR1-2 cells and in the other patient. RNA-seq and EGFR signaling antibody array revealed that the EGFR pathway was significantly upregulated in HCC78CR3 versus HCC78 cells. Cells with the ROS1 mutation and upregulated EGFR were sensitive to foretinib, an inhibitor of c-MET, VEGFR2, and ROS1 and irreversible EGFR tyrosine kinase inhibitors plus crizotinib, respectively. Molecular changes associated with acquired crizotinib resistance in ROS1-rearranged NSCLC are heterogeneous, including ROS1 tyrosine kinase mutations, EGFR activation, and epithelial-to-mesenchymal transition.

An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket.

Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer.

ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required. The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor-resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1. We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations. We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance.

Abstract 3717: Therapeutic strategies to overcome the crizotinib resistance in ROS1-rearranged lung cancers

Abstract ROS1 fusion oncogene represent a novel molecular target in a small subset of non-small cell lung cancers (NSCLC).The ROS1 fusion oncogene, such as CD74-ROS1, leads to constitutive ROS1 activation with potent transforming activity. In the ongoing phase 1 clinical trial, the response rate of crizotinib, ALK/MET/ROS1 tyrosine kinase inhibitor (TKI), in the patients with NSCLC harboring ROS1 fusions was around 60%. However, despite crizotinib has shown remarkable initial responses, cancers eventually develop resistance to crizotinib. In a recent report of a clinical resistance to crizotinib, G2032R mutation in ROS1 kinase domain was identified, which is analogous to the G1202R mutation found in crizotinib-resistant ALK-rearranged lung cancers. To further identify the mechanism of resistance to crizotinib in ROS1 fusion positive NSCLC, we established a model of acquired resistance by ENU mutagenesis screening using CD74-ROS1 addicted Ba/F3 cell line models. As the results, we identified several novel crizotinib resistance mutations including G2032R mutation. To overcome the resistance, we performed the drug screening with small molecular inhibitors, and identified several inhibitors including Foretinib, cMET/VEGFR inhibitor, effectively inhibit the survival of CD74-ROS1 expressing cells. Furthermore, we also identified that some of those inhibitors can overcome the resistance caused by G2032R mutation. Thus, we have developed a comprehensive model of acquired resistance to crizotinib in ROS1 fusion harboring NSCLC and identified therapeutic strategies to overcome the resistance. Citation Format: Ryohei Katayama, Yuka Kobayashi, Sumie Koike, Naoya Fujita. Therapeutic strategies to overcome the crizotinib resistance in ROS1-rearranged lung cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3717. doi:10.1158/1538-7445.AM2014-3717

Abstract LB-218: Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC)

Abstract Background: Although ROS1-rearranged NSCLC is sensitive to crizotinib, a dual inhibitor of c-MET and ALK, an acquired resistance is inevitable. Recently, ROS1 G2032R mutation was identified in a patient with CD74-ROS1 rearranged NSCLC who developed an acquired resistance to crizotinib. Here, we identified novel molecular changes in crizotinib-resistant tumors from a NSCLC patient with CD74-ROS1 rearrangement and in HCC78 cells harboring SLC34A2-ROS1 fusion that showed crizotinib resistance (HCC78CR1, CR2 and CR3). Methods: HCC78CR1, -2 and -3 cells were generated by increasing dose of crizotinib to 1 μM. ROS1 kinase domain mutations were examined in fresh tumor tissues from a patient and in HCC78CR1, -2 and -3 cells by direct sequencing of individual clones. Whole-transcriptome sequencing (RNA-seq) was performed to identify up-regulated pathway in HCC78CR3 compared with HCC78. Cell proliferation, phosphorylation of RTKs and ROS1 downstream signaling pathways were compared among HCC78 and HCC78CR1, -2, -3 cell lines. BaF3 cells that constantly express ROS1 variants were constructed to evaluate a crizotinib resistance. Results: ROS1 G2032R in crizotinib-resistant tumor from a patient and L2155S from HCC78CR1, -2 cells were identified by direct sequencing of individual clones. ROS1 G2032R mutation was identified in 100% of cDNA clones of crizotinib-resistant tumors from a patient. However, this was not found in normal tissues and crizotinib-pretreatment tumor tissues. cDNA clones of HCC78CR1 and 2 cells harbored ROS1 L2155S mutations at a frequency of 73.3% and 76.2% respectively. Each BaF3 cells expressing ROS1 G2032R or L2155S mutation exhibited 128 and 10 fold higher IC50 value to crizotinib than those expressing wild-type CD74-ROS1, respectively. Downstream signaling molecules such as ERK and SHP2 were more activated in HCC78CR1, -2 and -3 cells than in HCC78 cells. In addition, HCC78CR1 and -2 cells displayed a spindle cell-like morphology with decreased E-cadherin and increased vimentin and fibronectin expression, which was not observed in HCC78 cells. RNA-seq and qRT-PCR demonstrated that EGFR mRNA expression levels were 2.6 and 5.1-fold up-regulated in HCC78CR3 cells than in HCC78 cells, respectively. EGFR signaling antibody array also revealed that EGFR pathway is significantly up-regulated in HCC78CR3 cells than in HCC78 cells. Conclusions: ROS1 G2032R and L2155S mutations are associated with acquired resistance to crizotinib in ROS1-rearranged NSCLC. In addition, epithelial-to-mesenchymal transition-like changes and EGFR activation may confer an acquired resistance to crizotinib. Citation Format: Ahn ah Song. Molecular changes associated with acquired resistance to crizotinib in ROS1-rearranged non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2014-LB-218

