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. 2017 Oct 18;16(20):1861-1869.
doi: 10.1080/15384101.2017.1288325. Epub 2017 Sep 8.

PARPi potentiates with current conventional therapy in MLL leukemia

Lu Zhao  1 , Chi Wai Eric So  1
Affiliations

PARPi potentiates with current conventional therapy in MLL leukemia

Lu Zhao et al. Cell Cycle. .

Abstract

Acute myeloid leukemias driven by MLL fusion proteins are commonly associated with poor prognosis and refractory treatment. Here, we provide evidence that olaparib can potentiate sensitivity of MLL leukemia cells to conventional chemotherapy and DNMT inhibitors offering new potential therapeutic strategies for MLL rearranged leukemias Using the primary mouse leukemia cells and human MLL-AF9 leukemic cell line, we demonstrate that treatment of MLL-AF9 leukemic cells with DNMT inhibitors or chemotherapies in combination with olaparib results in significant reduction in colony formation or cell growth while the single agent treatments had little impacts. Combining olaparib with DNMT inhibitor induce cell cycle block and apoptosis. Furthermore, we observe a significant increase in proportion of cells with >10 γH2AX DNA damage foci and double stranded breaks when treated with DNMT inhibitors or chemotherapy agents in combination with olaparib, thus providing possible mechanistic insights for the synergism.

Keywords: AML; DNA damage; DNMT inhibitor; MLL; PARP; chemotherapy; olaparib; synthetic lethality.

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Figures

Figure 1.
Figure 1.
Olaparib potentiates anti-leukaemogenic activity of DNMT inhibitor in MLL leukemia. (a) Quantification of the number of colonies formed by MLL-AF9 LSCs in varying concentration of DNMT inhibitor and/or in combination with 1 µM olaparib. Unpaired t-test was performed between samples. Statistical significances are as indicated, * p < 0.05, *** p < 0.001. (b) Cell morphology of MLL-AF9 LSCs treated with DNMT inhibitor, olaparib or in combination. (c) Data shows percentage of cell that undergoing differentiation characterized by NBT-positive following treatment with DNMT inhibitor, olaparib or in combination. (d) Summary of cell cycle analysis showing relative percentage of cells in G0/G1, S and G2/M phases. Unpaired t-test was performed between samples. Statistical significances are as indicated, * p < 0.05, ** p < 0.01. (e) Quantification of percentage of Annexin V positive cells treated with chemotherapy treatments and/or in combination with olaparib Unpaired t-test was performed between samples. Statistical significances are as indicated, ** p < 0.01, ***p < 0.001. (f) Relative proliferation of patient-derived MLL-AF9 leukemic cell line, MOLM13 treated with DNMT inhibitor, olaparib or in combination Unpaired t-test was performed between samples. Statistical significances are as indicated, *p < 0.05, ** p < 0.01. (g) Summary of cell cycle analysis showing relative percentage of MOLM13 cells in G0/G1, S and G2/M phases after treatment. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, *p < 0.05, ** p < 0.01. (h) Quantification of percentage of Annexin V positive MOLM13 cells treated DNMT inhibitor, olaparib or in combination. Unpaired t-test was performed between samples. Statistical significances are as indicated, *p < 0.05, ** p < 0.01.
Figure 2.
Figure 2.
Olaparib enhances DNA damage induced with DNMT inhibitor in MLL leukemia. (a) Representative picture of γH2AX and RAD51 immunofluorescent staining of MLL-AF9 LSC treated with DNMT inhibitor, olaparib or in combination for 24 h. (b-c) Quantification of percentage of MLL-AF9 leukemic cells with greater than 10 γH2AX and greater than 6 RAD51 foci following treatment as indicated. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, *p < 0.05, ** p < 0.01, ***p < 0.001. (d) Quantification of comet tail formation in MLL-AF9 LSC following 24 h treatment of DNMT inhibitor, olaparib or in combination. Results are presented tail moment. At least 50 cells were analyzed for each condition. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, *p < 0.05, ** p < 0.01, ***p < 0.001.
Figure 3.
Figure 3.
Olaparib potentiates anti-leukaemogenic activity of conventional chemotherapy in MLL leukemia. (a) Quantification of the number of colonies formed by MLL-AF9 LSCs in varying concentration of chemotherapy agents and/or in combination with 1 µM olaparib. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, *p < 0.05, ***p < 0.001. (b) Cell morphology of MLL-AF9 LSCs treated with chemotherapy agents, olaparib or in combination. (c) Data shows percentage of cell that undergoing differentiation characterized by NBT-positive following treatment with chemotherapy agents, olaparib or in combination. (d) Summary of cell cycle analysis showing relative percentage of cells in G0/G1, S and G2/M phases are indicted. Unpaired t-test was performed between samples, Statistical significances are as indicated, *p < 0.05, ** p < 0.01. (e) Quantification of percentage of Annexin V positive cells treated with chemotherapy treatments and/or in combination with olaparib. (f) Relative proliferation of patient-derived MLL-AF9 leukemic cell line, MOLM13 treated with chemotherapy, olaparib or in combination. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, ** p < 0.01, ***p < 0.001. (g) Summary of cell cycle analysis showing relative percentage of MOLM13 cells in G0/G1, S and G2/M phases after treatment. (h) Quantification of percentage of Annexin V positive MOLM13 cells treated with chemotherapy, olaparib or in combination. Unpaired t-test was performed between samples. Statistical significances are as indicated ** p < 0.01, ***p < 0.001.
Figure 4.
Figure 4.
Olaparib enhances DNA damage induced with conventional chemotherapy in MLL leukemia. (a) Representative picture of γH2AX and RAD51 immunofluorescent staining of MLL-AF9 LSC treated with chemotherapy, olaparib or in combination for 24 h. (b-c) Quantification of percentage of MLL-AF9 leukemic cells with greater than 10 γH2AX and greater than 6 RAD51 foci upon treatment. Unpaired t-test was performed between samples. Statistical significances are as indicated *p < 0.05, ** p < 0.01, ***p < 0.001. (d) Quantification of comet tail formation in MLL-AF9 LSC following 24 h treatment of chemotherapy, olaparib or in combination. Results are presented tail moment. At least 50 cells were analyzed for each condition. Unpaired t-test was performed between indicated samples. Statistical significances are as indicated, ***p < 0.001.
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