Augmented HR Repair Mediates Acquired Temozolomide Resistance in Glioblastoma

Abstract

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults and is universally fatal. The DNA alkylating agent temozolomide is part of the standard-of-care for GBM. However, these tumors eventually develop therapy-driven resistance and inevitably recur. While loss of mismatch repair (MMR) and re-expression of MGMT have been shown to underlie chemoresistance in a fraction of GBMs, resistance mechanisms operating in the remaining GBMs are not well understood. To better understand the molecular basis for therapy-driven temozolomide resistance, mice bearing orthotopic GBM xenografts were subjected to protracted temozolomide treatment, and cell lines were generated from the primary (untreated) and recurrent (temozolomide-treated) tumors. As expected, the cells derived from primary tumors were sensitive to temozolomide, whereas the cells from the recurrent tumors were significantly resistant to the drug. Importantly, the acquired resistance to temozolomide in the recurrent lines was not driven by re-expression of MGMT or loss of MMR but was due to accelerated repair of temozolomide-induced DNA double-strand breaks (DSB). Temozolomide induces DNA replication-associated DSBs that are primarily repaired by the homologous recombination (HR) pathway. Augmented HR appears to underpin temozolomide resistance in the recurrent lines, as these cells were cross-resistant to other agents that induced replication-associated DSBs, exhibited faster resolution of damage-induced Rad51 foci, and displayed higher levels of sister chromatid exchanges (SCE). Furthermore, in light of recent studies demonstrating that CDK1 and CDK2 promote HR, it was found that CDK1/2 inhibitors countered the heightened HR in recurrent tumors and sensitized these therapy-resistant tumor cells to temozolomide.

Implications: Augmented HR repair is a novel mechanism underlying acquired temozolomide resistance in GBM, and this raises the possibility of improving the therapeutic response to temozolomide by targeting HR with small-molecule inhibitors of CDK1/2. Mol Cancer Res; 14(10); 928-40. ©2016 AACR.

Figure 1. Protracted TMZ treatment leads to acquired-TMZ resistance in GBM cells in vitro

A–D. TMZ sensitivities of glioma lines and their TMZ-treated counterparts (T12) were assessed by colony formation assays (T98G, U138, and U251), or by the neurosphere formation assay (GBM9). The fraction of surviving colonies (A–C), or neurospheres (D) (y-axis) is plotted against the corresponding TMZ dose (x-axis). Error bars, S.D. ****, p<0.0001. Lysates from corresponding cell lines treated with DMSO or 50 µM TMZ were probed for MGMT expression by Western blotting.

Figure 2. Protracted TMZ treatment leads to acquired-TMZ resistance in GBM cells in vivo

A. GBM9 cells were injected intra-cranially in nude mice. Brain tumors from vehicle-treated (primary) or TMZ-treated (recurrent) mice were harvested when the mice became moribund, and ex-vivo cultures were generated for further characterization. B. Primary (plots in black: 414, 483 and 484) and recurrent (plots in green: 419, 479 and 481) cultures were assessed for TMZ sensitivity by the colony formation assay. The fraction of surviving colonies (y-axis) is plotted against the corresponding TMZ dose (x-axis). Error bars, S.D. ****, p<0.0001 (419 vs. any primary culture); ***, p=0.0003 (479 vs. any primary culture); **, p=0.0017 (481 vs. any primary culture). C. Primary (483 and 484) and recurrent (419 and 481) cultures were treated with 50 µM TMZ for 48 hours, and cell cycle distribution was assessed by single-parameter flow cytometry after propidium iodide staining for DNA content (x axis).

Figure 3. Accelerated DNA double-strand break repair underlies acquired-TMZ resistance

A. Lysates from GBM9 cells and derivative ex-vivo cultures were probed for MGMT expression by Western blotting. MGMT-expressing T98G cells serve as a positive control. B. Lysates from GBM9 cells and derivative ex-vivo cultures were assessed for expression of mismatch repair proteins by Western blotting. C. Microsatellite instability was assayed by PCR amplification of BAT25, BAT26, BAT40 and D2S123 loci encompassing microsatellites to assess MMR proficiency. Low passage primary human skin fibroblasts (HSF) serve as a wild type reference cell line. A human colon cancer line - HCT116 - serves as a MMR-deficient control cell line. Please note matching band patterns (black arrow) for the HSF and GBM9-derived lines, and altered band pattern (red arrow) for the HCT116 line. D. Primary (483 and 484) and recurrent cultures (419 and 481) were pulsed with 10 µM TMZ for 48 hours, fixed at the indicated time points post-treatment, immunofluorescence-stained for 53BP1 foci (red), and imaged at 40× magnification. Nuclei are stained with DAPI (blue). Representative images are shown. Rates of repair of TMZ-induced DSBs were determined by scoring 53BP1 foci. Average number of 53BP1 foci per nucleus (y-axis) is plotted against the corresponding time post-treatment (x-axis). Error bars, S.E.M. ****, p<0.0001 (for any recurrent versus any primary culture at 48hr).

