Abstract 5317: A novel approach to the treatment of ATRA/ATO resistant acute promyelocytic leukemia

Abstract

Acute Promyelocytic Leukemia (APL) is characterized by proliferation of immature promyelocytes. Combination therapy using retinoic acid (RA) and arsenic trioxide (ATO) is considered standard treatment in the clinical environment. Resistance to both RA and ATO is associated with an increased risk of mortality. In addition, relapse occurs in approximately 10-20% of patients undergoing treatment for APL. Growth Hormone Releasing Hormone (GHRH) is a hypothalamic peptide that stimulates the release of Growth Hormone from the anterior pituitary. Our previous studies have demonstrated that human AML cell lines (K-562, THP-1, and KG-1a) display the GHRH receptor (GHRH-R). Incubation with GHRH-R antagonist MIA-602, inhibited proliferation and induced apoptosis. The purpose of this study is to examine the effects of GHRH-R antagonist MIA-602, on the NB4 Human Promyelocytic Leukemia cell line and its ATRA and ATO resistant sub-clone. NB4 cells expressed both GHRH-R and SV1 variants via western blot analysis. Wild type NB4 cells, as well as NB4 cells resistant to both ATRA and ATO (NB4-RAA), were cultured in suspension in RPMI and 10% fetal calf serum. NB4-RAA were established in Dr. Jimenez's lab from the wild type NB4 parent cell line. NB4 and NB4-RAA were plated in triplicate on multi-wells at 50,000 cells/mL. Cell lysates were prepared after incubation with 0.05-5 µmol/L of MIA-602. Cell viability was measured at 24 and 48h. Flow cytometry was also performed on the naïve NB4 parent cell line in addition to NB4-RAA to assess expression of CD-56 the cell surface marker. Upregulation of CD-56, also known as Neural cell adhesion molecule 1 (NCAM-1), has been shown to confer drug resistance in AML and is an important prognostic marker in APL. The viability of both cell lines decreased to similar levels at 24 h and 48 h when exposed to concentrations of MIA-602 higher than 0.05 μmol/L (p < 0.05). A significant decrease in cell viability was seen above 0.5 μmol/L after 48h. No viable cells were found in either cell line after 48h when exposed to 5 μmol/L of MIA-602. No difference in cell viability was found between naïve NB4 and resistant NB4-RAA when exposed to the same concentrations of MIA-602. MIA-602 induced significant changes in the expression level of many death-related genes including CASP9 (>1.5-fold; P<0.05). Results of flow cytometry revealed significantly increased expression of CD-56 (>5.8-fold; p,0.05) in NB4-RAA cells as compared to the parent cell line. These results indicate that resistance to ATRA and ATO in APL cells does not confer subsequent resistance to MIA-602. As a result, MIA-602 targets a pathway that is distinct from that of ATRA & ATO. More importantly, upregulation of pro-apoptotic genes by MIA-602 indicates an alternative pathway elicited by the GHRH-R antagonist in the setting of resistant APL cells. Our results indicate that the resistance to frontline APL therapy can be effectively overcome by using MIA-602. Citation Format: Ravinder S. Chale, Stephanie M. Almeida, Ivan Jozic, Andrew V. Schally, Joaquin J. Jimenez. A novel approach to the treatment of ATRA/ATO resistant acute promyelocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5317.