Targeting Cell Adhesion Molecules in IDH Mutant Myeloid Malignances

Neomorphic mutations in the genes encoding isocitrate dehydrogenase (IDH) are common in human cancers including myeloid malignancies. The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by mutant IDH promotes leukemia development by competitively inhibiting α-ketoglutarate (α-KG)-dependent dioxygenases, causing epigenetic dysregulation of histone and DNA methylation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA-approved allosteric inhibitors (ivosidenib and enasidenib targeting mutant IDH1 and IDH2 respectively) are used for treating newly diagnosed and/or relapsed/refractory AML patients with IDH mutations. However, the low response rate and primary resistance to IDH^mut inhibitors are major clinical challenges. Moreover, acquired resistance to IDH inhibition can occur through multiple mechanisms, including R-2-HG-restoring second-site IDH mutations and the selection of sub-clonal mutations in RUNX1/CEBPA or RAS-RTK pathway. These findings underscore the importance of developing new and more effective approaches for the treatment of IDH-mutant MDS and AML patients.

To identify mutant IDH-mediated alterations and actionable dependencies, we generated isogenic TF-1 myeloid leukemia cell lines containing endogenous, hotspot IDH mutations (IDH1^R132H, IDH2^R140Q, IDH2^R140W, and IDH2^R172K) by CRISPR/Cas9-mediated base-editing. By comparative transcriptomic (RNA-seq) and epigenomic (ATAC-seq and ChIP-seq) profiling of base-edited versus parental isogenic leukemia cells, we identified cell adhesion molecules, especially CD44, as the top upregulated genes in IDH-mutant leukemia cells. CD44 expression is significantly upregulated in IDH-mutant AML patient samples relative to normal CD34⁺ hematopoietic stem/progenitor cells (HSPCs) or other AML samples without IDH mutations. Increased CD44 expression is also associated with overall poorer survival and therapy resistance in independent AML cohorts. Importantly, we found that ectopic CD44 expression promotes cytokine-independent cell growth of TF-1 myeloid leukemia cells, resembling the effect observed upon expression of mutant IDH. Moreover, CD44 depletion led to decreased R-2-HG levels in base-edited IDH-mutant TF-1 leukemia cells, and impaired the propagation of IDH-mutant leukemia cells in vitro and in xenotransplanted recipient mice. These studies establish direct evidence that IDH mutations cause aberrant activation of cell adhesion molecules such as CD44, and CD44 is required for the propagation of IDH-mutant myeloid leukemia cells.

Mechanistically, we found that the aberrantly activated CD44 is first cleaved by matrix metalloproteinases MMP2 and MMP9 for proteolytic release of its ectodomain from membrane, followed by γ-secretase-mediated intramembranous cleavage to produce the CD44 intracellular domain (CD44ICD). CD44ICD subsequently activates the pentose phosphate pathway (PPP) for the production of NADPH, a critical cofactor for mutant IDH enzymes to convert α-KG to R-2-HG. Hence, mutant IDH-mediated activation of CD44 functions to rewire intracellular metabolism to facilitate IDH^mut-catalyzed R-2-HG production, supporting a model for mutant IDH-induced feed-forward oncogenic programs. CD44 also activates AKT/ERK signaling pathway to promote the proliferation and cytokine-independent growth of IDH-mutant leukemia cells. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combination regimens consisting of IDH^mut inhibitors and humanized monoclonal antibody specific for CD44 (RG7536) or MMP2/9 inhibitors (SB-3CT) invitro and invivo. Taken together, our findings uncover the cell adhesion molecule-mediated metabolic signaling as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies.

No relevant conflicts of interest to declare.