A Cyclin-Dependent Kinase Inhibitor, Dinaciclib, Impairs Homologous Recombination and Sensitizes Multiple Myeloma Cells to PARP Inhibition

Abstract

PARP1/2 are required for single-strand break repair, and their inhibition causes DNA replication fork collapse and double-strand break (DSB) formation. These DSBs are primarily repaired via homologous recombination (HR), a high-fidelity repair pathway. Should HR be deficient, DSBs may be repaired via error-prone nonhomologous end-joining mechanisms, or may persist, ultimately resulting in cell death. The combined disruption of PARP and HR activities thus produces synthetic lethality. Multiple myeloma cells are characterized by chromosomal instability and pervasive DNA damage, implicating aberrant DNA repair. Cyclin-dependent kinases (CDK), upstream modulators of HR, are dysregulated in multiple myeloma. Here, we show that a CDK inhibitor, dinaciclib, impairs HR repair and sensitizes multiple myeloma cells to the PARP1/2 inhibitor ABT-888. Dinaciclib abolishes ABT-888-induced BRCA1 and RAD51 foci and potentiates DNA damage, indicated by increased γH2AX foci. Dinaciclib treatment reduces expression of HR repair genes, including Rad51, and blocks BRCA1 phosphorylation, a modification required for HR repair, thus inhibiting HR repair of chromosome DSBs. Cotreatment with dinaciclib and ABT-888 in vitro resulted in synthetic lethality of multiple myeloma cells, but not normal CD19(+) B cells, and slowed growth of multiple myeloma xenografts in SCID mice almost two-fold. These findings support combining dinaciclib with PARP inhibitors for multiple myeloma therapy. Mol Cancer Ther; 15(2); 241-50. ©2015 AACR.

Figure 1. Synthetic lethality of MM cells, but not normal B cells, co-treated with the CDK inhibitor dinaciclib and the PARP1/2 inhibitor ABT-888

MM cell lines (A–C) or CD19⁺ B cells from normal peripheral blood (D) were treated with dinaciclib (SCH), ABT-888 (ABT), or the two combined (S+A), or B02 ± ABT (G), at the indicated doses. Cell viability was measured by WST-1 assay after 72 hours. Clonogenic survival assays were also run for MM.1S (E) and H929 (F) myeloma cells treated with 20-nM SCH, 20-μM ABT, or their combination. Mean survival or colony formation ± SEM are shown for 3 replicate experiments normalized to the untreated controls. *, ** and *** indicate p<0.05, 0.01 and 0.001, respectively, for 2-tailed paired t-tests against SCH treatment alone (open bar, DOX = 0 nM), with the exception of SCH treatment, which was compared to its vehicle-only control (i.e. contrasting the filled vs. open bar at DOX = 0 nM).

Figure 2. Dinaciclib impairs recruitment of BRCA1 and RAD51 proteins to DSB sites

MM.1S cells, exposed 24 h to DMSO, dinaciclib (SCH; 20 nM), ABT-888 (ABT; 20 μM) or SCH + ABT, were examined by immunofluorescence to identify foci, and DAPI staining to define nuclei. (A) Representative images of RAD51 and γH2AX foci in cells exposed to drugs indicated at left. (B) Mean percent of cells with ≥5 RAD51 or γH2AX foci, ±SEM, after the exposures indicated; data were combined from three experiments. (C) Integrated signal intensity per nucleus, after subtraction of peripheral background. (B, C) *, **, *** and **** indicate p < 0.05, p<0.01, p<0.001 and p<0.0001, respectively, for 2-tailed t-tests on the effect of drug treatment relative to DMSO (vehicle).

Figure 3. Dinaciclib reduces HR repair of chromosome DSBs in MM cells

(A) HR repair reporter substrate: The HR reporter DR-GFP is stably integrated in the genome of the MM.1S myeloma cell line (MM.1S-DR-GFP). The sceGFP is a GFP gene that contains an I-SceI endonuclease site within the coding region; its cleavage in vivo and repair by HR, using the downstream iGFP repeat as template, results in GFP⁺ cells. (B–D) Examples of flow-cytometric analysis of MM.1S-DR-GFP cells, wherein GFP fluorescence (x-axis signal) beyond the control boundary (segmented line) indicates HR repair. (E) Summary of combined data from runs such as those illustrated in (B–D), for cells without I-SceI transfection (mock-treated), cells treated with vehicle (DMSO), or 20-nM SCH, for 24h after transient transfection with I-SceI expression adenovirus (AdNUGS24i). HR data combined from three experiments are presented as mean ± SEM. Statistical significance of the difference between groups (SCH vs. DMSO, each n=3) was determined by a two-tailed t -test; ****p <0.0001.

Figure 4. Dinaciclib reduces the mRNA levels of RAD51, its paralogs and BRCA1, and reduces the protein levels of RAD51 and phosphorylated BRCA1 (ser1497)

(A) Reduction in the quantity of mRNA for Rad51, its paralogs and Brca1 as determined by RT-qPCR; (B, C) dose-dependent decrease in RAD51 protein levels; and (D, E) blockage of doxorubicin-induced phosphorylation of BRCA1 on S1497, as shown in western blots. Data were combined from at least two independent experiments (with n=3 per group in each experiment), shown as means ± SEM. *, **, *** and **** indicate p<0.05, 0.01, 0.001 and 0.0001, respectively, by 2-tailed t tests for the significance of differences between each treatment and its control (DMSO).

Figure 5. Dinaciclib induces S-phase accumulation and reduces G2/M-phase accumulation

MM.1S cells were treated as indicated for 24h, and (A, B) cell-cycle distribution was determined by propidium iodide (PI) staining. (C, D) BrdU incorporation was assessed to determine the percent of cells with S phase DNA content (as determined by PI staining) that are BrdU-positive or -negative. (E, F) After the indicated treatments, cells were co-stained for DNA content with PI, and for phospho-histone H3 (S10) with Alexa Fluor 488-conjugated antibody to phospho-histone H3 (S10). Panels (E) show representative flow-cytometry scatter plots. (F) Averaged results are shown from independent experiments. Each treatment differed from the DMSO control by 2-tailed t test at least p<0.001.

Figure 6. Effect of dinaciclib and ABT-888 on tumor growth and survival in SCID mice bearing MM xenografts

CB.17 SCID mice were inoculated subcutaneously with 5×10⁶ MM.1S cells, and mice were randomized into 4 groups (n=10) when tumors became measurable (100–150 mm³). Groups were treated with vehicle (DMSO), dinaciclib (SCH; 35 mg/kg i.p twice weekly), ABT-888 (ABT; 50 mg/kg p.o. twice daily, five times a week) or the combined treatments of SCH and ABT-888. Tumor volumes measured every 3 days as indicated in Materials and Methods. (A) Tumor volumes were evaluated on the third week of treatment and significance of differences in tumor volume among groups was determined by ANOVA followed by the Tukey's post hoc test; * and ** indicate p<0.05 and 0.01, respectively, for the indicated comparisons. (B) Kaplan-Meier survival was evaluated from the day treatment started to the day tumor volume reached the pre-determined termination point (see Materials and Methods). Significance of difference in survival between groups was determined by log-rank test; * and ** indicate p<0.05 and 0.01, respectively, relative to DMSO control.