A Novel Allosteric Inhibitor Targeting IMPDH2 at Y233 Overcomes Resistance to Tyrosine Kinase Inhibitors in Lymphoma

Inosine monophosphate dehydrogenase-2 (IMPDH2) is a rate-limiting enzyme in the de novo biosynthesis of guanine nucleotides and is often overexpressed in hematologic malignancies and solid tumors. However, its regulatory mechanisms in cancer remain poorly understood. Here, we identify IMPDH2 as a direct substrate of the oncogenic kinases ALK and SRC, which phosphorylate tyrosine 233 (Y233) within the enzyme’s allosteric Bateman domain. Using peptide-based mutagenesis and enzymatic assays, we demonstrate that Y233 phosphorylation is essential for IMPDH2 activity. We found that phosphoinositide-3-phosphate (PI3P), a signaling phospholipid, selectively binds to and inhibits IMPDH2, but not its isoform IMPDH1, revealing a novel lipid-mediated, isoform-specific regulatory mechanism. Next, we conducted structure-based virtual screening and identified a potent allosteric inhibitor of IMPDH2, compound 10 (comp-10), with an IC₅₀ of 260 nM. Comp-10 significantly impairs cell proliferation in ALK-positive anaplastic large cell lymphoma (ALCL) cell lines, including those resistant to the ALK inhibitors crizotinib and lorlatinib, and outperformed the FDA-approved IMPDH inhibitor mycophenolic acid. These findings reveal the dual regulation of IMPDH2 through tyrosine phosphorylation and binding to PI3P, and describe the discovery of a new IMPDH2 inhibitor, suggesting a potential therapeutic strategy to overcome resistance to tyrosine kinase inhibitors.