Bafilomycin A1 Targets B-Cell Acute Lymphoblastic Leukemia

Objectives:B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of pediatric leukemia. Despite improved remission rates, current treatment regimens for pediatric B-cell acute lymphoblastic leukemia are often associated with adverse effects and central nervous system relapse, necessitating more effective and safer agents. Bafilomycin A1 (Baf-A1), which belongs to the broad spectrum macrolide antibiotic family, has been used in clinical treatment on septicemia, but its treatment study on leukemia remains unexplored. This study aims at investigating the therapeutic effect and its underlying mechanism by which Baf-Al fights against B-ALL cells to provide necessary preclinical experiment data and rationale for its possible application as a B-ALL-targeted therapy drug. Specifically, this project is designed to (1) investigate the selective killing effect of Baf-Al on the B-ALL cells. (2) determine the curative effect of Baf-Al on B-ALL animal models. (3) investigate the clinical significance of Baf-Al on the B-ALL primary cells. (4) explore the molecular and cellular mechanisms by which Baf-A1 works on the treatment of B-ALL cells.Methods:Firstly, CCK8, CFSE, PI and PI/AnnexinV double dye detection were carried out to observe in vitro effect of Baf-A1 on cell growth, proliferation, cell cycle and apoptosis. Secondly, B-ALL patient samples were obtained from hosipital. Then CCK8 and PI/AnnexinV double dye detection on the B-ALL patient primary cells were carried out to observe ex vivo effect of Baf-A1 on cell growth and apoptosis.Thridly, B-ALL mice model was established. Male NOD/SCID mice were reared under specific-pathogen free (SPF) conditions. B-ALL mice model was established by injecting GFP-697 cells (5 x 106 cells/0.2 ml) into NOD/SCID mice through the tail vein. Changes of weight, dietetic state and activity of mice model were examined. On the day 30, the mice were killed. The standards of successfully established B-ALL mouse model:a) decreased weight, activity, and diet consumption; b) paralysis of the limbs in mice; c) abnomally increased number of white blood cells in peripheral blood and bone marrow; a larger number of immature cells in blood smear; d) a larger number of white blood cells infiltration in liver, spleen and stomach; e) transcription of E2A/Pbx1 fusion gene in the blood and bone marrow.Then, the effects of the treatment of Baf-A1 on the B-ALL mice model were examined. NOD/SCID mice were divided into four groups:control group (C), model group (M), treatment group 1 (T1) and treatment group 2 (T2). A week after injection of B-ALL 697 cells, the treatment group was injected with Baf-A1. The NOD/SCID mice of the treatment group 1 were injected with 0.1 mg/kg Baf-A1 by peritoneal injection. The NOD/SCID mice of treatment group 2 were injected with 1 mg/kg Baf-Al. At the same time, the mice of control group and model group were injected with normal saline. All groups were injected once a day, for 3 consecutive days. Evaluation criteria of therapeutic effect:a) longer survival of mice; b) reduction in the rates of limb paralysis of mice; c) reduction of leukemia cells invasion for tissue; d) the blood and bone marrow recovered to normal.To determine the single-dose maximum tolerated dose, Baf-A1 was administered daily to C57BL/6J mice by intraperitoneal injection for 3 days at doses ranging from 0.1 to 25 mg/kg. Two weeks after the injection, no deaths have been observed. On the 14th day after injection, the mice were sacrificed, and followed by analysis on morphology of blood and bone marrow as well as pathology of the liver tissue of C57 mice.Furthermore, western blotting, flow cytometry analysis, confocal microscopy and siRNA interference was used to understand the molecular mechanisms of Baf-A1 on the treatment of B-ALL cells.Results:Baf-Al preferentially inhibits in vitro growth of pediatric B-cell acute lympho blastic leukemia cells. In contrast, acute and chronic myeloid leukemia cell lines were virtually insensitive to the action of this growth inhibitory agent. Baf-A1 extended in vivo survival in the Baf-A1-treated B-ALL mice with advanced disease compared with control mice. The mice in the B-ALL model group had severe hepatosplenomegaly compared with the control group. However, the size and weight of livers and spleens in the Baf-Al-treated group were normalized compared with those in the disease model group.At a dose of 10 mg/kg, the body weight, peripheral complete blood counts and the organ coefficients of mice with different doses of Baf-A1 in C57BL/6J mice were not significantly different from the control mice. And histological examination of the liver did not reveal signs of tissue damage or liver toxicity in the mice treated with 10 mg/kg Baf-A1. Thus, doses of Baf-A1 up to 10 mg/kg were well tolerated with no detectable toxicity. In line with the results from the in vitro system and animal model, in which profound inhibition of the growth of the B-ALL cells was shown, significant inhibition of the growth of B-ALL primary cells was found after 72 h of treatment with 1 nM Baf-A1.Baf-A1 is an inhibitor of vacuolar H+-ATPase that is frequently used at high concentration to block late-phase autophagy. Here, we show that Baf-A1 at a low concentration (1 nM) effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. It targeted both early and late stages of the autophagy pathway by activating mammalian target of rapamycin signaling and by disassociating the Beclin 1-Vps34 complex, as well as by inhibiting the formation of autolysosomes, all of which attenuated functional autophagy, which had not been reported before.Moreover, the treatment of Baf-A1 targeted mitochondria and induced caspase independent apoptosis by inducing the translocation of apoptosis-inducing factor from mitochondria to the nucleus In addition, Baf-A1 induced the binding of Beclin1 to Bcl-2, which further inhibited autophagy and promoted apoptotic cell death.Conclusions:1) Baf-A1 preferentially inhibits in vitro growth of pediatric B-cell acute lymphoblastic leukemia cells.2) In primary cells from pediatric patients with B-cell acute lymphoblastic leukemia and a xenograft model, Baf-A1 specifically targeted leukemia cells while sparing normal cells.3) An in vivo mouse toxicity assay confirmed that Baf-A1 is safe.4) Baf-A1 at low concentration effectively inhibits autophagy via multiple targets but induces caspase-independent but apoptosis-inducing factor-dependent apoptosis of pediatric B-cell acute lymphoblastic leukemia cells.Our data thus suggest that Baf-A1 is a promising candidate drug for the treatment of pediatric B-cell acute lymphoblastic leukemia.