A Novel Chemical Compound Inhibiting Hematopoietic Cell Kinase (iHCK) Has a Synergic Effect with Azacytidine (Aza) or Cytarabine (Ara-C) for Acute Myeloid Leukemia Treatment

Introduction.Hematopoietic cell kinase (HCK) belongs to the Src kinase family (SFK) involved in the oncogenic process and hematological malignancy. Some SFK inhibitors are currently under investigation in clinical trials for leukemia after demonstrating efficacy in patients with solid tumors. We have previously reported that HCK is overexpressed in leukemic cells and its inhibition by lentivirus resulted in reduction of cell growth and increased cell death (Roversi et al. BBA Mol Basis Dis. 2017, 1863(2):450-61). In light of the genomic and molecular diversity of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), the development of chemical compounds specific for new molecular targets is currently an important subject.

Aims. To investigate the in vitro and in vivo effects of a new chemical compound targeting HCK inhibition (iHCK), alone or in combination with the most used drugs for treatment of MDS and AML (Azacytidine - Aza - or Cytarabine - Ara-C).

Methods. After iHCK development, we tested its activity alone or in combination with Aza or Ara-C in CD34⁺ cells isolated from AML patients (n=5) as well as in a panel of myeloid leukemia cell lines (KG1, HL-60, HEL and K562). Additionally, we tested the iHCK in normal and malignant cells cultured in a 3D bioscaffold obtained by decellularization of bovine bone marrow (Bianco et al. Biomat Sci 2019, 7(4):1516-28), in order to mimic the bone marrow niche. After informed written consent and approval of the Ethical Committee of University of Campinas (CAAE 1000.0.146.00-11), in accordance to the Helsinki Declaration, CD34⁺ cells were isolated from bone marrows of healthy donors (HD), MDS and AML patients and were treated with iHCK or vehicle (DMSO) in liquid culture, for three days. Meanwhile, HS-5 mesenchymal cells were cultured into the 3D bioscaffold. iHCK or vehicle treated CD34⁺ cells were introduced into the 3D bioscaffold containing HS-5 and evaluated after 7 and 14 days, by light microscopy (hematoxilin and eosin regular staining) and immunohistochemistry (expression of CD34 and CD90 antigens). NOD.CB17-Prkdc^scid/J mice received 2 Gy irradiation followed by transplantation with caudal intravenous injection of leukemia cells obtained from hCG-PML-RARα transgenic mice. After acute promyelocytic leukemia (APL) establishment, animals were treated or not with intraperitoneally iHCK and peripheral blood was collected for hematological analysis and protein was extracted from spleen and bone marrows for Western Blot analysis. ANOVA and Student's T-Test were used.

Results.In leukemia cell lines and primary cells, the combinatory treatment of iHCK and Cytarabine (1μM) or 5-Azacitidine (1μM) demonstrated synergistic effects, compared to either drug alone, on the reduction of growth and induction of cell death (P<0.001; Figure 1). Further, Western blot revealed increased BAX expression and decreased BCL-XL expression. Moreover, iHCK treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to severe reduction of ERK, AKT and p70S6 phosphorylation. Treatment with iHCK reduced CD34⁺ MDS and AML cells proliferation cultured into the 3D bioscaffold but had no effect upon normal CD34⁺cells. In vivo analysis showed that APL mice treated with iHCK (5μM) for 48h had reduced leukocyte number compared to APL mice treated with vehicle (13.2±1.1 vs 49.4±18.8; P<0.001). No alterations in hemoglobin levels and platelet were found. Likewise, the in vivo iHCK (2.5μM, 5.0μM or 10.0μM) treatment decreased the phosphorylation of ERK, AKT and P70S6K proteins of leukemic cells (Figure 2).

Conclusion.The iHCK pharmacological inhibitor has an antiproliferative activity in leukemic cells without altering cell death and survival rate of normal cells, demonstrating on-target malignant cell killing activity as a single agent or in combination with Azacytidine (Aza) or Cytarabine (Ara-C).

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