Inhibition of miR-1193 leads to synthetic lethality in glioblastoma multiforme cells deficient of DNA-PKcs

Abstract

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and has the highest mortality rate among cancers and high resistance to radiation and cytotoxic chemotherapy. Although some targeted therapies can partially inhibit oncogenic mutation-driven proliferation of GBM cells, therapies harnessing synthetic lethality are 'coincidental' treatments with high effectiveness in cancers with gene mutations, such as GBM, which frequently exhibits DNA-PKcs mutation. By implementing a highly efficient high-throughput screening (HTS) platform using an in-house-constructed genome-wide human microRNA inhibitor library, we demonstrated that miR-1193 inhibition sensitized GBM tumor cells with DNA-PKcs deficiency. Furthermore, we found that miR-1193 directly targets YY1AP1, leading to subsequent inhibition of FEN1, an important factor in DNA damage repair. Inhibition of miR-1193 resulted in accumulation of DNA double-strand breaks and thus increased genomic instability. RPA-coated ssDNA structures enhanced ATR checkpoint kinase activity, subsequently activating the CHK1/p53/apoptosis axis. These data provide a preclinical theory for the application of miR-1193 inhibition as a potential synthetic lethal approach targeting GBM cancer cells with DNA-PKcs deficiency.

Fig. 1. miR-1193 was identified from an automated, high-throughput screen as a specific synthetic lethal interacting partner with DNA-PKcs in M059J cells.

a Workflow of the anti-miRNA screening procedure. GBM cells (M059J and U118MG) were seeded into seven 384-well plates in 50 μl of medium on the first day. In-house designed antisense oligonucleotides targeting 2590 human miRNAs were individually transfected to knockdown the corresponding miRNAs. Cells were fixed, and nuclei were stained with Hoechst on day 4. Automated nuclei counting was performed with an acumen® Cellista laser scanning imaging cytometer. b Z scores were calculated from the screen of all antisense oligonucleotides targeting 2590 human miRNAs in both U118MG cells and DNA-PKcs-deficient M059J cells. The Z scores for miR-1193 are indicated in red and identified by black arrows. c Effects of anti-miR-1193 on the proliferation of U118MG, M059J and several other cell lines. d The survival fractions were calculated. The data are presented as the mean ± SD values from triplicate biological experiments. *P < 0.05, **P < 0.01, ***P < 0.005. NS not significant: p > 0.05.

Fig. 2. Synthetic lethality of miR-1193 in GBM cells treated with DNA-PKcs inhibitors.

Images of colony formation by M059K (a) and U251 cells (b) treated with the DNA-PK-inhibitors VX-984 (left panel; 0, 0.1, 0.5, and 1 µM) and NU-7441 (right panel; 0, 0.5, 1, and 2 µM) and transfected with anti-miR-1193 or anti-miR-NC. c–f Survival fractions were calculated. g–j Cell viability was measured by a CCK8 assay after 4 days of culture. The data are presented as the mean ± SD values from triplicate biological experiments. **P < 0.01, ***P < 0.005. NS not significant: p > 0.05.

Fig. 3. Synthetic lethality between miR-1193 and DNA-PKcs in DNA-PKcs-deficient M059J cells.

Isogenic M059J (DNA-PKcs −/−) and M059K (DNA-PKcs+/+) cells were cultured for 4 days after transfection with antisense oligonucleotides targeting miR-1193 (anti-miR-1193) or nontargeting antisense oligonucleotides (anti-miR-NC). a Image of colonies from the clonogenic assay in six-well plates. b Bar plots of clonogenic survival data. c Time course of cell viability using the CCK8 reagent kit. d Assessment of apoptosis by a TUNEL assay. e Quantification of the apoptotic fraction from d. f Assessment of apoptosis by an Annexin-V/PI assay. g Quantification of the apoptotic fraction from f. h Cell death proteins were assayed by western blotting. Images and blots are representative of three independent biological replicates. The data are presented as the mean ± SD values, and the error bars represent data from triplicate biological experiments. *P < 0.05, **P < 0.01, ***P < 0.005. NS not significant: p > 0.05.

