GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress
Mullen MM, Lomonosova E, Toboni MD, Oplt A, Cybulla E, Blachut B, Zhao P, Noia H, Wilke D, Rankin EB, Kuroki LM, Hagemann AR, Hagemann IS, McCourt CK, Thaker PH, Mutch DG, Powell MA, Mosammaparast N, Vindigni A and Fuh KC
GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress
Mullen MM, Lomonosova E, Toboni MD, Oplt A, Cybulla E, Blachut B, Zhao P, Noia H, Wilke D, Rankin EB, Kuroki LM, Hagemann AR, Hagemann IS, McCourt CK, Thaker PH, Mutch DG, Powell MA, Mosammaparast N, Vindigni A and Fuh KC
Over 80% of women with high-grade serous ovarian cancer (HGSOC) develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to first-line platinum- and taxane-based chemotherapy or to PARP inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors. We found that GAS6 levels in patient tumor and serum samples collected before chemotherapy correlated with ovarian cancer chemoresponse and patient survival. Compared with chemotherapy alone, AVB-500 plus carboplatin and/or paclitaxel led to decreased ovarian cancer-cell survival and tumor burden . Cells treated with AVB-500 plus carboplatin had more DNA damage, slower DNA replication fork progression, and fewer RAD51 foci than cells treated with carboplatin alone, indicating AVB-500 impaired homologous recombination (HR). Finally, treatment with the PARP inhibitor olaparib plus AVB-500 led to decreased ovarian cancer-cell survival and less tumor burden . Importantly, this effect was seen in HR-proficient and HR-deficient ovarian cancer cells. Collectively, our findings suggest that GAS6 levels could be used to predict response to carboplatin and AVB-500 could be used to treat platinum-resistant, HR-proficient HGSOC. IMPLICATIONS: GAS6/AXL is a novel target to sensitize ovarian cancers to carboplatin and olaparib. Additionally, GAS6 levels can be associated with response to carboplatin treatment.
Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability
Graziano S, Coll-Bonfill N, Teodoro-Castro B, Kuppa S, Jackson J, Shashkova E, Mahajan U, Vindigni A, Antony E and Gonzalo S
Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability
Graziano S, Coll-Bonfill N, Teodoro-Castro B, Kuppa S, Jackson J, Shashkova E, Mahajan U, Vindigni A, Antony E and Gonzalo S
Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease-mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination-deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.
Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells
Tirman S, Quinet A, Wood M, Meroni A, Cybulla E, Jackson J, Pegoraro S, Simoneau A, Zou L and Vindigni A
Temporally distinct post-replicative repair mechanisms fill PRIMPOL-dependent ssDNA gaps in human cells
Tirman S, Quinet A, Wood M, Meroni A, Cybulla E, Jackson J, Pegoraro S, Simoneau A, Zou L and Vindigni A
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Aberrant RNA methylation triggers recruitment of an alkylation repair complex
Tsao N, Brickner JR, Rodell R, Ganguly A, Wood M, Oyeniran C, Ahmad T, Sun H, Bacolla A, Zhang L, Lukinović V, Soll JM, Townley BA, Casanova AG, Tainer JA, He C, Vindigni A, Reynoird N and Mosammaparast N
Aberrant RNA methylation triggers recruitment of an alkylation repair complex
Tsao N, Brickner JR, Rodell R, Ganguly A, Wood M, Oyeniran C, Ahmad T, Sun H, Bacolla A, Zhang L, Lukinović V, Soll JM, Townley BA, Casanova AG, Tainer JA, He C, Vindigni A, Reynoird N and Mosammaparast N
Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.
RADX prevents genome instability by confining replication fork reversal to stalled forks
Krishnamoorthy A, Jackson J, Mohamed T, Adolph M, Vindigni A and Cortez D
RADX prevents genome instability by confining replication fork reversal to stalled forks
Krishnamoorthy A, Jackson J, Mohamed T, Adolph M, Vindigni A and Cortez D
RAD51 facilitates replication fork reversal and protects reversed forks from nuclease degradation. Although potentially a useful replication stress response mechanism, unregulated fork reversal can cause genome instability. Here we show that RADX, a single-strand DNA binding protein that binds to and destabilizes RAD51 nucleofilaments, can either inhibit or promote fork reversal depending on replication stress levels. RADX inhibits fork reversal at elongating forks, thereby preventing fork slowing and collapse. Paradoxically, in the presence of persistent replication stress, RADX localizes to stalled forks to generate reversed fork structures. Consequently, inactivating RADX prevents fork-reversal-dependent telomere dysfunction in the absence of RTEL1 and blocks nascent strand degradation when fork protection factors are inactivated. Addition of RADX increases SMARCAL1-dependent fork reversal in conditions in which pre-binding RAD51 to a model fork substrate is inhibitory. Thus, RADX directly interacts with RAD51 and single-strand DNA to confine fork reversal to persistently stalled forks.