Abstract 4745: Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode

Abstract Chromosomal rearrangements resulting in oncogenic fusion proteins containing the ROS1 receptor tyrosine kinase have been described in subsets of a variety of human malignancies including non-small-cell lung cancer (NSCLC), cholangiocarcinoma, and glioblastoma multiforme. Promising clinical responses have been observed in patients bearing tumors with ROS1 fusions treated with Xalkori, an ALK/MET kinase inhibitor that also inhibits Ros1. However, resistance has been observed and a ROS1 kinase domain mutation, G2032R, was identified in a ROS1 fusion positive NSCLC patient who developed resistance to Xalkori treatment. Clinical experience with other receptor tyrosine kinase inhibitors suggests that additional resistance mutations are likely to arise, highlighting the need for therapeutic agents that can overcome this type of resistance. We identified a novel chemical series of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. The structure of human Ros1 in a complex with a ligand from this chemical class was confirmed by X-ray crystallography. Here we describe the structure-activity relationships and synthesis route for this chemical series. A representative compound from this series inhibited isolated recombinant Ros1 kinase activity with an IC50 of approximately 30 nM. This compound inhibited less than 6% of kinases in a panel of 400 at 1 μM concentration. Growth of Ba/F3 cells engineered to express Ros1 autophosphorylation in HCC78 NSCLC cells that harbor a SLC34A2-ROS1 fusion were inhibited at similar concentrations as the isolated protein. This activity translated into potent Ba/F3-Ros1 tumor growth inhibition in mice. This compound was also active on Ba/F3 cells containing Ros1 with a mutation in the gatekeeper residue, L2026M, in cell growth assays in vitro and tumor growth in vivo. Furthermore, this unique binding mode provides scope for activity on additional Ros1 mutations that confer resistance to Xalkori, such as the clinically relevant G2032R mutation. The results shown here describe a novel chemical series with a unique binding mode that has potential for activity in Ros1 driven tumors with mutations that confer resistance to Xalkori. Citation Format: Laurence Mevellec, Berthold Wroblowski, Ron Gilissen, Sophie Descamps, Elisabeth Pasquier, Christophe Adelinet, Marine Bourgeois, Guillaume Mercey, Matthieu Jeanty, Thierry Jousseaume, Aurélie Luguern, Javier Astray Gandara, Said Akzinnay, Etienne Daras, Inge Boeckx, Nele Van Slycken, Mariette Bekkers, Jeroen Van De Ven, Tinne Verhulst, Lieven Meerpoel, Jorge Vialard. Discovery of potent and selective Ros1 inhibitors with a unique DFG-out binding mode. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2014-4745

Next-Generation Sequencing Reveals a Novel NSCLC ALK F1174V Mutation and Confirms ALK G1202R Mutation Confers High-Level Resistance to Alectinib (CH5424802/RO5424802) in ALK-Rearranged NSCLC Patients Who Progressed on Crizotinib

Acquired secondary mutations in the anaplastic lymphoma kinase (ALK) gene have been identified in ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC) patients who developed disease progression while on crizotinib treatment. Here, we identified a novel secondary acquired NSCLC ALK F1174V mutation by comprehensive next-generation sequencing in one ALK+ NSCLC patient who progressed on crizotinib after a prolonged partial response to crizotinib. In a second case, we identified a secondary acquired ALK G1202R, which also confers resistance to alectinib (CH5424802/RO5424802), a second-generation ALK inhibitor that can inhibit ALK gatekeeper L1196M mutation in vitro. ALK G1202R is located at the solvent front of the ALK kinase domain and exhibits a high level of resistance to all other ALK inhibitors currently in clinical development in vitro. Comprehensive genomic profiling of resistant tumor is increasingly important in tailoring treatment decisions after disease progression on crizotinib in ALK+ NSCLC given the promise of second-generation ALK inhibitors and other therapeutic strategies.