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 8

PI3K inhibition reverts Dnmt3a loss–induced changes in the expression of genes involved in cell migration and inflammation and alters the expression of fetal liver HSC genes.

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PI3K inhibition reverts Dnmt3a loss–induced changes in the expression of...
BM cells were collected from vehicle or PI3K αβ inhibitor–treated mice bearing Dnmt3a–/– malignant cells. RNA was isolated and subjected to next-generation sequencing, and gene expression analysis was performed. (A) Heatmap showing that PI3K αβ inhibitor treatment reduces the expression of genes involved in cell motility, cell attachment, inflammatory cytokines, and chemokines. (B and C) Quantitative assessment of the level of expression of IL-1α and IL-6 in drug-treated mice versus controls. n = 3, mean ± SEM, EdgeR DE analysis, *P = 0.05, ***P = 0.0005. (D) Heatmap showing gene expression involved in the development of fetal liver HSCs downregulated as a result of PI3K αβ inhibitor treatment in the Dnmt3a–/– cells versus controls. (E and F) Analysis of GMP progenitor gene expression data from a study involving Dnmt3a–/– and WT mice (13) was compared with the BM-derived gene expression data in the current study from vehicle- and PI3K αβ inhibitor–treated mice bearing Dnmt3a–/– cells. Venn diagram shows that 93 genes were found to be differentially regulated in both data sets. Comparative assessment of gene expression fold changes of indicated genes (F) in Dnmt3a–/–/WT and PI3Kαβ/vehicle treatment groups.