Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors

Abstract

Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1-and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1 G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1-and ALK-driven NSCLC and other malignancies.onstitutively activated kinase fusion proteins that arise from somatic chromosomal rearrangements are frequent drivers of malignant transformation in cancer and represent a targetable vulnerability for clinical intervention. The clinical success of the tyrosine kinase inhibitor (TKI) imatinib in targeting the oncogenic BCR-ABL1 fusion protein in chronic myeloid leukemia (CML) motivated efforts to identify and target oncogenic kinases in other cancers (1-3). One such setting is non-small cell lung cancer (NSCLC), where chromosomal rearrangements of the receptor tyrosine kinase (RTK) anaplastic lymphoma kinase (ALK) are found in 4-5% of patients (4, 5). The validation of rearranged ALK as an oncogenic driver prompted the discovery and clinical implementation of crizotinib as the first clinical targeted inhibitor for use in ALK fusionpositive NSCLC (6, 7).Fusion proteins involving the highly related kinase ROS1, an orphan RTK of the insulin receptor family, are present in ∼1% of NSCLC patients. ROS1 rearrangements span a variety of fusion partners across several other epithelial malignancies, including cholangiocarcinoma, gastric cancer, and ovarian cancer (4, 8). CD74-ROS1 is the most frequent ROS1 fusion detected in NSCLC. ROS1 fusion proteins are transforming drivers that contribute to tumorigenesis or tumor progression in multiple experimental model systems (9)(10)(11).Approximately 75,000 and 15,000 new NSCLC patients per year are anticipated to harbor tumors...

Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14 Mutations and Overcomes Mutation-mediated Resistance to Type I MET Inhibitors in Nonclinical Models

“…In contrast, the increased allelic cfDNA burden for MET D1228N and G1163R, increased MET amplification, and emergence of MET L1195V suggest that subclonal lesions harboring these genetic variants were nonresponsive to glesatinib treatment. The observation of MET G1163R prior to glesatinib treatment is consistent with the decreased activity of crizotinib against this MET mutation, which is analogous to the ALK G1202R and ROS1 G2302R mutations implicated in crizotinib resistance in ALK and ROS1 rearrangement positive cancers, respectively (40). The emergence of the L1195V mutation during glesatinib treatment is consistent with the 5-7-fold loss of potency for both glesatinib and crizotinib and suggestive of the continued progression of the lesion(s) harboring this variant despite continued treatment.…”

Recent Advances in Targeting ROS1 in Lung Cancer

“…Like crizotinib and ceritinib, it also has anti-ROS1 activity based on preclinical studies, with an IC 50 of 7.5 nM (versus anti-ALK IC 50 of 9.8 nM) in CD74-ROS1 -expressing Ba/F3 cells (80). In a recently reported phase 1/2 study of brigatinib, 3 patients with ROS1 -rearranged NSCLC were enrolled.…”

“…The activity of brigatinib against resistant ROS1 mutants has thus far appeared comparable to ceritinib based on Ba/F3 models. In vitro , brigatinib inhibits the L2026M mutation, but not G2032R, D2033N, or L1951R (64, 67, 80, 81), raising the concern that similarly to ceritinib, it likely has limited activity against crizotinib-resistant ROS1 -driven tumors (Figure 4B). The most common treatment-emergent AEs for brigatinib have consisted of nausea, diarrhea, headache, and cough; however, it has notably been associated with early pulmonary events including radiographic changes consistent with pneumonitis (78, 79).…”

“…However, lorlatinib’s activity against the ROS1 G2032R mutation may be limited in the clinic. Based on preclinical studies, G2032R appears to significantly reduce the cellular potency of lorlatinib, with IC 50 ranging from 177 nM to 508 nM in ROS1 -rearranged Ba/F3 models (as compared to cellular IC 50 of 0.5–1 nM against wild-type ROS1) (65, 80, 81). Therefore, lorlatinib could be clinically active in select patients post-crizotinib, but may have limited efficacy in the subset of patients with tumors known to harbor G2032R.…”

Stratified Treatment in Lung Cancer

“…Following the same biological rules, targeted therapy in ROS1 -positive patients results in the acquisition of resistance [64,65,66,67,68]. Unlike EGFR and ALK TKI resistance, resistance to crizotinib in ROS1 -positive patients is not well understood.…”