Using a CRISPR-Cas Whole-Genome Screen to Identify Genetic Factors Linked to expression of Cancer-Associated O-glycans

TY - CONF

T1 - Using a CRISPR-Cas Whole-Genome Screen to Identify Genetic Factors Linked to expression of Cancer-Associated O-glycans

AU - Rømer, Troels Boldt

AU - Dylander, August

AU - Horn, Sabrina

AU - Pallesen, Emil Marek Heymans

AU - Wandall, Hans H.

PY - 2018/11/6

Y1 - 2018/11/6

N2 - Truncated O-glycans, designated Tn and STn, have been recognized as cancer-associated antigens correlated with worsened patient outcomes for more than four decades. In epithelial cancers, truncated O-glycans are identified in 70-90 % of cases and are often seen in premalignant lesions, making them attractive therapeutic targets as well as interesting biological entities potentially linked to fundamentally important processes in carcinogenesis. However, little is still known of the exact mechanism(s) resulting in the expression of truncated O-glycans in cancer cells. Several hypotheses have been proposed, including mutations of the CORE1 and COSMC genes, pH-dependent regulation, methylation of gene promotors, relocation of GalNAc-transferases in the secretory pathway and substrate deficiencies, but none of these hypotheses have yet to explain more than a limited subset of cases. To investigate potential genetic factors linked to Tn and STn expression, we therefore conducted a whole-genome genetic perturbation screen in the human keratinocyte HaCaT cell line with stable expression of the Cas9-protein and no expression of truncated O-glycans. Employing the glycan-specific monoclonal antibodies to Tn (mAb 5F4) and STn (mAb 3F1), we subsequently used FACS to separate cells with perturbated genes resulting in Tn or STn expression from keratinocytes not expressing truncated O-glycans. We found a subset (approximately 0.05 %) of the genetically modified cells from the whole-genome screen to be Tn- or STn-positive when analyzed på flow cytometry, with a larger proportion of Tn-positive cells as compared to STn-positive cells. We are currently using Next Generation Sequencing (NGS) on PCR products amplified from the signature sgRNA sequences imbedded in the lentiviral inserts from the Brunello library to identify candidate genes which – when knocked out – results in expression of truncated O-glycans in the Tn- and STn-positive cell populations. We aim to validate the candidate genes by conducting single-gene knock outs of the genes targeted by the most abundant sgRNAs in the Tn- and STn-positive cell populations. Subsequently, we seek to evaluate the single-cell knock outs by antibody staining, lectin staining and MS/MS to confirm the expression of Tn- and STn-antigens, as implied by the findings in the whole-genome screen. In conclusion, we conducted a whole-genome screen investigating the effect on expression of truncated O-glycans from perturbation of single genes by CRISPR-Cas9 induced knock outs in a human cell line. We find that the whole-genome screen results in expression of Tn- and STn-expression in a small number of cells – but a larger number than what can be explained from COSMC and CORE1 perturbations exclusively – and aim to present candidate genes and validated genetic targets involved in expression of truncated O-glycans in a human setting.

AB - Truncated O-glycans, designated Tn and STn, have been recognized as cancer-associated antigens correlated with worsened patient outcomes for more than four decades. In epithelial cancers, truncated O-glycans are identified in 70-90 % of cases and are often seen in premalignant lesions, making them attractive therapeutic targets as well as interesting biological entities potentially linked to fundamentally important processes in carcinogenesis. However, little is still known of the exact mechanism(s) resulting in the expression of truncated O-glycans in cancer cells. Several hypotheses have been proposed, including mutations of the CORE1 and COSMC genes, pH-dependent regulation, methylation of gene promotors, relocation of GalNAc-transferases in the secretory pathway and substrate deficiencies, but none of these hypotheses have yet to explain more than a limited subset of cases. To investigate potential genetic factors linked to Tn and STn expression, we therefore conducted a whole-genome genetic perturbation screen in the human keratinocyte HaCaT cell line with stable expression of the Cas9-protein and no expression of truncated O-glycans. Employing the glycan-specific monoclonal antibodies to Tn (mAb 5F4) and STn (mAb 3F1), we subsequently used FACS to separate cells with perturbated genes resulting in Tn or STn expression from keratinocytes not expressing truncated O-glycans. We found a subset (approximately 0.05 %) of the genetically modified cells from the whole-genome screen to be Tn- or STn-positive when analyzed på flow cytometry, with a larger proportion of Tn-positive cells as compared to STn-positive cells. We are currently using Next Generation Sequencing (NGS) on PCR products amplified from the signature sgRNA sequences imbedded in the lentiviral inserts from the Brunello library to identify candidate genes which – when knocked out – results in expression of truncated O-glycans in the Tn- and STn-positive cell populations. We aim to validate the candidate genes by conducting single-gene knock outs of the genes targeted by the most abundant sgRNAs in the Tn- and STn-positive cell populations. Subsequently, we seek to evaluate the single-cell knock outs by antibody staining, lectin staining and MS/MS to confirm the expression of Tn- and STn-antigens, as implied by the findings in the whole-genome screen. In conclusion, we conducted a whole-genome screen investigating the effect on expression of truncated O-glycans from perturbation of single genes by CRISPR-Cas9 induced knock outs in a human cell line. We find that the whole-genome screen results in expression of Tn- and STn-expression in a small number of cells – but a larger number than what can be explained from COSMC and CORE1 perturbations exclusively – and aim to present candidate genes and validated genetic targets involved in expression of truncated O-glycans in a human setting.

M3 - Poster

T2 - Annual Meeting - Society for Glycobiology

Y2 - 5 November 2018 through 7 November 2018

ER -