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Evolved resistance to tyrosine kinase inhibitor (TKI)-targeted therapies remains a major clinical challenge. In epidermal growth factor receptor (EGFR) mutant non-small-cell lung cancer (NSCLC), failure of EGFR TKIs can result from both genetic and epigenetic mechanisms of acquired drug resistance. Widespread reports of histologic and gene expression changes consistent with an epithelial-to-mesenchymal transition (EMT) have been associated with initially surviving drug-tolerant persister cells, which can seed bona fide genetic mechanisms of resistance to EGFR TKIs. While therapeutic approaches targeting fully resistant cells, such as those harboring an EGFRT790M mutation, have been developed, a clinical strategy for preventing the emergence of persister cells remains elusive. Using mesenchymal cell lines derived from biopsies of patients who progressed on EGFR TKI as surrogates for persister populations, we performed whole-genome CRISPR screening and identified fibroblast growth factor receptor 1 (FGFR1) as the top target promoting survival of mesenchymal EGFR mutant cancers. Although numerous previous reports of FGFR signaling contributing to EGFR TKI resistance in vitro exist, the data have not yet been sufficiently compelling to instigate a clinical trial testing this hypothesis, nor has the role of FGFR in promoting the survival of persister cells been elucidated. In this study, we find that combining EGFR and FGFR inhibitors inhibited the survival and expansion of EGFR mutant drug-tolerant cells over long time periods, preventing the development of fully resistant cancers in multiple vitro models and in vivo. These results suggest that dual EGFR and FGFR blockade may be a promising clinical strategy for both preventing and overcoming EMT-associated acquired drug resistance and provide motivation for the clinical study of combined EGFR and FGFR inhibition in EGFR-mutated NSCLCs.

BackgroundResistance to therapy is a major problem in treating head and neck squamous cell carcinomas (HNSCC). Complement system inhibition has been shown to reduce tumor growth, metastasis, and therapeutic resistance in other tumor models, but has yet to be explored in the context of HNSCC. Here, we tested the effects of complement inhibition and its therapeutic potential in HNSCC.MethodsWe conducted our studies using two Human Papilloma Virus (HPV)-negative HNSCC orthotopic mouse models. Complement C3aR and C5aR1 receptor antagonists were paired with radiation therapy (RT). Tumor growth was measured and immune populations from tumor, lymph node, and peripheral blood were compared among various treatment groups. Genetically engineered mouse models DEREG and C3-/- were used in addition to standard wild type models. Flow cytometry, clinical gene sets, and in vitro assays were used to evaluate the role complement receptor blockade has on the immunological makeup of the tumor microenvironment.ResultsIn contrast to established literature, inhibition of complement C3a and C5a signaling using receptor antagonists accelerated tumor growth in multiple HNSCC cell lines and corresponded with increased frequency of regulatory T cell (Treg) populations. Local C3a and C5a signaling has importance for CD4 T cell homeostasis and eventual development into effector phenotypes. Interruption of this signaling axis drives a phenotypic conversion of CD4+ T cells into Tregs, characterized by enhanced expression of Foxp3. Depletion of Tregs reversed tumor growth, and combination of Treg depletion and C3a and C5a receptor inhibition decreased tumor growth below that of the control groups. Complete knockout of C3 does not harbor the expected effect on tumor growth, indicating a still undetermined compensatory mechanism. Dexamethasone is frequently prescribed to patients undergoing RT and inhibits complement activation. We report no deleterious effects associated with dexamethasone due to complement inhibition.ConclusionsOur data establish Tregs as a pro-tumorigenic driver during complement inhibition and provide evidence that targeted C3a and C5a receptor inhibition may add therapeutic advantage when coupled with anti-Treg therapy.

Purpose: KRAS-mutant lung cancers have been recalcitrant to treatments including those targeting the MAPK pathway. Covalent inhibitors of KRAS p.G12C allele allow for direct and specific inhibition of mutant KRAS in cancer cells. However, as for other targeted therapies, the therapeutic potential of these inhibitors can be impaired by intrinsic resistance mechanisms. Therefore, combination strategies are likely needed to improve efficacy. Experimental Design: To identify strategies to maximally leverage direct KRAS inhibition we defined the response of a panel of NSCLC models bearing the KRAS G12C–activating mutation in vitro and in vivo. We used a second-generation KRAS G12C inhibitor, ARS1620 with improved bioavailability over the first generation. We analyzed KRAS downstream effectors signaling to identify mechanisms underlying differential response. To identify candidate combination strategies, we performed a high-throughput drug screening across 112 drugs in combination with ARS1620. We validated the top hits in vitro and in vivo including patient-derived xenograft models. Results: Response to direct KRAS G12C inhibition was heterogeneous across models. Adaptive resistance mechanisms involving reactivation of MAPK pathway and failure to induce PI3K–AKT pathway inactivation were identified as likely resistance events. We identified several model-specific effective combinations as well as a broad-sensitizing effect of PI3K-AKT–mTOR pathway inhibitors. The G12Ci+PI3Ki combination was effective in vitro and in vivo on models resistant to single-agent ARS1620 including patient-derived xenografts models. Conclusions: Our findings suggest that signaling adaptation can in some instances limit the efficacy of ARS1620 but combination with PI3K inhibitors can overcome this resistance.

