Abstract 1939: Follicular Lymphoma-associated mutations in the V-ATPase assembly factor VMA21 activate autophagic flux

Abstract

Abstract Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the Western world. FL carries a high incidence of gene mutations in various V-ATPase subunits and regulators (ATP6V1B2, ATP6AP1, and VMA21) accounting for a combined 25-30% of FL cases. We have previously demonstrated that an FL-associated mutation in the V-ATPase V1 subunit ATP6V1B2 induces autophagic flux, thus conferring a survival addiction on mutated FL B cells. Here, we report results from functional analysis of the common hotspot truncating mutations p.R93* in the V-ATPase assembly factor VMA21 in FL. Methods: We measured autophagic flux using a variety of complementary assays in recombinant cell lines expressing inducible VMA21 WT and MUT (p.R93*). Lysosome acidification was measured using pH-sensitive dyes linked to dextran and flow-cytometry. The subcellular localization of VAM21 was investigated using confocal microscopy. The VMA21 WT and MUT-associated proteome was analyzed using immunoprecipitation followed by quantitative mass spectrometry-based protein profiling (IP-MS). Results: The inducible expression of VMA21 p.R93* activated autophagic flux as measured through various assays, including: i) increased autolysosome numbers measured via electron microscopy, ii) elevated LC3-II and elevated free GFP in HEK293T and lymphoma cells expressing a GFP-LC3-RFP probe, iii) elevated autophagosomes and autolysosomes detected via confocal microscopy in HEK293T cells, which were ablated by ULK1 and PIK3C3/VPS34 inhibitors, iv) elevated autophagic flux in various yeast assays. The inducible expression of mutant VMA21 partially impaired lysosomal acidification, implicating inhibitory effects on the V-ATPase holoenzyme. Analysis of subcellular localization of WT and MUT VMA21 using lysosomal-IPs and confocal microscopy showed that VMA21 MUT mislocalized to lysosomes and did not shuttle back to the ER. Using quantitative IP-MS, we detected elevated amounts of V-ATPase components in the VMA21 MUT immunoprecipitates, indicating increased complex formation between MUT VMA21 and fully assembled V-ATPase. Studies are underway measuring V-ATPase activity in lysosomes in VMA21 WT and MUT cells and effects on the lysosomal metabolome. In addition, we are testing the effects of small molecule autophagy inhibitors on the survival of VMA21 MUT primary FL cells. Conclusion: Our data support the hypothesis that the FL-associated VMA21 p.R93* has altered subcellular trafficking. As a consequence, more MUT VMA21 associates with the V-ATPase holoenzyme resulting in partial impairment of V-ATPase activity. Similar to our previous findings on ATP6V1B2 mutations, the MUT VMA21 aberrantly activates autophagic flux possibly as compensation to impaired V-ATPase function. This study supports future investigations into therapeutic targeting of aberrantly activated autophagy in FL. Citation Format: Fangyang Wang, Ying Yang, Gabriel Boudagh, Eeva-Liisa Eskelinen, Daniel J. Klionsky, Sami N. Malek. Follicular Lymphoma-associated mutations in the V-ATPase assembly factor VMA21 activate autophagic flux [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1939.