SLFN11 and ATR as targets for overcoming cisplatin resistance in ovarian cancer cells

The levels and activities of the DNA/RNA helicase schlafen11 (SLFN11) and the serine/threonine-protein kinase ataxia telangiectasia and Rad3-related protein (ATR) may determine cancer cell sensitivity to DNA damaging agents, including platinum drugs. Here, we studied the roles of SLFN11 and ATR in cisplatin resistance of ovarian cancer using cell lines displaying acquired or intrinsic cisplatin resistance. W1CR, the cisplatin-resistant subline of W1 ovarian cancer cells, displayed reduced SLFN11 levels. HDAC inhibition using entinostat returned an epigenetic downregulation of SLFN11 in W1CR cells, caused SLFN11 re-expression and re-sensitized these cells to cisplatin. Moreover, entinostat also sensitized intrinsically resistant EFO21 ovarian cancer cells to cisplatin by upregulating SLFN11. However, SLFN11 was not involved in cisplatin resistance in all other cell models. Thus, SLFN11 expression is not a general cisplatin resistance marker in ovarian cancer. In contrast, inhibition of the DNA damage repair master regulator ATR using sub-toxic concentrations of elimusertib sensitized parental cell lines as well as intrinsically resistant EFO21 cells to cisplatin, and fully reversed acquired cisplatin resistance in cisplatin-adapted sublines W1CR, A2780cis, and KuramochirCDDP2000. Mechanisms underlying ATR-mediated cisplatin resistance differed between the cell lines and included CHK1/WEE1 signaling and induction of homologous recombination. In conclusion, SLFN11 and ATR are involved in ovarian cancer cisplatin resistance. Although our data identify ATR as key target for tackling cisplatin resistance in ovarian cancer, future studies are needed to identify biomarkers that indicate, which individual ovarian cancers benefit from SLFN11 re-activation and/or ATR inhibition

Stochastic variation in the FOXM1 transcription program mediates replication stress tolerance

Oncogene-induced replication stress (RS) is a vulnerability of cancer cells that forces reliance on the intra-S-phase checkpoint to ensure faithful genome duplication. Inhibitors of the crucial intra-S-phase checkpoint kinases ATR and CHK1 have been developed, but persistent proliferation and resistance to these drugs remain problematic. Understanding drug tolerance mechanisms is impeded by analysis of bulk samples, which neglect tumor heterogeneity and often fail to accurately interpret cell cycle-mediated resistance. Here, by combining intracellular immunostaining and RNA-sequencing of single cells, we characterized the transcriptomes of oncogenic RAS-expressing cells that exhibit variable levels of RS when challenged with a CHK1 inhibitor in combination with the chemotherapeutic drug gemcitabine. We identified 40 genes differentially expressed between tolerant and sensitive cells, including several FOXM1 target genes. While complete knockdown of FOXM1 impeded cell proliferation, a partial knockdown protected cells against DNA damage, and improved recovery from drug-induced RS. Our results suggest that low levels of FOXM1 expression protects subsets of oncogenic RAS-expressing cells against DNA damage during drug-induced replication stress

Combining ATR inhibition with topoisomerase I inhibitors for the treatment of colorectal cancer

Metastatic colorectal cancers are commonly treated with irinotecan, a topoisomerase 1 (TOP1) inhibitor. TOP1 inhibition results in the formation of TOP1-DNA cleavage complexes, which induce replication stress, and lead to activation of the Ataxia Telangiectasia and Rad-3 related (ATR) signalling pathway. Activation of ATR leads to cell cycle arrest and DNA repair, reducing the cytotoxicity of TOP1 inhibition. Using a panel of colorectal cancer (CRC) cell lines and patient derived organoids (PDOs), a synergistic interaction was identified between SN38 (active irinotecan metabolite) and VX-970 (ATR inhibitor). Treating CRC cell lines and PDOs with a combination of TOP1 and ATR inhibition resulted in increased ƔH2AX accumulation and the activation of double strand break (DSB) repair kinases, DNA-PK and ATM. Increased DNA DSBs and the formation of micronuclei in combination treated cells, further demonstrated the elevated levels of DNA damage. Colocalisation of cGAS with micronuclei was detected in combination treated cells, suggesting increased DNA damage may result in immune activation. Trastuzumab deruxtecan (DS-8201a) is a novel HER2 targeting antibody drug conjugate (ADC), carrying a potent TOP1 inhibitor (Dxd) warhead. Previous studies have reported minimal sensitivity of HER2-low expressing CRCs to DS-8201a therapy. In this study, several HER2-low CRC cell lines and PDOs were sensitised to DS-8201a by ATR inhibition (VX-970 and AZD6738). Loss of replication arrest and increased DNA-damage were identified as the mechanisms for the synergy observed. ATR inhibition was also explored as a possible mechanism to overcome irinotecan resistance. However, neither ATR inhibition combined with SN38, or ATR inhibition combined with DS-8201a was effective at reducing survival in two SN38 resistant cell lines. Understanding the interaction between TOP1 and ATR inhibition will better inform the treatment of CRC

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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