Abstract
Understanding the molecular basis of therapy-resistant DLBCL is a critical unmet need. We explored whether the family of Sirtuin proteins might contribute to such effects. Analysis of four independent clinically annotated patient cohorts revealed that higher SIRT3 expression was linked to inferior outcome (p=4.7e-5). This was not the case for any other of the sirtuins. SIRT3 mRNA and protein expression were also much higher in DLBCL patients as compared to normal germinal center (GC) B-cells. Among the seven sirtuins, only SIRT3 depletion universally suppressed proliferation, induced cell cycle arrest, suppressed colony formation, and induced apoptosis in a large panel of DLBCL cell lines regardless of cell of origin, OxPhos status, or somatic mutation profiles.
Constitutive Sirt3-/- mice manifested completely normal GC formation after T-cell dependent antigen immunization. However SIRT3 depleted human DLBCL cells manifested inferior engraftment and tumor formation in mice (p=0.023 for hairpin#1, p=0.045 for hairpin#2). Sirt3 inducible knockdown caused strong regression of established DLBCL xenografts. We examined whether SIRT3 was important in lymphoma initiation by crossing VavP-Bcl2 mice with Sirt3-/- animals. As compared to VavP-Bcl2 controls, the VavP-Bcl2/Sirt3-/- mice manifested significantly longer overall survival (P=0.0035), and greatly reduced tumor burden and systemic lymphoma infiltration of organs.
SIRT3 is exclusively localized to mitochondria and hence its actions are likely metabolic. We therefore performed metabolomic profiling in SIRT 3 depleted DLBCL cell lines. This analysis revealed profound suppression of the TCA (tricarboxylic acid) cycle, with reduced TCA metabolites such as citrate, alpha-ketoglutarate, succinate, fumarate, malate, etc. SIRT3 depletion caused significant reduction in acetyl-CoA pools as measured by solid phase extraction and LC-MS, indicating that SIRT3 is required to maintain the production of key metabolic intermediates from the TCA cycle. To define the nature of the TCA defect we performed metabolic tracing studies using 13C-labeled glutamine and glucose. The results revealed that SIRT3 drives the TCA cycle through glutaminolysis. We showed that SIRT3 mediates this effect by directly deacetylating and hence hyper-activating the enzymatic activity of mitochondrial glutamine dehydrogenase (GDH). Indeed GDH overexpression could fully rescue the collapse of the TCA, cell proliferation arrest and apoptosis induced by SIRT3 depletion. SIRT3 knockdown was also rescued by feeding cells DMKG (which mimics alpha-ketoglutarate) and hence bypasses the need for SIRT3 mediated glutaminolysis.
Because SIRT3 depletion caused metabolic collapse, DLBCL cells manifested potent induction of autophagy, as shown by ratios of LC3II/LC3I in DLBCL cells and using a mCherry-EGFP-LC3 reporter to measure autophagic flux. This autophagy effect was rescued by feeding cells with DMKG or by overexpressing GDH, which uncouple the TCA cycle from SIRT3 dependency. Notably the ratio of LC3II/LCI and perturbed autophagy flux was also Increased in lymphoma cells from VavP-Bcl2;sirt3-/- vs. VavP-Bcl2;sirt3+/+ mice.
These data nominate SIRT3 as a putative therapeutic target. Therefore we designed a nanomolar-potency SIRT3 selective small molecule inhibitor including a mitochondrial-targeting motif that concentrates drug in the mitochondrial matrix. This compound (called YC8-02), phenocopied all the effects of SIRT3 depletion including proliferation arrest, apoptosis, TCA collapse by metabolomics study, hyperacetylation of mitochondrial proteins, suppression of GDH activity, and induction of autophagy. Yet YC8-02 had no effect on normal B-cells. Moreover, YC8-02 treatment of chemotherapy resistant DLBCL cell lines restored their sensitivity to clinically relevant doxorubicin concentrations.
In summary, SIRT3 is a novel metabolic oncoprotein widely required for DLBCL cells to satisfy their metabolic needs by enhancing the activity of the TCA cycle through glutaminolysis. SIRT3 is a crucial new therapeutic vulnerability especially impactful for the most resistant DLBCLs regardless of their somatic mutations. YC8-02 and its newer derivatives are a promising and entirely new mechanism-based approach to help these patients.
Disclosures
Cerchietti: Leukemia and Lymphoma Society: Research Funding; Lymphoma Research Foundation: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment; Celgene: Research Funding.
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