Inosine Monophosphate Dehydrogenase-2 (IMPDH2) As the Potential Novel Therapeutic Target in Mantle Cell Lymphoma (MCL): The Underlying Tyrosine Phosphorylation-Based Activation Mechanism and Translational Implications

Introduction: Mantle cell lymphoma (MCL) is a non-curable form of B-cell lymphoma with a median overall survival of ~3-5 years. MCL is characterized by chromosomal translocation involving the CCND1 and MYC genes, contributing to drug resistance to chemo-immunotherapy and tyrosine kinase inhibitors. The SRC kinase family members, such as LYN, HCK, and SRC, are involved in BCR-signaling in normal B lymphocytes and certain lymphomas, including MCL. IMPDH2 is one of the rate-limiting enzymes in de novo purine biosynthesis, and its expression has been studied extensively in solid tumors, but the post-translational modification status, such as tyrosine phosphorylation, remains unknown.

Methods: In our study of IMPDH2 tyrosine phosphorylation, we used an in-vitro kinase assay with SRC kinase and mass-spectrometry-based LC-MS/MS phosphoproteomics analysis. Subsequently, we synthesized mutated IMPDH2 constructs with tyrosine residues (Y) replaced by phenylalanine (F), tagged them with HA-GFP, and expressed in HEK293T cells. We performed co-immunoprecipitations (Co-IP) using HA-conjugated magnetic beads and measured IMPDH2 enzymatic activity to identify specific phosphosites regulated by SRC kinase. Additionally, we developed a phosphospecific antibody targeting an IMPDH2 Y233-containing peptide and utilized it in immunoblot analyses on MCL cell lines. We then performed Immunohistochemistry (IHC) on MCL and DLBCL biopsies. Additionally, we treated MCL cell lines and primary cells with mycophenolic acid (MPA), an FDA-approved small molecule inhibitor of IMPDH1/2, and performed transcriptomics and global metabolomics multi-omic analysis. Next, we performed functional studies, including cell proliferation, cell death (annexin V, caspase3 cleavage, tunnel assay), and cell cycle analysis.

Results:

Our study found that several SRC tyrosine kinase family members directly phosphorylates IMPDH2. This was confirmed by both in-vitro kinase assay and western blotting with a pan-tyrosine-specific (pY100) antibody. Further, in-depth studies using active SRC kinase in a large-scale in-vitro kinase assay followed by LC-MS/MS phosphoproteomics study revealed multiple tyrosine phosphorylation sites on IMPDH2, including Y110, Y233, Y294, Y348, Y430, and Y484. Subsequent experiments involving the expression of lentivirus plasmids containing IMPDH-WT and IMPDH2-Y-F mutant in 293T cells, followed by an enzyme activity assay, demonstrated a significant decrease in enzyme activity with the IMDH2 Y233 mutation compared to other tyrosine residues. Analysis of IHC studies of MCL biopsies showed a significant correlation between IMPDH2 and phospho-IMPDH2 Y233 phosphorylation. Treatment of several MCL cell lines and primary cells with mycophenolic acid (MPA), a small molecule inhibitor of IMPDH1/2, universally inhibited their growth. In addition, multi-omic analysis of MCL cell lines treated with MPA revealed intriguing gene and metabolite expression changes associated with nucleotide synthesis and metabolic pathways. Notably, MPA treatment decreased the expression of the critical oncogene CCND1/Cyclin D1, both at the gene and protein levels, associated with cell cycle arrest at the G0/G1 stage, alterations in metabolite concentrations, including increased IMP, Inosine and Hypoxanthine, as well as reductions in GMP and GTP metabolites. Finally, the genetic and pharmacological approaches: CRISPR-Cas9-mediated IMPDH2 deletion and MPA treatment on MCL cell lines inhibited cell proliferation and induced apoptotic cell death as detected by caspase-3 cleavage, annexin V expression, and DNA fragmentation (TUNEL assay).

Conclusion:

Our study revealed a previously undiscovered oncogenic mechanism, showing that the SRC family kinases directly phosphorylate IMPDH2 at the specific tyrosine 233 site and documenting the pathogenic role of IMPDH2 in MCL. These findings enhance our understanding of IMPDH2 regulation and open avenues for developing targeted therapies to disrupt tumor metabolism in MCL and, possibly, other tyrosine-kinase-driven malignancies.