6 research outputs found
MV4-11 (FMS-like tyrosine kinase (FLT)3-internal tandem duplication (ITD) ๋ณ์ด ๋ฐ BCL2 ๊ณผ๋ฐํ) ์ธํฌ์ฃผ์์ midostaruin๊ณผ ABT199์ ์ํ ํ์ ํญ์์น๋ฃ
ํ์๋
ผ๋ฌธ (์์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ : ์ฝํ๋ํ ์ฝํ๊ณผ, 2019. 2. Diederich, Marc.๊ธ์ฑ ๊ณจ์์ฑ ๋ฐฑํ๋ณ (Acute myeloid leukemia, AML)์ ๊ณจ์์ฑ ํ์ก ์์ผ๋ก์, ์ ์ ํ์ , ์๋ฆฌํ์ ์ด์์ผ๋ก ๋ฐ๋ณ๋๋ ๊ฒ์ผ๋ก ๋ณด๊ณ ๋์ด ์๋ค. ํนํ, FMS-like tyrosine kinase 3 (FLT3)๋ ์ธํฌ ์์กด, ์ฆ์ ๋ฐ ๋ถํ์ ํต์ฌ์ ์ธ ์ญํ ์ ํ๋ ์์ฉ์ฒด๋ก, ํนํ ์ฌ๋ฐํ์์์ ๋ํ๋๋ FLT3-ITD ๋์ฐ๋ณ์ด๋ ๊ทธ ์ํ๋ฅผ ์
ํํ๋ค.
๋ณธ ์ฐ๊ตฌ์์๋ ๋จ๋ฐฑ์ง์ ๊ธฐ์ด ๋ฐํ ์์ค์ ๋ค์ํ AML ์ธํฌ์ฃผ์์ ํ์ธํ๊ณ FLT3 ์ธ์ฐํ ๋ฐ Bcl-2 ๊ด๋ จ ๋จ๋ฐฑ์ง์ ๊ณผ๋ฐํ ํน์ฑ์ ๊ฐ์ง MV4-11 ์ธํฌ์ฃผ๋ฅผ ์ ํํ์๋ค.
๋ณด๋ค ํจ์จ์ ์ธ ํ์ ํํ ์๋ฒ์ ์ ์ํ๊ธฐ ์ํด FLT3-ITD์ BCL-2๋ฅผ ์ ํ์ ์ผ๋ก ์ต์ ํ๋ midostaurin (Rydapt)๊ณผ ABT199 (Venetoclax)์ sub-toxicํ ๋๋๋ฅผ ์ฐ์ถํ์๋ค.
๋ ํํฉ๋ฌผ์ Sub-toxicํ ๋๋๋ ๊ฐ๊ฐ MV4-11 ์ธํฌ์ฃผ์์ ์์กด๋ฅ๋ ฅ์ ํฐ ์ํฅ์ ๋ฏธ์น์ง ์์ ๊ฒ๊ณผ ๋์กฐ์ ์ผ๋ก, ๋๋ฐ์ฒ๋ฆฌ ํ caspase์์กด์ฑ ์ธํฌ์ฌ๋ฉธ๊ธฐ์ ์ ์ ๋ํจ์ผ๋ก์จ ์ ์๋ฏธํ ์์นํจ๊ณผ๋ฅผ ํ์ธํ์๋ค. ๋ฟ๋ง ์๋๋ผ, ์ด ์กฐํฉ์ ์ ์ ์ธํฌ์ฃผ์์ ์ ์๋ฏธํ ์ธํฌ๋
์ฑ์ ๋ํ๋ด์ง ์์๋ค. ์ด ๊ฒฐ๊ณผ๋ฅผ ๋ฐํ์ผ๋ก ํต๊ณ์ ์ผ๋ก Chou-Talalay ์ CompuSyn ์ํํธ์จ์ด๋ฅผ ์ด์ฉํ์ฌ combination index๋ฅผ ์ฐ์ถํ์๋ค.
๋ํ, ๋ ์กฐํฉ์ ์ํ ์ธํฌ๋
์ฑ์ annexin V/PI ์ผ์์ ํตํด flow cytometry๋ก ์ ๋ ํ์๊ณ , ์ ์ฒด caspase ๋ฅผ ์ต์ ํ๋ z-VAD-FMK ์ฒ๋ฆฌ๋ฅผ ํตํด Caspase ์์กด์ฑ ์ธํฌ์ฌ๋ฉธ ๊ธฐ์ ์ ๊ฒ์ฆํ์๋ค.Acute myeloid leukemia (AML) is a heterogenous disease with low survival rate, and relapsed AML with FMS-like tyrosine kinase (FLT)3 (FLT3)-internal tandem duplication (ITD) mutations worsens the prognosis of the patients. After an initial profiling of AML cell lines, we selected the cell line MV4-11 presenting both the FLT3-ITD mutation as well as overexpression of the antiapoptotic BCL2 resistance protein. In an attempt to suggest a more efficient targeted chemotherapeutic approach, we determined sub-toxic doses of midostaurin targeting the FLT3-ITD and ABT199 (Venetoclax), a specific inhibitor of BCL2. Whereas subtoxic doses of both compounds did not strongly affect viability of MV4-11 cells, a combination treatment synergistically triggered caspase-dependent apoptotic cell death compared to AML cells lines without FLT3 mutation or cells with a healthy phenotype. Our results could be validated by calculation of the combination index according to Chou-Talalay. Moreover, the cytotoxic effect of the combination treatment was evaluated by Annexin-PI staining and flow cytometry and detection of caspase activation with or without pre-treatment of the pan-caspase inhibitor z-VAD-FMK. Importantly, the combination of midostaurin and ABT199 reduced the tumor formation in in vitro colony formation assays and zebrafish xenografts. Altogether, we suggest a novel, more efficient chemotherapeutic approach for AML patients expressing both elevated levels of antiapoptotic BCL2 and mutated FLT3.Table of Contents
1. INTRODUCTION 1
2. MATERIAL AND METHODS 4
2.1 Compounds 4
2.2 Cell culture and treatments 4
2.3 Cell Proliferation and viability 5
2.4 Protein extraction and western blots 5
2.5 Quantification of apoptosis 5
2.6 Colony formation assay 6
2.7 Investigation of cellular morphology 6
2.8 Zebrafish xenografts 7
2.9 Statistical analysis 8
3. RESULTS 9
3.1 Effect of midostaurin on the viability of AML cell lines with differential phosphorylation levels of FLT3 and BCL2 protein patterns 9
3.2 Midostaurin and ABT199 induce caspase-dependent apoptosis 18
3.3 Synergistic apoptotic effect of a combination of midostaurin and ABT199 21
3.4 Validation of synergistic combination treatments by colony formation assays and in vivo zebrafish xenografts 28
4. DISCUSSION 31
5. REFERENCES 37
6. ๊ตญ๋ฌธ์ด๋ก 40Maste