Hematopoietic Cell Kinase (HCK) Is Highly Expressed in Myelodysplastic Syndrome (MDS) and Regulates Proliferation, Cell Cycle Progression and Hematopoietic Differentiation

Abstract 3813

The pathogenesis of MDS is complex and remains elusive. The molecular characterization of this disease has been a tool to obtain a better understanding. Our group recently identified new possible target genes involved in MDS pathophysiology through the microarray analysis of CD34⁺ cells from MDS patients. One of the interesting overexpressed genes found was the HCK gene, a Src kinase family member. This family integrates signaling from a variety of cell-surface receptors, such as RTK, EPO receptor, chemokine receptor, MET and CXCR4, to regulate diverse cellular responses including proliferation, differentiation, apoptosis and migration. HCK is restrictedly expressed in hematopoietic cells and overexpressed in some types of solid cancer and in acute lymphoid leukemia; however, functional role of HCK in MDS and myeloid cells is still unknown. Here, using qPCR we investigated the HCK mRNA expression in CD34⁺ and total bone marrow (BM) cells from 54 MDS patients (FAB 39 low-risk and 15 high-risk), receiving no treatment, and 24 normal donors. We evaluated the role of HCK, studying its effect on proliferation, cell cycle and apoptosis by silencing this gene in U937 myeloid cell line. To inhibit HCK, specific shRNA expressing lentiviral vectors targeting the HCK gene or no specific sequence were used. Cell growth was measured by MTT colorimetric assay. Annexin-PI was used to evaluate apoptosis and cell cycle was analyzed by flow cytometry. These assays were carried out in lentiviral transduced cells treated, or not, with rapamycin(10 or 100nM) and LY294002(50nM). We further analyzed the HCK expression during granulocytic and erythroid differentiation of established leukemia cell lines models. Granulocytic differentiation was induced in NB4 or HL60 cells by treatment with ATRA(10⁻⁶ M) for 4 days. Erythroid differentiation was induced in KU812 cells by treatment with HE(50μM) and HU(100μM) for 4 days. We observed a significant increase in HCK mRNA expression of MDS CD34⁺ cells (P=0.002) and BM cells (P=0.03) compared to normal cells. HCK mRNA expression in low-risk MDS cells was higher compared to high-risk MDS cells. In U937 cells, HCK inhibition resulted in a significant decrease in proliferation compared to control cells (P<0.0001). Interestingly, the combination of HCK inhibition/rapamycin or LY treatment, inhibitors of PI3K/mTOR pathway, showed no reduction in cell proliferation compared to HCK inhibition alone. HCK inhibition induced cell cycle delay in G0/G1 phase and a consequent decrease in cell percentages in the S phase. The combination of HCK inhibition/rapamycin or LY treatment showed a significant decrease in cell number in G0/G1 phase (P=0.007) and increase in S phase (P=0.01) compared to HCK inhibited alone. Apoptosis was unaffected by HCK knockdown. HCK expression was significantly increased in NB4 or HL60 cells after granulocytic differentiation and in KU812 cells after erythroid differentiation (P<0.01). Furthermore, HCK mRNA was significantly up-regulated during erythroid differentiation of MDS low-risk CD34⁺ cells but not in normal CD34⁺ cells. Considering that some transmembrane receptor (CXCR4, MET, chemokine) expressions are altered in MDS patients and their downstream pathway is mediated by Scr family/PI3K, our results suggest that HCK overexpression in MDS may be related to alteration in the expression of some of these cell-surface receptors. Loss of HCK expression may contribute to disease progression by skewing the balance between proliferation and differentiation, perhaps giving malignant cells a proliferative advantage. Further, HCK inhibition in U937 cells potently decreases proliferation through PI3K/mTOR pathway since the combination of HCK inhibition/rapamycin or LY treatment showed no alteration in the proliferation. In addition, HCK protein might have role in cell cycle control by modulating cell cycle gene expression in G0/G1 phase check point. Moreover, granulocyte and erythroid differentiation results in an HCK overexpression probably because this gene is an adaptor protein that is essential for intermediating downstream signaling of activated cell-surface receptors. Our findings also indicate that HCK plays a role in myeloid cell abnormalities, through different cell activities, including proliferation, cell cycle and differentiation, suggesting that HCK represents a possible therapeutic target in MDS. Support: FAPESP, CNPq.