Abstract
Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3-ITD) are among the most common mutations in Acute Myeloid Leukemia (AML). Resulting in constitutive activation of the kinase, FLT3-ITD portends a particularly poor prognosis, with reduced overall survival and increased rates of relapse. We previously generated a knock-in mouse harboring an internal tandem duplication at the endogenous Flt3 locus, that develops a fatal myeloproliferative neoplasm (MPN), but fails to develop full blown leukemia, suggesting additional mutations are necessary for transformation. Global genomic sequencing studies have identified a substantial subset of patients in which FLT3-ITD and DNMT3A mutations are concomitantly present. Moreover, the co-occurrence of these mutations is significantly associated with adverse clinical outcomes (Patel JP, et al. NEJM. 2012;366:1079-89). Based on these observations, we investigated the potential cooperativity of FLT3-ITD and mutant DNMT3A to drive leukemogenesis using genetically engineered mouse strains.
In accordance with mounting evidence that DNMT3A mutations result in a loss of function, we used a mouse model harboring floxed Dnmt3a alleles (Dnmt3af/f), and a lymphocyte specific Cre transgene (Mx1-Cre+), which is activated upon injection with Polyinosinic-polycytidylic acid (PiPC). We used a substrain of our Flt3-ITD knock-in mice, which retains a floxed Neomycin (Neo) selection cassette from the initial targeting (Flt3ITDneo/+), and reduces expression of the mutant allele until PiPC treatment. Mice were bred to contain both types of mutation and were injected with PiPC intraperitoneally at 8 weeks of age and monitored for disease development. Mice containing both the FLT3/ITD and Dnmt3af/f developed leukemias. Hempatopoietic tissues were examined morphologically and by flow cytometry. Interestingly, deletion of Dnmt3a significantly reduced median survival of Flt3-ITD mice in a dose-dependent manner, with median survival of 162 days and 260 days for Dnmt3af/f;Flt3ITDneo/+ and Dnmt3af/+;Flt3ITDneo/+, respectively (Figure 1). Both genotypes confer a significantly shorter survival time compared to Flt3ITDneo/+ alone controls, which have a median survival of 412 days.
Moribund mice exhibited elevated white blood cell counts and splenomegaly, and developed a spectrum of diseases. As expected, Flt3ITDneo/+ mice solely developed MPN, while Dnmt3af/f;Flt3ITDneo/+ and Dnmt3af/+;Flt3ITDneo/+ developed a spectrum of neoplasms including MPN, T-ALL, and AML. Dnmt3a dosage influences the disease phenotype, as Dnmt3af/f;Flt3ITDneo/+ (n=18) develop T-ALL (46%), AML (31%), and MPN (23%), while Dnmt3af/+;Flt3ITDneo/+ mice (n=17) present with T-ALL (14%), T cell Lymphoma (14%), AML (43%), and MPN (29%).
Recent work has demonstrated that Dnmt3a deletion in hematopoietic stem cells (HSC) promotes self renewal and expansion of the LT-HSC pool (Challan GA, et al. Nat Genet. 2011;44:23-31). Conversely, Flt3-ITD disrupts LT-HSC quiescence, resulting in depletion of this compartment (Heiser D & Chu SH, et al. Cell Stem Cell. 2012;11:346-58). To investigate if Dnmt3a deletion might restore or expand the LT-HSC compartment when combined with Flt3-ITD, we examined the bone marrow of mice 8 weeks post PiPC injection, and found that Dnmt3af/f;Flt3ITDneo/+ mice displayed an expansion of the LT-HSCs, ST-HSCs, and Multipotent Progenitors compared to wild type, Dnmt3af/f;Flt3+/+, and Flt3-ITD mice. The HSC populations of Dnmt3af/+;Flt3ITDneo/+ mice exhibit similar proportions compared with Flt3-ITD mice, with a modest increase in the MPP population.
These results illustrate, for the first time, that Dnmt3a loss cooperates with Flt3-ITD to generate myriad hematopoietic neoplasms, including AML. In combination with Flt3-ITD, homozygous Dnmt3a knock-out results in reduced time to disease onset, LT-HSC expansion, and a higher incidence of T-ALL compared with loss of just one allele. The co-occurrence of FLT3 and Dnmt3a mutations in AML, as well as subsets of T-ALL, suggests the Dnmt3af/f;Flt3ITDneo/+ model may serve as a valuable resource for delineating effective therapeutic strategies in two clinically relevant contexts.
Kaplan-Meier Survival Curve. Median survival: Dnmt3af/f;Flt3ITDneo/+ = 162 days (n=24), Dnmt3af/+;Flt3ITDneo/+ =260 days (n=20), Flt3ITDneo/+ = 412 days(n=12).
Kaplan-Meier Survival Curve. Median survival: Dnmt3af/f;Flt3ITDneo/+ = 162 days (n=24), Dnmt3af/+;Flt3ITDneo/+ =260 days (n=20), Flt3ITDneo/+ = 412 days(n=12).
No relevant conflicts of interest to declare.
