Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway
Lakshmi Reddy Palam, Baskar Ramdas, Katelyn Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce Selman, Utpal P. Dave, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur
Lakshmi Reddy Palam, Baskar Ramdas, Katelyn Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce Selman, Utpal P. Dave, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur
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Research Article Hematology

Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway

Abstract

Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which — when combined with other genetic lesions — result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a–/– HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a–/– LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation–driven myeloid malignancies.

Authors

Lakshmi Reddy Palam, Baskar Ramdas, Katelyn Pickerell, Santhosh Kumar Pasupuleti, Rahul Kanumuri, Annamaria Cesarano, Megan Szymanski, Bryce Selman, Utpal P. Dave, George Sandusky, Fabiana Perna, Sophie Paczesny, Reuben Kapur

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Figure 1

Loss of Dnmt3a in BMNC results in increased cell proliferation via the PI3K pathway.

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Loss of Dnmt3a in BMNC results in increased cell proliferation via the P...
(A) WT and Dnmt3afl/fl:Mx-Cre mice were administered poly I:C at 12–16 weeks of age, and BMNCs were subjected to Western blot analysis to detect the presence of DNMT3a protein. Two independent WT and Dnmt3a–/– mouse–derived BMNCs were utilized for these experiments. (B) BMNCs from WT or Dnmt3a–/– mice were cultured in media supplemented with SCF (50 ng/mL) or IL-3 (10 ng/mL) or no growth factor for 48 hours. Cell proliferation was evaluated by [3H] thymidine incorporation. Counts per minute (CPM) are shown. Three independent experiments, n = 6, mean ± SD, ****P < 0.00005. BM cells collected from WT or Dnmt3a–/– mice were subjected to RNA isolation and, subsequently, next-generation sequencing. (C and D) GSEA revealed an enrichment for genes in the PI3K signaling pathway (C), and upregulation of specific genes in the PI3K pathway are shown in the heatmap (D). (E) BMNCs from WT or Dnmt3a–/– mice were starved of growth factors and stimulated with SCF, followed by Western blot analysis. (F) Class I PI3K complex is composed of p85 regulatory subunit and p110α, p110β, and p110δ catalytic subunits. Activated PI3K signaling regulates AKT phosphorylation, which in turn promotes cell grow, cell survival, and proliferation. (G) WT, Dnmt3afl/fl:Mx-Cre, and Dnmt3afl/fl:p85αfl/fl:Mx-Cre mice were administered poly I:C at 12–16 weeks of age. BMNCs were collected from 3 different mice of each genotype and stimulated with SCF (25 ng/mL or 50 ng/mL) or in the absence of growth factors for 48 hours. Cell proliferation was evaluated by [3H] thymidine incorporation. Counts per minute (CPM) are shown. Experiment performed 3 times, and representative experiment shown with n = 4, mean ± SD, *P < 0.05, **P < 0.005, ****P < 0.00005. Two-way ANOVA, Tukey’s multiple-comparison test (B and C).