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. 2016 Oct 10;30(4):637-650.
doi: 10.1016/j.ccell.2016.09.002.

Enhancing the Cytotoxic Effects of PARP Inhibitors With DNA Demethylating Agents - A Potential Therapy for Cancer

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Free PMC article

Enhancing the Cytotoxic Effects of PARP Inhibitors With DNA Demethylating Agents - A Potential Therapy for Cancer

Nidal E Muvarak et al. Cancer Cell. .
Free PMC article

Abstract

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.

Keywords: AML; DNA damage; DNA double-strand break; DNA repair; DNMT inhibitor; DNMT1; PARP; PARP inhibitor; PARP trapping; breast cancer.

Conflict of interest statement

DISCLOSURES C.R., F.V.R. and S.B.B. share co-inventor ship on US Provisional Patent Application Number: 61/929,680 for the concept of the combinatorial therapy.

Figures

Figure 1
Figure 1. PARP1 interacts with DNMT1 and DNMTi-PARPi combination binds PARP1 tightly in chromatin
Co-immunoprecipitation of DNMT1 or PARP1 in chromatin in (A) MDA-MB-231 or (B) MV411 cells untreated or treated with camptothecin (CPT, 1 µM) or methyl methanesulphate (MMS, 0.01%). Left panels: Western blotting for input proteins; right panels: co-IPs. IgG served as negative control. Note: in (A), lower panel (PARP1 IP), the DNMT1 band was cropped and moved next to IgG lane from the same gel. (C–F) PARP1, DNMT1 and histone H3 (loading control) in chromatin (nuclear insoluble) fractions from untreated and drug-treated cells. Upper panels, representative blots; middle/lower panels, quantitation of PARP1 and/or DNMT1 trapping. MOLM14 cells were collected post 72 hr treatment with (C) BMN 673- 1, 2.5, 5 nM or (D) DAC- 1, 2.5, 5 nM. (E) Combination treatments in MOLM14 with DAC and BMN 673 with indicated doses for 72 hr. (F) Combination treatment in MDA-MB-231 with AZA and BMN 673 with indicated doses for 72 hr followed by 4 Gy IR. Cells were collected 4 hr post IR for Western blotting. Experiments in triplicates are represented, mean ± SD (represented by error bars), # p<0.05 by t-test, single treatments vs. control, * p<0.05, by t-test, combination treatments vs. control and single treatments. See also Figures S1 and, S2.
Figure 2
Figure 2. Combination of AZA and BMN 673 increases the retention of DNMT1 and PARP1 at laser-induced DNA damage sites and increases cytotoxic DSBs
(A) MDA-MB-231 cells were treated with AZA (150 nM) and BMN 673 (10 nM) alone or in combination for 72 hr, followed by laser-induced DNA damage, then fixed at the indicated time points. Localization of DNMT1 and PARP1 to DNA damage sites was examined by immunofluorescence staining. Left panel, representative images at indicated time points. Right panel, graph of percentage cells with co-localization of PARP1 and DNMT1 at damage sites with single and combination drug treatments. Experiments in triplicates are represented. 30 cells per time point and treatment were analyzed for each experiment. * p<0.05, by t-test. Scale bar (upper left white bar) =10µm. (B,C) Graphs represent percentages of cells with γH2AX micro-irradiation tracts that have visible accumulation of DNMT1 (B) or PARP1 (C) co-localizing with γH2AX. Experiments in triplicates are represented. (D,E) Quantitation of the mean intensities of DNMT1 or PARP1 co-localized to γH2AX tracts that were quantified in single cells at the indicated time points after laser treatment. Experiments in triplicates are represented. 30 cells per time point and treatment were analyzed for each experiment * p<0.05, by t-test. (F,G) Graphs of γH2AX foci examined by immunofluorescence in untreated, AZA, BMN 673 and combination treated MDA-MB-231 cells (F) at indicated time points after IR (4 Gy). MOLM14 AML cells (G) untreated or treated with DAC, BMN 673 or the drug combination. Scale bars (lower left white bar) = 5 µm. Experiments in triplicate are represented; percent mean ± SD (represented by error bars), * p<0.05, ** p<0.01. See also Figures S3 and S4.
Figure 3
Figure 3. DNMTi and PARPi act synergistically to produce cytotoxicity
(A, B) MDA-MB-231 cells treated with (A) AZA (50–400 nM) and BMN 673 (2.5–20 nM), or (B) DAC (2.5–20 nM) and BMN 673 (2.5–20 nM). (C, D) MOLM14 cells treated with (C) AZA (50–400 nM) and BMN 673 (2.5–20 nM), or (D) DAC (2.5–20 nM) and BMN 673 (2.5–20 nM). Cells were treated daily for 7 days with the indicated drug combinations, followed by MTS assay to determine cytotoxicity. Upper panel, x-axis; Fa = fraction of cells affected; y-axis = combination index (CI). Combinations below red line are synergistic. Middle and bottom panels, survival of cells treated with DNMTi (DAC or AZA) or BMN 673 alone or in combination. Data are from 3 independent experiments, mean ± SD (represented by error bars). * p<0.05, ** p<0.01, by t-test, combination treatments vs single treatments.
