Evaluation Of Acadesine, a Drug Stimulating Cell Autophagy, In Azacitidine(AZA)-Resistant Myelodysplastic Syndromes (MDS)
Authors: Cluzeau Thomas , Robert Guillaume , Karsenti Jean Michel , Luciano Frederic , Mounier Nicolas , Cassuto Jill Patrice , Ades Lionel , Raynaud Sophie , Fenaux Pierre , Auberger Patrick 
Published: October 2019

Abstract

Background
AZA is currently the first line treatment for intermediate-2 and high-risk IPSS myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with 20 to 30% of marrow blasts. We have previously reported that cells from AZA-resistant patients exhibit impaired mitochondrial apoptosis but maintain functional autophagy (Cluzeau et al. Cell Cycle 2011, Oncotarget 2012). Acadesine (ACA), also known as AICAR or Aica-Riboside is a nucleoside analogue that has been shown to trigger autophagy in AZA resistant cells.

Methods
In vitro effect: We used an AZA-resistant MDS/AML cell line (SKM1-R) as a tool to decipher AZA resistance. Cells were treated with increasing doses of ACA (0.5-2mM) or with a maximally efficient dose of AZA (1µM) and induction of cell death was assessed by cell metabolism and Propidium Iodide (PI) assays. In vivo effect: The effect of ACA was also assessed in a mouse xenograft model of SKM1-R cells. When tumors reached 100 mm3, mice were treated daily with an intra-peritoneal injection of the vehicle alone, 5 mg/kg AZA, or 200 or 400 mg/kg ACA. Ex vivo effect: We finally used primary bone marrow cells from AZA-resistant MDS or AML-resistant patients (n=12) (clinicalTrials.gov identifier: NCT01210274) to perform cell metabolism assays.

Results
In vitro effect: Only a slight decrease of cell metabolism and a moderate increase of PI staining were detected following stimulation with 1µM AZA confirming the resistance of SKM1-R cells to AZA. In identical conditions, ACA induced a robust increase of cell death in AZA-resistant cells with a maximal effect at 2mM. Induction of cell death by ACA was independent of apoptosis but relied on autophagy induction, as shown by the conversion of LC3-I to LC3-II and an increase of cathepsin B activity, that are respectively early and late markers of autophagy. In vivo effect: As expected, AZA failed to trigger tumor regression of AZA-resistant SKM1-R cells in vivo compared to vehicle alone, whereas ACA was found to induce a statistically significant inhibition of tumor growth at both tested concentrations. Ex vivo effect:Bonferroni’s Multiple Comparison Test performed in 6 AZA-resistant MDS patients showed significant reduction of cell metabolism between ACA and untreated cells (66% and 78% at 1 and 2 mM of ACA) and between ACA and AZA-treated cells (60% and 72% at 1 and 2mM of ACA,). Identical results were found in 6 AML AZA-resistant AML patients with a significant reduction of cell metabolism between ACA and untreated cells (63% and 81% at 1 and 2mM of ACA) and ACA and AZA-treated cells (56% and 75% at 1 and 2 mM of ACA).

Conclusion
Our results show the high efficacy of Acadesine in vitro, in vivo and ex vivo in AZA-resistant MDS and AML cell lines and patient’s bone marrow cells. Induction of cell death by autophagy seems to be the main mechanism by which ACA circumvents AZA resistance in MDS and AML cells. These encouraging results prompted us to initiate a multicenter phase I/II clinical trial with the French MDS Group (GFM) to assess the safety and efficacy of ACA in MDS and AML patients with 20 to 30% of marrow blasts not responding or relapsing after AZA treatment (clinicalTrials.gov identifier: NCT01813838).

Disclosures:
Cluzeau: Advancell: Research Funding. Robert:Advancell: Research Funding. Auberger:Advancell: Research Funding.