Targeting ORP4L With LYZ-81 Selectively Eliminates Leukemia Stem Cells

Acute myeloid leukemia（AML）is a myeloid progenitor cells and clonal expansion in the blood,bone marrow,and other tissues,and a common type of leukemias among adults.The leukemia stem cells（LSC）of leukemia patients have self-renewal and proliferative capacity,which is the main reason of that many leukemia patients will still relapse after treatment.ORP4L is a member of the Oxysterol Binding Protein-Related Protein Family（ORPs）and is selectively expressed in LSCs but not in HSCs.Previous studies have shown that OSW-1 can selectively target ORP4L and OSBP and eliminate tumor cells,but the cytotoxicity is too large,it will not become a good medicine for treating leukemia.So we have modified it,and LYZ-81 has obtained an analogue by modifying OSW-1.To date,no cure for leukemia has been created worldwide.We observed the mortality and colony-forming ability of LSCs will be decreased If ORP4L was silenced by small molecule interference in LSCs.Based on this phenomenon,we judged that ORP4L plays a crucial role in the survival and proliferation of LSCs.We employed surface plasmon resonance（SPR）assays to determine the binding affinity of these analogues to ORP4L and OSBP.By screening,we uncovered an analogue termed LYZ-81 that binds ORP4L with markedly greater affinity than OSBP.The IC50 test results showed that the cytotoxicity of LYZ-81 was much lower than that of OSW-1.The relate reasults of LYZ-81stimulates LSCs or Overexpression of ORP4L in the LSCs that were stimulated by LYZ-81and the results of ORP4L knockdown in LSCs,these results support the notion that ORP4L protected the cells against the cytotoxicity of LYZ-81.It was concluded that small molecule compounds selectively eliminate LSCs by targeting ORP4L.ORP4L interacts with PLCβ3 and Gα_q/11 to control Ca²⁺release from the ER and subsequent parallel cytosolic and mitochondrial oscillations and thus maintains active OXPHOS in LSCs’energy homeostasis.Western blot analysis of LC3 II indicated that the autophagy induced by LYZ-81 treatment.According to the results,We found that LYZ-81targets ORP4L,disassembles the ORP4L/Gα_q/11/PLCβ3 complex,disturbs Ca²⁺signaling and OXPHOS,resulting in LSCs death.In conclusion,ORP4L is considered as a potential new target for drug development.Since the destruction of ORP4L function by LYZ-81 can selectively eradicate LSCs,LYZ-81may become the starting point for drug development for the cure of AML or other leukemias.