We present a cohort of 41 patients with osimertinib resistance biopsies, including two with an acquired CCDC6-RET fusion. While RET fusions have been identified in resistant EGFR-mutant NSCLC, their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6-RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6-RET was sufficient to confer resistance to EGFR-TKIs. The selective RET inhibitors BLU-667 or cabozantinib resensitized CCDC6-RET-expressing cells to EGFR inhibition. Finally, we treated two patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well-tolerated and led to rapid radiographic response in both patients. This study provides proof-of-concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients.

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions,-ethyl--nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single mutations. In similar screens with EML4-ALK containing single resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance..

SummaryPersonalized cancer therapy is based on a patient’s tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient’s living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.

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Highlights d A living biobank of CAFs from NSCLC patients recapitulates clinical CAF heterogeneity d Therapeutic profiling of the NSCLC CAFs reveals three distinctive functional subtypes d Subtype I and II CAFs have high HGF and FGF7 expression and protect cancer cells d Subtype III CAFs associate with better clinical response and immune cell migration

Traumatic brain injury (TBI) results in systemic inflammatory responses that affect the lung. This is especially critical in the setting of lung transplantation where more than half of donor allografts are obtained postmortem from individuals with TBI. The mechanism by which TBI causes pulmonary dysfunction remains unclear but may involve the interaction of high mobility group box 1 (HMGB1) protein with the receptor for advanced glycation end products (RAGE). To investigate the role of HMGB1 and RAGE in TBI-induced lung dysfunction, RAGE sufficient (wildtype) or deficient (RAGE−/−) C57BL/6 mice were subjected to TBI through controlled cortical impact and studied for cardio-pulmonary injury. Compared to control animals, TBI induced systemic hypoxia, acute lung injury, pulmonary neutrophilia and decreased compliance, all of which were attenuated in RAGE −/− mice. Neutralizing systemic HMGB1, induced by TBI, reversed hypoxia and improved lung compliance. Compared to wildtype donors, lungs from RAGE−/− TBI donors did not develop acute lung injury after transplantation. In a study of clinical transplantation, elevated systemic HMGB1 in donors correlated with impaired systemic oxygenation of the donor lung pre-transplantation and predicted impaired oxygenation post-transplantation. These data suggest that the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces lung dysfunction and that targeting this pathway prior to transplant may improve recipient outcomes following lung transplantation.

Background Renal sympathetic denervation (RD) is a promising method of neuromodulation for the management of cardiac arrhythmia. Objective We tested the hypothesis that RD is antiarrhythmic in ambulatory dogs because it reduces the stellate ganglion nerve activity (SGNA) by remodeling the stellate ganglion (SG) and brain stem. Methods We implanted a radiotransmitter to record SGNA and electrocardiogram in 9 ambulatory dogs for 2 weeks, followed by a 2nd surgery for RD and 2 months SGNA recording. Cell death was probed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results Integrated SGNA at baseline, 1 and 2 months after RD were 14.0±4.0, 9.3±2.8 and 9.6±2.0 μV, respectively (p=0.042). The SG from RD but not normal control (N=5) dogs showed confluent damage. An average of 41±10% and 40±16% of ganglion cells in the left and right SG, respectively, were TUNEL-positive in RD dogs compared with 0% in controls dogs (p= 0.005 for both). Left and right SG from RD dogs had more tyrosine hydroxylase-negative ganglion cells than left SG of control dogs (p= 0.028 and 0.047 respectively). Extensive TUNEL positive neurons and glial cells were also noted in the medulla, associated with strongly positive glial fibrillary acidic protein staining. The distribution was heterogeneous, with more cell death in the medial than lateral aspects of the medulla. Conclusion Bilateral RD caused significant central and peripheral sympathetic nerve remodeling and reduced SGNA in ambulatory dogs. These findings may in part explain the antiarrhythmic effects of RD.

CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.