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FLT3-ITD leukemia, DOCK2 and DNA damage response
MK1775 (0.1 μM) or B02 (20 μM) (Figure 4B).
This assay indicates not only the overall percentage of cells that harbored elevated DNA damage, but also the extent of the damage as measured by the mean fluores- cence intensity of gH2AX. The differences in these meas- urements were particularly notable when cells were treat- ed with ara-C with or without DDR inhibitors. In the presence of ara-C, the percentage of cells with elevated gH2AX level was much higher among DOCK2 KD cells than among the control cells. In contrast, the mean fluo- rescence intensity of gH2AX was not markedly increased in the DOCK2 KD cells as compared to the control cells. This indicates that the DDR was activated in both control and DOCK2 KD cells after a low level of DNA damage was induced by ara-C, resulting in the arrest of DNA repli- cation and cell cycle to prevent further damage. As shown in Online Supplementary Figure S3, after ara-C treatment, control cells were able to overcome the cell cycle arrest due to higher DNA repair activity. In contrast, DOCK2 KD cells were unable to repair the damage and remained arrested. Therefore, at the point of measurement (16 h after ara-C treatment), when the majority of control cells had repaired the damage and resumed DNA replication and cell cycling, a much higher percentage of DOCK2 KD cells still harbored DNA damage. As expected, when a DDR inhibitor (MK8776 or MK1775) was added, the number of cells (gH2AX %) that exhibited DNA damage increased significantly over that following treatment with ara-C alone. Notably, the extent of DNA damage (gH2AX mean fluorescence intensity) in DOCK2 KD cells showed a marked increase over that of cells treated with ara-C alone, while a modest increase was observed in control cells. These findings are consistent with an increase in DNA damage level and a loss of DNA damage checkpoint response in cells treated with both ara-C and a DDR inhibitor, which are enhanced by suppression of DOCK2
(Figure 4B, Online Supplementary Table S2).
We further investigated whether suppression of Rac1
activity affects sensitivity to ara-C in primary mouse leukemic samples. Whole bone marrow cells from mori- bund Flt3+/ITD; NHD1345 and Flt3+/+; NHD13 mice46 that had developed acute leukemia were treated in vitro with ara-C and the Rac1 inhibitor NSC23766. As shown in Online Supplementary Figure S5, NSC23766 and ara-C acted syner- gistically to promote apoptosis in Flt3+/ITD; NHD13 leukemic bone marrow cells, but not in Flt3+/+; NHD13 bone marrow cells.
DOCK2 knockdown enhances the efficacy of ara-C treatment in a mouse xenograft model of FLT3-ITD acute myeloid leukemia, both alone and
in combination with MK8776
As previously reported, NSG mice transplanted with MV4;11 cells displayed markedly extended survival when expression of DOCK2 was suppressed.7 Since DOCK2 KD MV4;11 cells exhibit significantly increased sensitivity to treatments with ara-C and DDR inhibitors in vitro, we fur- ther investigated the effects of DOCK2 KD on the sensi- tivity of FLT3-ITD leukemic cells to these treatments in a mouse xenograft model. Mice were injected with 0.6 x 106 MV4;11 cells with or without DOCK2 KD cells via a lat- eral tail vein, and engraftment of the cells was monitored over time. Treatment with ara-C and/or DDR inhibitors was initiated when mice transplanted with control and DOCK2 KD cells reached similar levels of engraftment
(day 12 after transplantation for control mice and day 49 after transplantation for DOCK2 KD mice) (Online Supplementary Figure S6). Each mouse received daily intraperitoneal injections of vehicle, ara-C (50 mg/kg), MK8776 (10 mg/kg), MK1775 (15 mg/kg), ara- C+MK8776, or ara-C+MK1775 for 3 consecutive days. DOCK2 KD mice treated with ara-C showed extended survival that was statistically significant as compared with vehicle-treated mice (Figure 5). Furthermore, DOCK2 KD mice treated with ara-C+MK8776 showed slightly pro- longed survival that was statistically significant as com- pared with mice treated with either single agent alone (Figure 5A). Examination of the bone marrow 7 days after the start of treatment revealed a significantly reduced blast percentage in DOCK2 KD mice treated with the combina- tion of ara-C and MK8776, as compared with mice in other treatment groups (Figure 5B). In contrast, no signifi- cant difference in survival (Figure 5A) or bone marrow blast percentage (Figure 5B) was observed among mice transplanted with control MV4;11 cells and treated with any of the individual drugs or combinations.
Discussion
The treatment of AML with FLT3-ITD mutations repre- sents a significant clinical challenge. Although remission in patients harboring FLT3-ITD mutations can be achieved with cytarabine-based conventional induction chemother- apy with a frequency similar to other AML patients, the remission is often shorter and the relapse rates are higher.
One well-established mechanism of chemoresistance is the enhancement of DNA damage repair activity by onco- genic kinases, which promotes cancer cell survival in the presence of genotoxic stress. The elevated FLT3 kinase activity in FLT3-ITD leukemic cells leads to increased STAT5 activity, which regulates the activity of several key DDR regulators, including PIM-1, CHK1, WEE1, and RAD51. Furthermore, ERK, another downstream target of FLT3-ITD signaling, regulates expression of MMR factors via AP-1. Accordingly, we found that exogenous expres- sion of FLT3-ITD in TF-1 cells led to elevated activity of Rac1, increased expression of CHK1, WEE1, RAD51 and MMR factors, as well as significantly increased resistance to ara-C treatment. The increased expression of these MMR and DDR pathway components in FLT3-ITD cells is likely crucial for the cells’ survival, since FLT3-ITD drives an increase in reactive oxygen species resulting in increased DNA damage.
Our previous study revealed that decreased DOCK2 expression in FLT3-ITD leukemic cells leads to increased sensitivity to ara-C treatment. FLT3-ITD is known to acti- vate Rac1, which controls a variety of cellular functions.47 Of particular interest, Rac1 has been implicated in chemoresistance in cancer cells due to its regulatory roles in DDR pathways.48 Since DOCK2 functions as a guanine nucleotide exchange factor for Rac1, DOCK2 KD results in decreased Rac1 activity, thereby decreasing STAT5 and ERK phosphorylation, as well as markedly reducing the expression of downstream DDR factors. Interestingly, KD of DOCK2 also resulted in reduced expression and activity of FLT3-ITD. The mechanism by which FLT3-ITD is reg- ulated by DOCK2 is not completely clear. However, the expression of Meis1 and Myb, two known transcription regulators of FLT3, was also significantly downregulated
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