Abstract B33: Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia
Authors: Almeida Luciana Yamamoto , Silva Cleide Lúcia Araújo , Weinhaüser Isabel , Cândido Larissa Ananias , Scheucher Priscila Santos , Bonaldo Camila Cristina Oliveira Menezes , Santana Bárbara Amélia Aparecida , Yamada Ana Sílvia Gouvêa Lima , Rego Eduardo Magalhães 
Published: May 2019

Abstract
Gefitinib and erlotinib are well-known epidermal growth factor receptor (EGFR) inhibitors approved by the Food and Drug Administration for the treatment of non-small cell lung cancer (NSCLC). Interestingly, patients with synchronous NSCLC and acute myeloid leukemia (AML) treated with erlotinib presented regression of both neoplasms, although AML myeloblasts were shown to lack expression of EGFR. In addition, preclinical studies have shown that gefitinib alone or combined with arsenic trioxide (ATO) or all-trans retinoic acid (ATRA) was able to induce differentiation in the EGFR-negative cell lines of acute promyelocytic leukemia (APL), which is a subtype of AML characterized by the t(15;17)/PML/RARA rearrangement. Our previous study demonstrated that EGFR gene expression levels were associated with prognostic outcomes of APL patients treated according to the International Consortium on APL protocol. However, the EGFR levels were not assessed at protein level. Two phase 3 trials reported that ATRA and ATO therapy was associated with excellent outcomes in APL. The two drugs induced the degradation of the PML/RARA oncoprotein through different mechanisms, with ATRA acting through the proteasome pathway, and ATO functioning through the PML-transformation related protein 53 (Trp53) axis. The purpose of this study was to investigate whether gefitinib or erlotinib has synergistic effects with ATO or ATRA in the treatment of APL. To this end, the APL cell lines NB4 and NB4-R2 were treated with gefitinib (C22H24ClFN4O; Selleck Chemicals, #S7786) and erlotinib (C22H23N3O4; Selleck-Chemicals, #S1025) alone or combined with ATO (As2O3; Sigma-Aldrich, #202673). The stock solutions of gefitinib and erlotinib were prepared in DMSO while ATO was dissolved in 1M NaOH solution. Then each drug was diluted with RPMI medium to the desired final concentration. The median effective dose (ED50) for gefitinib was calculated to be 20.97 and 27.06 µM for NB4 and NB4-R2, respectively. ATO was a potent inducer of apoptosis with an ED50 of 2.27 µM for NB4 and 1.73 µM for NB4-R2 cells. ED50 of ATRA (C20H28O2; Sigma-Aldrich, #R2625) was not calculated because ATRA is a potent differentiation inducer but did not induce cell death of APL blasts. Due to the high value of ED50 for NB4 (78.97 µM) and NB4-R2 (111.36 µM) after 24 h of erlotinib treatment, the analysis of this drug was discontinued. The interaction between gefitinib and ATO demonstrated a moderate synergism for NB4 and NB4-R2 with a combination index values of 0.88 and 0.83, respectively. These in vitro results encouraged us to investigate the in vivo effects of gefitinib alone or in combination with ATO and/or ATRA. To this second end, we developed a syngeneic transplant murine model using leukemic cells obtained from transgenic mice hCG-PML/RARA, which develop a form of leukemia that closely recapitulates the human disease. The engraftment of leukemic cells from hCG-PML-RARα transgenic mice transplanted to wild-type littermate recipients was evaluated by morphology and flow cytometry analysis of the peripheral blood (PB), bone marrow (BM), and spleen. Recipient mice were euthanized and evaluated for signs of engraftment 14 (n=3) and 20 (n=3) days after transplantation. Of note, the morphologic analysis revealed &gt;20% of blasts among nucleated cells in the BM samples. The flow cytometric analysis showed the presence of CD11b+ CD117+ cells in PB (median 10.50, range 8.81-12.20), BM (median 18.30, range 17.80-18.80), and spleen (median 16.45, range 16.00-16.90). These results demonstrate that this rapid and robust murine model is useful to assess the efficacy of EGFR inhibitors in APL. Taken together, our preliminary in vitro results suggest that gefitinib and ATO may have potential application for the APL treatment and that this current syngeneic transplant mouse model of APL is suitable to test this hypothesis.
Citation Format: Luciana Yamamoto Almeida, Cleide Lúcia Araújo Silva, Isabel Weinhaüser, Larissa Ananias Cândido, Priscila Santos Scheucher, Camila Cristina Oliveira Menezes Bonaldo, Bárbara Amélia Aparecida Santana, Ana Sílvia Gouvêa Lima Yamada, Eduardo Magalhães Rego. Synergistic effect of gefitinib and arsenic trioxide in acute promyelocytic leukemia [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B33.