Hsp90 at the Hub of Metabolic Homeostasis in Malignant B Cells

Reprogramming of tumor cell metabolism contributes to disease aggressiveness and chemoresistance, but how this process is regulated on the molecular levels is unclear. Hsp90 regulates diverse cellular processes through its interaction with co-chaperones and client proteins. Although basally expressed, Hsp90 is almost universally overexpressed in malignant cells due to increased levels of internal (oxidants, genomic instability, unfolded proteins) and external (hypoxia, nutrients limitation, drugs) stressors. Indeed, Hsp90 is starting to be acknowledged also for its role as an integrator of nutrient sensing, tumor bioenergetics and metabolic stress response (Chae et al, Cancer Cell, 2012). Here we report that active cytosolic Hsp90 binds and stabilizes enzymes regulating central metabolic processes in DLBCL, and provides an actionable target for this disease.

Despite its overexpression, not all DLBCL are equally responsive to Hsp90 inhibitors. Using a panel of 10 DLBCL cell lines we found that those with higher glycolytic activity were more likely to respond to the Hsp90 inhibitors 17-DMAG and PU-H71. Global metabolomics profiling (GC-MS and UPLC-MS) of Hsp90-dependent (OCI-Ly7, OCI-Ly1, SU-DHL6, OCI-Ly10) vs. Hsp90-independent (Karpas422) cells identified “purine metabolism” (p=0.000027) and “pyrimidine metabolism” (p=0.0017) as upregulated, whereas “TCA cycle” was downregulated (p=0.000027). We validated these findings by short-term (6 h) exposure of OCI-Ly1 and OCI-Ly7 cells with non-cytotoxic doses of PU-H71 and found a highly reduced oxygen consumption rate (OCR) as well as glycolytic rate (ECAR). This was accompanied by a sharp decrease in glucose consumption and lactate production and by only a slight reduction in glutamine consumption. Unexpectedly, the drop in glycolysis and respiration in OCI-Ly1 and OCI-Ly7 did not lead to a significant decrease either in ATP or reducing power, suggesting that actually both the production and the utilization of ATP and redox equivalents are shut down when Hsp90 activity (an ATP-hydrolyzing enzyme) is inhibited. To identify the molecular basis for these differences, we characterized the active Hsp90-associated proteome in DLBCL by using chemical affinity purification followed by MS in cytosolic fractions of two Hsp90-dependent DLBCL cell lines (OCI-Ly1 and OCI-Ly7) in duplicates. This method pulls down only active Hsp90 multichaperone complexes (Moulick et al, Nat Chem Biol, 2011). Pathway enrichment analysis identified “cellular metabolism” as a significant process actively chaperoned by Hsp90 (ratio of enrichment 4.35, p=8.22E-15). Specifically, “nucleotides” and “carbohydrates” metabolism were the two most represented pathways, suggesting their critical role in Hsp90-dependent DLBCL cells. To functionally validate these findings, we integrated the proteomics analysis with serum metabolomics obtained from 10 OCI-Ly7-xenografted mice treated with PU-H71 vs vehicle (75 mg/m² for 24 h (n=5)). Bioinformatic analysis revealed that PU-H71-treated mice had significant changes in 122 metabolites including lower levels of xanthine, hypoxanthine, adenosine and inosine, suggesting an alteration of the purines metabolism. IMPDH1/2 is the enzyme catalyzing the first committed and rate-limiting step of de novo guanine nucleotide biosynthesis. To determine whether IMPDH stability depends on Hsp90 activity, we treated a panel of 6 DLBCL cell lines (including OCI-Ly7) with their GI50 for PU-H71 or 17-DMAG for up to 24 h and found a time-dependent decrease in IMPDH2 protein levels. Finally, SCID mice were xenografted with OCI-Ly1 and SU-DHL6 and treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of the IMPDH inhibitor mycophenolic acid) and the combination of PU-H71 and MMF. We found that the combination of drugs exhibited a greater antitumor effect than each drug alone (p=0.002 for SU-DHL6 and P<0.001 for OCI-Ly1, T-test day 10). There were no toxic effects.

Altogether, our work supports a role for Hsp90 as a necessary component in a subset of DLBCLs to build-up the metabolic features that allow these tumor cells to meet the requirements for their unrestrained growth and proliferation.