Functional Analyses of V-Atpase Mutations in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. Recently, frequent mutations (RRAGC, V-ATPase) in the amino acid sensing pathway connected to MTOR signaling have been described in FL. The RRAGC mutations activate MTOR. The V-ATPase is a multi-protein complex primarily involved in proton pumping and acidification of cellular compartments. Here, we report initial results from functional analyses of the common hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2.

Methods: We introduced the ATP6V1B2 hotspot mutations by site-directed mutagenesis. The cDNAs were cloned into lentiviral or retroviral vectors and used to generate stable lymphoma cells lines and HEK293T cells. We also generated recombinant S. cerevisiae expressing a mutation homologous to human ATP6V1B2 p.R400Q at the endogenous VMA2locus (R381Q). We modeled the location of the mutations onto the existing 3D structures.

Results: Some of the stable cell lines lost the expression of the ATP6V1B2 mutants over time, while others were more permissive. Given the critical role of V-ATPase in lysosomal acidification and autophagy we measured LC3-II levels in various recombinant cell lines and detected very elevated LC3-II levels in the V-ATPase mutant lines. Highly elevated LC3-II levels at steady state could indicate impairments in LC3-II metabolism as seen with chemical V-ATPase inhibition (bafilomycin A₁ treatment) or elevated autophagic initiation and flux. We resorted to multiple experimental approaches to resolve this question: i) chemical MTOR inhibition strongly activates autophagy. Using rapamycin and Torin 2, we induced LC3-II to levels comparable to those seen with the V-ATPase mutants; ii) using inhibitors of lysosomal LC3-II degradation, we further increased already elevated LC3-II levels in V-ATPase mutant HEK293T cell lines; iii) in S. cerevisiae Vma2^R381Qcells we found elevated autophagy activity based on analysis of the LC3 homolog Atg8, vacuolar/lysosomal processing of a GFP-Atg8 chimera, and enzymatic analysis of an autophagy marker, Pho8∆60. Together, these findings currently allow for the working hypothesis that V-ATPase mutants strongly increase autophagic flux.

We performed studies of the effects of V-ATPase mutants on MTOR activity as measured through RPS6KB/S6-kinase phosphorylation. In transient transfections in HEK293T cells and in stable HEK293T cells lines achieving expression slightly above endogenous V-ATPase levels, performed under conditions of leucine starvation or sufficiency, we detected MTOR activation.

We mapped the mutated p.Y371C and p.R400Q residues onto the published electron microscopy-derived structures of yeast V-ATPase. We found that both residues are closely spaced and are located at the interface of the V1B2 and V1A subunits; the latter with potential effects on subunit interactions, the conformational state of the V1 complex (open, loose or tight) and possibly effects on ATPase activity.

To gain insights into the physiological consequences of these findings, we measured growth and viability of a recombinant lymphoma cell line (SUDHL4) expressing wild type and mutant V-ATPase in the presence of lowered leucine concentration. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. In a parallel experiment, we induced cellular stress by intentionally overgrowing these lines and measured survival over time. We found improved growth and survival of cells expressing the ATP6V1B2 mutations. Complementary measurements of the biochemical and functional consequences of V-ATPase mutations in isolated primary FL B cells are ongoing and will be updated at the meeting.

Conclusion: Our initial efforts at functional characterization of the common FL-associated hotspot mutations p.Y371C and p.R400Q in the V-ATPase subunit ATP6V1B2 uncovered substantially elevated autophagic flux. To our knowledge, this is the first report of mutational activation of autophagy in follicular lymphoma. Our current working model is that this elevated flux promotes lysosomal amino acid generation that subsequently activates MTOR through previously proposed inside-out signaling pathways. Combined, these changes may allow for improved survival of FL cells under conditions of nutrient stress. These findings may allow for future therapeutic targeting of this pathway in FL.