A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

Abstract

The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization.

Figure 1

Screening of a siRNA library of genes involved in DNA repair in SQ20B and MRC5 cells. A) Irradiated SQ20B cells. Z-scores of the top 30 genes associated with elevated γH2AX foci twenty-four hours after 4Gy radiation. B) Irradiated MRC5 cells. Z-scores of the top 30 genes associated with elevated γH2AX foci twenty-four hours after 4Gy radiation. C) Radiosensitisation of MRC5 cells with APEX2 depletion. Clonogenic assay in MRC5 cells treated with 50nM NT or APEX2 siRNA. **, P < 0.01 unpaired two sided t-test (left). Demonstration of effective knockdown of APEX2 by qRT-PCR. Gene expression normalised to cells treated with NT siRNA (right). D) Unirradiated SQ20B cells. Z-scores of the top 30 genes associated with elevated γH2AX foci in cells transfected with siRNA pools.

Figure 2

Effects of POLQ knockdown on γH2AX foci. A) In unirradiated SQ20B cells POLQ knockdown has no effect on γH2AX foci formation. B) γH2AX foci in SQ20B cells transfected with either NT or POLQ siRNA fixed 24 hours after receiving 4Gy. C) Increase in proportion of cells with >7 γH2AX foci in irradiated SQ20B cells with POLQ knockdown. **, P < 0.01 unpaired two sided t-test.

Figure 3

Radiosensitisation of tumour cells following POLQ depletion. A) Radiosensitisation of SQ20B (SER=1.18) and HeLa (SER=1.28) cells following POLQ siRNA transfection. **, P < 0.01 unpaired two sided t-test. B) Effective POLQ knockdown confirmed by qRT-PCR with SQ20B and HeLa cells.

Figure 4

Demonstration of the absence of off-target effects. A) T24 cells transfected with either 50nM POLQ pool of siRNA (SER=1.18), or 25nM of each individual siRNA strand. **, P < 0.01 unpaired two sided t-test. B) Relative expression of POLQ normalised to cells treated with NT siRNA as determined by qRT-PCR.

Figure 5

Effect of POLQ siRNA on normal cells. A) POC cells treated with POLQ siRNA were not sensitised to IR. B) MRC5 cells show modest sensitisation to POLQ depletion only at high doses of IR. **, P < 0.01 unpaired two sided t-test. C) Effective knockdown of POLQ in MRC5 cells treated with 50nM POLQ siRNA as determined by qRT-PCR. D) Relative expression of POLQ in untransfected T24 and MRC5 cells as determined by qRT-PCR. The expression of POLQ was expressed as a ratio relative to the presence of GAPDH in each cell line.

Figure 6

Effect of temozolomide treatment after POLQ knockdown. A) Clonogenic assays performed with SQ20B cells transfected with either NT or POLQ siRNA. Survival following temozolomide treatment expressed as a fraction relative to cells not exposed to temozolomide. B) Confirmation of effective knockdown of POLQ in SQ20B cells as determined by qRT-PCR. C) Effects of temozolomide treatment on the percentage of unirradiated SQ20B cells containing >7 γH2AX foci per cell. D) Percentage of SQ20B cells containing >7 γH2AX foci per cell following 4Gy irradiation.