Figure 4
Figure 4. DNMTis in combination with PARPis decreases clonogenicity
(A) Colony formation of non-tumorigenic MCF10A and TNBCs (SUM149PT, HCC1937, MDA-MB-231, MDA-MB-468). Cells were treated daily for 7 days with the indicated drug treatments. Results expressed as % survival relative to mock (Ctrl) treated groups. (B) Colony formation of AML (MOLM-14, MV411, KASUMI, MOLM13) cell lines and (C) primary AML cells untreated or treated with DAC (10–20 nM), AZA (50–100 nM), BMN 673 (1–5 nM) or combination. Cell lines were treated daily for 72 hr with indicated doses of DNMTis, followed by a 24 hr recovery period without DNMTis, then plated in methylcellulose with or without the indicated doses of BMN 673 and incubated for 10–14 days. Primary AML samples were treated as AML cell lines, except they were plated in Methocult for 14 days. Samples #081, #090, #107, #110 were treated with AZA, and samples #29, #34, #086, #092, #109 were treated with DAC. Experiments performed in triplicates; mean ± SD (represented by error bars). * p<0.05 by t-test, combination vs control and single treatments. See also Figures S5 and S6.
Figure 5
Figure 5. Anti-tumorigenic effect in xenograft models of TNBCs
(A–C) SUM149PT xenograft model. (A) Tumor volume (mm3) measurements (mean ± SD) in vehicle and drug treated groups with time for the duration of the experiment and euthanasia. Significant difference in tumor volume from Day 28 post treatment to the end of study is denoted by the asterisk and arrow. (B) Graphs of % survival with time (until tumor volume reached 1500 mm3) for the duration of the experiment. (C) Graph of mean ± SD % body weight change vs time for the duration of the experiment and euthanasia. (D–G) MDA-MB-231 xenograft model. (D) Graph of tumor volume (mm3) measurements (mean ± SD) in vehicle and drug treated groups with time for the duration of the experiment and euthanasia. Significant difference in tumor volume from Day 24 post treatment to the end of study is denoted by the asterisk and arrow. (E) Graph of % survival with time (until tumor volume reached 1500 mm3 or showed necrosis) for the duration of the experiment. Triangles denote mice removed from study due to necrosis of tumor. (F) Graph of mean ± SD % body weight change vs time for the duration of the experiment and euthanasia. (G) Tight binding of PARP1 into chromatin in tumors from euthanized MDA-MB-231 xenograft mice from control and treatment groups. Upper panel, PARP1 levels in chromatin. H3: loading control. Lower panel, quantitation of PARP1 levels. In all of the above, error bars represent the SD. See also Figure S7.
Figure 6
Figure 6. Anti-leukemic effect of BMN 673 and AZA in a systemic IV leukemia model MV411
(A) Bioluminescence measurements (mean ± SD) of photon intensity showing relative leukemia burden. (B) Graph of photon intensity with time post drug treatment up to 47 days and euthanasia. (C) Graph of spleen size (mm3) in different treatment groups post euthanasia. Measurements represent mean ± SD. Representative pictures of spleens are placed above graph for each group. (D) Graph of % blasts in the peripheral blood of mice from different treatment groups post euthanasia. (E) % body weight change vs time. Measurements represent mean ± SD. In all of the above, error bars represent the SD. See also Figure S7.
FIGURE 7
FIGURE 7. Anti-leukemic effect of BMN 673 and AZA in a systemic IV leukemia model MOLM14
(A) Bioluminescence measurements of photon intensity showing leukemia burden for duration of the experiment. Measurements represent mean ± SD. (B) Graph of photon intensity with time post drug treatment up to 61 days and euthanasia. Measurements represent mean ± SD (C) Graph of % survival with time post therapy. (D) % body weight change vs time. Measurements represent mean ± SD. In all of the above, error bars represent the SD. See also Figures S7 and S8.

Comment in

  • Synthetic lethality and beyond.
    Beltran H. Beltran H. Sci Transl Med. 2016 Nov 16;8(365):365ec182. doi: 10.1126/scitranslmed.aal0070. Sci Transl Med. 2016. PMID: 27856791 No abstract available.

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