Fig. 4. miR-1193 directly targets YY1AP1 and exhibits synthetic lethality with DNA-PKcs through the YY1-FEN1 pathway.

a Predicted miR-1193 binding sequence in the 3′-UTR of YY1AP1 (wild-type YY1AP1-3′-UTR) and in a mutant containing seven altered nucleotides indicated in red (YY1AP1-3′-UTR-mut). b Luciferase activity of the reporter constructs containing the 3′UTR of YY1AP1 with the wild-type or mutated miR-1193 binding site in 293 T cells after co-transfection with the control or miR-1193 expression constructs. The mRNA levels of miR-1193 (c) and YY1AP1 (d) were measured by RT-qPCR in M059K or M059J cells transfected with anti-miR-NC or anti-miR-1193. U6 RNA was used as the internal control. e Protein expression levels of YY1AP1, YY1, and FEN1 in M059K and M059J cells transfected with anti-miR-NC or anti-miR-1193. GAPDH was used as the loading control. f Colony formation assay of M059K and M059J cells transfected with shNC and shFEN1 and cultured for 4 days. The expression levels of FEN1 and GAPDH are presented. g The viability of transfected M059K and M059J cells was measured with a CCK8 kit across a time course. h Cell proliferation was measured by an EdU analysis of FACS. i Apoptosis was measured by a TUNEL assay and quantified. j, k The percentage of cells in S-phase and apoptosis under each condition was quantified. The images are representative of three independent biological replicates. The data are presented as the mean ± SD values, and the error bars represent data from triplicate biological experiments. *P < 0.05, **P < 0.01, ***P < 0.005. NS not significant: p > 0.05.

Fig. 5. miR-1193 inhibition in DNA-PKcs-deficient cells (M059J cells) leads to DSB repair defects and the formation of RPA-coated ssDNA intermediates.

DNA-PKcs-proficient M059K cells and anti-miR-NC were used as controls. a Treatment of M059J cells with anti-miR-1193 resulted in DSBs, as indicated by nuclear γH2AX and 53BP1 staining. b RPA foci were visualized by immunofluorescence. c–e Quantification of γH2AX, 53BP1, and RPA foci, respectively. Images are representative of three independent biological replicates. The data are presented as the mean ± SD values, and the error bars represent data from triplicate biological experiments. *P < 0.05, **P < 0.01, ***P < 0.005.

Fig. 6. miR-1193 inhibition in DNA-PKcs-deficient cells (M059J cells) results in activation of apoptosis through the ATR/Chk1/p53 axis and promotes chromosomal instability.

DNA-PKcs-proficient M059K cells and anti-miR-NC were used as controls. a Western blot of p-ATR, ATR, p-CHK1, CHK1, p-p53, p53, and GAPDH. Images of mitotic spread (b) and chromosomal abnormalities (d) were acquired and quantified. c, e The images are representative of three independent biological replicates. The data are presented as the mean ± SD values, and the error bars represent data from triplicate biological experiments. *P < 0.05, **P < 0.01, ***P < 0.005. NS not significant: p > 0.05.

Fig. 7. Model of synthetic lethality between miR-1193 inhibition and DNA-PKcs deficiency.

a Interference with either DNA-PKcs or miR-1193 alone did not reduce the viability of cancer cells, while cell death was achieved by combined abolition of DNA-PKcs and miR-1193. b Function loss of DNA-PKcs resulted in dysregulation of the NHEJ-mediated DSB repair pathway (left panel). Inhibition of miR-1193 led to blockade of the FEN1-mediated HR/MMEJ repair pathway (middle panel). Combined abolition of DNA-PKcs and miR-1193 promoted DSB repair deficiency and induced apoptosis regulated by the ATR/CHK1/p53 axis (right panel).