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Updated: Jun 21, 2025
Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
CRISPR-Cas9 Knockout Screens Identify DNA Damage Response Pathways and BTK as Essential for Cisplatin Response in
Issa Ismail Issa1,2, Hanne Due1, Rasmus Froberg Brøndum3
1Department of Hematology, Clinical Cancer Research Center, Aalborg University Hospital, 9000 Aalborg, Denmark.
Abstract:
The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3-5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27-54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL.
Insights
This study identified DNA damage response genes XPA and ERCC6, and BTK, as crucial for platinum drug sensitivity in diffuse large B-cell lymphoma (DLBCL). Targeting these genes could improve treatment outcomes for relapsed or refractory DLBCL.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Diffuse large B-cell lymphoma (DLBCL) recurrence affects 40% of cases, with poor prognosis for refractory/relapsed (RR-DLBCL) patients despite standard platinum-based chemotherapy.
- Identifying genetic factors influencing platinum drug response is critical for improving RR-DLBCL treatment efficacy.
Purpose of the Study:
- To identify genes that modulate cisplatin sensitivity in DLBCL using whole-genome CRISPR-Cas9 screening.
- To investigate the role of DNA damage response (DDR) pathways and Bruton's tyrosine kinase (BTK) in platinum drug response in DLBCL.
- To assess the clinical relevance of identified genes in DLBCL patient cohorts.
Main Methods:
- Conducted whole-genome CRISPR-Cas9 knockout screens with cisplatin treatment in DLBCL models.
- Performed functional analysis to assess DNA damage, cell cycle distribution, and drug synergy.
- Analyzed mutation frequencies of identified genes (XPA, ERCC6, BTK) in local and external DLBCL patient cohorts.
Main Results:
- DNA damage response (DDR) pathways were enriched among genes sensitizing DLBCL cells to cisplatin.
- Knockout of nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs, increasing DNA damage and altering cell cycle.
- BTK knockout enhanced cisplatin sensitivity, and ibrutinib (a BTK inhibitor) showed synergistic effects with platinum drugs.
- BTK mutations were frequent (3-5%) in DLBCL, with XPA and ERCC6 also mutated; 27-54% of DLBCL samples had mutations in cisplatin-sensitizing pathways.
Conclusions:
- XPA and ERCC6 are essential for cisplatin sensitivity in DLBCL by regulating DNA repair.
- BTK plays a significant role in platinum drug response, suggesting therapeutic potential for BTK inhibitors in combination therapy.
- Genetic alterations in DDR and BTK pathways are clinically relevant and impact platinum-based treatment efficacy in DLBCL.
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