HSP90 Facilitates Oncogene-Induced Metabolic Reprogramming in B-Cell Lymphomas
Authors: Calvo Vidal Maria Nieves , Krumsiek Jan , Patel Jayeshkumar , Yang Shao Ning , Phillip Jude M , Marullo Rossella , Taldone Tony , Inghirami Giorgio , Melnick Ari , Chiosis Gabriela , Cerchietti Leandro 
Published: April 2021

Abstract
The chaperone HSP90 is used by B-cell lymphomas to support the stability of proteins involved in oncogenic processes such as signaling and anti-apoptosis. While HSP90 inhibitors decrease the levels of these client proteins favoring cell death they also prompt cellular counter-regulatory mechanisms that diminish the efficacy of these drugs. Improving the clinical activity of HSP90 inhibitors will depend on understanding the complexity of HSP90 functions. Here we show that HSP90 facilitates the function of MYC by improving the efficiency of metabolic pathways through the orchestration of enzymatic networks, and that HSP90 inhibition impairs the metabolic fitness of DLBCLs without client protein degradation. Moreover, drugs inducing sub-lethal metabolic stress in DLBCL cells cause apoptosis upon HSP90 inhibition.
To identify metabolic enzymes actively chaperoned by HSP90 we integrated the information from proteomics and metabolomics in DLBCL cell lines. Proteomics was performed from the cytoplasmic fraction of OCI-Ly1 and OCI-Ly7 cells chemically precipitated with PU-H71, an HSP90 inhibitor that selectively binds to HSP90 contained in active multi-chaperone complexes.STRING network analysis of the metabolic client proteins identified several hubs highly enriched for enzymes involved in metabolism of nucleotides (e.g. IMPDH2, CTPS1, CAD), carbohydrates (e.g. G6PD, HK2) and proteins (e.g. MTHFR, ASNS). Functionality of the network was assessed by metabolomics from OCI-Ly1 cells treated with PU-H71 500 nM for 6 h (sub-lethal). This dose and timing assured HSP90 inhibition but no client protein degradation. The proteomics and metabolomics mapping into KEGG pathways showed a significant overlap, indicating that HSP90 preferentially interacts with proteins representing regulatory hubs to coordinate their committed activity and thus secure the flow of the pathway.
We quantified the effect of HSP90 on the activity of metabolic networks by measuring glycolysis (by lactate production and medium acidification) and mitochondrial respiration (by oxygen consumption) in OCI-LY1 and OCI-LY7 cell lines upon PU-H71. We found that inhibition of HSP90 decreased glycolysis by 20-25% and respiration by 25% (p&lt;0.01 for both). There were no changes in ATP levels. Given that in proliferating cells respiration serves intermediates for crucial anabolic roles, we assessed nucleotides and protein syntheses by using uridine and methionine analogs, respectively. We found that inhibition of HSP90 decreases biosynthesis of nucleotides by 10-20% and proteins by 20-30% (p&lt;0.05 for both). Altogether these results suggest that HSP90 contributes to the channeling of metabolic intermediates into the mitochondria and from there to critical biosynthetic pathways. Further supporting this notion, we found HSP90 inhibition caused an increase in the number of the so-called "rods and rings", enzyme assemblies composed of CTPS1 and IMPDH2, two key enzymes in the synthesis of GTP and CTP. These enzymatic "polymers" form under metabolic stress conditions to increase the cell's nucleotides biosynthetic efficiency.
To understand the mechanistic relevance of these findings to lymphomagenesis, we analyzed the HSP90 metabolic proteome for common features and found it was significantly enriched (chi-square p&lt;0.0001) for MYC target genes. Moreover, it correlated to MYC expression in DLBCL and Burkitt lymphoma (BL) cell lines and patient samples. Chemical precipitation of active HSP90 in BL and DLBCL patient samples showed that HSP90 does not interact directly with MYC but with enzymes that are MYC target genes such as CTPS1 and CAD, in agreement with HSP90 supporting MYC oncogenesis by improving the efficiency of metabolic networks. Remarkably, the expression of MYC (by IHC) in 18 DLBCL tumors associated with only nucleotides (e.g. IMP) and amino acids (e.g. glutamine) (by intracellular metabolomics), which are more reliant on HSP90 as we showed before. We capitalized on this by xenografting a MYC-amplified DLBCL cell line into 20 mice and treating them with the IMPDH2 inhibitor MMF to induce nucleotides stress in presence or absence of PU-H71. We found that under HSP90 inhibition, MMF significantly decreased lymphoma volume better than each drug alone (p&lt;0.001). In sum, we describe a novel function of HSP90 in establishing higher efficiency anabolic networks to support the metabolic stress imposed by MYC in lymphomas.

Disclosures
Yang: Regeneron Pharmaceuticals: Employment. Cerchietti: Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment; Celgene: Research Funding.