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.

Základní údaje

Originální název

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

Autoři

KOZLOVÁ, Veronika (203 Česká republika, domácí), Aneta LEDEREROVÁ (203 Česká republika, domácí), Veronika MANČÍKOVÁ (703 Slovensko, domácí), Michael DOUBEK (203 Česká republika, domácí), Jiří MAYER (203 Česká republika, domácí), Šárka POSPÍŠILOVÁ (203 Česká republika, domácí) a Michal ŠMÍDA (203 Česká republika, domácí)

Vydání

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

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Kód RIV

RIV/00216224:14740/20:00118157

Organizační jednotka

Středoevropský technologický institut

Klíčová slova anglicky

CRISPR KNOCKOUT SCREEN

Štítky

rivok

Změněno: 18. 3. 2021 20:06, Mgr. Pavla Foltynová, Ph.D.

Anotace

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.

Návaznosti

LQ1601, projekt VaV

Název: CEITEC 2020 (Akronym: CEITEC2020)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, CEITEC 2020

MUNI/A/1395/2019, interní kód MU

Název: Nové přístupy ve výzkumu, diagnostice a terapii hematologických malignit VII (Akronym: VýDiTeHeMa VII)

Investor: Masarykova univerzita, Nové přístupy ve výzkumu, diagnostice a terapii hematologických malignit VII, DO R. 2020_Kategorie A - Specifický výzkum - Studentské výzkumné projekty

Citovat

KOZLOVÁ, Veronika, Aneta LEDEREROVÁ, Veronika MANČÍKOVÁ, Michael DOUBEK, Jiří MAYER, Šárka POSPÍŠILOVÁ a 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 a 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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