Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets are functionally dependent on different survival mechanisms and exhibit distinct metabolic fingerprints. One of DLBCLs subtypes relies on mitochondrial oxidative phosphorylation and nutrient utilization pathways that provide pro-survival advantage independent of B-cell receptor (BCR) signaling. These OxPhos-DLBCLs are resistant to BCR inhibition and remain functionally poorly defined.
We and others found that OxPhos-DLBCLs, compared to the BCR-dependent DLBCLs, overexpress heat shock protein HSP90alpha (Br J Haematol 2009; 144:358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. We found that HSP90alpha gene/protein expression correlated with SIRT1 protein level in DLBCL cell lines. SIRT1 knockdown or chemical inhibition reduced HSP90alpha expression in a mechanism involving HSF1, whereas HSP90 inhibitor (17AAG) reduced SIRT1 protein stability suggesting a chaperone function of HSP90alpha toward SIRT1 and a functional link between these proteins. We confirmed the SIRT1-HSP90alpha interaction in DLBCL cells using proximity ligation assay (PLA). The number of PLA complexes was significantly higher in OxPhos- (Ly4, K422, Toledo) than BCR- (Ly1, DHL4, Ly7, DHL6) DLBCL cell lines (p<0.001). Importantly, the number of PLA complexes increased markedly in mitotic when compared to interphase cells, indicating that the interaction between these proteins plays a mitosis-specific role. For this reason, we next assessed the chromosome segregation of DLBCL cells in the presence and absence of SIRT1 and/or HSP90alpha activity. Both genetic (siRNA) and chemical (EX-527) inhibition of SIRT1 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes- respectively 50%, 26% and 20% of all observed abnormal mitotic incidents). Similarly, chemical (17AAG) or genetic (siRNA) inhibition of HSP90alpha disturbed chromosome segregation, albeit to a lesser extent than SIRT1 disruption. Concurrent chemical or genetic ablation of SIRT1 and HSP90alpha synergistically increased the number of mitotic cells with chromosome segregation errors in OxPhos-DLBCLs. In the BCR-dependent cell lines, neither of the inhibitors used separately had a significant impact on the number of cells with improper chromosome segregation events, but the simultaneous inhibition of SIRT1 (EX-527) and HSP90alpha (17AAG) significantly increased the number of cells with aberrant mitosis, although to a lesser extent than in OxPhos-DLBCLs (15.4-17.1% for BCR-DLBCL versus 28.37-48% for OxPhos-DLBCL, respectively). Consistent with the postulated role of SIRT1 in chromosome dynamics during mitosis, we found downregulated expression of SIRT1-dependent genes in the recently characterized DLBCL subset characterized by chromosome instability (C2, Chapuy et al., Nat Medicine 2018).
While low rates of chromosomal instability induces tumorigenesis, increased chromosomal missegregaton leads to cell death and suppresses cancer development. SIRT1 inhibitors (EX-527, cambinol, tenovin-6) induced dose-dependent cytotoxicity in DLBCL cell lines, while simultaneous addition of HSP90 inhibitor (17AAG) produced synergistic or additive effect in OxPhos-, but not BCR-DLBCLs. Taken together, our findings define a new OxPhos DLBCL-specific pathogenetic mechanism involving SIRT1 and HSP90alpha that regulates chromosome dynamics during mitosis and may be exploited therapeutically.
Juszczynski:Ryvu Therapeutics: Other: member of advisory board.
Asterisk with author names denotes non-ASH members.