Abstract A08: Genetic drug resistance screen identifies LZTR1 as regulator of RAS ubiquitination and signaling
Authors: Bigenzahn Johannes W. , Collu Giovanna M. , Kartnig Felix , Pieraks Melanie , Vladimer Gregory I. , Heinz Leonhard X. , Sedlyarov Vitaly , Schischlik Fiorella , Fauster Astrid , Rebsamen Manuele , Parapatics Katja , Blomen Vincent A. , Müller André C. , Winter Georg E. , Kralovics Robert , Brummelkamp Thijn R. , Mlodzik Marek , Superti-Furga Giulio 
Published: June 2021

Abstract
Small-molecule tyrosine kinase inhibitor (TKI)-based treatment of chronic myeloid leukemia (CML), directed by the presence of the Philadelphia chromosome (Ph+) encoded BCR-ABL tyrosine kinase, is a paradigm of targeted cancer therapy. However, the development of TKI resistance limits the long-term success of these therapeutics. We used a genetic screening approach in the near-haploid CML cell line KBM-7 to identify six genes whose individual loss of function led to TKI drug resistance. We investigated in detail the role of the leucine zipper-like transcription regulator 1 (LZTR1) gene, as it was mechanistically enigmatic despite its involvement in many human developmental and oncologic diseases including glioblastoma, schwannomatosis and Noonan syndrome. We found that LZTR1 inactivation led to enhanced MAPK pathway activity and reduced TKI sensitivity of CML cells in a RAS-dependent way. LZTR1 missense mutations identified in human diseases failed to revert the loss-of-function phenotype. Knockdown of the LZTR1 orthologue CG3711 in Drosophila led to an increase in ectopic wing vein formation that could be rescued by impairment of RAS gene function, confirming the epistatic relationship and suggesting an evolutionary conserved role for LZTR1 in RAS regulation. Endogenous LZTR1 protein associated with the four main RAS isoforms KRAS4A, KRAS4B, NRAS and HRAS in proximity biotinylation proteomic and immunoprecipitation experiments in a manner that for KRAS required farnesylation and palmitoylation. Ectopic expression of LZTR1 along with all four RAS proteins led to their ubiquitination, compatible with the proposed role of LZTR1 as a cullin (CUL)-3 E3 ligase adaptor. Loss of LZTR1 led to reduced ubiquitination of endogenous KRAS and its increased abundance as well as enhanced localization at the plasma membrane. Together with the work on the role of LZTR1 in diseases driven by the dysregulation of RAS ubiquitination and signaling (Steklov, Pandolfi, Baietti et al., Anna Sablina lab, KU Leuven; see separate abstract), we propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation, providing a mechanistic explanation to its genetic involvement across a variety of human pathologies.
Citation Format: Johannes W. Bigenzahn, Giovanna M. Collu, Felix Kartnig, Melanie Pieraks, Gregory I. Vladimer, Leonhard X. Heinz, Vitaly Sedlyarov, Fiorella Schischlik, Astrid Fauster, Manuele Rebsamen, Katja Parapatics, Vincent A. Blomen, André C. Müller, Georg E. Winter, Robert Kralovics, Thijn R. Brummelkamp, Marek Mlodzik, Giulio Superti-Furga. Genetic drug resistance screen identifies LZTR1 as regulator of RAS ubiquitination and signaling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A08.