DOCK2 Knockdown Mitigates Disease Progression in Murine Models Harboring a FLT3/ITD Mutation

Abstract

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts in the bone marrow and other tissues. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis.Previous work in the laboratory has demonstrated that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is also expressed in the leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity, and leads to decreased survival of leukemic cells that carry a FLT3/ITD mutation. This effect is seen in both in vitro studies and a mouse xenograft model, and is enhanced by treatment with cytarabine. Initial studies have shown that the anti-leukemic effect of DOCK2 KD may, in part, be due to reduced expression of both FLT3 and key DNA Damage Response (DDR) factors. In order to further investigate the relationship between DOCK2 and FLT3/ITD in hematopoietic neoplasms, we utilized mice that harbor a spontaneous Dock2 mutation (Dock2/Hsd). This mutation results in a substantial reduction in Dock2 mRNA and protein expression in hematolymphoid cells.Flt3/ITD mice develop a myeloproliferative neoplasm (MPN) that includes greatly increased spleen size. In contrast, the spleens of progeny of Flt3/ITD mice bred with Dock2/Hsd mice were greatly reduced in size. Flow cytometric analysis of the bone marrow also demonstrated that DOCK2 KD results in at least a partial reversal of the Flt3/ITD phenotype. Flt3/ITD mice characteristically demonstrate an increased fraction of immature hematopoietic stem/progenitor cells (Lin-Sca-1+c-KIT+ cells or LSKs); however, the fraction of LSKs in Flt3/ITD-Dock2/Hsd mice is similar to that seen in the marrow of WT mice. While the Flt3/ITD-Dock2/Hsd mice continued to show a disruption in the early to late Pro-B transition similar to that described in Flt3/ITD mice, the Flt3/ITD-Dock2/Hsd mice had a small but significant increase in the late Pre-B cell population, suggesting a less stringent block in B-cell development. Additionally, as compared with Flt3/ITD mice, the bone marrow of Flt3/ITD-Dock2/Hsd mice exhibited a decreased fraction of Mac1+Gr-1+ myeloid cells with a concomitant increase in Ter119+CD41- erythroid precursors, to levels that are indistinguishable from WT mice. These findings indicate that DOCK2 KD mitigates the myeloproliferative phenotype of Flt3/ITD mice.Since experiments using FLT3/ITD cell lines have suggested that DOCK2 KD may act by reducing expression of the FLT3 protein itself and also by inhibiting the DNA Damage Response, we evaluated these factors in the Flt3/ITD-Dock2/Hsd mice. Quantitative PCR of mouse whole bone marrow revealed that the mRNA levels of the Flt3 transcription regulators Meis1 and c-Myb were decreased in the Flt3/ITD-Dock2/Hsd mice, compared with Flt3/ITD mice, and the expression of Flt3 itself was markedly decreased as well. Moreover, reduced expression of key DDR factors including Chk1, Wee1, Rad51, Pim1, MSH2, MSH6 and MLH1 was also observed in Flt3/ITD-Dock2/Hsd mice.Finally, to assess the role of DOCK2 in leukemogenesis and disease progression, the Dock2/Hsd mutation was introduced into a murine AML model that carries both Flt3/ITD and the Nup98-HoxD13 (NHD13) fusion gene. The Flt3/ITD-NHD13 mice develop AML with 100% penetrance and short latency; however, the expression of Dock2/Hsd in Flt3/ITD-NHD13 mice extended the median survival of these mice from approximately 110 days to over 155 days (and counting).These findings suggest that DOCK2 may cooperate with FLT3/ITD and play an important role in disease progression of FLT3/ITD leukemia. Thus, pharmacologic inhibition of DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. Additionally, since DOCK2 KD results in decreased expression of DDR factors, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML. DisclosuresNo relevant conflicts of interest to declare.