Diffuse large B‐cell lymphoma (DLBCL) is characterized by extensive genetic heterogeneity, and this results in unpredictable responses to the current treatment, R‐CHOP, which consists of a cancer drug combination supplemented with the humanized CD20‐targeting monoclonal antibody rituximab. Despite improvements in the patient response rate through rituximab addition to the treatment plan, up to 40% of DLBCL patients end in a relapsed or refractory state due to inherent or acquired resistance to the regimen. Here, we employ a lentiviral genome‐wide clustered regularly interspaced short palindromic repeats library screening approach to identify genes involved in facilitating the rituximab response in cancerous B cells. Along with the CD20‐encoding MS4A1 gene, we identify genes related to B‐cell receptor (BCR) signaling as mediators of the intracellular signaling response to rituximab. More specifically, the B‐cell linker protein (BLNK) and Bruton's tyrosine kinase (BTK) genes stand out as pivotal genes in facilitating direct rituximab‐induced apoptosis through mechanisms that occur alongside complement‐dependent cytotoxicity (CDC). Our findings demonstrate that rituximab triggers BCR signaling in a BLNK‐ and BTK‐dependent manner and support the existing notion that intertwined CD20 and BCR signaling pathways in germinal center B‐cell‐like‐subtype DLBCL lead to programmed cell death.

SummaryThe frontline therapy R‐CHOP for patients with diffuse large B‐cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome‐wide CRISPR screening to uncover the cellular response to one of the components of R‐CHOP, vincristine, in the DLBCL cell line SU‐DHL‐5. We discover important pathways and subnetworks using gene‐set enrichment analysis and protein–protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB‐1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9‐directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine‐induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28‐53BP1‐p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. Inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 for which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response involving the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

Background: Overtreatment of indolent prostate cancer (PC) and delayed treatment of aggressive PC is common due to suboptimal risk stratification tools, thus warranting identification of novel prognostic biomarkers. Although a few long non-coding RNAs (lncRNAs) with biomarker potential in PC are known, the majority of lncRNAs remain uncharacterized. Here, we aimed to identify novel lncRNA biomarker candidates. We hypothesized that strong candidates would have a functional role in driving PC progression in addition to their expression being linked to PC prognosis, and we therefore combined functional CRISPR screening with lncRNA expression profiling of PC patients. Methods: Total RNA sequencing (RNAseq) data was generated from 31 adjacent normal (AN) and 125 tumor samples from 141 clinically localized PC patients, along with 17 primary tumor samples from metastatic PC patients. Raw reads were mapped to the hg38 reference genome and kallisto was used for quantification. CRISPR activating (CRISPRa) and CRISPR interference (CRISPRi) screens were performed in the LNCaP PC cell line stably expressing either dCas9-VP64 or dCas9-KRAB, respectively. Cells were transduced in duplicate with custom single guide RNA (sgRNA) libraries targeting 20,306 and 20,474 lncRNA transcripts of interest using 72,281 and 72,360 sgRNAs (CRISPRa and CRISPRi, respectively). Cells were harvested and DNA extracted from an early (day 4 post-transduction) and a late (day 17-21) timepoint and next-generation sequenced. MAGeCK was used for data analysis. Results: To identify lncRNAs with biomarker potential in PC, we analyzed lncRNA expression in total RNAseq data from 158 PC patients. Using differential expression analysis and cox regression analysis with biochemical recurrence as endpoint, we identified 6,928 lncRNAs with biomarker potential. To investigate if any of these had a functional role in driving PC progression, we performed CRISPRa and CRISPRi screens to assess how lncRNA activation/inhibition affected PC cell proliferation. Based on the screens, lncRNA candidates with the most prominent phenotypes (normalized read count difference >200 and log-fold change >33% between the early and late timepoint for ≥3 sgRNAs in both replicates) were selected for individual validation. This identified 7 (CRISPRa) and 8 (CRISPRi) negative hits (decreased cell proliferation) along with 5 (CRISPRa) and 2 (CRISPRi) positive hits (increased cell proliferation). Individually activated/inhibited LNCaP cell lines have been established for the 22 candidate lncRNAs and proliferation assays are performed to validate their functional role in PC progression. Conclusion: We identified numerous lncRNAs with biomarker potential and a possible driver role in PC progression. Citation Format: Simone Weiss, Allegra Lord, Bernhard Schmierer, Anne B. Rovsing, Emil A. Thomsen, Jacob G. Mikkelsen, Benedicte Ulhøi, Jakob S. Pedersen, Michael Borre, Karina D. Sørensen. Genome-Scale CRISPRa and CRISPRi screening for lncRNA drivers of prostate cancer progression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3778.

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