Abstract Background: The HER2 tyrosine kinase inhibitor (TKI) neratinib has exhibited clinical activity in patients with metastatic HER2-mutant cancers. However, responses are heterogeneous across tumor types and not generally prolonged, suggesting mechanisms of de novo and acquired drug resistance. Methods: Neratinib-resistant 5637 (HER2S310F) bladder cancer and OVCAR8 (HER2G776V) ovarian cancer cells were developed after gradual dose escalation. Candidate pathways associated with drug resistance identified by RNA sequencing were validated in a panel of HER2-mutant cell lines and in the SUMMIT basket trial in patients with HER2-mutant cancers. Results: Neratinib-resistant 5637 and OVCAR8 cells were cross-resistant to the HER2 TKIs afatinib and lapatinib. Immunoblot analysis showed that neratinib was still able to suppress HER2, EGFR and HER3 phosphorylation. Gene Set Enrichment and Connectivity Map analyses of RNA-seq data suggested mTORC1 signaling as a druggable pathway driving neratinib resistance. Immunoblot analysis of drug-resistant cells revealed a striking increase in S6K and S6 phosphorylation compared to parental cells. P-S6 levels and viability of drug resistant cells/tumors were ablated upon combining neratinib with the TORC1i everolimus both in vitro and in vivo. Similar results were obtained in cells transfected with Raptor or Rheb siRNAs. Further, neratinib resistance was induced by TSC2 knockdown and resultant TORC1 hyperactivation in parental 5637, OVCAR8, and MCF7 cells expressing L755S or V777L HER2 mutations. RNA-seq also revealed significant enrichment of RAS pathway in neratinib resistant cells which was confirmed by RAS-GTP pulldown. Pharmacological inhibition of RAS signaling using the PI3Ki buparlisib and the MEKi trametinib, or genetic suppression using H-, K-, and N-RAS isoform-specific siRNAs, ablated P-S6 and viability of neratinib resistant cells, suggesting RAS is causally associated with TORC1 hyperactivity and drug resistance. Further, intrinsically neratinib-resistant HER2-mutant cell lines with KRAS or PIK3CA co-mutations [DV90 (ERBB2V842I, KRASG13D), SNUC2A (ERBB2R678Q, KRASG12D, TSC2P1521T), MCF7 (HER2L755S/V777L, PIK3CAH1047R)] were sensitized to neratinib upon the addition of everolimus. Finally, DNA sequencing of tumors (MSK-IMPACT panel; 410 genes) from 141 patients enrolled in the SUMMIT trial showed enrichment of somatic alterations associated with aberrant activation of TORC1 pathway (KRAS, NRAS, NF1, PIK3CA, PIK3R1, AKT1/2, PTEN) in patients exhibiting primary resistance to neratinib. Conclusions: These data suggest that hyperactivation of TORC1 pathway promotes de novo and acquired resistance to neratinib across histologically distinct HER2-mutant cancers. Thus, we propose the combination of neratinib with TORC1 inhibitors is worthy of investigation in patients with HER2-mutant cancers. Citation Format: Dhivya R. Sudhan, Angel Guerrero-Zotano, Helen Won, Paula Gonzales Ericsson, Qi Liu, Teresa Dugger, James Koch, Alison Schram, Alberto Servetto, Richard Cutler, Alshad Lalani, Richard Bryce, Alan Auerbach, Ariella Hanker, Carlos L. Arteaga. Hyperactivation of mTORC1 drives acquired resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 329.