Mechanisms of Heme Oxygenase 1-Induced Resistance to Imatinib In CML Cells.
Authors: Tibullo Daniele , Barbagallo Ignazio , Branca Antonio , Giallongo Cesarina , Cava Piera La , Parrinello Nunziatina , Volti Giovanni Li , Vanella Angelo , Stagno Fabio , Palumbo Giuseppe A , Raimondo Francesco Di 
Published: October 2019

Abstract
Abstract 3385
Heme oxygenase 1 (HO-1) is a rate-limiting enzyme in heme degradation (from hemoproteins and hemoglobin), leading to the generation of free iron, biliverdin and carbon monoxide. Recent studies have reported that HO-1 expression may represent an important protective endogenous mechanism against physical, chemical and biological stress. The cytoprotective role of HO-1 has been demonstrated for several solid tumors and acute leukemias. In addition, HO-1 is considered to play an important role as a survival molecule in CML cells, and an overexpression of HO-1 has been found to inhibit apoptosis induced by imatinib. In our experiments, K562 cells were incubated for 24 hrs with imatinib (1 uM) alone, or with an inductor of HO-1 (Cobalt protoporphyrin, CoPP, 10uM), or with inhibitor of HO-1 activity (Tin- mesoporphyrin, TIN) and viability of cells was evaluated by the ATP-lite1step assay (PerkinElmer). As expected, addition of CoPP was able to overcome the inhibitory effect of IM on K562 cells (p<0.002) while the cytotoxic effect of IM was restored by adding an inhibitor of HO-1 (Tin 10uM) to the combination (p<0.002). In order to examine the mechanism of this protective effect, we repeated the experiments with the HO-1 catabolites but neither biliverdin nor carbon monoxide (the latter induced by CORM3 and CORM3A) were able to protect K562 cells from imatinib. The protective effect of HO-1 inductor (CoPP) was inhibited by the addition of a protease inhibitor (Ed64), able to stop migration of HO-1 to the nucleous (p<0.002). We also analyzed 96 kinase genes by Taqman low density array, (Applied Biosystems, 7900 Fast Real Time PCR) and we found that at least for seven kinases a significant increase of gene expression was found when CoPP was added to IM while they were decreased in cultures with IM alone or IM + TIN (HO-1 inhibitor). The kinase genes induced by CoPP were Ephrin receptor EphA3 (EPHA3), ephrin receptor EphA5 (EPHA5), insulin receptor (INSR), VEGF-R2 (KDR), leukocyte receptor tyrosine kinase (LTK), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), and SLIT and NTRK-like family, member 6 (SLITRK6). All of them are able to activate mitogenic signals (Tab.1). We also evaluated stress oxidative production by measuring the ROS formation by flow cytometry and we found that IM 1uM was able to increase ROS formation, and this effect was inhibit by CoPP and restored in presence of TIN. (CTRL 32.28 IFM; IM 66.25 IFM; IM+CoPP 37.0 IFM; IM+TIN 64.6 IFM). In conclusion, we found that the protective effect of HO-1 on IM cytotoxicity was not associated with its catabolites, but with the ability to move into the nucleus after proteolytic cleavage. Migration to the nucleous activates several kinases able to induce mitogenic signals. Ho-1 is also able to reduce the oxidative stress induced by IM.
Table 1 - Relative quantification of kinase genes expression. EPHA3 (fold) EPHA5 (fold) INSR (fold) KDR (fold) LTK (fold) NTRK1 (fold) SLITRK6 (fold) CTRL 1 1 1 1 1 1 1 Imatinib 1uM 0.03 0 0.13 0 0 0.002 0.05 Imatinib+CoPP 1.89 21 8.42 21.9 92.8 2.89 2.076 Imatinib+Tin 0.04 0.00001 0.2 0.0003 0.00004 0.6 0.01

Disclosures:
No relevant conflicts of interest to declare.