The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Valentina Gaidano,Paola Circosta,Nicoletta Vitale,Marco Lucio Lolli,Stefania Rapelli,Stefano Sainas,Giovanna Carrà,Marta Giorgis,Alessandro Morotti,Alessandro Cignetti,Agnese Chiara Pippione,Mohammad Houshmand,Giuseppe Saglio,Valerio Tenace,Daniela Cilloni,Donatella Boschi

doi:10.3390/cancers13051003

Abstract

Simple SummaryIn this study, we investigated and boosted the pro-apoptotic and pro-differentiating activity of MEDS433 in acute myeloid leukemia (AML). MEDS433 is an inhibitor of Dihydroorotate Dehydrogenase, a fundamental enzyme in the de novo pyrimidine biosynthesis. We discovered that MEDS433 alone and in combination with classical antileukemic agents had a good apoptotic activity, but it could be reduced in vivo due to the physiological presence of uridine. On the contrary, the combination of MEDS433 and dipyridamole, a blocker of the pyrimidine salvage pathway, induced metabolic lethality and myeloid differentiation in all our AML models, while being characterized by a limited toxicity on non-AML cells.Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Highlights

The inhibition of Dihydroorotate Dehydrogenase (DHODH) has recently been found to induce differentiation in several models of acute myeloid leukemia (AML) [1]
While the exact mechanisms triggered by DHODH inhibitors on leukemic cells have not been fully elucidated [11], transcriptome analyses revealed that leukemic cells treated with DHODH inhibitors upregulate genes related to apoptosis and differentiation, and downregulate protein translationrelated genes, impairing protein synthesis [6,7]
We show that MEDS433 has a strong pro-apoptotic effect on several AML cell lines, as demonstrated by: (i) the Annexin V assay (Figure 1); (ii) the increased caspase3/7 activity (Figure 2A) and the increased levels of cleaved caspase 3 (Figure 2B and Figure S8) after exposure to MEDS433; (iii) the reduction in apoptosis when experiments were performed in the presence of a caspase inhibitor (Z-VAD-fmk, data not shown); and (iv) the morphologic evaluation of cells treated with MEDS433 (Figure 2C)

Summary

Introduction

The inhibition of Dihydroorotate Dehydrogenase (DHODH) has recently been found to induce differentiation in several models of acute myeloid leukemia (AML) [1]. Evidence [3] suggests that DHODH inhibition acts through pyrimidine starvation, rather than cellular respiration impairment; the counter-proof is that high levels of uridine, a downstream product of DHODH in the pyrimidine biosynthesis, are able to abolish the effect of DHODH inhibitors on AML cells [1]. From this seminal discovery, several academic and industrial research groups, including ours, have designed new and more potent DHODH inhibitors, confirming original results and extending the knowledge about this topic [4–10]. Pyrimidines are crucial for the proliferation of living entities, and the depletion of the intracellular pyrimidine pool results in cell cycle arrest in S-phase [12]

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