Potent and EGFRWT Sparing HER2 Inhibitors for the Treatment of HER2 Exon 20 Insertion-Driven Tumors

Activating mutations in ERBB receptors act as oncogenes in non-small cell lung cancer (NSCLC). Besides the more prevalent epidermal growth factor receptor (EGFR) activating mutations, oncogenic variants in human epidermal growth factor receptor 2 (HER2) occur in approximately 2% of NSCLC patients. HER2 oncogenic mutations in NSCLC predominantly affect the tyrosine kinase domain and cluster in exon 20 of the ERBB2 gene. Most clinically approved and currently tested tyrosine kinase inhibitors are limited by either insufficient potency on exon 20 altered HER2 and/or their insufficient selectivity against EGFR wild type (EGFR WT) – a major cause of dose limiting toxicity. We report herein the discovery of covalent tyrosine kinase inhibitors that potently inhibit HER2 exon 20 mutants while sparing EGFR WT. The new inhibitors presented in this study reduce tumor cell survival and proliferation in vitro, and result in regressions in in vivo preclinical xenograft models of HER2 exon 20 mutant NSCLC as well as inhibition of downstream signaling. Our results suggest that HER2 exon 20 insertion driven tumors can be effectively treated by a potent and highly selective HER2 inhibitor while sparing EGFR WT. The new inhibitors described in this study pave the way for testing potent HER2 selective inhibitors in HER2 mutant NSCLC patients and may offer an additional therapeutic option for these patients. In-vitro (cell lines: NCI-H2170 HER2-YVMA, NCI-H1781, OE19, and AU565) and in-vivo (patient-derived xenograft: CTG-2543) gene expression profiling under compound treatment. Groups include control (DMSO, Natrosol) and compound treated samples (Poziotinib, BI-4142, and BI-1622). CRISPR/Cas9 genome editing was used to generate NCI-H2170 HER2-YVMA cells. The HER2-YVMA variant was knocked into the endogenous HER2 locus in the HER2 amplified (HER2amp) NCI-H2170 cells to overexpress this variant. Sampling time points, compound concentrations, and biological replicates are detailed out in the respective sample descriptions. All samples were subjected to RNA isolation, followed by library preparation (QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina, Lexogen), and messenger RNA sequencing on an Illumina NextSeq 500 system.