Journal of Blood Disorders & Transfusion

Histamine receptor mediates EGFR-independent anti-leukemic activity of an EGFR inhibitor â??gefitinibâ?

Hematologists Global Summit 2018

July 13-14, 2018 Sydney, Australia

Manisha Yadav

Jawaharlal Nehru University, India

Posters & Accepted Abstracts: J Blood Disord Transfus

Abstract:

Background: Epidermal growth factor receptor (EGFR) inhibitor gefitinib (Iressa) is a synthetic anilinoquinazoline, which is extensively used for treating non-small cell lung cancer. Apart from its anti-cancer activities against several cancers, gefitinib was also reported to show MAPK dependent anti-leukemic effects in EGFR-deficient acute myeloid leukemia (AML) cell line HL-60 and patients. However, it is still unclear to how gefitinib induces these effects in cells that lacks its target. Therefore our aim was to decipher the underlying mechanism of action of gefitinib in AML cells. Methods: We used diverse cell and molecular techniques to study the cytostatic and differentiation inducing effects of gefitinib in HL-60 cells. MTT cell viability assay, BrdU incorporation assay and trypan blue cell count were used to assess the inhibition in growth of cells. FACS based Annexin V-FITC - PI labeling was also performed for determining the extent of apoptosis induced by gefitinib and expression of differentiation markers CD14 and CD11b that tells us the maturation stage of cells. Intracellular cyclic AMP (cAMP) was measured using FRET based Cisbio HTRF kit. The GPCR profiling and radio-ligand binding assays were outsourced from Eurofins. Western blot and co-immunoprecipitation assay was done to study the interaction between histamine receptors and gefitinib. Results & Conclusion: Gefitinib was found to reduce intracellular cyclic AMP and induce cytostatic effects in EGFR-rich and EGFR-deficient cell lines both. As a next logical step, a GPCR profiling study was done that revealed that gefitinib regulates a number of GPCRs. However, the radio-ligand competition and functional assays suggested that gefitinib inhibits histamine receptors. This was further confirmed by the activation of histamine receptors by pharmacological agonists followed by assessment of gefitinib mediated effects in AML cells, which suggested that histamine receptor inhibition is necessary for gefitinib mediated effects in AML cells. The co-immunoprecipitation studies also validated the interaction of EGFR and Histamine receptor 4. In the end we concluded that our results indicate the involvement of histamine receptors in gefitinib mediated EGFR-independent anti-proliferative effects. Thus, our report provides new insights into gefitinib mode of action, and also indicates that other EGFR inhibitors displaying anti-leukemic properties may display similar signaling attributes and can contribute towards gefitinib related non-targeted response.