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 2

PI3K αβ inhibition modulates Dnmt3a loss–induced myeloid leukemia development.

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PI3K αβ inhibition modulates Dnmt3a loss–induced myeloid leukemia develo...
(A) Donor cells from Dnmt3a–/– mice were mixed with BoyJ cells in 1:1 ratio (0.5 × 106 versus 0.5 × 106) for a competitive transplantation assay. Six weeks after transplantation, mice were treated with vehicle or the PI3K αβ inhibitor (Bay1082439; 7 mg/kg body weight) or PI3K αδ inhibitor (GDC-0941; 75 mg/kg body weight) for 30 days, and mice were analyzed. (B) Representative images of liver and spleen from vehicle- and drug-treated mice are shown. (C) Quantitative assessment of spleen and liver weights from the indicated groups. n = 3–5, mean ± SD, **P < 0.005, ****P < 0.00005. (D) PB counts from mice described in B and C before they were sacrificed. n = 3–5, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005. The boxes shown with lower and upper quartiles separated by the median (horizontal line), and the whiskers extend to the minimum and maximum values. (E and F) Mice described in A were analyzed for PB chimerism 2 weeks after drug treatment and after 30 days after drug treatment for liver chimerism. Chimerism was assessed by staining the cells using an anti-CD45.2 antibody and flow cytometry. n = 3–5, mean ± SEM, *P < 0.05, **P < 0.005. One-way ANOVA in C, D, and F; 2-way ANOVA in E with Tukey’s multiple comparison test performed.