DLBCL is a molecularly heterogeneous disease usually treated with chemoimmunotherapy ultimately curing ∼65% of pts. In order to improve therapy for these pts, the identification of broadly relevant therapeutic targets is critical. One such target is HSP90. Tumor cells are enriched for a fraction of HSP90 found in higher-order multi-chaperone complexes. Tumor-enriched HSP90 (teHSP90) displays higher affinity for HSP90 inhibitors than normal tissues, which contain latent, uncomplexed HSP90. Many client proteins are depleted upon exposure to teHSP90 inhibitors. PU-H71 is a highly teHSP90 selective inhibitor with an excellent toxicity profile currently being tested for DLBCL in clinical trials. Combination therapies involving teHSP90 inhibitors may synergize with drugs targeting client proteins of teHSP90 by more powerfully inhibit survival pathways. Cell replication involves substantial metabolic demands and PU-H71-induced degradation of enzymatic client proteins may affect critical metabolic pathways. We hypothesized that by identifying PU-H71-induced metabolic changes, metabolomics could point to potential new targets for combinatorial therapeutic intervention.
We therefore analyzed the global metabolic consequence of teHSP90 inhibition in DLBCL by PU-H71. The metabolome was analyzed in the serum of LY7 DLBCL xenografted mice treated with 75 mg/m2 of PU-H71 for 24 h (n=5) or vehicle (n=5) by HPLC/MS. Bioinformatic analysis revealed significant changes in 122 metabolites in PUH-71- vs. vehicle-treated mice; including significantly lower levels of xanthine, hypoxanthine, adenosine, xanthosine monophosphate (XMP), depletion of the guanine nucleoside pool, together with higher levels of inosine and inosine monophosphate (IMP). These metabolic changes pointed towards possible PU-H71 mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH catalyzes the NAD-dependent oxidation of IMP to XMP, which is the committed step in de novo guanosine nucleotide biosynthesis. This reaction is particularly important to lymphocytes, which depend on IMPDH activity to generate the guanosine nucleotide levels needed to initiate a proliferative response to antigen. Increased IMPDH activity has also been observed in leukemia and lymphoma, mostly as consequence of up-regulation of the IMPDH2 isoform. In order to determine whether IMPDH1/2 stability depends on teHSP90, we treated a panel of 6 DLBCL cell lines (including LY7) with the mean GI50 of PU-H71 (1 μM) for up to 24 h and checked for IMPDH1/2 abundance. We found a time-dependent decrease in IMPDH2 protein levels. Similar results were obtained with the chemically unrelated HSP90 inhibitor 17-DMAG. To confirm that IMPDH2 binds to teHSP90, we took advantage of an affinity-based PUH-71 pull-down method we recently developed. In this assay PU-H71-beads preferentially bind to teHSP90 complexes pecipitating cancer-related client proteins. By using this assay, we determined that IMPDH2 was indeed a teHSP90 client in DLBCL cells. We also found that PU-H71 (and 17-DMAG) inhibited the activity of IMPDH in DLBCL cells by shortening its half-live (IMPDH t1/2from 2 h to 45 min). To determine whether the combination of teHSP90 inhibitors will synergize with IMPDH inhibitors in killing DLBCL, we treated a panel of 10 DLBCL cell lines with the combination of PU-H71 and two IMPDH inhibitors in clinical use, mycophenolic acid (MPA, an uncompetitive inhibitor) and ribavirin (RIB, a competitive inhibitor). We found that most cell lines showed synergistic killing effect when treated with the combination of drugs compared to each drug alone (determined by isobologram method). This prompted us to test the combination in vivo. SCID mice were xenografted with LY7 and SUDHL6 and once tumor developed, they were treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of MPA), RIB and the combination of PU-H71 and MMF or RIB. We found that mice treated with the combination of drugs exhibited greater effect that each drug alone (p=0.002 for SU-DHL6 and p>0.001 for LY7 for PU-H71+MMF, and p=0.01 for SU-DHL6 and p=0.02 for LY7 for PU-H71+RIB, all T-test day 10). There were no toxic effects.
In sum, our work uses serum metabolomics to provide new insights into the pharmacological targets of a particular HSP90 inhibitor, and unveiled a critical survival pathway in DLBCL that was harnessed to develop a rationally combined targeted therapy.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.