Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F20%3A00118157" target="_blank" >RIV/00216224:14740/20:00118157 - isvavai.cz</a>

Výsledek na webu

<a href="http://www.interdisciplinarymeeting.cz/" target="_blank" >http://www.interdisciplinarymeeting.cz/</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement

Popis výsledku v původním jazyce

Standard of care for B-lymphoid malignancies nowadays still relies on the administration of monoclonal antibodies, with CD20 antigen being the prime target. Although effective at first, repeated cycles of anti-CD20 treatment, e.g. Rituximab (RTX), often result in the loss of CD20 from the surface of malignant B cells and consequently in therapy resistance1,2. In our first approach, we mimicked the situation in patients through chronic exposure of B-lymphoid cell line (Ramos) to gradually increasing doses of anti-CD20 antibody RTX. In this way, we have generated cell lines that are resistant to additional treatment with anti-CD20 antibodies and have low CD20 expression. Since epigenetic changes were predicted to play a role in CD20 regulation3,4, we aimed to uncover which epigenetic modifiers could enhance the expression levels of CD20 and recover its presence on the cell surface. Therefore, we screened our resistant CD20-low cells against a library consisting of 182 small-molecule compounds targeting various epigenetic modifying enzymes to determine surface CD20 expression changes by flow cytometry. In our second approach, we used CRISPR/Cas9 system for functional knockout screen of WT Ramos cells. We infected them with the CRISPR screen library to generate a genome-scale collection of single gene knockouts. Infected cells were either treated with RTX or used as control without RTX treatment and cultured for 2 weeks. The abundance of single gene knockout at the beginning versus the end of the incubation period was compared using next-generation sequencing. This approach allowed us to reveal enriched and lost gene knockouts after RTX administration. These genes could play a role in RTX therapy response. Unbiased combination of epigenetic drug screen and CRISPR knockout screen helped us to reveal possible treatment targets. Our results indicate the role of Aurora kinases in CD20 regulation and possible involvement of vesicle trafficking i n RTX antibody therapy resistance. Further analysis of mechanisms regulating CD20 expression is ongoing in order to validate the effect of detected targets.

Název v anglickém jazyce

Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement

Popis výsledku anglicky

Standard of care for B-lymphoid malignancies nowadays still relies on the administration of monoclonal antibodies, with CD20 antigen being the prime target. Although effective at first, repeated cycles of anti-CD20 treatment, e.g. Rituximab (RTX), often result in the loss of CD20 from the surface of malignant B cells and consequently in therapy resistance1,2. In our first approach, we mimicked the situation in patients through chronic exposure of B-lymphoid cell line (Ramos) to gradually increasing doses of anti-CD20 antibody RTX. In this way, we have generated cell lines that are resistant to additional treatment with anti-CD20 antibodies and have low CD20 expression. Since epigenetic changes were predicted to play a role in CD20 regulation3,4, we aimed to uncover which epigenetic modifiers could enhance the expression levels of CD20 and recover its presence on the cell surface. Therefore, we screened our resistant CD20-low cells against a library consisting of 182 small-molecule compounds targeting various epigenetic modifying enzymes to determine surface CD20 expression changes by flow cytometry. In our second approach, we used CRISPR/Cas9 system for functional knockout screen of WT Ramos cells. We infected them with the CRISPR screen library to generate a genome-scale collection of single gene knockouts. Infected cells were either treated with RTX or used as control without RTX treatment and cultured for 2 weeks. The abundance of single gene knockout at the beginning versus the end of the incubation period was compared using next-generation sequencing. This approach allowed us to reveal enriched and lost gene knockouts after RTX administration. These genes could play a role in RTX therapy response. Unbiased combination of epigenetic drug screen and CRISPR knockout screen helped us to reveal possible treatment targets. Our results indicate the role of Aurora kinases in CD20 regulation and possible involvement of vesicle trafficking i n RTX antibody therapy resistance. Further analysis of mechanisms regulating CD20 expression is ongoing in order to validate the effect of detected targets.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LQ1601" target="_blank" >LQ1601: CEITEC 2020</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů