Hematopoietic Cell Kinase (HCK) Is Involved in Myelodysplastic Syndromes and Acute Leukemia Pathophysiology By Modulating PI3K Pathway and Enhancing Erythroid Differentiation

Introduction: Src family non-receptor tyrosine kinase regulates diverse cellular responses mainly by activating PI3K signaling, an important pathway that contributes to many cancers pathology. HCK, a Src family member, is restricted expressed in hematopoietic cells; however, high levels of HCK have been reported in solid tumors and hematologic neoplasms and have been associated with a worse prognosis. Identification of unbalanced pathways and upregulated proteins and a better knowledge of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) pathogenesis could help identify potential druggable targets and new prognostic biomarkers. The clinical and biological role of HCK in MDS and AML pathophysiology has not yet been elucidated and may be a novel therapeutic approach.

Aims: To evaluate HCK expression in hematopoietic stem cells (HSC/CD34⁺) and in total bone marrow (BM) cells from healthy donors and MDS and AML patients. We further attempted to analyze the HCK knockdown and drug inhibition on differentiation, proliferation, apoptosis and migration in HSC/CD34⁺ and in a panel of human leukemia cell lines (KG1a, U937, HL60, P39).

Materials and Methods: A total of 34 healthy donors and 139 patients at diagnosis (MDS=75 [low-risk=46, high-risk=29], AML with Myelodysplastic related changes (AML-MRC)=21 and de novo AML=43), were included in the study. HCK gene and protein expression was analyzed by qPCR and Western blot. Results are expressed as median (minimum–maximum) after appropriate statistical analysis. For functional analysis, HCK was inhibited with specific lentiviral vector system in HSC/CD34⁺ (isolated from umbilical cord blood units and BM), KG1a, U937, HL60 and P39 cells. Apoptosis was evaluated by Annexin-V/PI, cell growth by CellTiter assay, migration by Transwell, CXCR4 expression and actin polymerization by flow cytometry. We also verified the effects of a HCK specific inhibitor, ASN05260065, kindly provided by ASINEX and Dr Maurizzio Bottas.

Results: HCK mRNA were significantly increased in HSC/CD34⁺ from patients with MDS (4.87 [0.03-14.75]; P=.003) and AML (8.36 [0.70-24.47]; P=.003) compared to healthy donors (1.19 [0.06-3.02]). When patients were stratified according to WHO classification, HCK expression was significantly higher in low-risk MDS compared to high-risk and AML-MRC (8.10 [2.30–14.75] vs 3.22 [0.03–6.33] and 1.02 [0.70–2.78], P<.001). Similar results were found in BM cells: HCK transcripts were significantly increased in MDS compared to healthy donors, and in low-risk compared to high-risk MDS (P<.0001). Interestingly, 5 of 5 MDS patients showed a significant decrease in HCK expression after disease progression. Also, a negative correlation was observed between BM blast percentage and HCK expression in MDS patients. In HSC/CD34⁺ cells, BFU-E colony number was significantly lower after HCK inhibition. HCK depletion reduced cell death in HSC/CD34⁺, P39 and KG1a cells and diminished proliferation in KG1a and U937 cells. Moreover, HCK knockdown decreased the phosphorylation of AKT, mTOR (PI3K pathway members) and ERK and treatment of HCK silenced cells with PI3K inhibitors had no synergic effect. Confirming the HCK lentivirus silencing results, HCK specific inhibitor treatment downregulated PI3K and ERK signaling. In addition, HCK inhibition significantly reduced migration towards CXCL12 chemoattractive stimuli by regulating CXCR4 expression and F-actin polymerization. Attempts have been made to transduct CD34⁺ from low-risk MDS patients however more than 80% cells were apoptotic and impossible to perform further assays.

Conclusions:HCK is overexpressed in HSC/CD34⁺ and BM cells from MDS, mainly in low-risk MDS, and de novo AML. During MDS progression, HCK expression reduction correlates with increasing blast percentage and could indicate less dependence on microenvironment. HCK inhibition reduced BFU-E colony formation and our previous results showed that HCK expression increases during erythroid differentiation of non-transduced low-risk MDS CD34⁺ cells. Thus, HCK seems to play a role as a downstream effector in erythropoietin (Epo) signaling which could explain the higher HCK expression in low-risk MDS. Besides, HCK is an upstream regulator of PI3K and MAPK pathway that enhances cell apoptosis, survival and proliferation. Our study described new insights to the role of HCK in Epo/PI3K signaling and in leukemogenesis.