Signaling Pathways of Mutant IDH1 Independent of R-2-Hydroxyglutarate

#Michael Heuser and Anuhar Chaturvedi share senior authorship

Background: Isocitrate dehydrogenase-1 (IDH1) is mutated in about 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, thereby linking metabolism to tumorigenesis. We recently reported that at comparable intracellular R-2HG levels, mice receiving transplants of IDH1 mutant cells died significantly earlier than R-2HG treated mice in the context of HOXA9 overexpression. This suggests oncogenic functions of mutant IDH1 beyond R-2HG production. We employed a splice variant of mutated IDH1 that does not produce R-2HG (IDH1mutantΔ7) to decipher R-2HG independent signaling pathways that may contribute towards leukemogenesis.

Methods: Bone marrow cells from mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt), IDH1mutant (IDH1mut), IDH1wildtypeΔ7 (IDH1wtΔ7) and IDH1mutΔ7, were constitutively expressed and the leukemogenic potential was evaluated in vivo. Intracellular R-2HG was measured by enantiomer-specific quantification. Deletion of exon 7 from IDH1mut leads to a frameshift that creates a premature stop codon in the 9th exon, finally producing a 119 amino acids truncated protein, IDH1mutΔ7. This splice variant does not produce increased levels of R-2HG. The signaling pathways were explored by immunoblotting and immunofluorescence.

Results: Mice receiving cells with IDH1mutΔ7 had the same short latency to leukemia as mice receiving cells with full-length mutant IDH1, while IDH1wt and IDH1wtΔ7 cells died with significantly longer latency. The WBC count increased over time in IDH1mutΔ7 mice similar to IDH1mut mice, whereas WBC counts in IDH1wtΔ7 mice remained normal. IDH1mutΔ7 mice died from monocytic leukemia that was phenotypically and morphologically indistinguishable from IDH1mut mice. HoxA9 IDH1mutΔ7 cells were readily transplantable into secondary recipients. During in vivo cell cycle analysis, we observed that the proportion of cells in S/G2/M phases was significantly higher in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. These data suggest that mutant IDH1 enhances myeloproliferation even in the absence of R-2HG.

To identify R-2HG independent signaling pathways mediated by the mutant IDH1 protein, we first analyzed the gene expression of important regulators of cell cycle, differentiation, cell signaling and transcription by quantitative RT-PCR. Several genes (Ccnd1, Slc2a, Hdac3, Tgif2,and c-myc) were upregulated in IDH1mut and IDH1mutΔ7 cells compared to IDH1wt cells. Interestingly, we found a specific up-regulation of Ctnnb1 and Nfkb genes in IDH1mutΔ7 cells over both IDH1mut and IDH1wt cells. We next validated our mRNA expression results by immunoblotting and found that NFKB and ERK signaling were upregulated in both IDH1mut and IDH1mutΔ7 compared to IDH1wt and IDH1wtΔ7 cells. Interestingly, the protein level of β-catenin, STAT3 and STAT5 were many fold higher in IDH1mutΔ7 compared to IDH1mut and IDH1wt cells. β-catenin is known to be transactivated via c-Src, which is phosphorylated by EGFR to promote β-catenin nuclear localization and signaling. We traced this pathway for its relevance in our cells and found that IDH1mutΔ7 cells indeed showed higher levels of both EGFR and c-Src phosphorylation compared to IDH1mut cells. We performed immunofluorescence and cellular fractionation for β-catenin and found it to be partially localized in the nucleus in IDH1mutΔ7 but not in IDH1mut cells. We also observed an up-regulated STAT3 phosphorylation in IDH1mutΔ7 cells over IDH1mut.

Conclusions: In summary, mutant IDH1 activates ERK and NFKB signaling, which is attributed to both R-2HG dependent and independent mechanisms of leukemogenesis. Interestingly, IDH1mutΔ7 employs R-2HG independent EGFR/β-catenin and JAK/STAT signaling for oncogenesis. This R-2HG-independent leukemogenesis reveals a novel signaling dynamic of IDH1mut which should be evaluated for its therapeutic potential.