PF398 KNOCKDOWN OF LASP1 IN CXCR4 EXPRESSING CML CELLS PROMOTES CELL PERSISTENCE, PROLIFERATION AND TKI RESISTANCE

Background: Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by a fusion protein, the constitutively active BCR–ABL tyrosine kinase, leading to expansion of differentiated granulocytes. Although tyrosine kinase inhibitors (TKI) have significantly improved patient care, minimal residual disease (MRD) remains a clinical challenge. Recently, the LIM and SH3 domain protein 1 (LASP1) has been identified as a novel BCR–ABL substrate, which is hyperphosphorylated at Y171 in CML cells. LASP1 is associated with proliferation, migration, tumorigenesis and chemoresistance. Furthermore, the protein can bind to the chemokine receptor 4 (CXCR4), known to be involved in homing of leukemic stem cells and relapse of CML patients. Aims: These two LASP1‐influenced disease promoting effects prompted us to investigate the effect of LASP1 and CXCR4 on processes involved in initiation, progression and maintenance of CML in order to identify and exploit BCR‐ABL‐independent pathways and other extrinsic factors, which may help to eradicate MRD. Methods: Using a CRISPR/Cas9‐based LASP1 knockout system and a lentivirus‐mediated CXCR4 overexpression in the CML cell line K562, the biological influence of CXCR4 and LASP1 was characterized in apoptosis, viability, cell cycle, migration and adhesion assays in the presence of nilotinib. Furthermore, comprehensive transcriptomic microarray data from a public database, immunofluorescence imaging and biochemical analysis were employed to elucidate the roles of LASP1 in CML. Results: Microarray analysis revealed that LASP1 is significantly lower expressed in samples of CML patients not responding to imatinib therapy. The in vitro data indicate that not only the overexpression of CXCR4, but also knockdown of LASP1 contributed to proliferation, reduced apoptosis and impaired cell migration. Additionally, we show that LASP1 depletion leads to decreased cytokine release and reduced natural killer (NK)‐cell mediated cytotoxicity. Finally, we describe a phosphorylation‐dependent loss of direct binding between pLASP1‐Y171 and CXCR4 in CML, which most likely results in receptor destabilization. Summary/Conclusion: Taken together, our results indicate that in CML, reduced levels of LASP1 alone and especially in combination with high CXCR4 expression contribute to TKI resistance and stem cell persistence.