The role of DNA damage and repair in decitabine-mediated apoptosis in multiple myeloma

Abstract

DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.

Figure 1. Decitabine has in vitroand in vivo anti-MM activity

A: Cells were treated with different concentrations of decitabine (DAC) for indicated timepoints. Apoptosis was determined by flow cytometry using AnnexinV-FITC/7'AAD staining. Apoptotic cell percentage is the sum of annexinV+ and AnnexinV+/7'AAD+ cell percentage. Dots and error bars represent mean and SD of 3 independent experiments. B: Cells were treated for 4 days. Total protein lysates were analyzed by western blot for the presence of caspase-9, -8, -3, PARP-1, BIM and MCL-1. tot. = total; cl. = cleaved. C: C57BL/KaLwRij mice were inoculated with 5T33MM cells and treated from day 1. The experiment was terminated when the first mice showed signs of morbidity. Treatment groups were vehicle (n=5), 0.1 (n=6), 0.2 (n=5) and 0.5mpk decitabine (n=6). After sacrification, BM from hind legs was isolated. Cytospins were made and stained with May Grünwald-Giemsa. BM plasmacytosis was quantified by manual counting. Total blood was collected and the serum M-spike was measured using serum electrophoresis. * indicates p<0.05 and ** indicates p<0.001 vs. vehicle. D: Mice were treated 1 day after inoculation with 5T33MM cells. Treatment groups were vehicle (n=9), 0.2 (n=9) and 0.5mpk decitabine (n=9). Mice were sacrified individually when showing signs of morbidity. Kaplan-Meier curves were constructed and significance was evaluated by a log-rank test.

Figure 2. Decitabine negatively affects cell cycle progression

A-B: OPM-2, NCI-H929, RPMI-8226 and JJN3 cells were treated with decitabine (DAC) for respectively 3, 3, 2 and 1 day(s). A: 2 hours prior to harvest, BrdU was added to the culture wells. Next, cells were stained with PI and anti-BrdU-FITC and analyzed by flow cytometry for DNA content and BrdU incorporation. Top: flow cytometry profiles of OPM-2 and RPMI-8226 cells. Bottom: Percentage of BrdU positive cells. B: Cell cycle profiles based on DNA content were obtained from PI histograms. Bars and error bars are mean and SD of 3 independent experiments. * indicates p<0.05 compared to control. C: Cells were treated for 3 days and total protein lysates were analyzed by western blot for the presence of p27 and SKP-2. ACTIN was used as loading control.

Figure 3. Decitabine induces gamma-H2AX foci formation

A-B: Cells were treated with decitabine (DAC; 1µM) for 24 and 48 hours. Melphalan (5µM, 24 hours) was used as positive control. Next, cytospins were made and stained for gamma-H2AX. A: Immunofluorescent pictures of OPM-2 and RPMI-8226 cells after staining for gamma-H2AX and DAPI. Scale bar= 50µm. B: Quantification of the gamma-H2AX foci using ImageJ macro PZ-FociEZ. At least 100 nuclei were analyzed and nuclei with at least 10 foci were scored as positive. Data are shown as mean ± SD of 3 experiments. * indicates p<0.05 compared to basal conditions. ‡ indicates p<0.05 compared to decitabine.

Figure 4. JNJ-585 enhances decitabine-mediated anti-MM effects

A-C: Cells were treated with decitabine (DAC) and/or JNJ-585 for 3 days (2 days for JJN3) (A) or 2 days (1 day for JJN3) (B, C). Doses used for OPM-2 were 1µM decitabine and 2.5nM JNJ-585; for RPMI-8226 1µM decitabine and 5nM JNJ-585; for JJN3 0.5µM decitabine and 10nM JNJ-585. A: Apoptosis was determined by flow cytometry using AnnexinV-FITC/7'AAD staining. Apoptotic cell percentage is the sum of annexinV+ and AnnexinV+/7'AAD+ cell percentage. Bars and error bars are mean ± SD of 3 experiments. B: Total protein lysates were subjected to western blot analysis for the expression of caspase-9, -8, -3, PARP-1, BIM and MCL-1. ACTIN was used as loading control. C: Samples were stained with PI and cell cycle profiles based on DNA content were obtained by flow cytometry. Bars and error bars are mean ± SD of 3 experiments. D: C57BL/KaLwRij mice were inoculated with 5T33MM cells and treated from day 1. The experiment was terminated upon first signs of morbidity of the mice. Treatment groups were vehicle (n=9), 0.2mpk decitabine (n=9), 1.5mpk JNJ-585 (n=9) or the combination (n=9). After sacrification, BM from hind legs was isolated. Cytospins were stained with May Grünwald-Giemsa and BM plasmacytosis was quantified by manual counting. Total blood was collected and the serum M-spike was measured using serum electrophoresis. E: Mice were treated 1 day after inoculation with purified 5T33MM cells. Treatment groups were as follows: vehicle (n=10), 0.2mpk decitabine (n=10), 1.5mpk JNJ-585 (n=10) and the combination (n=9). Mice were sacrified individually when showing signs of morbidity. Kaplan-Meier curves were constructed and significance was evaluated by a log-rank test. * indicates p<0.05 compared to control. ‡ indicates p<0.05 compared to decitabine. † indicates p<0.05 vs. single agents.

Figure 5. JNJ-585 affects the repair response elicited by decitabine

A-B: Cells were treated with decitabine (DAC) and/or JNJ-585 for 1 day. Doses for OPM-2 were 1µM decitabine and 2.5nM JNJ-585; for RPMI-8226 1µM decitabine and 5nM JNJ-585. Next, cytospins were made and stained for gamma-H2AX (A), RAD51 and 53BP1 (B). Images were quantified using ImageJ PZFociEZ plugin. A: Quantification of gamma-H2AX foci. B: Quantification of RAD51 and 53BP1 foci. At least 100 nuclei were analyzed and nuclei with at least 10 foci were scored as positive. C: Cells were treated with JNJ-585 for 1 day. Samples were processed and used for qRT-PCR to analyze expression of RAD51, BRCA1 and BRCA2. ABL-1 was used as housekeeping gene. D: Cells were treated with decitabine (1µM) and/or B02 (10µM) or ABT-888 (10µM) for 2 or 3 days. Next, apoptosis was determined by flow cytometry using AnnexinV-FITC/7'AAD staining. Apoptotic cell percentage is the sum of annexinV+ and AnnexinV+/7'AAD+ cell percentage. Data is shown as mean ± SD of 3 experiments. * indicates p<0.05 compared to untreated conditions. ‡ indicates p<0.05 compared to decitabine. † indicates p<0.05 compared to single agents.