Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51

Abstract

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

“…Results from this study indicate that sensitization to prexasertib by RAD51 or MRE11 inhibitors involves the boost of replication stress, which reinstates CRC-SC dependence on the function of the ATR-CHK1 pathway of the replication stress response. This is in line with previous evidence showing enhanced anti-CSC activity of ATR-CHK1 inhibitors due to the induction of replication stress by agents including (but not limited to) gemcitabine, irinotecan and inhibitors of PARP1, RRM2 or WEE1 [20,25,26,[32][33][34]. However, here, we showed that the two identified prexasertib-based regimens exerted a rather broad impact on the DDR of CRC-SCs, simultaneously impairing several cellular processes for the preservation of genomic stability.…”

“…Irrespective of these mechanistic unknowns, we demonstrated that mitotic catastrophe by RAD51+CHK1 or MRE11+CHK1 inhibitors resulted in tumorsphere disaggregation and CRC-SC death via caspase-dependent apoptosis. The particular proneness of CRC-SCs to mitotic catastrophe suggested by this and our previous study [26] can be potentially exploited for the design of effective colorectal cancer therapy.…”

“…We recently established distinct pairs of primary CRC-SCs sensitive (SENS) and resistant (RES) to pharmacological inhibitors of ATR and CHK1 kinases [26], the principal transducers of the cellular response to replication stress. From this panel, we selected two pairs of CRC-SCs: #1SENS/#1RES and #19SENS/#19RES, and used them to identify novel actionable targets improving the sensitivity and/or reverting the resistance to CHK1 inhibitors.…”

“…We then analyzed the impact of combined inhibition of CHK1 and RAD51 (by prexas-ertib+B02) and CHK1 and MRE11 (by prexasertib+mirin) on DNA replication and DNA damage in CRC-SCs. Through western-blot studies, we provided evidence that these two prexasertib-based regimens promoted an elevated increase in the phosphorylation of RPA32 (pRPA32) (Figure 2A), a marker of ongoing replication stress [20,26,29], as compared to prexasertib alone. As expected, replication stress was not augmented by the administration of mirin or B02 alone (Figure 2A; Figure S1).…”

“…Although most colorectal CSCs (CRC-SCs) are sensitive to inhibitors of the ATR-CHK1 cascade ultimately succumbing via replication catastrophe subsequent to the induction of intolerable or lethal levels of replication stress, a significant fraction of CSCs survives this regimen [20]. As a further limitation of these strategies, we recently revealed a mechanism of resistance to ATR-CHK1 inhibitors in CRC-SCs based on upregulation of poly(ADPribose) polymerase 1 (PARP1) [26], a DDR player with pleiotropic roles in DNA damage repair, the response to replication stress, and regulated cell death [27].…”