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Studies of New Signal Transduction Modulators in Acute Myeloid Leukemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 823
Keyword [en]
Acute myeloid leukemia, Targeted therapies, Drug development, Tyrosine kinase inhibition, AKN-032, AKN-028
National Category
Hematology Pharmacology and Toxicology
Research subject
Internal Medicine
Identifiers
URN: urn:nbn:se:uu:diva-182440ISBN: 978-91-554-8495-8 (print)OAI: oai:DiVA.org:uu-182440DiVA: diva2:559974
Public defence
2012-11-23, Enghoffsalen, Akademiska Sjukhuset, Ingång 50, bv, Akademiska Sjukhuset, Sjukhusvägen, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2012-11-01 Created: 2012-10-10 Last updated: 2013-01-23Bibliographically approved
List of papers
1. Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia
Open this publication in new window or tab >>Significant cytotoxic activity in vitro of the EGFR tyrosine kinase inhibitor gefitinib in acute myeloblastic leukaemia
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2008 (English)In: European Journal of Haematology, ISSN 0902-4441, E-ISSN 1600-0609, Vol. 81, no 5, 344-353 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: 

Gefitinib inhibits epidermal growth factor receptor (EGFR) signalling, but may also act by non-EGFR dependent mechanisms. We have investigated the activity of gefitinib in haematological tumour cells, in particular acute myeloblastic leukaemia (AML).

Methods: 

Cytotoxic activity of gefitinib, alone or in combination with standard anti-leukaemic drugs, was assessed by the short-term fluorometric microculture cytotoxicity assay in tumour cells from 117 patients representing five haematological and five non-haematological malignancies. In AML, the EGFR status was analysed by immunochemistry. Gefitinib-induced apoptosis was investigated in a subset of AML samples, as well as in the leukaemia cell line MV-4-11, using a multiparametric high content screening assay. To confirm activation of caspase-3 in cells treated with gefitinib, a blocking test was carried out in which MV4-11 cells were pretreated with the specific caspase inhibitor DEVD-FMK.

Results: 

Gefitinib showed highest cytotoxic activity in AML (= 19) with many samples being sensitive at concentrations achievable in clinical practice (<10 μM), and no difference between previously untreated and relapsed patients. No correlation between the activity of gefitinib and standard antileukaemic drugs (cytarabine, doxorubicin, etoposide) was observed. Combining gefitinib with these drugs resulted in mainly additive or synergistic (etoposide) effects, with no evidence of sequence dependency. The AML cells did not express the EGFR. Gefitinib induced apoptosis, which was at least partly mediated by activation of the caspase-3 pathway.

Conclusion: 

In vitro, gefitinib has significant cytotoxic activity in AML by inducing apoptosis through non-EGFR dependent pathways.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102372 (URN)10.1111/j.1600-0609.2008.01120.x (DOI)000260185700002 ()18637032 (PubMedID)
Available from: 2009-05-06 Created: 2009-05-06 Last updated: 2017-12-13Bibliographically approved
2. Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia
Open this publication in new window or tab >>Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia
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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.

Keyword
Acute myeloid leukemia, New drug development, Tyrosine kinase inhibitor, FLT3
National Category
Medical and Health Sciences Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-134176 (URN)10.1016/j.bcp.2010.08.002 (DOI)000282850900006 ()
Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
3. The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia
Open this publication in new window or tab >>The novel tyrosine kinase inhibitor AKN-028 has significant antileukemic activity in cell lines and primary cultures of acute myeloid leukemia
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2012 (English)In: Blood Cancer Journal, ISSN 2044-5385, E-ISSN 2044-5385, Vol. 2, e81- p.Article in journal (Refereed) Published
Abstract [en]

Aberrantly expressed tyrosine kinases have emerged as promising targets for drug development in acute myeloid leukemia (AML). We report that AKN-028, a novel tyrosine kinase inhibitor (TKI), is a potent FMS-like receptor tyrosine kinase 3 (FLT3) inhibitor (IC50=6 nM), causing dose-dependent inhibition of FLT3 autophosphorylation. Inhibition of KIT autophosphorylation was shown in a human megakaryoblastic leukemia cell line overexpressing KIT. In a panel of 17 cell lines, AKN-028 showed cytotoxic activity in all five AML cell lines included. AKN-028 triggered apoptosis in MV4-11 by activation of caspase 3. In primary AML samples (n=15), AKN-028 induced a clear dose-dependent cytotoxic response (mean IC50 1 μM). However, no correlation between antileukemic activity and FLT3 mutation status, or to the quantitative expression of FLT3, was observed. Combination studies showed synergistic activity when cytarabine or daunorubicin was added simultaneously or 24 h before AKN-028. In mice, AKN-028 demonstrated high oral bioavailability and antileukemic effect in primary AML and MV4-11 cells, with no major toxicity observed in the experiment. In conclusion, AKN-028 is a novel TKI with significant preclinical antileukemic activity in AML. Possible sequence-dependent synergy with standard AML drugs and good oral bioavailability has made it a candidate drug for clinical trials (ongoing).

National Category
Medical and Health Sciences Hematology Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-182062 (URN)10.1038/bcj.2012.28 (DOI)000308664500001 ()22864397 (PubMedID)
Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
4. AKN-028 induces cell cycle arrest, downregulation of Myc associated genes and a dose dependent reduction of kinase activity in acute myeloid leukemia
Open this publication in new window or tab >>AKN-028 induces cell cycle arrest, downregulation of Myc associated genes and a dose dependent reduction of kinase activity in acute myeloid leukemia
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2014 (English)In: Biochemical Pharmacology, ISSN 0006-2952, E-ISSN 1356-1839, Vol. 87, no 2, 284-291 p.Article in journal (Refereed) Published
Abstract [en]

AKN-028 is a novel tyrosine kinase inhibitor with preclinical activity in acute myeloid leukemia (AML), presently undergoing investigation in a phase I/II study. It is a potent inhibitor of the FMS-like kinase 3 (FLT3) but shows in vitro activity in a wide range of AML samples. In the present study, we have characterized the effects of AKN-028 on AML cells in more detail. AKN-028 induced a dose-dependent G(0)/arrest in AML cell line MV4-11. Treatment with AKN-028 caused significantly altered gene expression in all AML cell types tested (430 downregulated, 280 upregulated transcripts). Subsequent gene set enrichment analysis revealed enrichment of genes associated with the proto-oncogene and cell cycle regulator c-Myc among the downregulated genes in both AKN-028 and midostaurin treated cells. Kinase activity profiling in AML cell lines and primary AML samples showed that tyrosine kinase activity, but not serine/threonine kinase activity, was inhibited by AKN-028 in a dose dependent manner in all samples tested, reaching approximately the same level of kinase activity. Cells sensitive to AKN-028 showed a higher overall tyrosine kinase activity than more resistant ones, whereas serine/threonine kinase activity was similar for all primary AML samples. In summary, AKN-028 induces cell cycle arrest in AML cells, downregulates Myc-associated genes and affect several signaling pathways. AML cells with high global tyrosine kinase activity seem to be more sensitive to the cytotoxic effect of AKN-028 in vitro.

Place, publisher, year, edition, pages
Elsevier, 2014
Keyword
Acute myeloid leukemia, AKN-028, Tyrosine kinase inhibitor, Signal transduction
National Category
Hematology Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-182065 (URN)10.1016/j.bcp.2013.10.022 (DOI)000330332800006 ()
Available from: 2012-10-10 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved

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