HCK Inhibition Displays Antineoplastic Activity on Acute Leukemia and Promotes Immune Regulation of the Bone Marrow Tumor Microenvironment

Abstract

Background. The hematopoietic cell kinase (HCK), a Src tyrosine kinase family member, is mainly expressed in myeloid cells and lymphocytes and regulates oncogenic signal transduction pathways acting on pivotal biological processes. Increased activity of HCK, a common feature in several hematological neoplasms, modulates immune cell recruitment and activity in the tumor microenvironment (TME). HCK is also associated with the development of acute leukemia and correlates with poor prognosis, particularly, in acute myeloid leukemia (AML) patients relapsed from standard chemotherapy. In light of the urgent need for advancing targeted therapeutic options for acute leukemia, we investigated the effects of a novel HCK inhibitor. Aims. To analyze HCK gene and protein expression in leukemia patients and their impact on clinical outcomes, and to investigate preclinical effects of a novel HCK inhibitor compound, an iHCK-37 subproduct (Dr Maurizio Botta, Siena University). Methods. Analysis of HCK gene expression was performed in mononuclear cells isolated from 163 leukemia patients (median age 57.5 years [range: 18-88]) (TCGA study). HCK protein levels were analyzed in 25 bone marrow (BM) aspirates from healthy donors (HD) and untreated patients with Myelodysplastic syndrome (MDS), AML with MDS-related changes (AML-MRC), de novo AML, and acute lymphoblastic leukemia (ALL) by flow cytometry and immunofluorescence. To further explore the clinical potential of the HCK inhibitor, we conducted functional assays in 9 leukemia cell lines: AML (OCI-AML3, MOLM13, U937, KG1a), ALL (RS4;11, Jurkat), chronic myeloid leukemia (K562), and erythroleukemia (Hel). We also tested the HCK inhibitor action on 12 BM hematopoietic stem cells isolated from HD, MDS, AML-MRC and AML to verify their colony-forming potential and to investigate the mechanisms in the TME using a 3D-coculture system containing mesenchymal cells and 2D-coculture with a monocyte monolayer. In vivo, we developed a xenograft mouse model in which leukemia cells were inoculated into NOD/SCID mice flank to form tumors. Statistical analyzes were performed using Kruskal-Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. HCK gene expression was increased in cells from M4 and M5 AML patients compared to patients with other AML (p<.0001). Additionally, HCK protein levels were higher in BM cells from MDS, AML-MRC, AML and ALL compared to HD. The HCK inhibitor exhibits a potent cell growth inhibition with doses ranging from 0.3 to 9.8μM in leukemia cells (KG1a: 2.0μM, HL60: 0.5μM, U937: 0.3μM, OCI-AML3: 4.2μM, MOLM13: 8.5μM, Jurkat: 3.9μM; RS4;11: 2.1μM; K562: 9.8μM; Hel: 5.0μM). Preclinical in vitro experiments demonstrated that treatment with the HCK inhibitor for 48h was sufficient to drive acute leukemia cells into irreversible cell growth inhibition and death (p<.0001). The ability of BM hematopoietic progenitors to proliferate and differentiate into colonies was significantly suppressed by the HCK inhibitor in MDS, AML-MRC and AML (p<.001) but not in HD. The HCK inhibitor led to increased apoptosis in a 3D-system of leukemia cell lines or AML patients’ cells, but had no effect on HD cells. In a 2D-coculture, programmed monocytes potentialized leukemia cell apoptosis (p<.001). In vivo xenograft studies showed a significant reduction of tumor growth (U937: 62%; OCI-AML3: 66%) and volume (U937: 68%; OCI-AML3: 71%) in HCK inhibitor-treated mice compared to the vehicle-treated group. In addition, both in vitro and in vivo protein analyses showed an abrogation of PI3K and MAPK signaling activation by a downregulation on AKT and ERK phosphorylation levels after the HCK inhibitor treatment. Conclusion. The HCK inhibitor has an antineoplastic activity onto leukemia cells without altering normal cell survival, demonstrating on-target malignant cell killing action probably through reducing the activation of the oncogenic pathways PI3K and MAPK. Interestingly, nowadays the inefficacy of standard chemotherapy is related to non-specificity between leukemic and normal hematopoietic cells and to the advantages of neoplasm cell survival due to TME protection. In this scenario, our results altogether demonstrate the efficacy of HCK inhibition and identify this compound as a promising therapeutic opportunity that could enhance the ability of reversing chemotherapy resistance. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.