KRAS GTPases are activated in one-third of cancers and KRAS G12C is the most common activating alteration in lung adenocarcinoma 1,2 . KRAS G12C inhibitors 3,4 are in Phase-I clinical trials and early data show partial responses in ~50% of lung cancer patients. 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][5][6] , and the inhibitors only bind to the latter, we tested if isogenic cell populations respond non-uniformly by studying the effect of treatment at Reprints and permissions information is available at www.nature.com/reprints.

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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.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplifi cation 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 coamplifi cation 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 amplifi cation or with acquisition of MET amplifi cation, 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 coamplifi ed 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-amplifi ed EG cancer. SIGNIFICANCE: Analysis of patients with ERBB2-amplifi ed, 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 coamplifi cation and MET amplifi cation, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones.

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 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 and as well 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.