Enforced Differentiation of Dnmt3a-Null Bone Marrow Leads to Failure with c-Kit Mutations Driving Leukemic Transformation
Authors: Celik Hamza , Mallaney Cates , Kothari Alok , Miller Christopher A , Hundal Jasreet , Klco Jeffery M. , Challen Grant A 
Published: October 2019

Abstract
Genome sequencing studies of patient samples have implicated the involvement of various components of the epigenetic machinery in myeloid diseases, including the de novo DNA methyltransferase DNMT3A (Cancer Genome Atlas Research, N Engl J Med, 2013). We have recently shown that Dnmt3a is essential for normal hematopoietic stem cell (HSC) differentiation. Genetic ablation of Dnmt3a resulted in HSCs that showed diminished capacity for peripheral blood generation after serial transplantation (on a per-HSC basis), while phenotypically-defined HSCs accumulated in the bone marrow (Challen et al., Nature Genetics, 2012). Although this differentiation arrest was insufficient to cause overt disease, in these competitive transplants the presence of wild-type whole bone marrow may have suppressed malignant transformation of the mutant HSCs. Dnmt3a-null HSCs were less proliferative than counterpart control HSCs in this transplantation setting, suggesting that the cellular turnover threshold necessary to generate additional genetic and/or epigenetic lesions required for leukemogenesis was not achieved. To further understand the contribution of Dnmt3a loss-of-function in hematopoiesis, we performed non-competitive transplantation of Dnmt3a-null bone marrow. This forces the mutant HSCs to divide in vivoto regenerate the hematopoietic system following lethal irradiation, and should uncover any predispositions to transformation.
Mice transplanted with Dnmt3a-null bone marrow in the absence of wild-type support cells succumbed principally to bone marrow failure (median survival 328 days) characteristic of myelodysplastic syndromes (MDS) with symptoms including anemia, neutropenia, bone marrow hypercellularity and splenomegaly with myeloid infiltration. 2/25 mice developed myeloid leukemia with >20% blasts in the blood and bone marrow. 4/25 primary mice succumbed to myeloproliferative disorders, some of which progressed to secondary leukemia after long latency. Exome sequencing was performed to identify co-operating mutations that drove leukemic transformation, and revealed c-Kit mutations found only in the Dnmt3a-null AML samples. As DNMT3A and KIT mutations can co-occur in AML and mastocytosis, we tested whether these two pathways could co-operate in vivo by ectopic introduction of c-Kit variants into hematopoietic progenitors followed by bone marrow transplantation (Figure 1). As previously reported, expression of c-KitD814V in wild-type cells lead to development of B-cell acute lymphoblastic leukemia (B-ALL). However, expression of c-KitD814V in a Dnmt3a-null background lead to acute leukemia with a much shorter latency (median survival 67 days), implicating a synergism between these pathways in vivo. Moreover, the absence of Dnmt3a also distorted the spectrum of leukemia resulting from enforced c-Kit signaling. While some of the mice transplanted with Dnmt3a-null c-KitD814V cells also succumbed to a B-ALL, 4/13 (31%) developed mastocytosis with involvement of myeloid blasts, and 4/13 (31%) mice developed a T-cell acute lymphoblastic leukemia (T-ALL). We show for the first time that these pathways can co-operate to accelerate transformation in vivo. This Dnmt3a/c-Kit disease model resembles the classical “two-hit” model of leukemogenesis in which one mutation in a hematopoietic progenitor cell inhibits differentiation (Dnmt3a loss-of-function), whilst another drives proliferation (c-Kit gain-of-function). Such mouse models present a unique opportunity to study the sequence of early events leading to HSC transformation following Dnmt3a-inactivation.
Figure 1 Kaplan-Meier survival curve of mice transplanted with control or Dnmt3a-KO bone marrow progenitor cells transduced with a lentivirus expressing c-KitD814V. *** p <0.001. Figure 1. Kaplan-Meier survival curve of mice transplanted with control or Dnmt3a-KO bone marrow progenitor cells transduced with a lentivirus expressing c-KitD814V. *** p <0.001.

Disclosures
No relevant conflicts of interest to declare.