Purpose Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential. Experimental Design We report the discovery of a novel, solvent-front ROS1D2033N mutation in a patient with CD74-ROS1-rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib-resistance of CD74-ROS1D2033N was functionally evaluated using cell based assays and structural modelling. Results In biochemical and cell-based assays, the CD74-ROS1D2033N mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near complete radiographic response in this patient. Conclusions These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer.

One of the greatest challenges in biomedical research, drug discovery and diagnostics is understanding how seemingly identical cells can respond differently to perturbagens including drugs for disease treatment. Although heterogeneity has become an accepted characteristic of a population of cells, in drug discovery it is not routinely evaluated or reported. The standard practice for cell-based, high content assays has been to assume a normal distribution and to report a well-to-well average value with a standard deviation. To address this important issue we sought to define a method that could be readily implemented to identify, quantify and characterize heterogeneity in cellular and small organism assays to guide decisions during drug discovery and experimental cell/tissue profiling. Our study revealed that heterogeneity can be effectively identified and quantified with three indices that indicate diversity, non-normality and percent outliers. The indices were evaluated using the induction and inhibition of STAT3 activation in five cell lines where the systems response including sample preparation and instrument performance were well characterized and controlled. These heterogeneity indices provide a standardized method that can easily be integrated into small and large scale screening or profiling projects to guide interpretation of the biology, as well as the development of therapeutics and diagnostics. Understanding the heterogeneity in the response to perturbagens will become a critical factor in designing strategies for the development of therapeutics including targeted polypharmacology.

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...

Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies. Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an murine xenograft study. analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate-fusions from TCGA glioblastoma dataset. fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC-ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L-ROS1 and GOPC-ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line. Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.