Inhibition of the DOT1L/MPL/JAK2 Signaling Axis Selectively Targets <i>TET2</i> Mutation-Driven Clonal Hematopoiesis
Bibliographic record
Abstract
Abstract Clonal Hematopoiesis of Indeterminate Potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations associated with hematologic malignancies in individuals without evidence of a blood disorder. CHIP carriers are at a 10-fold higher risk of developing blood cancers relative to non-carriers. The time interval from acquisition of CHIP to overt neoplasia can span many years. This finding suggests a potential window of opportunity to intervene to prevent or delay malignant transformation; however, this goal is not yet possible due to the lack of known effective interventions. One of the most commonly mutated genes in CHIP is TET2, which encodes an epigenetic modifier involved in DNA demethylation. Tet2 knockout mouse models have an expansion of HSPCs and are at increased risk of development of myeloid malignancies, thus establishing TET2 mutation as a bona fide driver of pre-leukemia. To identify drugs that selectively target Tet2-deficient HSPCs, we generated isogenic murine cell lines by overexpressing HOXB4 in Sca-1 + HSPCs derived from a Tet2 knockout (Tet2-/-) mouse and a Tet2 wildtype (Tet2 +/+) littermate. HOXB4 overexpression has previously been shown to expand and immortalize HSPCs indefinitely without causing leukemogenesis. Using this isogenic cell line system termed HPC HOXB4, we conducted a drug screen of 37 small-molecule inhibitors of epigenetic signaling to find compounds that selectively reduced the proportion of Tet2-/- to Tet2+/+ HPC HOXB4 cells in competition assays. Through this screen, we discovered that DOT1L inhibitors, including SGC0946 and pinometostat, preferentially reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Importantly, SGC0946 treatment also reduced the competitive advantage of unmodified Tet2 -/- murine HSPCs over wildtype cells in short-term competition assays. Mechanistic studies revealed that DOT1L inhibition induced higher levels of apoptosis in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells and promoted the differentiation of Tet2-/- HPC HOXB4 cells. Genetic knockdown of Dot1l expression using shRNAs phenocopied the effects of pharmacologic DOT1L inhibition. DOT1L is a histone methyltransferase that catalyzes histone H3K79 methylation. Unexpectedly, we found that global H3K79me2 and H3K79me3 levels were higher in Tet2 -/- than in Tet2+/+ HPC HOXB4 cells. SGC0946 treatment effectively removed the H3K79me2/3 mark in Tet2 -/- cells. Next, we conducted RNA-seq analysis to gain insights into the transcriptomic changes in Tet2 -/- HPC HOXB4 after DOT1L inhibition. We found that expression of Mpl, which encodes the thrombopoietin receptor (TPOR), was over 20-fold higher in Tet2 -/- HPC HOXB4 than in wildtype cells at baseline and was strongly decreased after SGC0946 treatment. In contrast, SGC0946 had no effect on Mpl expression in Tet2 +/+ HPC HOXB4 cells. Mpl expression was also elevated in unmodified Tet2 -/- HSPCs compared with Tet2+/+ cells. Given that TPO signaling promotes HSC self-renewal and proliferation, we hypothesized that the effects of Dot1L inhibition are mediated through suppression of Mpl. Consistent with our hypothesis, enforced expression of Mpl in Tet2 -/- HPC HOXB4 cells completely rescued the effects of DOT1L inhibition, and downregulation of Mpl expression using shRNAs phenocopied the effects of DOT1L inhibition on Tet2 -/- HPC HOXB4 cells. Based on the findings above, we hypothesized that inhibition of TPOR signaling would selectively target Tet2 -/- over Tet2+/+ HSPCs. Given that Janus Kinase 2 (JAK2) is required to transduce signals downstream of the TPOR, we studied the effects of two potent JAK2 inhibitors, fedratinib and AZD1480, along with a JAK1/2 inhibitor, ruxolitinib, on Tet2 -/- and Tet2+/+ HPC HOXB4 cells in competition assays. In line with our hypothesis, all three compounds reduced the competitive advantage of Tet2 -/- HPC HOXB4 cells. Ruxolitinib also reduced the competitive advantage of unmodified Tet2 -/- HPSCs cells over wildtype cells in a dose-dependent manner. In summary, our data demonstrate that 1) the TPOR signaling pathway is upregulated in TET2-mutated HSPCs through epigenetic dysregulation by DOT1L, and 2) inhibition of DOT1L or the MPL/JAK2 signaling axis selectively targets TET2-mutated HSPCs. Our findings have important implications in the development of pharmacologic interventions against TET2-mutation driven CHIP. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding.
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".