Follicular lymphoma–associated mutations in vacuolar ATPase ATP6V1B2 activate autophagic flux and mTOR
Fangyang Wang, … , Daniel J. Klionsky, Sami N. Malek
Fangyang Wang, … , Daniel J. Klionsky, Sami N. Malek
Published February 5, 2019
Citation Information: J Clin Invest. 2019;129(4):1626-1640. https://doi.org/10.1172/JCI98288.
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Follicular lymphoma–associated mutations in vacuolar ATPase ATP6V1B2 activate autophagic flux and mTOR

Abstract

The discovery of recurrent mutations in subunits of the vacuolar-type H+-translocating ATPase (v-ATPase) in follicular lymphoma (FL) highlights a role for the amino acid– and energy-sensing pathway to mTOR in the pathogenesis of this disease. Here, through the use of complementary experimental approaches involving mammalian cells and Saccharomyces cerevisiae, we have demonstrated that mutations in the human v-ATPase subunit ATP6V1B2 (also known as Vma2 in yeast) activate autophagic flux and maintain mTOR/TOR in an active state. Engineered lymphoma cell lines and primary FL B cells carrying mutated ATP6V1B2 demonstrated a remarkable ability to survive low leucine concentrations. The treatment of primary FL B cells with inhibitors of autophagy uncovered an addiction for survival for FL B cells harboring ATP6V1B2 mutations. These data support the idea of mutational activation of autophagic flux by recurrent hotspot mutations in ATP6V1B2 as an adaptive mechanism in FL pathogenesis and as a possible new therapeutically targetable pathway.

Authors

Fangyang Wang, Damián Gatica, Zhang Xiao Ying, Luke F. Peterson, Peter Kim, Denzil Bernard, Kamlai Saiya-Cork, Shaomeng Wang, Mark S. Kaminski, Alfred E. Chang, Tycel Phillips, Daniel J. Klionsky, Sami N. Malek

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Figure 4

An intact autophagy pathway and lysosomal amino acid sensing and transport are necessary for delayed activation of mTOR by mutant ATP6V1B2.

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An intact autophagy pathway and lysosomal amino acid sensing and transpo...
(A and B) ATG7–/– HEK293T cells were generated using CRISPR-Cas9 targeting, and independent single-cell clones were expanded. Control ATG7 WT cells were expanded from cells that were subjected to CRISPR-Cas9 targeting but remained intact for ATG7. All clones were subsequently infected with lentiviruses carrying doxycycline-inducible WT or mutant ATP6V1B2. Cells were induced with doxycycline for 72 hours, and cell lysates were prepared for immunoblotting using the indicated antibodies. (B) Results of quantification (p-S6K/t-S6K) of 4 independent experiments using data from all lanes labeled WT, Y371C, and R400Q. Statistical analysis compared the difference of the differences between measurements under ATG7 WT and ATG7–/– conditions (see Methods). P = 0.06 for Y371C and P = 0.04 for R400Q for ATG7–/–/ATG7 comparisons (standard regression-based Wald-type tests). Error bars indicate the SD. (C and D) SLC38A9–/– HEK293T cells were generated using CRISPR-Cas9 targeting, and independent single-cell clones were expanded. Control SLC38A9 WT cells were expanded from cells that were subjected to CRISPR-Cas9 targeting but remained intact for SLC38A9. All clones were infected with lentiviruses carrying doxycycline-inducible WT or mutant ATP6V1B2. Cells were induced with doxycycline for 72 hours, and cell lysates were prepared for immunoblotting using the indicated antibodies. (D) Results of quantification (p-S6K/t-S6K) of 3 independent experiments using data from all lanes labeled WT, Y371C, and R400Q. Statistical analysis compared the difference of the differences between measurements under SLC38A9 WT and SLC38A9–/– conditions (see Methods). P = 0.01 for Y371C and P = 0.01 for R400Q for SLC38A9–/– and SLC38A9 comparisons (standard regression-based Wald-type tests). (B and D) *P < 0.05, by standard regression-based Wald-type tests. Error bars indicate the SD.