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

Year 2021, , 149 - 155, 08.12.2021

Hande Nur Şahin , Aysun Adan

https://doi.org/10.26650/experimed.2021.974943

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

References

• 1. Blum WG, Mims AS. Treating acute myeloid leukemia in the modern era: A primer. Cancer 2020; 126(21): 4668-77. [CrossRef] google scholar
• 2. Ambinder AJ, Levis M. Potential targeting of FLT3 acute myeloid leukemia. Haematologica 2021; 106(3): 671-81. [CrossRef] google scholar
• 3. Kennedy VE, Smith CC. FLT3 Mutations in acute myeloid leukemia: Key concepts and emerging controversies. Front Oncol 2020; 10: 612880. [CrossRef] google scholar
• 4. Daver N, Schlenk RF, Russell NH, Levis MJ. Targeting FLT3 muta-tions in AML: review of current knowledge and evidence. Leuke-mia 2019; 33: 299-312. [CrossRef] google scholar
• 5. Antar AI, Otrock ZK, Jabbour E, Mohty M, Bazarbachi A. FLT3 inhib-itors in acute myeloid leukemia: ten frequently asked questions. Leukemia 2020; 34(3): 682-96. [CrossRef] google scholar
• 6. Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer S, et al. Mi-dostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Eng J Med 2017; 377(5): 454-64. [CrossRef] google scholar
• 7. Fischer T, Stone RM, Deangelo DJ, Galinsky I, Estey E, et al. Phase IIB trial of oral Midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syn-drome with either wild-type or mutated FLT3. J Clin Oncol 2010; 28(28): 4339-45. [CrossRef] google scholar
• 8. Stone RM, Fischer T, Paquette R, Schiller G, Schiffer CA, et al. Phase IB study of the FLT3 kinase inhibitor midostaurin with chemother-apy in younger newly diagnosed adult patients with acute my-eloid leukemia. Leukemia 2012; 26(9): 2061-8. [CrossRef] google scholar
• 9. Weisberg E, Meng C, Case AE, Tiv HL, Gokhale PC et al. Effects of the multi-kinase inhibitor midostaurin in combination with che-motherapy in models of acute myeloid leukaemia. J Cell Mol Med 2020; 24(5): 2968-80. [CrossRef] google scholar
• 10. Morales ML, Arenas A, Ortiz-Ruiz A, Leivas A, Rapado I, et al. MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia. Sci Rep 2019; 9(1): 18630. [CrossRef] google scholar
• 11. Giussani P, Tringali C, Riboni L, Viani P, Venerando B. Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug re-sistance. Int J Mol Sci 2014; 15: 4356-92. [CrossRef] google scholar
• 12. Truman JP, Garcia-Barros M, Obeid LM, Hannun YA. Evolving con-cepts in cancer therapy through targeting sphingolipid metabo-lism. Biochim Biophys Acta 2014; 1841: 1174-88. [CrossRef] google scholar
• 13. Morad SA, Cabot MC. Ceramide-orchestrated signalling in cancer cells. Nat Rev Cancer 2012; 13: 51-65. [CrossRef] google scholar
• 14. Kartal Yandım M, Apohan E, Baran Y. Therapeutic potential of tar-geting ceramide/glucosylceramide pathway in cancer. Cancer Chemother Pharmacol 2013; 71(1): 13-20. [CrossRef] google scholar
• 15. Roh JL, Kim EH, Park JY, Kim JW. Inhibition of Glucosylceramide Synthase Sensitizes Head and Neck Cancer to Cisplatin. Mol Can-cer Ther 2015; 14(8): 1907-15. [CrossRef] google scholar
• 16. Baran Y, Bielawski J, Gunduz U, Ogretmen B. Targeting glucosyl-ceramide synthase sensitizes imatinib-resistant chronic myeloid leukemia cells via endogenous ceramide accumulation. Cancer Res Clin Oncol 2011; 137(10): 1535-44. [CrossRef] google scholar
• 17. Watters RJ, Fox TE, Tan SF, Shanmugavelandy S, Choby JE, et al. Tar-geting glucosylceramide synthase synergizes with C6-ceramide nanoliposomes to induce apoptosis in natural killer cell leukemia. Leuk Lymphoma 2013; 54(6): 1288-96. [CrossRef] google scholar
• 18. Adan A, Baran Y. The pleiotropic effects of fisetin and hespere-tin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks. Tumor Biology 2015; 36(11): 8973-84. [CrossRef] google scholar
• 19. Chou TC. Drug combination studies and their synergy quantifica-tion using the Chou-Talalay method. Cancer Res 2010; 70: 440-6. [CrossRef] google scholar
• 20. Casagrande N, Borghese C, Favero A, Vicenzetto C, Aldinucci D. Trabectedin overcomes doxorubicin-resistance, counteracts tu-mor-immunosuppressive reprogramming of monocytes and decreases xenograft growth in Hodgkin lymphoma. Cancer Lett 2021; 500: 182-93. [CrossRef] google scholar
• 21. Sahin HN, Adan A. Combinatorial effect of midostaurin and sphin-gosine kinase-1 inhibitor on FLT3 wild type acute myeloid leuke-mia cells. Turkish Journal of Biochemistry 2021; in press google scholar
• 22. Kroll A, Cho HE, Kang MH. Antineoplastic agents targeting target-ing sphingolipid pathways. Front Oncol 2020; 10: 833. [CrossRef] google scholar
• 23. Levis M. Midostaurin approved for FLT3-mutated AML. Blood 2017; 129(26): 3403-6. [CrossRef] google scholar
• 24. Dohner H, Sierra J, Stone R, Hoenekopp A, Berkowitz N, et al. Trial in progress: A phase 3, randomized, double-blind study of mido-staurin in combination with chemotherapy and as single-agent maintenance therapy in newly diagnosed patients with FLT3 mu-tation-negative acute myeloid leukemia (AML). Clin Lymphoma Myeloma Leuk 2018; 18(Suppl 1): S206-7. [CrossRef] google scholar
• 25. Salustiano EJ, da Costa KM, Freire-de-Lima L, Mendonça-Previato L, Previato JO. Inhibition of glycosphingolipid biosynthesis reverts multidrug resistance by differentially modulating ABC transport-ers in chronic myeloid leukemias. J Biol Chem 2020; 295(19): 645771. [CrossRef] google scholar
• 26. Bassoy EY, Baran Y. Bioactive sphingolipids in docetaxel-induced apoptosis in human prostate cancer cells. Biomed Pharmacother 2012; 66(2): 103-10. [CrossRef] google scholar
• 27. Gencer EB, Ural AU, Avcu F, Baran Y. A novel mechanism of dasat-inib-induced apoptosis in chronic myeloid leukemia; ceramide synthase and ceramide clearance genes. Ann Hematol 2011; 90(11): 1265-75. [CrossRef] google scholar
• 28. Odgerel T, Kikuchi J, Wada T, Shimizu R, Futaki K, et al. The FLT3 inhibitor PKC412 exerts differential cell cycle effects on leukemic cells depending on the presence of FLT3 mutations. Oncogene 2008; 27(22): 3102-10. [CrossRef] google scholar
• 29. Olshefski RS, Ladisch S. Glucosylceramide synthase inhibition en-hances vincristine-induced cytotoxicity. Int J Cancer 2001; 93(1): 131-8. [CrossRef] google scholar
• 30. Zhu Y, Wang C, Zhou Y, Ma N, Zhou J. C6 ceramide motivates the anticancer sensibility induced by PKC412 in preclinical head and neck squamous cell carcinoma models. J Cell Physiol 2018; 233(12): 9437-46. [CrossRef] google scholar