Glucosylceramide Synthase Is a Novel Biomarker of Midostaurin-Induced Cytotoxicity in Non-Mutant FLT3 Positive Acute Myeloid Leukemia Cells

Abstract

Objective: Glucosylceramide (GC) synthesized by glucosylce-ramide synthase (GCS) favors cell survival and proliferation in many cancers. However, it’s role in Fms-like tyrosine kinase 3 (FLT3) non-mutant Acute Myeloid Leukemia (AML) pathogenesis is not clarified. Midostaurin, a multi-kinase inhibitor, clinically benefits FLT3-mutated AML, however, its clinical efficacy is under-estimat-ed in FLT3 non-mutant AML. This study aimed to investigate the efficacy of combination of midostaurin with GCS inhibitor in FLT3 AML cell carrying wild-type FLT3 and the underlying molecular mechanisms.

Material and Method: Cytotoxic and cytostatic effects of mido-staurin, PDMP (GCS inhibitor) alone and in combination on THP1 cells were determined by MTT assay and flow cytometric propidi-um iodide (PI) staining, respectively. Calcusyn software was used to calculate combination indexes (CIs). GCS expression was checked by western blot.

Results: Midostaurin downregulated GCS. Simultaneous inhibi-tion of FLT3 and GCS resulted in suppression of cell proliferation as compared to untreated control. Combinations showed synergistic cytotoxic effects (CI<1). Co-treatments increased cell cycle popula-tion at G2/M phase.

Conclusion: Inhibition of GCS enhances the efficacy of midostau-rin in FLT3 non-mutant AML, which could be a novel therapeutic approach to increase midostaurin’s limited usage in the clinic after detailed mechanistic studies.

Keywords

• Cell cycle
• FLT3 non-mutant AML
• glucosylceramide synthase
• midostaurin

Glukozilseramid Sentaz Mutant Olmayan FLT3 Pozitif Akut Miyeloid Lösemi Hücrelerinde Midostaurin İlişkili Sitotoksisitenin Yeni Bir Biyobelirtecidir

Abstract

Amaç: Glukozilseramid sentaz (GSS) tarafından sentezlenen gluko-zilseramid (GS) birçok kanser türünde hücre yaşamını ve proliferas-yonunu sağlamaktadır. Ancak, mutant olmayan Fms-benzeri tirozin kinase 3 (FLT3) pozitif akut miyeloid lösemi (AML) patogenezindeki rolü açıklanmamıştır. Çoklu kinaz inhibitörü olan midostaurin mutant FLT3 AML tedavisinde etkili olmasına rağmen mutant olmayan FLT3 pozitif AML’deki klinik etkisi gözden kaçırılmıştır. Bu çalışmada, midostaurinin GSS inhibitörü ile kombinasyonunun yabanıl tip FLT3 ifadesine sahip AML hücrelerindeki etkisinin belirlenmesi ve moleküler mekanizmalarının açıklanması amaçlanmıştır.

Gereç ve Yöntem: Midostaurin, PDMP (GSS inhibitörü) ve kombi-nasyonların THP1 hücreleri üzerindeki sitotoksik ve sitostatik etkileri sırasıyla MTT testi ve PI boyaması ile akım sitometri kullanılarak belirlenmiştir. Kombinasyon indeksleri (CI) Calcusyn programı ile hesaplanmıştır. GSS ifadesi western blot ile belirlenmiştir.

Bulgular: Midostaurin GSS ifadesini baskılamıştır. FLT3 ve GSS’ın birlikte inhibe edilmesi kontrolle karşılaştırıldığında hücre çoğalmasını baskılamıştır. Kombinasyonlar sinerjistik sitotoksik etki göstermiştir (CI<1). Kombinasyon hücre döngüsünün G2/M fazındaki hücre populasyonunu arttırmıştır.

Sonuç: Mutant olmayan FLT3 AML’de GSS inhibisyonunun midostaurin’in etkisini arttırdığı saptanmıştır. Detaylı mekanizma çalışmaları yapıldıktan sonra kombinasyon tedavisinin midostaurin’in sınırlı klinik kullanımını arttırması açısından yeni bir yaklaşım olabileceği düşünülmektedir.

Keywords

• Hücre döngüsü
• FLT3 mutant olmayan AML
• glukosilseramid sentaz
• midostaurin

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