We present a cohort of 41 patients with osimertinib resistance biopsies, including 2 with an acquired CCDC6–RET fusion. Although RET fusions have been identified in resistant EGFR-mutant non–small cell lung cancer (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 tyrosine kinase inhibitors (TKI). The selective RET inhibitors BLU-667 and cabozantinib resensitized CCDC6–RET-expressing cells to EGFR inhibition. Finally, we treated 2 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. Significance: The role of RET fusions in resistant EGFR-mutant cancers is unknown. We report that RET fusions mediate resistance to EGFR inhibitors and demonstrate that this bypass track can be effectively targeted with a selective RET inhibitor (BLU-667) in the clinic. This article is highlighted in the In This Issue feature, p. 1494

BH3 mimetic drugs, which inhibit prosurvival BCL2 family proteins, have limited single-agent activity in solid tumor models. The potential of BH3 mimetics for these cancers may depend on their ability to potentiate the apoptotic response to chemotherapy and targeted therapies. Using a novel class of potent and selective MCL1 inhibitors, we demonstrate that concurrent MEK + MCL1 inhibition induces apoptosis and tumor regression in KRAS-mutant non–small cell lung cancer (NSCLC) models, which respond poorly to MEK inhibition alone. Susceptibility to BH3 mimetics that target either MCL1 or BCL-xL was determined by the differential binding of proapoptotic BCL2 proteins to MCL1 or BCL-xL, respectively. The efficacy of dual MEK + MCL1 blockade was augmented by prior transient exposure to BCL-xL inhibitors, which promotes the binding of proapoptotic BCL2 proteins to MCL1. This suggests a novel strategy for integrating BH3 mimetics that target different BCL2 family proteins for KRAS-mutant NSCLC. Significance: Defining the molecular basis for MCL1 versus BCL-xL dependency will be essential for effective prioritization of BH3 mimetic combination therapies in the clinic. We discover a novel strategy for integrating BCL-xL and MCL1 inhibitors to drive and subsequently exploit apoptotic dependencies of KRAS-mutant NSCLCs treated with MEK inhibitors. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

BackgroundNumerous trials combining radiation therapy (RT) and immunotherapy in head and neck squamous cell carcinoma (HNSCC) are failing. Using preclinical immune cold models of HNSCC resistant to RT-immune checkpoint inhibitors, we investigate therapeutic approaches of overcoming such resistance by examining the differential microenvironmental response to RT.MethodsWe subjected two HPV-negative orthotopic mouse models of HNSCC to combination RT, regulatory T cells (Treg) depletion, and/or CD137 agonism. Tumor growth was measured and intratumorous and lymph node immune populations were compared among treatment groups. Human gene sets, genetically engineered mouse models DEREG and BATF3–/–, flow and time-of-flight cytometry, RNA-Seq, Treg adoptive transfer studies, and in vitro experiments were used to further evaluate the role of dendritic cells (DCs) and Tregs in these treatments.ResultsIn MOC2 orthotopic tumors, we find no therapeutic benefit to targeting classically defined immunosuppressive myeloids, which increase with RT. In these radioresistant tumors, supplementing combination RT and Treg depletion with anti-CD137 agonism stimulates CD103+ DC activation in tumor-draining lymph nodes as characterized by increases in CD80+ and CCR7+ DCs, resulting in a CD8 T cell-dependent response. Simultaneously, Tregs are reprogrammed to an effector phenotype demonstrated by increases in interferonγ+, tumor necrosis factorα+, PI3K+, pAKT+ and Eomes+ populations as well as decreases in CTLA4+ and NRP-1+ populations. Tumor eradication is observed when RT is increased to an 8 Gy x 5 hypofractionated regimen and combined with anti-CD25+ anti-CD137 treatment. In a human gene set from oral squamous cell carcinoma tumors, high Treg number is associated with earlier recurrence.ConclusionsRegulating Treg functionality and DC activation status within the lymph node is critical for generating a T cell effector response in these highly radioresistant tumors. These findings underscore the plasticity of Tregs and represent a new therapeutic opportunity for reprogramming the tumor microenvironment in HNSCCs resistant to conventional radioimmunotherapy approaches.