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FLT3-ITD leukemia, DOCK2 and DNA damage response
sive accumulation of cells in the early S phase of the cell cycle throughout the 26 h observation period. The DOCK2 KD MV4;11 cells showed only a slight increase in the percentage of cells in S phase at later time points when treated with 5-FU (Figure 1C, Online Supplementary Figure S3B). These findings indicate that when leukemic cells are stressed via treatment with cytotoxic agents, DOCK2 KD affects cell proliferation and cell cycle differently in FLT3- ITD versus WT FLT3 cells.
DOCK2 and FLT3-ITD cooperate to regulate the DNA damage response in FLT3-ITD leukemic cells
5-FU is a thymidylate synthase inhibitor that blocks the synthesis of thymidine, and is utilized in the treatment of solid tumors including colorectal adenocarcinoma. MMR- deficient colorectal adenocarcinoma cells are reported to exhibit markedly decreased sensitivity to 5-FU treatment with a concurrent increase in sensitivity to ara-C, which is a profile similar to that seen in FLT3-ITD leukemic cells with DOCK2 KD.31,32 This suggests that DOCK2 may exert its effects on FLT3-ITD leukemic cell growth via DDR path- ways. To verify this, we evaluated the effects of DOCK2 KD on components of MMR and DDR in FLT3-ITD cells.
We first investigated the effects of DOCK2 KD on mRNA levels of key MMR and DDR factors. Decreased DOCK2 expression in MV4;11 cells resulted in significantly reduced mRNA levels of key MMR factors MLH1, MSH2 and MSH6, as well as DDR factors including CHK1, WEE1, RAD51 and PIM-1, although MLL (KMT2A) was not affect- ed (Figure 2A). Accordingly, western blot analysis demon- strated that protein levels of MLH1, MSH2, RAD51, PIM-1, CHK1, WEE1 and JUN were also markedly decreased in DOCK2 KD MV4;11 cells, as was the expression of activat- ed (phosphorylated) CHK1, WEE1 and JUN (Figure 2B, D). Of note, JUN is part of the AP1 complex that regulates the transcription of MMR factors.33
DOCK2 KD MV4;11 cells also exhibited significantly reduced expression of MEIS1 and MYB, which are known regulators of FLT3 expression (Figure 2A, B, D).34,35 Accordingly, the binding of MEIS1/2 and MYB to the regu- latory element located -15 kb from the FLT3 initiating codon was significantly reduced, as indicated by chromatin immunoprecipitation assays (Figure 2C), and the expression level and activity of FLT3 were markedly decreased in DOCK2 KD cells (Figure 2A, B, D). Similar changes in expression levels of FLT3 and DDR factors were also observed in the FLT3-ITD-positive Molm14 leukemia cell line (Online Supplementary Figure S4A). However, the expres- sion of most of the DDR factors examined was not signifi- cantly altered in REH cells, which express WT FLT3 (Online Supplementary Figure S4B).
Since DOCK2 KD leads to decreased Rac1 activity and FLT3 expression in MV4;11 cells, we investigated whether a pharmacological reduction in Rac1 and FLT3 activity would also lead to downregulation of DDR factors. After treatment with the Rac1 inhibitor NSC23766 (40 μM) or the FLT3 inhibitor sorafenib (25 nM), MV4;11 cells exhibit- ed a similar profile of protein expression changes as those seen in DOCK2 KD cells, including decreased phospho- STAT5 as well as AP1 and DDR factors (Figure 2D). These findings suggest that the downregulation of DDR activity observed in DOCK2 KD MV4;11 cells is likely due to reduced Rac1 and/or FLT3 activity in these cells. Furthermore, this observation is consistent with our previ- ous finding that FLT3 inhibitors markedly sensitized
DOCK2 KD MV4;11 cells to ara-C treatment, while control cells were not significantly affected.7
We further investigated the downstream effects of the reduction of MMR and DDR factors seen in association with DOCK2 KD in FLT3-ITD leukemic cells by assessing the phosphorylation of histone H2AX (gH2AX), which is triggered by DNA damage. Western blot analysis revealed a significantly reduced level of gH2AX in DOCK2 KD MV4;11 cells compared with the level in control cells (Figure 2B), suggesting a lower level of DNA damage and/or decreased baseline DNA repair activity in DOCK2 KD cells. This finding was confirmed by flow cytometric analysis of cellular gH2AX levels, indicating a greater percentage of cells with a high gH2AX signal (above the baseline level observed during normal DNA replication) in control MV4;11 cells versus DOCK2 KD cells (Figure 2E). Control MV4;11 cells showed increased DNA damage upon treat- ment with either ara-C (3 μM; 18 h) or 5-FU (0.5 μM; 18 h). In contrast, DOCK2 KD MV4;11 cells exhibited a signifi- cant increase in the gH2AX-high proportion only in response to ara-C but not 5-FU treatment. The 5-FU-treated DOCK2 KD MV4;11 cells demonstrated a gH2AX profile similar to that of untreated cells (Figure 2E). Meanwhile, both control and DOCK2 KD REH cells exhibited similar levels of gH2AX after treatment with either ara-C or 5-FU (Figure 2E). These data indicate that DOCK2 KD impedes the cells’ ability to repair damaged DNA upon ara-C but not 5-FU treatment.
In order to confirm that FLT3-ITD affects expression of DDR factors, we utilized a TF-1 leukemia cell line that does not normally express FLT3. Consistent with previous reports, TF-1 cells exogenously expressing a moderate (TF- 1-ITD-A) or relatively high level of FLT3-ITD (TF-1-ITD-B) both exhibited elevated Rac1 activity and an increase in the level of reactive oxygen species (Figure 3A). Downstream targets of FLT3-ITD signaling include STAT5 and ERK1/2. The STAT5 pathway is known to regulate the expression of DDR factors (CHK1, WEE1, RAD51), and the ERK1/2 path- way is known to affect the generation of MMR and DDR factors. Thus, we would expect both DDR and MMR fac- tors to be enhanced by exogenous expression of FLT3-ITD in TF-1 cells.33,36-41 Quantitative reverse transcriptase poly- merase chain reaction studies of TF-1 cell lines confirmed that expression of FLT3-ITD resulted in a significant increase in CHK1, WEE1, MSH2, MSH6, MLH1 and RAD51 expression, which positively correlated with the level of FLT3-ITD expression in these cells (Figure 3B). In contrast, relatively high expression of WT FLT3 in TF-1 cells resulted in only minor increases in the expression of MMR factors (MSH2, MSH6, MLH1), with no change in CHK1, WEE1 or RAD51 expression (Figure 3B). Consistent with increased DNA repair activity in FLT3-ITD-expressing TF-1 cells, these cells exhibited markedly increased resistance to ara-C treatment, which also correlated positively with the level of expression of FLT3-ITD (Figure 3C).
Taken together, these results indicate that DOCK2 expression affects the level of FLT3-ITD expression, with associated changes in the expression of DDR factors and DNA damage.
DOCK2 knockdown renders MV4;11 cells more sensitive to treatment with DNA damage response inhibitors
Since DOCK2 KD MV4;11 cells exhibit downregulation of CHK1, WEE1 and RAD51, we further investigated
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