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Neurofibromin (NF1) is a negative regulator of RAS signaling, frequently mutated in cancer. NF1-mutant melanoma is a highly malignant tumor for which targeted therapies are lacking. Here, we use biochemical and pharmacological assays on patient-derived models and isogenic cell lines to identify potential pharmacologic targets, revealing that NF1-null melanomas are dependent on RAS activation and that MEK inhibition relieves ERK-dependent negative feedback, increasing RAS signaling. MEK inhibition with avutometinib abrogates the adaptive rebound in ERK signaling, but the antitumor effects are limited. However, concurrent inhibition of MEK and SOS1 abrogates ERK activation, induces cell death, and suppresses tumor growth. In contrast to the NF1-deficient setting, concurrent SOS1 and SOS2 depletion is required to completely inhibit RAS signaling in NF1 wild-type cells. In sum, our data provide a mechanistic rationale for enhancing the therapeutic efficacy of MEK inhibitors by exploiting the lower residual SOS activity in NF1-null tumor cells., Competing Interests: Declaration of interests M.H.H., D.G., and F.S. are employees of Boehringer Ingelheim. T.M. is a consultant for Immunos Therapeutics, Daiichi Sankyo Co, TigaTX, Normunity, and Pfizer; is a cofounder of and equity holder in IMVAQ Therapeutics; has received research grants funding from Surface Oncology, Kyn Therapeutics, Infinity Pharmaceuticals, Peregrine Pharmaceuticals, Adaptive Biotechnologies, Leap Therapeutics, and Aprea Therapeutics; currently receives research funding from Bristol Myers Squibb, Enterome SA, and ReAlta Life Sciences; and is an inventor on patent applications related to work on oncolytic viral therapy, alpha virus-based vaccine, neoantigen modeling, CD40, GITR, OX40, PD-1, and CTLA-4. J.D.W. is a consultant for Apricity, Ascentage Pharma, AstraZeneca, BeiGene, Bicara Therapeutics, Bristol Myers Squibb, Daiichi Sankyo, Dragonfly, Imvaq, Larkspur, Takeda, Tizona, Trishula Therapeutics, Immunocore (on their data safety board), and Scancell; received grant/research support from Bristol Myers Squibb and Enterome; has equity in Apricity, Arsenal IO/CellCarta, Ascentage, Imvaq, Linneaus, Larkspur, Georgiamune, Maverick, Tizona Therapeutics, and XenImmune; and is an inventor on the following patents: Xenogeneic DNA Vaccines, Newcastle Disease Viruses for Cancer Therapy, Myeloid-Derived Suppressor Cell Assay, Prediction of Responsiveness to Treatment with Immunomodulatory Therapeutics and Method of Monitoring Abscopal Effects During Such Treatment, Anti-PD1 Antibody, Anti-CTLA4 Antibodies, and Anti-GITR Antibodies and Methods of Use Thereof. Z.Y. is a past scientific advisory board (SAB) member of MapKure and currently an employee of Loxo Oncology at Lilly. D.S. reports personal fees from Pfizer, Vividion Therapeutics, BridgeBio, FogPharma, FORE Therapeutics, Scorpion Therapeutics, and Rain Therapeutics outside the submitted work. S.M. reports grants from Boehringer Ingelheim and Daiichi Sankyo outside the submitted work and is a SAB member of Bionseek. N.R. is on the SAB and owns equity in BeiGene, Zai Labs, MapKure, Ribon, and Effector; is also on the SAB of AstraZeneca and Chugai and a past SAB member of Novartis, Millennium-Takeda, Kura, and Araxes; and is a consultant to Revolution Medicines, Tarveda, Array-Pfizer, Boehringer Ingelheim, and Eli Lilly. He receives research funding from Revolution Medicines, AstraZeneca, Array Pfizer, and Boehringer Ingelheim and owns equity in Kura Oncology and Fortress., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Increased insight into the mutational landscape of squamous cell lung cancers (LUSCs) in the past decade has not translated into effective targeted therapies for patients with this disease. NRF2, encoded by NFE2L2, and its upstream regulator, KEAP1, control key aspects of redox balance and are frequently mutated in NSCLCs., Methods: Here, we describe the specific potent activity of TAK-228, a TORC1/2 inhibitor, in NSCLC models harboring NRF2-activating alterations and results of a phase 2 clinical trial of TAK-228 in patients with advanced NSCLC harboring NRF2-activating alterations including three cohorts (NFE2L2-mutated LUSC, KEAP1-mutated LUSC, KRAS/NFE2L2- or KEAP1-mutated NSCLC)., Results: TAK-228 was most efficacious in a LUSC cohort with NFE2L2 alterations; the overall response rate was 25% and median progression-free survival was 8.9 months. Additional data suggest that concurrent inhibition of glutaminase with the glutaminase inhibitor CB-839 might overcome metabolic resistance to therapy in these patients., Conclusions: TAK-228 has single-agent activity in patients with NRF2-activated LUSC. This study reframes oncogenic alterations as biologically relevant based on their downstream effects on metabolism. This trial represents, to the best of our knowledge, the first successful attempt at metabolically targeting NSCLC and identifies a promising targeted therapy for patients with LUSC, who are bereft of genotype-directed therapies., (Copyright © 2022 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression. Serial analysis of patient blood samples on treatment demonstrates that most of these alterations are detected at a low frequency except for KRASG12C amplification, a recurrent resistance mechanism that rises in step with clinical progression. Upon drug withdrawal, resistant cells with KRASG12C amplification undergo oncogene-induced senescence, and progressing patients experience a rapid fall in levels of this alteration in circulating DNA. In this new state, drug resumption is ineffective as mTOR signaling is elevated. However, our work exposes a potential therapeutic vulnerability, whereby therapies that target the senescence response may overcome acquired resistance., Significance: Clinical resistance to KRASG12C-EGFR inhibition primarily prevents suppression of ERK signaling. Most resistance mechanisms are subclonal, whereas KRASG12C amplification rises over time to drive a higher portion of resistance. This recurrent resistance mechanism leads to oncogene-induced senescence upon drug withdrawal and creates a potential vulnerability to senolytic approaches. This article is highlighted in the In This Issue feature, p. 1., (©2022 The Authors; Published by the American Association for Cancer Research.)

