DOCK2 Expression Affects Leukemogenesis and Disease Progression in a Murine Model of FLT3/ITD Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and an internal tandem duplication (ITD) mutation portends a poor prognosis. FLT3/ITD activates Rac1 GTPase, and both proteins play key roles in DNA damage response (DDR). Disease progression in FLT3/ITD AML is often heralded by copy-neutral loss of heterozygosity (CN-LOH) at the FLT3 locus, which is typically caused by duplication of the mutant allele through homologous recombination with loss of the wild-type (WT) allele.

We previously identified dedicator of cytokinesis 2 (DOCK2) as a FLT3/ITD-interacting protein. The DOCK family of proteins acts as guanine nucleotide exchange factors for the Rho family of GTPases, including Rac1. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts in FLT3/ITD AML. Knockdown of DOCK2 in FLT3/ITD cell lines results in reduced Rac1 activity, decreased expression and activity of FLT3 and DDR factors, and increased sensitivity to cytarabine.

In this study, we used mouse models to further investigate the relationship between DOCK2 and FLT3/ITD. Mice with a Dock2 mutation (Dock2/Hsd) that results in a marked reduction in Dock2 expression were bred with mice that carry a Flt3/ITD mutation (Flt3^+/ITD). Quantitative PCR of mouse bone marrow (BM) revealed that homozygous expression of Dock2/Hsd markedly decreased the expression of Flt3 and key DDR factors in both WT and Flt3^+/ITD mice. Flow cytometric analysis of the BM demonstrated that Dock2/Hsd resulted in partial reversal of the myeloproliferative neoplasm that develops in Flt3^+/ITD mice, with a significant reduction in multipotent progenitors (MPP) in Flt3^+/ITD;Dock2^Hsd/Hsd (0.026%) vs. Flt3^+/ITD mice (0.075%; P=0.048) at 3 months.

To assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine model of FLT3/ITD acute leukemia that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. It was previously shown that Flt3^+/ITD;NHD13 mice develop acute leukemia with 100% penetrance and short latency. We found that the median survival of Flt3^+/ITD;NHD13 mice (117 days) was not significantly affected by heterozygous expression of Dock2/Hsd (123 days); however, the median survival of Flt3^+/ITD; NHD13; Dock2^Hsd/Hsd mice was extended to 198 days (P=0.001). In contrast, Dock2/Hsd provided no survival benefit to mice that carried either Flt3^+/ITD or NHD13 alone. The prolonged survival of Flt3^+/ITD;NHD13;Dock2^Hsd/Hsd mice was accompanied by delayed onset of LOH at the Flt3/ITD locus. At 3 months, approximately half of Flt3^+/ITD;NHD13;Dock2^+/+ (47%; n=19) and Flt3^+/ITD;NHD13;Dock2^+/Hsd (57%; n=14) mice exhibited LOH in the peripheral blood, whereas only 11% of Flt3^+/ITD; NHD13;Dock2^Hsd/Hsd mice showed LOH (n=18; P=0.008 vs. Dock2^+/Hsd and P=0.029 vs. Dock2^+/+). Most of the moribund Flt3^+/ITD;NHD13 mice (n=13) developed AML with minimal maturation (70%), and the remainder developed T-lymphoblastic leukemia with an early T-precursor phenotype (ETP-ALL). In contrast, moribund Flt3^+/ITD;NHD13;Dock2^Hsd/Hsd mice (n=14) exhibited a more diverse disease spectrum: 30% mixed phenotype acute leukemia, 21% AML with minimal maturation, 21% ETP-ALL, 21% AML with maturation, and 7% acute erythroid leukemia (AEL). All moribund Flt3^+/ITD;NHD13 mice showed complete LOH in BM; however, only 68% of moribund Flt3^+/ITD;NHD13;Dock2^Hsd/Hsd mice exhibited complete LOH, with the rest showing partial or no LOH. Notably, all mice that exhibited partial or no LOH developed AEL or AML with maturation, diseases that were present in NHD13 mice but were not observed in Flt3^+/ITD;NHD13 mice. This suggests that suppression of Dock2 mitigates the effects of Flt3/ITD, allowing the NHD13 mutation to drive the development of disease.

These findings demonstrate that decreased DOCK2/Rac1 activity delays LOH at the FLT3 locus and disease progression in a murine model of FLT3/ITD leukemia. Thus, pharmacologic inhibition of the Rac1 signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML.