Metformintreatment Overcomes ATRA-Resistance in Acute Promyelocytic Leukemia and Increases FOXO3A Expression
Authors: Pereira-Martins Diego Antonio , Ortiz Cesar , Franca-Neto Pedro Luis , Weinhäuser Isabel , Schiavinato Josiane Lilian , Palma Patrícia Vianna Bonini , Bonaldo Camila , Koury Luisa C A , Do Nascimento Mariane Cristina , Melo Raul Antônio Morais , Bittencourt Rosane , Pagnano Katia B B , Pasquini Ricardo , Fagundes Evandro Maranhão , Gloria Ana Beatriz F. , Kerbauy Fabio R. , Chauffaille Maria de Lourdes , Keating Armand , Tallman Martin S. , Ribeiro Raul , Dillon Richard , Ganser Arnold , Lowenberg Bob , Valk Peter , Lo-Coco Francesco , Sanz Miguel A. , Berliner Nancy , Lucena-Araujo Antonio R. , Rego Eduardo Magalhães 
Published: October 2019

Abstract
Introduction: FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P<.05), compared to only 0.5-fold reduction rate using ATRA alone. In the clinical setting, APL patients expressed a lower absolute level of FOXO3A than normal BMMC (P<.001). These results were corroborated in an external validation cohort (P=.004). The retrospective analysis of patients enrolled in the ICAPL2006 study revealed that the baseline characteristics were similar between patients with low and high FOXO3A levels. Low FOXO3A expression was associated with lower DFS (84%; 95% CI: 63-94%)(HR: 1.25, 95% CI: 0.64-10.4) and OS (76%; 95% CI: 63-84%) (HR: 1.43, 95% CI: 0.12-1.48). The overexpression of FOXO3A in APL cell lines was associated with reduced basal cell viability (P=.02), clonogenicity (P=.001), and proliferation (P=.001) in both APL cell lines. Subsequent cell cycle analysis showed no significant difference between FOXO3A-cells and controls in normal conditions and when treated with ATRA. Using a nonlinear regression analysis, IC50 for ATO was significantly lower for NB4-FOXO3A cells (0.27μM) than control (2.27µM), with no corresponding response for NB4R2 cell line (2µM for empty vector vs 1.46μM for NB4R2 FOXO3A). NB4 (IC50: 14mM) and NB4R2 (IC50: 4.2mM) FOXO3A cells presented higher sensibility to metformin treatment versus control group (IC50: 23mM for NB4; IC50: 22mM for NB4R2). For ATRA treatment alone, only NB4-FOXO3A presented increased sensitivity to ATRA (P=.001). In accordance, ATRA treatment (alone or in combination with metformin) increased the drug-induced apoptosis in a time-dependent manner (P<.05) in NB4-FOXO3A cells, but had no effect on NB4R2-FOXO3A, which presented increased apoptosis rate only with metformin treatment. The differentiation rate was higher in FOXO3A-expressing cells (P<.05). Conclusion: Based on clinical and functional data, the FOXO3A pathway could be an interesting target to overcome ATRA resistance in APL in offsetting where ATO is unavailable in low- and middle-income countries.

Disclosures
Tallman: BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.