Table 1.
Proteins related to replicative stress and DSB processing: situation of inhibitor studies (in vitro) and relevant biomarkers.
| Inhibitor | Biomarkers | Combined Therapy or Deficiency | Model of Study | Results | Biological Explanation | Reference |
|---|---|---|---|---|---|---|
| Inhibitors in perspective for clinical trial in CRC (inhibitors already tested in clinical trial for another tumor type or condition) | ||||||
| PARPi (ABT-888) | Presence of MSI | MRE11 deficiency | Homozygous MRE11 mutant CRC cell lines (HCT116, LoVo, RKO, SW48) and wild type cell lines (HCT15, SW403, HT29, SW620) | Cell lines with homozygous MRE11 mutation had increased sensitivity to PARPi. | PARPi induces DSB during replication, which is repaired mainly by HR repair. However, the presence of MSI is associated with HR impairment by MRE11 deficiency, which sensitizes cells to PARPi. | [101] |
| ATRi (VX-970) | - | CHK1i (V158411) | CRC cell line (HT29 and U2OS) | Both inhibitors induced loss of viability and the combination of them in low doses increased sensitivity. | Both proteins act on the same cell cycle control axis-repair of DNA DSB forming during S and G2 phases. The inhibition of both reinforces the blocking of the axis, generating replicative stress. | [144] |
| CHK1i (LY2606368) | TP53 mutation and RS markers | - | Primary CRC enriched for cancer stem cells | Sensitive cells displayed signs of replicative stress (P-RPA32, γ-H2AX, and P-ATM) and the effect did not depend on RAS mutation status. | Cells that lack p53 depend on the ATR-CHK1 axis to promote cell cycle arrest when DSB is formed. Therefore, the inhibition of CHK1 kinase in these cells sensitizes them to DSB. Replicative stress induces DSB, therefore cells ongoing RS are sensitive to CHK1i. | [145] |
| WEE1i (AZD1775) | WEE1 overexpression | - | Primary CRC liver metastases endothelial cells | Induction of apoptosis, increase in DNA DSB markers and inhibition of tube formation. | The WEE1 protein is upregulated in CRC liver metastases compared to endothelial cells of the normal adjacent liver and has functional importance by protecting against DNA DSB. The inhibition of WEE1 makes cells vulnerable to DSB. | [146] |
| WEE1i (AZD1775) | TP53 mutation | 5-FU | CRC cell line (HT29) | The WEE1i single treatment had cytotoxic effects by induction of DNA DSB. The co-treatment with 5-FU increased DSB marker (γ-H2AX) and apoptosis compared to the 5-FU single treatment. | The lack of functional p53 prevents G1 arrest, so the cell depends on the G2 checkpoint to repair DNA damage. WEE1 is a kinase that regulates the G2 checkpoint to repair DNA damage, so targeting WEE1 increases DNA damage sensitivity. | [147] |
| WEE1i (AZD1775) | - | PARGi (PDDX-004/PDD00017272) | CRC cell line (HCT116) | The co-treatment increased DNA DSB marker (γ-H2AX) in S-phase dependent manner. | WEE1i induces replication stress and DNA damage while PARGi delays the replication fork restart during replication stress. The combination of both increases DNA damage. | [148] |
| WEE1i (MK1775) | TP53 mutation | Irinotecan | CRC cell line (HT29 and SW480) | The co-treatment increased DNA DSB marker (γ-H2AX) and apoptosis compared to single treatments. | The lack of functional p53 prevents G1 arrest, so the cell depends on the G2 checkpoint to repair DNA damage. WEE1 is a kinase that regulates the G2 checkpoint to repair DNA damage, so targeting WEE1 increases DNA damage sensitivity. | [149] |
| Inhibitors in progress in CRC in vitro studies | ||||||
| PARPi (LT-626) | Presence of MSI | MRE11 deficiency | CRC cell line with a biallelic mutation in MRE11 (HCT116, HCT116/CS, HCT116/C3, RKO, SW48, and LoVo), with monoallelic mutation (DLD1), and wild type (SW837, HT29, and SW480) | Cell lines with a biallelic mutation in MRE11 showed higher sensitivity to PARPi and the knocked-down of MRE11 increased sensitivity to PARPi. | PARPi induces DNA DSB during replication, which is repaired mainly by HR repair. However, the presence of MSI is associated with HR impairment by MRE11 deficiency, which sensitizes cells to PARPi. | [103] |
| RAD51i (RI-1) | KRAS mutation | - | CRC cell line (HCT116, HKe-3) | KRAS-mutant cell (HCT116) was more sensitive to RI-1 than the isogenic wild type HKe-3, which showed a limited response. | KRAS-mutated CRC cells show stalling of the replication fork, which increases HR repair signaling. This occurs because of the hyperactivation of c-MYC. Targeting RAD51, a key protein in HR repair, thus may sensitize KRAS-mutated CRC cells. | [150] |
| RAD51i (B02) | - | - | CRC cell line (SW480) | Induction of apoptosis. | RAD51 expression levels are upregulated in biopsy samples of CRC and may thus support cancer progression. | [48] |
