Mutated IDH1 Has 2-Hydroxyglutarate-Independent Functions in Leukemogenesis
Bibliographic record
Abstract
Abstract Abstract 770 Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in glioma and AML patients. Mutant IDH produces R-2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R-2HG is required for transformation. To investigate the function of mutated IDH1 we established an in vivo mouse model, which allowed us to study R-2HG-dependent and –independent functions of mutated IDH1. We cloned wildtype IDH1 (IDH1wt) and mutated IDH1 (IDH1mut, R132C) from AML and MDS patients and identified a mutated splice variant that lacked exon 7 (IDH1mutΔ7) in several IDH1 mutated patients. To evaluate whether the mutated proteins produce the oncometabolite R-2HG, we expressed IDH1mut, IDH1mutΔ7, and IDH1wt in immortalized murine bone marrow cells and measured intracellular levels of 2HG by enantiomer-specific quantification. IDH1mut expressing cells produced high levels of R-2HG, but not IDH1 mutated cells that lacked exon 7 of IDH1, control-vector transduced cells (CTL), or IDH1 wildtype cells (P<.001). To functionally analyze mutated IDH1, we transduced IDH1wt, IDH1wtΔ7, IDH1mut, IDH1mutΔ7, or empty vector CTL in HoxA9-immortalized murine bone marrow cells and transplanted sorted cells in lethally irradiated mice. Mice receiving transplants with IDH1mut and IDH1mutΔ7 transduced cells had higher engraftment levels at 4, 8, and 12 weeks after transplantation (P<.001) than IDH1wt, IDH1wtΔ7, and CTL mice, and developed severe leukocytosis, anemia and thrombocytopenia, whereas IDH1wt, IDH1wtΔ7, and CTL mice had normal blood counts at 12 weeks. Mice with mutated IDH1, whether producing 2HG (IDH1mut) or not (IDH1mutΔ7), died with a median latency of 83 and 80 days after transplantation, respectively, whereas IDH1wt and CTL mice survived for a median of 167 and 210 days, respectively, and IDH1wtΔ7 mice were alive at 200 days (pooled data from 3 independently transduced cell populations that were transplanted at 3 different time points, P<.001). IDH1mut and IDH1mutΔ7 mice died with a myeloproliferative-like disease with high white blood cell counts, large spleen, anemia and thrombocytopenia. Bone marrow cells from moribund mice were readily transplantable in secondary animals, and rapidly induced disease. These data demonstrate that myeloproliferation is accelerated by an alternatively spliced mutant IDH1 independent of the metabolite R-2HG. In vivo cell cycle analysis showed a significantly higher proportion of cells in S/G2/M phase in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. Also, cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) were markedly downregulated in IDH1mut and IDH1mutΔ7 cells when compared to IDH1wt cells. We next investigated, whether the promoters of the repressed Cdk inhibitors were hypermethylated. The CpG island in the promoter of Cdkn2a and Cdkn2b showed low levels of DNA methylation in IDH1wt, IDH1mut, IDH1mutΔ7 and CTL cells (0.6% to 4.3% average methylation), suggesting that Cdk inhibitors are repressed in cells with mutated IDH1 independently of DNA methylation. Gene set enrichment analysis from microarray data of transduced bone marrow cells showed that genes related to MAP-kinase signalling were highly enriched in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL transduced cells. By Western blot we found that pERK was highly upregulated in IDH1mut and IDHmutΔ7 cells compared to IDH1wt or CTL cells. Pharmacologic inhibition of transduced cells in vitro showed that IDH1mut and IDH1mutΔ7 cells were resistant to inhibition with the MEK1/2 inhibitor UO126, suggesting that MAP-kinase signalling is a relevant target in IDH1 mutated AML. In summary, we show that an alternatively spliced isoform of mutated IDH1 promotes leukemogenesis independently of R-2HG in a mouse model of transformed hematopoietic cells with the same kinetics as mutated full-length IDH1 through transcriptional repression of cyclin-dependent-kinase inhibitors p15 and p16 and activation of MAP-kinase signalling. Our mouse model of mutated IDH1 represents a powerful tool to test the efficacy of newly developed drugs targeting mutated IDH1 in a 2-HG-dependent and –independent manner. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".