Immune checkpoint blockade (ICB) has improved outcomes for patients with advanced cancer, but the determinants of response remain poorly understood. Here we report differential effects of mutations in the homologous recombination genes BRCA1 and BRCA2 on response to ICB in mouse and human tumors, and further show that truncating mutations in BRCA2 are associated with superior response compared to those in BRCA1. Mutations in BRCA1 and BRCA2 result in distinct mutational landscapes and differentially modulate the tumor-immune microenvironment, with gene expression programs related to both adaptive and innate immunity enriched in BRCA2-deficient tumors. Single-cell RNA sequencing further revealed distinct T cell, natural killer, macrophage, and dendritic cell populations enriched in BRCA2-deficient tumors. Taken together, our findings reveal the divergent effects of BRCA1 and BRCA2-deficiency on ICB outcome, and have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy.

Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively). To define the heterogeneity of tumors and their associated microenvironments across subtypes, we sequenced 155,098 transcriptomes from 21 human biospecimens, including 54,523 SCLC transcriptomes. We observe greater tumor diversity in SCLC than lung adenocarcinoma, driven by canonical, intermediate, and admixed subtypes. We discover a PLCG2-high SCLC phenotype with stem-like, pro-metastatic features that recurs across subtypes and predicts worse overall survival. SCLC exhibits greater immune sequestration and less immune infiltration than lung adenocarcinoma, and SCLC-N shows less immune infiltrate and greater T cell dysfunction than SCLC-A. We identify a profibrotic, immunosuppressive monocyte/macrophage population in SCLC tumors that is particularly associated with the recurrent, PLCG2-high subpopulation., Competing Interests: Declaration of interests J.M.C. reports an advisory role in VantAI. A.Q.-V. reports honoraria from AstraZeneca. M.O. reports advisory roles for PharMar, Novartis, and Targeted Oncology, and reports honoraria from Bristol-Myers Squibb and Merck Sharp & Dohme. C.M.R. has consulted regarding oncology drug development with AbbVie, Amgen, Ascentage, AstraZeneca, Bicycle, Celgene, Daiichi Sankyo, Genentech/Roche, Ipsen, Jazz, Lilly, Pfizer, PharmaMar, Syros, and Vavotek. C.M.R. serves on the scientific advisory boards of Bridge Medicines, Earli, and Harpoon Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: ROS1 tyrosine kinase inhibitors (TKI) provide significant benefit in lung adenocarcinoma patients with ROS1 fusions. However, as observed with all targeted therapies, resistance arises. Detecting mechanisms of acquired resistance (AR) is crucial to finding novel therapies and improve patient outcomes., Experimental Design: ROS1 fusions were expressed in HBEC and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, SLC34A2/ROS1) or CRISPR-Cas9-mediated genomic engineering (EZR/ROS1). We reviewed targeted large-panel sequencing data (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, and genetic alterations hypothesized to confer AR were modeled in these cell lines., Results: Eight of the 75 patients with a ROS1 fusion had a concurrent MAPK pathway alteration and this correlated with shorter overall survival. In addition, the induction of ROS1 fusions stimulated activation of MEK/ERK signaling with minimal effects on AKT signaling, suggesting the importance of the MAPK pathway in driving ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and 2 patients harbored NF1 loss-of-function mutations. Expression of MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion-positive cells activated MEK/ERK signaling and conferred resistance to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited growth of cells expressing both ROS1 fusion and MEK1del., Conclusions: We demonstrate that downstream activation of the MAPK pathway can mediate of innate acquired resistance to ROS1 TKIs and that patients harboring ROS1 fusion and concurrent downstream MAPK pathway alterations have worse survival. Our findings suggest a treatment strategy to target both aberrations., (©2020 American Association for Cancer Research.)

