RAD51C germline mutations can result in Fanconi anemia-like disorder and make cells more susceptible to breast and ovarian cancers. These mutants were found to be hypomorphic in their functions.
Nuclear protein Poly (ADP-ribose) polymerase (PARP1) plays important roles in SSB repair and restart of stalled replication forks. PARP1 inhibition can cause DSB accumulation by increasing amount of non-repaired SSBs and collapsed replication forks. Using specific inhibitors to target PARP in order to cause "synthetic lethality" in BRCA1/2-deficient cells in breast and ovarian cancers has been investigated in clinical trials. It was known that RAD51C deficient cells can be targeted by PARPi, but targeting hypomorphic RAD51C mutant cells by PARPi can be difficult.
Chinese hamster lung fibroblast cells:
CL-V4B (RAD51C -/-)
V79B (Parental)
PHEN (PARPi)
4-ANI (PARPi)
Camptothecins (CPT) activate S or G(2)-M arrest and the homologous recombination (HR) repair pathway in tumor cells.
"RAD51CC-deficient cells can be targeted by PARPi." CL-V4B cell survival was significantly decreased in dose-dependent manner (compared to wtV79B cells) after CL-V4b cells were treated with different 4-ANI doses (0.1-10 uM). Similar results were obtained in p53-defective HeLa cells and p53-proficient MCF7 cells. PHEN was demonstrated to generate the same effect in these cell lines. It was concluded that RAD51C-deficient cells are extremely sensitive to PARPi due to accumulation of CL-V4b cells in G2/M boundary and increased chromatid aberrations and radial chromosomes.
"Cells expressing RAD51C pathological mutants are sensitive to PARP inhibitors." Missense mutations RAD51C L138F, G125V, L219S and Q143R resulted significant reduction in HR activity, but RAD51C G264S, R366Q, D159N and T287A only show their hypomorphic function in HR. All of these 8 mutants showed sensitivity to CPT. To show if they were also sensitive to PARPi, CL-V4B cells were treated with 0.5 uM and 10 uM 4-ANI. RAD51C L138F, G125V, L219S and Q143R were found to be highly sensitive to 4-ANI and show high levels of G2/M accumulation. But, RAD51C G264S, R366Q, D159N and T287A did not show much to the sensitivity and did not show much of the G2/M accumulation.
"PARP is hyperactivated in RAD51C-deficient cells due to replicative stress." PARP1 levels were similar in CL-V4B and wtV79B, but PAR formation controlled by PARP was significantly increased in CL-V4B cells. This hyperactive PARP activity was reduced with PARPi. PARP activity was high in RAD51C L138F, G125V, L219S and Q143R compared to in RAD51C G264S, R366Q, D159N and T287A. To test if the increase in PARP activity was due to replicative stress, they treated CL-V4B cells with hydroxuyrea to induce replicative stress. Interestingly, PARP activity was significantly elevated in these cells compared to the control. All of these results combine, they concluded that deficiency of RAD51C causes replicative stress which elevates PARP activity. To see how PARPi can affect the the replication process, CL-4B cells were treated with PARPi 4-ANI. Accumulation of PARP1 on chromatin and formation of DSBs were observed. So I can conclude that PARPi can be used to reduce PARP activity in RAD51C-deficient cells in order to generate "synthetic death" in cancerous cells caused by RAD51C-deficiency.
"Inhibition of PARP in RAD51C-deficient cells leads to enhanced recruitment of NHEJ factors." RAD51C deficient cells showed significant reduction of RAD51 foci formation. When V79B cells were treated with PARPi 4-ANI, RAD51 foci formation was increased compared to that in CL-V4B cells. 4ANI-treated RAD51C-deficient cells showed reduced RAD51 recruitment on chromatin but higher amount of NHEJ proteins, KU70, KU80, Ligase IV and Artemis, there. In addition, the NHEJ downstream protein, DNA-PKcs, was also activated in these treated CLV4B cells.
"PARP inhibition induces error-prone NHEJ in RAD51C-deficient cells and suppression of NHEJ rescues PARP inhibitor-induced toxicity in CL-V4B cells." The NHEJ efficiency in vivo was measure using GFP-based NHEJ reporter. Linearization with HindIII produces cohesive 4 bp overhangs. But digestion with I-SceI produces an inverted overhang that required nucleolytic end process by certain proteins before end joining/circularization. In CL-V4B cells, HR pathway is non-functional, leading to NHEJ to be more dominant even though NHEJ is an error-prone pathway. RAD51C-XRCC2 double depletion led to increase NHEJ in PARPi-treated cells. However, depletion of KU70 increased survival of 4-ANI treated-RAD51-deficient cells and rescued CPT-induced damage in RAD51-deficient cells, but made RAD51C-depleted cells become more sensitive to IR damage. Similarly, inhibition of DNA-KPcs, also one of the proteins required for NHEJ activity, or Ligase IV depletion also reduced the toxicity of PARPi in CL-V4B cells.
"Stimulation of NHEJ synergizes 4-ANI-induced toxicity in cells that express pathological RAD51C mutants."Activation of DNA-PKcs in CL-V4b cells was increased after treatment of IR and PARP more than each treatment alone. They also tested the combined effect of Zeocin and PARP can affect NHEJ pathway with and without inhibition of DNA-PKcs in both V79B and CL-V4B cells. They found that Zeocin and 4-ANI simultaneously resulted in moderate increase in cells V79B and massive increase in CL-V4B cells in radial chromosome structures. However, these effects were rescued when DNA-PKcs was present. These results indicated that "synergersm"/combined effects in induction of genomic instability was by activating the NHEJ pathway.
Interestingly, CL-V4B cells that express some RAD51C mutants were resistant to PARPi even at high drug concentrations; also, low dose of IR(2.5 Gy) did not affect cell survival at all. Therefore, they tested if the combination of low dose IR and PARPi can change this result. As expected, this combination generated 10 fold cell death in RAD51C G264S, R366Q, D159N and T287A. [Why did not they test the cell survival in RAD51C L138F, G125V, L219S and Q143R?] When they overexpress these RAD51C missense mutants in U2OS-SCR24 cells, then treated these cells with IR and 4-ANI in combination, they observed that HR activity in RAD51C L138F, G125V, L219S and Q143R was reduced by 3 folds and HR activity in RAD51C G264S, R366Q, D159N and T287A was moderately reduced. Similar results of combined effects of IR and 4-ANI were obtained when using these mutants in HeLa cells. Notably, the levels of RAD51C expression in these mutants was compatible to wtRAD51C. These results helped them conclude that RAD51C mutants can be treated with PARPi to cause DSB accumulation, and this effect can be further induced by using low-dosed IR and PARPi in combination to force cells to undergo NHEJ error-prone pathway, which leads to synthetic genomic instability and cell death.
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