Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells.

Edit Kotogány,Lajos I Nagy,László G Puskás,Valeria Bertagnolo,Csaba Varga,Gábor J Szebeni,Renáta Szabó,Anita K Kovács,Gabriella Mezei,József Á Balog,Federica Brugnoli,András Demjén,Iván Kanizsai,Róbert Alföldi,Péter Batár

doi:10.3390/ijms21145135

Abstract

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

Highlights

Myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDS) are diseases of the hematopoietic stem cells in the bone marrow (BM) where an excess of immature cells is produced, which can frequently evolve to different myeloid leukemias [1,2]
Three agents—the calcitriol receptor agonist 1,25-dihydroxyvitamin D3 [32,33], the retinoic acid receptor agonist all-trans retinoic acid (ATRA) [34], and TLR7/8 agonist resiquimod [35]—have been verified to mature myeloid-derived suppressor cells (MDSCs) and reduce tumor nursing condition of immunosuppression [28]. Based on these findings we addressed to investigate the cytotoxic and differentiating effect of our imidazo[1,2-b]pyrazole-7-carboxamide derivative on splenic immature myeloid cells, both on M- and G-MDSCs accumulated in 4T1 breast cancer-bearing mice
We have previously observed the asynchronous response of HL-60 cells in terms of morphology treated with DU325 and decided to obtain single cell protein expression data in a subpopulation of cells by using flow cytometry

Summary

Introduction

Myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDS) are diseases of the hematopoietic stem cells in the bone marrow (BM) where an excess of immature cells is produced, which can frequently evolve to different myeloid leukemias [1,2]. The group of myeloid cells have been determined by the WHO classification, including granulocytic (neutrophil, eosinophil, and basophil), monocytic/macrophage, erythroid, megakaryocytic, and mast cell lineages [1]. The AMLs represent a group of heterogeneous forms of myeloid malignancies with diverse genetic abnormalities and different stages of myeloid differentiation. The AMLs originate from myeloid stem cells or myeloid blasts halted in an immature state during hematopoiesis where more than 20% of blasts can be detected in the BM which counteracts with the production of normal blood cells [4]

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