| ATRi (CBP-93872) | TP53 mutation | Oxaliplatin, cisplatin, and 5-FU | CRC cell line (HT29) | ATRi single treatment had no effect, however, the combination with compounds that induce DNA damage as DNA DSB (oxaliplatin and cisplatin) or replication fork arrest (5-FU) induced an increase in apoptosis compared to single therapies. | The lack of functional p53 prevents G1 arrest, so the cell depends on the G2 checkpoint to repair DNA damage. ATR activation regulates the G2 checkpoint to repair DNA damage, so targeting ATR activation may increase DNA damage sensitivity by suppressing the maintenance of the G2 checkpoint. | [151] |
| ATMi (KU55933) | - | PARPi (Olaparib) | CRC cell line (HCT116) | ATMi sensitizes cells to PAPRi and the deletion of p53 increased the co-treatment effect. | PARP1 induces DNA DSB during replication, which activates ATM and so ATR promotes cell cycle arrest and DNA repair. The sensitivity to PARPi increases when the cell lacks ATM levels. | [152] |
| ATMi (AZ31) | - | Irinotecan | CRC cell line (HCT15, HCT116, RKO, LoVo, LS132, and Caco2) and patient-derived xenografts (PDX) | Three (HCT15, HCT116, and RKO) of the six cell lines presented combinational sensitivity to AZ31 and irinotecan compared to single treatments. In CRC PDX models, the co-treatment was effective only in irinotecan-resistant tumors. | Irinotecan induces DNA DSB, which activates ATM to promote cell cycle arrest and DNA repair. The inhibition of ATM may sensitize cells to DNA DSB. | [153] |
| Inhibitors in perspective for in vitro analysis in CRC | ||||||
| MRE11i (mirin) |
RS markers | - | Human myeloma cell lines (MM1S, RPMI-8226, JJN3, U266) and B-cell (LINF903). | Only cells presenting RS markers (RAD51 and γ-H2AX signaling) showed sensitivity to mirin. | Human myeloma cell lines presenting DNA DSB signaling depend on DSB repair pathways for survival. To inhibit DSB repair may sensitize them. | [154] |
| MRE11i (mirin) |
MYCN amplified | - | Neuroblastoma cell line with MYCN-amplification (SHEP, GIMEN, and SK-N-SH) and MYCN single copy (LAN5, IMR32, and KELLY) and LAN5 xenograft in mice. | Only the cell lines with MYCN-amplification showed increased mRNA levels of MRE11 and sensitivity to mirin treatment. Mirin suppressed tumor growth in xenografted mice. | MRE11 is required to restrain replication stress induced by MYCN-amplification. MRE11 inhibition may trigger intolerable levels of RS. | [155] |
| MRE11i (mirin) |
PARP1 upregulation | RAD51i (B02) | CRC-stem cells with upregulation of PARP1, generated by CHK1i (prexasertib) treatment until acquired resistance. | The single treatments with Mirin or B02 were ineffective against CHK1i-resistant cells, but co-treatment killed the cells by induction of mitotic catastrophe and apoptosis. | CHK1i-resistant cells upregulate PARP1 to modulate fork speed and decrease RS levels. MRE11 and RAD51 may cooperate with PARP1 to deal with RS. | [156] |
| RAD52i (F79 aptamer, D-I03) | BRCA1 deficiency | PARPi (talazoparib) |
BRCA1-deficient primary acute myeloid leukemia (AML) xenograft in NSG mice, and BRCA1-deficient solid tumor growth in nude mice. | The combination of PARP inhibitor with RAD52 inhibitors selectively reduced BRCA1-deficient tumor growth. | PARPi induces DNA DSB during replication, which is repaired mainly by HR repair. However, BRCA-deficient cells have HR repair impairment, and single-strand annealing (SSA) is a backup pathway that may support survival. The RAD52 inhibition may sensitize cells to PARPi by the accumulation of DNA DSB in BRCA-deficient tumor cells. | [157] |
| RAD52i (F79 aptamer, 6-OH-Dopa, D-I03) |
BRCA deficiency | PARPi (olaparib, talazoparib) | Several human tumor cell lines | The combination of PARP inhibitors with RAD52 inhibitors selectively killed BRCA-deficient cells. | See description above. | [157] |
| Polθi (Novobiocin) |
BRCA deficiency | PARPi (Olaparib) | Xenograft mice derived from patients with germline BRCA1 mutation and acquired PARPi resistance, and HR-proficient PDX model. | Olaparib single treatment did not reduce tumor growth, while Polθi single treatment reduced tumor growth and the combined therapy was even more efficient. BRCA1 wild type PDX model was resistant to both single and co-treatment. Polθi toxicity depends on the accumulation of RAD51 foci. | PARPi induces DNA DSB during replication, which is repaired mainly by HR repair, but also by MMEJ repair. BRCA-deficient cells have HR repair impairment and respond to PARPi. However, MMEJ has emerged as a backup pathway in PARPi resistant cells. The Polθ inhibition may prevent MMEJ repair and increase PARPi sensitivity in BRCA-deficient tumor cells. | [158] |
MSI: microsatellite instability; CRC: colorectal cancer; DSB: double-strand break; HR: homologous recombination; 5-FU: 5-fluorouracil.