Acute Promyelocytic Leukemia (APL) is characterized by a block in differentiation where leukemic cells are halted at the promyelocyte stage. A characteristic balanced chromosomal translocation between chromosomes 15 and 17 t (15;17) (q24; q21) is seen in 95% of cases-the translocation results in the formation of the PML-RARA fusion protein. The introduction of retinoic acid (RA) and arsenic trioxide (ATO) has been responsible for initially remarkable cure rates. However, relapsed APL, particularly in the high-risk subset of patients, remains an important clinical problem. In addition, despite the success of ATRA & ATO, many clinicians still elect to use cytotoxic chemotherapy in the treatment of APL. Patients who become resistant to ATO have an increased risk of mortality. The probability of relapse is significantly higher in the high-risk subset of patients undergoing treatment for APL; overall approximately 10-20% of APL patients relapse regardless of their risk stratification. Furthermore, 20-25% of patients undergoing treatment will develop differentiation syndrome, a common side effect of differentiation agents. Recent evidence using in vitro models has shown that mutations in the B2 domain of the PML protein, mediate arsenic resistance. Alternative agents and approaches considering these clinical outcomes are needed to address ATO resistance as well as the relapse rate in high risk APL.

GHRH is a hypothalamic peptide shown to stimulate the proliferation of malignant cells in humans. We have previously shown that the use of GHRH antagonist MIA-602 successfully suppressed the growth of many human cancer cell lines, spanning more than 20 types of cancers. In this study, we demonstrate the presence of GHRH-R in the NB4, NB4-RAA, and K-562 model cell lines. Furthermore, we demonstrate the inhibited proliferation of all three cell lines in vitro after incubation with MIA-602. The treatment of xenografts of human APL cell lines with MIA-602 led to a significant reduction in tumor growth. Additionally, combination therapy with both doxorubicin (DOX) and MIA-602 showed a marked synergistic effect in reducing the proliferation of the K-562 AML cell line. These findings suggest that MIA-602 could be utilized to address resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) therapies, as well as in augmenting anthracycline-based regimens.

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.

Introduction: Acute Promyelocytic Leukemia (APL) accounts for 10%-15% of all acute myeloid leukemia's. The use of All-Trans Retinoic Acid/ Arsenic trioxide (ATRA/ATO) in the treatment regimens of low to intermediate-risk patients has led to remarkable cure rates (~80%). However, treatment guidelines of high-risk patients (WBC count≥ 10 x 109/L) remain unclear and these patients are at an increased risk of relapse. Design: Due to ambiguous treatment guidelines for high-risk APL patients as well as limited options for patients with arsenic resistant disease we sought to analyze clinical outcomes in this subset of patients. We conducted a literature search through Pubmed, EBSCO, and JSTOR to identify original articles that evaluated treatment protocols as well as outcomes for high-risk/refractory APL patients. We also evaluated the literature for clinical data involving patients who had arsenic resistant APL. Only relevant articles from 2000 to 2019 were included. Clinical trials where patients did not receive ATRA/ATO treatment were excluded. High risk patients have an increased mortality and decreased overall survival compared to intermediate and low risk patients. Although early death resulting from bleeding complications remains an important clinical hindrance, cases of arsenic resistant disease are becoming more prevalent. Resistant/refractory APL is a rare occurrence; however, no effective treatment exists to overcome resistance in this subset of patients. Of particular interest is the identification of variant translocations in APL; those that differ from the classical PML-RARA (~95%). 12 non-characteristic rearrangements have so far been described; of these, the ZBTB16-RARA and STAT5b-RARA are the most common variants. Both ZBTB16-RARA & STAT5b-RARA are insensitive to ATRA/ATO combination therapy, leaving these patients with limited options. Clinical outcomes have recently been described in a subset of 13 patients with arsenic resistant disease. Of these 13 patients with arsenic resistant disease, 11 eventually died. Mutations in a particular region of the PML protein have been shown to mediate arsenic resistant disease in both in vitro and in vivo models, suggesting a potential benefit of molecular screening to improve clinical outcomes. Neither ATO nor cytotoxic chemotherapy was successful in inducing remissions in this particular subset of patients. Conclusion: Well-defined treatment guidelines in high-risk APL and the need for alternative agents in patients resistant to ATO therapy are needed to improve clinical efficacy and outcomes. 1. Zhu, H.H.; Qin, Y.Z.; Huang, X.J. Resistance to arsenic therapy in acute promyelocytic leukemia. N Engl J Med 2014, 370, 1864-1866, doi:10.1056/NEJMc1316382. Citation Format: Ravinder Singh Chale, Joaquin J. Jimenez. Acute promyelocytic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 639.

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