AKN-028, a FLT-3 Kinase Inhibitor In Preclinical Development, Induces Significant Gene Regulation That Differs From PKC-412

Abstract 1837

AKN-028 (formerly BVT-II, abstract presented at EHA 2008, Eriksson A et al) is a tyrosine kinase inhibitor originally identified in a screen targeting FLT-3. In vitro testing by fluorometric microculture cytotoxicity assay (FMCA, Lindhagen E. et al, Nat Protoc 2008; 3:1364-9) on primary tumor cells from 29 patients with different haematological malignancies showed that the cytotoxic activity was most pronounced in acute myeloic leukaemia (AML). In vivo, efficacy was demonstrated in a hollow fiber mouse model using MV4-11 cells and primary tumor cells from AML patients.

Kinase inhibitors interacting with the ATP-binding pocket usually targets more than one kinase. PKC-412, presently in phase III trials in AML, is a staurosporine analogue targeting many kinases. In this study we show the kinase inhibition profile of AKN-028 when characterised on a broad panel of 320 different kinases. AKN-028 is relatively specific compared to the staurosporine analogues. To further analyse the difference in mechanism of action between the multi-targeting inhibitor PKC-412 and the more selective AKN-028, we have performed global gene expression analysis by Affymetrix arrays using two different leukaemia cell-lines, HL-60 and MV4-11 (expressing FLT3-ITD), and tumour cells from a patient with FLT3-ITDpos AML. The cells were treated with 10 μM of either compound or vehicle control for 6 h. Principal component (PC) analysis was used to visualise the global gene expression pattern, see fig 1. Searching for differential expression of mRNA levels showed that treatment with AKN-028 resulted in significantly altered gene expression in all three cell types tested, compared to vehicle control treated cells. 430 mRNAs were down-regulated, and 280 were up-regulated. By contrast, treatment with PKC-412 caused very few significant alterations of mRNA levels when compared to vehicle treated cells. Further analysis of gene expression patterns to elucidate the mechanism of action of AKN-028 is ongoing. In conclusion, even though AKN-028 is a relatively selective kinase inhibitor targeting FLT-3, it has more profound effects altering gene expression both in cultured AML cell-lines and a primary AML tumor sample than the multikinase inhibitor PKC-412.

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