Arsenic Trioxide (ATO) Interacts Synergistically with MEK Inhibitors to Induce Apoptosis in STI571-Resistant Bcr-Abl Mutants.
Authors: Lunghi Paolo , Mazzera Laura , Corradi Attilio , Cantoni Anna Maria , Jottini Stefano , Lombardi Guerino , Ricca Micaela , Arienti Davide , Rizzoli Vittorio , Deininger Michael WN , Bonati Antonio 
Published: October 2019

Abstract
Abstract 2177
Poster Board II-154
Resistance toward Imatinib and other Bcr-Abl tyrosine kinase inhibitors remains an increasing clinical problem in the treatment of advanced stages of chronic myeloid leukemia. Thus novel therapeutic strategies are needed to address the emerging problem of Imatinib resistance. Previous preclinical studies reported that the MEK inhibitors PD184352 or PD0325901 (Pfizer), strikingly enhances ATO-mediated apoptosis in Acute Myelogenous Leukemia and in Multiple myeloma. The aim of this study was to investigate whether the combined treatment with PD184352 (PD) and ATO has cytotoxic effects on murine Ba/F3 cells expressing wild-type (wt) or various imatinib-resistant mutant forms of Bcr-Abl, including T315I. We first analyzed the pharmacologic interactions between PD and ATO using a fixed-ratio experimental design in Bcr-Abl Ba/F3p210wt, Ba/F3p210T315I and Ba/F3p210Y253F cell lines and found that the combined treatment with PD plus ATO resulted in the synergistic induction of apoptosis in all cell lines tested (Chou-Talalay method): the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in PD plus ATO treated cells were 0.72± 0.19, 0.61± 0.04 and 0.69± 0.09 in BCR-ABL Ba/F3p210wt, Ba/F3p210T315I, and Ba/F3p210Y253F respectively. Synergistic interaction between ATO and PD0325901, a derivative of PD184352, was demonstrated in all tested cell lines. In order to investigate the molecular effectors involved in PD/ATO-induced apoptosis we first evaluated its effects on Bcr-Abl protein expression and CrkL phosphorylation, a well-known downstream target of Bcr-Abl. Immunoblotting analyses demonstrated that treatment for 24 to 48 hours of Ba/F3p210wt and Ba/F3p210T315I cells with PD (1μM) or ATO (1μM) alone or in combination had no effects on levels of total Bcr-Abl or phospho—CrkL thereby indicating that the combination PD/ATO does not act via Bcr-Abl oncogenic signaling. Therefore, we studied whether p53 and the p53-related gene p73 are molecular targets of the combined treatment in Ba/F3p210wt and Ba/F3p210T315I cell lines. We found that monotreatment with neither PD nor ATO 1 μM (or their combination) was able to induce p53 accumulation, whereas the combination PD/ATO promoted the accumulation of the proapoptotic and antiproliferative TA-p73α protein and reduced the levels of the antiapoptotic and proproliferative dominant-negative ΔN-p73α in both cell lines. Consistent with these results, we found that PD greatly enhanced the ATO-induced Puma expression, mitochondrial depolarization, caspase-3 activation, and apoptosis in Ba/F3p210wt and Ba/F3p210T315I cells and functional knock-out of p73 gene expression by small interfering (si)RNAs significantly reduced (P< .05 Dunnett test) the PD/ATO induced mitochondrial depolarization. To determine whether the PD plus ATO efficacy observed in vitro for BaF3 cells expressing mutant forms of Bcr-Abl was recapitulated in vivo, we studied PD/ATO combination in a mouse model of Imatinib-resistant, Bcr-Abl—dependent disease. Severe combined immunodeficient mice were injected intravenously with Ba/F3 cells expressing Bcr-Abl-T315I isoform. Mice with Bcr-Abl-T315I—induced leukemia were treated with the MEK inhibitor PD0325901 (10 mg/kg; orally) plus ATO (3.75 mg/kg; intraperitoneally) or Imatinib (50 mg/kg, twice daily; intraperitoneally) or vehicle for three weeks. Untreated or Imatinib-treated mice harboring the T315I isoform developed aggressive disease, with massive liver and splenic infiltration, typically resulting in death in 32 days. However, mice harboring the T315I isoform showed significantly prolonged survival when treated with PD/ATO (43 days, P=.001, Kaplan-Meier method and compared using the log-rank test). Moreover, histopathological analysis of 20 days Imatinib-treated mice revealed infiltration of the liver and spleen. In contrast, histopathological analysis of organs from PD/ATO-treated mice demonstrated normal tissue architecture. Consistent with these results immumoblottig analysis of the lysates from livers and spleens revealed a marked expression of Bcr-Abl protein in mice treated with Imatinib or vehicle. Our preclinical in vitro and in vivo studies suggest that a strategy combining ATO with disruption of MEK pathway could represent an effective therapeutic strategy for the treatment of Imatinib-resistant Bcr-Abl-positive leukemias, including those harboring the T315I mutation.

Disclosures:
No relevant conflicts of interest to declare.