Figure 4. Recurrent cultures exhibit heightened repair of replication-associated breaks

A–D. Primary (483 and 484) and recurrent (419 and 481) cultures were treated with 1 Gy of IR (A), pulsed with 2 µM ETO for 1 hour (B), pulsed with 1 µM MNNG for 48 hours (C), or pulsed with 50 nM CPT for 2 hours (D), fixed at the indicated time points, immunofluorescence-stained for 53BP1 foci (red), and imaged at 40× magnification. Nuclei are stained with DAPI (blue). Representative images are shown. DSB repair rates were determined by scoring 53BP1 foci. Average number of 53BP1 foci per nucleus (y-axis) is plotted against the corresponding time post-treatment (x-axis). Error bars, S.E.M. ****, p<0.0001 (any recurrent versus any primary culture at 48 hours in C, and 24 hours in D).

Figure 5. TMZ-resistant cultures exhibit augmented homologous recombination repair

A. Primary (483 and 484) and recurrent (419 and 481) cultures were pulsed with 50 nM CPT for 2 hours, and fixed 2 hours later. Cells were immunofluorescence-stained for RPA foci (red), and imaged at 40× magnification to assess DNA end resection. Nuclei are stained with DAPI (blue). Representative images are shown. Average number of RPA foci per nucleus is plotted for DMSO- or CPT-treated cells. Error bars, S.E.M. B. Primary (483 and 484) and recurrent (419 and 481) cultures were pulsed with 50 nM CPT for 2 hours, fixed at the indicated timepoints post-treatment, immunofluorescence-stained for Rad51 foci (red), and imaged at 40× magnification. Nuclei are stained with DAPI (blue). Representative images are shown. Average number of Rad51 foci per nucleus (y-axis) is plotted against the corresponding time post-treatment (x-axis). Error bars, S.E.M. ****, p<0.0001 (any recurrent versus any primary culture at 8 or 24 hr). C. Primary (483 and 484) and recurrent (419 and 481) cultures were labeled with BrdU, and treated with DMSO or CPT (50 nM) over two cell division cycles. Metaphase spreads were prepared from the treated cells, and slides were stained with acridine orange (green) and imaged at 40× magnification. Representative images are shown. Reciprocal exchange events (red arrows) were counted. Plot shows average number of SCEs per chromosome per metaphase for DMSO- or CPT-treated primary and recurrent cultures. Error bars, S.E.M. ****, p<0.0001 (any recurrent versus any primary culture treated with CPT).

Figure 6. Targeting HR via CDK inhibition re-sensitizes recurrent cultures to TMZ

A. Recurrent cultures (419 and 481) were treated with 10 µM TMZ for 48 hours followed by addition of either DMSO or CDK inhibitors (AZD5438 or Roscovitine). Cells were fixed at the indicated times post-TMZ treatment, immunofluorescence-stained for 53BP1 foci (red), and imaged at 40× magnification. Nuclei are stained with DAPI (blue). Representative images are shown. Rates of repair of TMZ-induced DSBs were determined by scoring 53BP1 foci. Average number of 53BP1 foci per nucleus (y-axis) is plotted against the corresponding time post-treatment (x-axis). Error bars, S.E.M. ****, p<0.0001 (TMZ+DMSO versus TMZ+AZD or TMZ+Roscovitine at 48 h). B. TMZ sensitivities of the recurrent cultures (419 and 481) treated with DMSO or with CDK inhibitors (AZD5438 or Roscovitine) were assessed by colony formation assays. The fraction of surviving colonies (y-axis) is plotted against the corresponding TMZ dose (x-axis). Error bars, S.D. *, p=0.0109. **, p=0.0019. ***, p=0.0006.