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ITD-positive leukemic cells led to a concomitant decrease of STAT5 and Rac1 activity, and that DOCK2 knockdown (KD) in a FLT3-ITD leukemia cell line prolonged disease progression in a mouse xenograft model.7 Additionally, we found that DOCK2 KD leads to increased sensitivity to the chemotherapeutic agent cytarabine (ara-C), which is the backbone of AML therapy.7
In the current study we further investigated the mecha- nisms by which Rac1/DOCK2 activity affects cell survival and response to ara-C in FLT3-ITD leukemia cells. We found that DOCK2 KD in FLT3-ITD cells resulted in decreased expression and activity of FLT3-ITD itself, as well as decreased expression of both mismatch repair (MMR) and DDR factors. Additionally, exogenous expres- sion of FLT3-ITD resulted in elevated expression of DDR factors, increased Rac1 activity, and increased resistance to ara-C in TF-1 cells. Furthermore, DOCK2 KD significantly enhanced the sensitivity of FLT3-ITD leukemic cells to combined treatment with ara-C and DDR inhibitors, both in vitro and in a mouse xenograft model. These findings suggest that FLT3-ITD and Rac1/DOCK2 are key modula- tors of a coordinated regulatory network that controls DDR activity in FLT3-ITD leukemic cells, and also indicate that modification of DDR pathways may be of value in the treatment of FLT3-ITD AML.
Methods
Additional methods are detailed in the Online Supplement.
Cell culture assays
All assays were performed according to the manufacturers’ instructions. To measure cell proliferation after drug treatments, 0.5 x 106 cells/mL were placed in 24-well plates in triplicate, and cell densities were measured. Apoptosis assays were performed using annexin V-APC and 7-amino-actinomycin D (7-AAD; BD Biosciences, San Jose, CA, USA). Late apoptosis was defined as cells positive for both 7-AAD and annexin V, and apoptotic cells were cells positive for annexin V. The half maximal inhibitory con- centration (IC50) values of the drugs for each cell line were deter- mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Diagnostics, Indianapolis, IN, USA) (Online Supplementary Figure S1). Rac-1 activation (Rac1-GTP) was assessed using the G-LISA activation assay (Cytoskeleton, Inc., Denver, CO, USA). The levels of reactive oxygen species in cells were measured using CM-H2DCFDA (ThermoFisher Scientific, Waltham, MA, USA). The cell cycle was analyzed using a BD PharmingenTM BrdU Flow Kit (BD Biosciences), and flow cytomet- ric analysis of cellular gH2AX level was performed using Alexa Fluor 647-anti phospho-Histone H2AX (S139) antibodies (613408; BD Biosciences) in combination with the BD PharmingenTM BrdU Flow Kit.
starting times for treatments were determined based on pilot experiments that revealed the difference in disease progression in these two groups of mice. Engraftment in peripheral blood was assessed immediately prior to the start of treatment to ensure that the two groups of mice had similar peripheral blood blast levels (Online Supplementary Figure S6). Each mouse was given 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. When administered in combi- nation with ara-C, MK8776 and MK1775 were injected 30 min after the ara-C injection. Each treatment group contained at least ten mice, three to five of which were sacrificed for bone marrow engraftment analysis 7 days after the start of treatment, and the rest were monitored for survival. All animal procedures were con- ducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institute of Health, Bethesda, MD, USA) and were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University.
Statistics
Statistical analyses were performed with the Student t test (two- tailed), repeated measure analysis of variance, and log-rank tests using GraphPad (GraphPad Software, Inc., La Jolla, CA, USA). Each data point represents the average of at least three biological replicates. All data are presented as the mean ± standard error of the mean. P values <0.05 were considered to be statistically signif- icant.
Results
Decreased DOCK2 expression in MV4;11 cells leads to differential responses to ara-C and 5-fluorouracil treatment
The antimetabolite ara-C interferes with the synthesis of DNA, and is the backbone of both induction and consolida- tion regimens in the treatment of AML. KD of DOCK2 expression via stable expression of a short hairpin (sh)RNA in the FLT3-ITD MV4;11 leukemic cell line resulted in increased sensitivity to ara-C (3 μM), as indicated by increased apoptosis (Figure 1A) and reduced cell prolifera- tion (Figure 1B). However, when the same cell lines were treated with the thymidylate synthase inhibitor 5-fluo- rouracil (5-FU; 0.5 μM) they exhibited a markedly different response to treatment, with DOCK2 KD MV4;11 cells showing decreased apoptosis and increased cell prolifera- tion. These differential effects were not seen in REH cells, a leukemia cell line that expresses wildtype (WT) FLT3 (Figure 1A,B), or K562 cells, a leukemia cell line that does not express FLT3 (Online Supplementary Figure S2), suggesting that the FLT3-ITD mutation is responsible for the effect.
We further investigated the differential effects of ara-C and 5-FU treatment in the proliferation and cell cycling of FLT3-ITD-positive cells using a bromodeoxyuridine (BrdU) incorporation assay. Both control and DOCK2 KD MV4;11 cells showed arrested DNA synthesis in response to ara-C (Figure 1C). While control cells continued to syn- thesize DNA, albeit following a brief partial arrest and at a reduced rate, DNA synthesis was completely abrogated in DOCK2 KD MV4;11 cells within 2 h of ara-C treat- ment. DNA replication recovered faster in the control MV4;11 cells, while an overall reduction in replication per- sisted in the DOCK2 KD cells throughout the 26 h obser- vation period (Online Supplementary Figure S3A). In con- trast, 5-FU treatment of control cells resulted in progres-
Mouse transplantation experiments
NSG (NOD/Shi-scid/IL-2Rgnull) mice were provided by the Johns Hopkins Research Animal Resources. Each mouse (female, 6-8 weeks) was injected with 0.6 x 106 cells via the lateral tail vein. Engraftment was assessed by flow cytometric measurement of human and mouse CD45 expression on the cell surface (APC mouse anti-human CD45 and FITC rat anti-mouse CD45, BD Biosciences). Treatments of mice transplanted with control MV4;11 (MV4;11-C) cells started on day 12 after transplantation, while treatments of mice transplanted with DOCK2 KD MV4;11 (MV4;11-KD) cells started on day 49 after transplantation. The
haematologica | 2019; 104(12)
FLT3-ITD leukemia, DOCK2 and DNA damage response
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