Halofuginone Exerts Antileukemic Effects in TP53 Mutated Cell Lines

Introduction: The application of the standardized therapy based on anthracyclines and cytarabine for all patients with AML regardless of which molecular aberration is present, results in a heterogeneous response exemplified by a worse prognosis for both young and elderly patients exhibiting TP53 mutation. Thus, the treatment of AML requires adjustments in order to improve the patient's therapeutic response. Recently, a new compound - Halofuginone (HF), revealing multitarget effects (antifibrotic, anti-inflammatory and pro-apoptotic) was described as a potential anticancerogenic drug. The diverse targets of HF have been studied in several tumors such as in multiple myeloma, where HF triggers the caspase dependent apoptotic pathway. Our group conducted a study in APL cell lines (NB4 and NB4-R2 - resistant to ATRA treatment), demonstrating HF anti-proliferative and anti-angiogenic effects in vitro suggesting that this molecule may become a new therapeutic agent.

Objective: Investigating the effect of HF in AML cell lines and characterize its impact on cellular signaling and function.

Methods: The AML cell lines (Kasumi-1, U937, OCI-AML3 and THP-1) were treated with HF in ascending concentrations (50-1000 nM) for 24 hours and the cell viability, apoptosis (annexin-V/PI and caspase 3/PARP1 cleavage) and cell cycle (flow cytometry) was evaluated. The expression of apoptosis related genes (BIRC5, BCL2, BAX and TNFR) and proteins was assessed by qPCR and western blot respectively, while the presence of phosphorylated proteins involved in signaling pathways was determined by the Proteome Profiler ^TM Array - HumanPhospho-Kinase Array. All experiments were performed using HF at the concentration of 125 nM at 3, 6, 12 and 24 hours. To study the anti-leukemic effect in vivo, NSG mice (n=16) were transplanted with either Kasumi-1 or THP-1 cells via mouse tail vein injection and subsequently treated intraperitoneally with HF 150 mg/kg/day for 14 days and followed for overall survival (OS) analysis. Statistical analyses were performed by Student's t-test or Mann-Whitnney test, as appropriate. For survival analyses, Kaplan-Meier method was applied and log-rank test was used to compare the curves.

Results: The cell viability of all cell lines was reduced upon HF treatment in a dose-dependent manner. Performing a nonlinear regression analysis, the IC50 for cytotoxicity was determined to be 125, 155, 282 and 323 nM for Kasumi-1, U937, OCI-AML3, and THP-1, respectively. Based on this data we decided to study the effects of HF in Kasumi-1 (HF sensitive cell line with a p53 p.R248Q mutation but with normal protein levels) and THP-1 (HF resistant cell line with a p53 p.R174fs) using a single HF concentration based on the IC50 value. The administration of HF in Kasumi-1 and THP-1 resulted in a decrease of S-phase cells for Kasumi-1 after 24h (P=.004). HF treatment significantly increased cleaved caspases 3, 9 and PARP in a time dependent manner, while the gene expression analysis of apoptosis related genes revealed only an elevated expression of the BIRC5 gene in Kasumi-1 cells after 24h. When screening for phospho-proteins, a decrease (0.5-fold) in phosphorylation was identified for the proteins: Phospholipase C gamma 1 Y783 (PLCγ1), Proline-rich tyrosine kinase 2 Y402, Endothelial nitric oxide synthase S1177 and Signal transducer and activator of transcription 3 (STAT3 Y705) in both cell lines upon HF treatment, suggesting that these proteins are primary targets of HF.

In addition, when treated with HF the levels of STAT3 S727 and STAT5a/b Y694/Y699 were decreased in Kasumi-1, whereas the level of TP53 S392/S46/S15 was diminished in THP-1. In vivo, NSG mice transplanted with Kasumi-1 cells and treated with HF exhibited significantly longer OS (mean value: 144 days), compared to vehicle- treated controls (mean value: 94.5 days; P=.006).

Conclusion: Our results demonstrated that HF exerts antileukemia activity, reducing the quantity of S-phase cells in sensitive cell lines and exhibiting a potent proapoptotic effect. The phosphoarray identified PLCγ1 and STAT 3/5 as targets of HF, corroborating the initiation of apoptosis upon treatment. In agreement with in vitro data, we demonstrated that NSG mice treated with HF presented longer OS, which provides new insights for the development of alternative/complementary therapeutic strategies for AML patients presenting a mutation in TP53 .