Amplification of and oncogenic mutations in ERBB2 , the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2 -amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2 . This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy. See related commentary by Rolfo and Russo, p. 643 . This article is highlighted in the In This Issue feature, p. 627 ., (©2020 American Association for Cancer Research.)

KRAS GTPases are activated in one-third of cancers, and KRAS(G12C) is one of the most common activating alterations in lung adenocarcinoma 1,2 . KRAS(G12C) inhibitors 3,4 are in phase-I clinical trials and early data show partial responses in nearly half of patients with lung cancer. How cancer cells bypass inhibition to prevent maximal response to therapy is not understood. Because KRAS(G12C) cycles between an active and inactive conformation 4-6 , and the inhibitors bind only to the latter, we tested whether isogenic cell populations respond in a non-uniform manner by studying the effect of treatment at a single-cell resolution. Here we report that, shortly after treatment, some cancer cells are sequestered in a quiescent state with low KRAS activity, whereas others bypass this effect to resume proliferation. This rapid divergent response occurs because some quiescent cells produce new KRAS(G12C) in response to suppressed mitogen-activated protein kinase output. New KRAS(G12C) is maintained in its active, drug-insensitive state by epidermal growth factor receptor and aurora kinase signalling. Cells without these adaptive changes-or cells in which these changes are pharmacologically inhibited-remain sensitive to drug treatment, because new KRAS(G12C) is either not available or exists in its inactive, drug-sensitive state. The direct targeting of KRAS oncoproteins has been a longstanding objective in precision oncology. Our study uncovers a flexible non-uniform fitness mechanism that enables groups of cells within a population to rapidly bypass the effect of treatment. This adaptive process must be overcome if we are to achieve complete and durable responses in the clinic.

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers 1 . Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer 1-3 . Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize 4-8 . We show here that PLX8394, a new RAF inhibitor 9 , inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF-CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the amino (N)-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants as well as BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2 . Heterogeneous 89 Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2 -amplified EG cancer. SIGNIFICANCE: Analysis of patients with ERBB2 -amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR / ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones. See related commentary by Klempner and Catenacci, p. 166 . This article is highlighted in the In This Issue feature, p. 151 ., (©2018 American Association for Cancer Research.)

Patient-derived xenograft (PDX) models have recently emerged as a highly desirable platform in oncology and are expected to substantially broaden the way in vivo studies are designed and executed and to reshape drug discovery programs. However, acquisition of patient-derived samples, and propagation, annotation and distribution of PDXs are complex processes that require a high degree of coordination among clinic, surgery and laboratory personnel, and are fraught with challenges that are administrative, procedural and technical. Here, we examine in detail the major aspects of this complex process and relate our experience in establishing a PDX Core Laboratory within a large academic institution.