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

a 2020

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

KOZLOVÁ, Veronika, Aneta LEDEREROVÁ, Veronika MANČÍKOVÁ, Michael DOUBEK, Jiří MAYER et. al.

Basic information

Original name

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

Authors

KOZLOVÁ, Veronika (203 Czech Republic, belonging to the institution), Aneta LEDEREROVÁ (203 Czech Republic, belonging to the institution), Veronika MANČÍKOVÁ (703 Slovakia, belonging to the institution), Michael DOUBEK (203 Czech Republic, belonging to the institution), Jiří MAYER (203 Czech Republic, belonging to the institution), Šárka POSPÍŠILOVÁ (203 Czech Republic, belonging to the institution) and Michal ŠMÍDA (203 Czech Republic, belonging to the institution)

Edition

XXth Interdisciplinary meeting of young researchs and students in the field of chemistry, biochemistry, molecular biology and biomaterials in Chemické listy, 2020

Other information

Language

English

Type of outcome

Conference abstract

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

Czech Republic

Confidentiality degree

is not subject to a state or trade secret

References:

URL

RIV identification code

RIV/00216224:14740/20:00118157

Organization unit

Central European Institute of Technology

Keywords in English

CRISPR KNOCKOUT SCREEN

Abstract

V originále

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.

Links

LQ1601, research and development project

Name: CEITEC 2020 (Acronym: CEITEC2020)

Investor: Ministry of Education, Youth and Sports of the CR

MUNI/A/1395/2019, interní kód MU

Name: Nové přístupy ve výzkumu, diagnostice a terapii hematologických malignit VII (Acronym: VýDiTeHeMa VII)

Investor: Masaryk University, Category A

Cite

KOZLOVÁ, Veronika, Aneta LEDEREROVÁ, Veronika MANČÍKOVÁ, Michael DOUBEK, Jiří MAYER, Šárka POSPÍŠILOVÁ and Michal ŠMÍDA. Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement. In XXth Interdisciplinary meeting of young researchs and students in the field of chemistry, biochemistry, molecular biology and biomaterials in Chemické listy. 2020.

@proceedings{1740859,
   author = {Kozlová, Veronika and Ledererová, Aneta and Mančíková, Veronika and Doubek, Michael and Mayer, Jiří and Pospíšilová, Šárka and Šmída, Michal},
   booktitle = {XXth Interdisciplinary meeting of young researchs and students in the field of chemistry, biochemistry, molecular biology and biomaterials in Chemické listy},
   keywords = {CRISPR KNOCKOUT SCREEN},
   language = {eng},
   title = {Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement},
   url = {http://www.interdisciplinarymeeting.cz/},
   year = {2020}
}

TY  - CONF
ID  - 1740859
AU  - Kozlová, Veronika - Ledererová, Aneta - Mančíková, Veronika - Doubek, Michael - Mayer, Jiří - Pospíšilová, Šárka - Šmída, Michal
PY  - 2020
TI  - Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement
KW  - CRISPR KNOCKOUT SCREEN
UR  - http://www.interdisciplinarymeeting.cz/
N2  - 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.
ER  -

KOZLOVÁ, Veronika, Aneta LEDEREROVÁ, Veronika MANČÍKOVÁ, Michael DOUBEK, Jiří MAYER, Šárka POSPÍŠILOVÁ and Michal ŠMÍDA. Combination of epigenetic drug screen and CRISPR knockout screen as an unbiased approach to reveal possible CD20 therapy improvement. In \textit{XXth Interdisciplinary meeting of young researchs and students in the field of chemistry, biochemistry, molecular biology and biomaterials in Chemické listy}. 2020.

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