DOCK2 Regulates DNA Damage Response and Modulates the Sensitivity to Chemotherapeutic Agents in FLT3/ITD Leukemic Cells

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 patients who harbor a FLT3/ITD mutation have a relatively poor prognosis.

Utilizing a co-immunoprecipitation/mass spectrometry analysis, we found 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 expressed in 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 with elevated FLT3 activity, both alone and in combination with cytarabine (Ara-C) treatment. Moreover, DOCK2 KD in transplanted FLT3/ITD leukemic cells prolongs disease progression in a mouse xenograft model.

We further investigated the mechanisms by which DOCK2 affects cell proliferation and response to chemotherapeutic agents in FLT3/ITD leukemic cells. DOCK2 KD via shRNA in a leukemia cell line that expresses FLT3/ITD (MV4;11) resulted in significantly elevated sensitivity towards Ara-C treatment, as indicated by a markedly increased apoptosis rate and reduced cell proliferation rate. Moreover, the survival of immunodeficient NSG mice transplanted with DOCK2 KD MV4;11 cells was prolonged after treatment with a relatively low dose of Ara-C, in contrast to no survival benefit observed in mice transplanted with control cells.  To investigate the mechanism of the increased sensitivity to Ara-C, we used a BrdU incorporation assay to investigate cell proliferation +/- drug treatment. Both control and DOCK2 KD cells showed arrested DNA synthesis and proliferation in response to Ara-C via the activation of DNA damage response (DDR), as evidenced by activation of the DDR effectors Chk1 and Wee1. The control cells continued to synthesize DNA and proliferate, albeit at a reduced rate, following a brief partial arrest; However, DNA synthesis was completely abrogated in DOCK2 KD MV4;11 cells within two hours of Ara-C treatment, and the arrest of DNA replication and cell proliferation persisted until apoptosis was initiated. As the resumption of DNA synthesis and cell proliferation requires efficient repair of damaged DNA, the persistent cell cycle arrest in DOCK2 KD cells suggests a deficiency in DNA repair.

To investigate the roles that DOCK2 plays in DNA repair, we quantified DNA damage via γH2AX levels. Western blot analysis revealed a progressive elevation of γH2AX levels in DOCK2 KD MV4;11 cells after Ara-C treatment, while the levels remained stable in control cells. Furthermore, DOCK2 deficiency in MV4;11 cells also led to reduced activity of Wee1 and Chk1, as well as decreased levels of Rad51, indicating multifaceted regulatory effects of DOCK2 on DNA repair pathways.

Another suggestion as to the function of the DOCK2/Rac1 pathway comes from the differential response of DOCK2 KD cells treated with Ara-C and 5-fluorouracil (5-FU).  While DOCK2 KD MV4;11 cells exhibited decreased proliferation and elevated apoptosis when treated with Ara-C, they demonstrated a significantly higher proliferation rate and lower apoptosis rate than control cells when treated with 5-FU. In contrast to Ara-C, treatment of DOCK2 KD MV4;11 cells with 5-FU resulted in stable γH2AX levels while γH2AX levels increased in control cells. These effects appear to be dependent on elevated FLT3 activity, as REH cells that express wild-type FLT3 did not show any observable difference in response to Ara-C or 5-FU. Interestingly, this differential response to Ara-C and 5-FU is characteristic of mismatch repair (MMR)-deficient colorectal cancer cells, which have decreased activity of key MMR components, such as MSH2 and MLH1. We found that levels of both MSH2 and MLH1 were significantly reduced in DOCK2 KD MV4;11 cells, suggesting that the MMR may also play a role in the response of FLT3/ITD leukemic cells to Ara-C.

Our observations suggest that FLT3/ITD-mediated differences in DDR and MMR may play a role in the relatively poor response of FLT3/ITD AML to standard AML chemotherapeutic regimens. Thus, the addition of DDR inhibitors may be useful in the treatment of FLT3/ITD AML, and pharmacologic inhibition of the Rac signaling pathways via DOCK2 provides a novel and promising therapeutic target for FLT3/ITD AML.