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Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Haematology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Cancer Pharmacology and Informatics/Rolf Larsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. (Cancer Pharmacology and Informatics/Rolf Larsson)
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2010 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 80, no 10, 1507-1516 p.Article in journal (Refereed) Published
Abstract [en]

Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC50 of 70 nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC50 = 0.4 mu M) and Kasumi-1 (IC50 = 2.3 mu M). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion. AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

Place, publisher, year, edition, pages
2010. Vol. 80, no 10, 1507-1516 p.
Keyword [en]
Acute myeloid leukemia, New drug development, Tyrosine kinase inhibitor, FLT3
National Category
Medical and Health Sciences Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-134176DOI: 10.1016/j.bcp.2010.08.002ISI: 000282850900006OAI: oai:DiVA.org:uu-134176DiVA: diva2:371762
Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2014-10-27Bibliographically approved
In thesis
1. Studies of New Signal Transduction Modulators in Acute Myeloid Leukemia
Open this publication in new window or tab >>Studies of New Signal Transduction Modulators in Acute Myeloid Leukemia
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development.

In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML.

AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study.

Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes.

Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 61 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 823
Acute myeloid leukemia, Targeted therapies, Drug development, Tyrosine kinase inhibition, AKN-032, AKN-028
National Category
Hematology Pharmacology and Toxicology
Research subject
Internal Medicine
urn:nbn:se:uu:diva-182440 (URN)978-91-554-8495-8 (ISBN)
Public defence
2012-11-23, Enghoffsalen, Akademiska Sjukhuset, Ingång 50, bv, Akademiska Sjukhuset, Sjukhusvägen, Uppsala, 09:15 (Swedish)
Available from: 2012-11-01 Created: 2012-10-10 Last updated: 2013-01-23Bibliographically approved

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