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 11

RAC/CDC42 pathway inhibition decreases Dnmt3a loss–induced myeloid skewing and improves B cells and T cells in mice.

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RAC/CDC42 pathway inhibition decreases Dnmt3a loss–induced myeloid skewi...
(A) BM cellularity quantitation for 2 femurs in each mouse from mice transplanted with Dnmt3a–/– cells and treated with the vehicle or MBQ-167. n = 5–6, mean ± SEM, *P = 0.05. (B) Flow cytometry analysis was performed on BM cells from mice transplanted with Dnmt3a–/– cells and treated with vehicle or MBQ-167. Quantitative data showing the absolute number of Lin–cKit+ myeloid progenitors, Lin–cKit+Sca-1+ (LSKs), CMPs, GMPs, and MEPs. n = 5–6, mean ± SEM, *P = 0.05. (C and D) Flow cytometry analysis was performed on BM cells from mice in A, and representative dot plots for CD150- and CD48-stained (LSK gated) HSCs as shown in F; quantitative data plotted for the frequency and absolute number of CD150–CD48+ cells is shown in G. n = 5–6, mean ± SEM, *P = 0.05. (E) Representative data showing the frequency of CLPs in BM cells from mice transplanted with Dnmt3a–/– cells and treated with vehicle or MBQ-167. n = 5–6, mean ± SEM, *P = 0.05. Flow cytometry was performed on BM cells from vehicle- or MBQ-167–treated mice as in Figure 10A, and a representative dot plot for Mac1/Gr1 expression is shown in F and B220/CD3 cells in I. Quantitative data showing decreased Gr1+Mac1+ in BM and PB fractions (G); increased CD3+ T cells in BM and spleen fractions (H); increased B220+ B cells in BM, spleen, and in PB fractions (J); and increased B220hiCD19hi mature B cells in BM fractions (K) of drug-treated mice compared with controls. n = 5–6, mean ± SEM, *P = 0.05, **P = 0.005, unpaired t test (2-tailed) performed (B, D, E, G, H, J, and K).