In this study, we report that AKN-028, a novel small molecular TKI identified in an extensive kinase screen as active against FLT3, inhibits the FLT3 enzyme in a dose-dependent manner at very low concentrations. Inhibition of wild-type as well as FLT3-mutated autophosphorylation was confirmed in cellular assays. In vitro
cytotoxic activity was further investigated in a cell line panel representing different malignancies and their drug-resistant sublines, as well as immortalized non-tumor cells. Notably, AKN-028 had a significant activity in all the five AML cell lines tested, whereas no or only marginal effect was observed in the other cell lines, suggesting a specific effect in AML. Interestingly, the FLT3-ITD-mutated cell lines, MV4-11 and MOLM-13, was by far the most sensitive cell lines with IC50
. Moreover, AKN-028 induced apoptosis in AML cell line MV4-11, at least partly mediated by activation of caspase 3.
However, cell lines may have limitations in predicting clinical activity of cytotoxic drugs, possibly owing to their acquisition of additional genetic changes in becoming immortalized and adapted to continuous growth.36
As primary cultures of patient tumor cells may be a model that is better suited for predicting clinical activity,37
we evaluated the cytotoxic effect of AKN-028 in leukemic blasts from AML patients, among those several cases with complex karyotype and chemotherapy-resistant disease. Importantly, AKN-028 showed a dose-dependent cytotoxic response in all patient samples tested, including those with no or only minor in vitro
response to AC220, a potent, selective FLT3 TKI having recently shown clinical efficacy in phase-II trials.38
The antileukemic effect was confirmed in vivo
using the hollow-fiber mouse model, where AKN-028 had significant effect in MV4-11, as well as in one of the primary AML samples. The hollow-fiber model was developed as a tool for anticancer drug screening. The method enables studies of hematological toxicity, pharmacokinetics and tumor effect in cell lines and primary patient samples within the same immunocompetent animal, which in turn reduces the number of animals needed. The hollow-fiber method is relatively resistant, with a risk of underestimating drug effect owing to low efficacy of drug delivery to the subcutaneously implanted fibers.39
Unexpectedly, we observed no difference in sensitivity to AKN-028 between FLT3-mutated and wild-type AML cases. As a pathogenetic role of the wild-type FLT3-receptor in AML has been proposed, we investigated whether a variance in quantitative FLT3 expression, regardless of mutation status, might explain a different response to AKN-028.6, 40
However, there was no clear correlation between quantitative FLT3 expression and cytotoxic activity of AKN-028. Although, the four patients (two FLT3-ITD, two FLT3-wt) with the highest FLT3/GUS × 100 ratio had all relatively low IC50
values, several cases with relatively low quantitative expression of FLT3 but with corresponding low IC50
values were present in our material.
Interestingly, AKN-028 showed ex vivo
efficacy in primary AML tumor cells that were resistant to treatment with the potent FLT3- and KIT-inhibitor AC220. This finding, together with the lack of correlation between FLT3 mutation status or quantitative expression of FLT3 and in vitro
response to AKN-028 in AML, indicates that the compound is also targeting other pathways than those directly associated with FLT3 inhibition. Using a radiometric protein kinase assay, we could exclude that AKN-028 inhibited the FLT3 downstream targets AKT 1-3 and ERK 1-2. Aberrant expression of receptor tyrosine kinases such as KIT, PDGF and JAK may have a role in leukemogenesis 41, 42, 43
and share in part the same downstream targets as FLT3.44
As many FLT3-inhibitors also target KIT, AKN-028 was tested in a human megakaryoblastic leukemia cell line overexpressing KIT, in which the substance induced a significant inhibition of KIT autophosphorylation. The inhibition was higher in this cell assay than predicted by the results from our kinase screen. A possible explanation for this discrepancy may be that only the active form of KIT is present in the kinase panel, whereas the cell assay might reflect effects on both the active and the inactive form of the kinase.
Our kinase-panel screen also revealed inhibition of RPS6- and CLK kinases. Recently, it has been shown that RPS6K, a downstream effector in the mammalian target of rapamycin (mTOR) signaling pathway, is activated in primary FLT3-mutated AML-cells.45
The mTOR pathway is involved in cellular growth and survival, and mTOR dysregulation has been implicated in several malignancies. Furthermore, mTOR inhibitors have been developed as anticancer treatment.46
AC220 did not inhibit RPS6K, in accordance with the published kinase-inhibition profile of this compound. Thus, inhibition of RPS6K could possibly be part of the differentiating effect between AKN-028 and AC220 on the cell kill induced in primary cells. CLK kinases have been reported to play a role in the AKT pathway,47
but their role (if any) in leukemogenesis is presently unknown. Further studies of AKN-028-mediated inhibition of KIT, and of other possible targets, are underway.
AML is biologically a heterogeneous disease. Hitherto, patients treated with FLT3 inhibitors have, with the possible exception of AC220, shown only modest and transient clinical response.48
Indeed, inhibition of FLT3 autophosphorylation does not always lead to cell death, and some leukemic cells may not be dependent on FLT3 signaling.18
In addition, monotherapy with TKIs is associated with the risk of developing resistance.49, 50
Thus, a TKI such as AKN-028, targeting other pathways apart from FLT3, may be potentially advantageous. Nevertheless, combinations with other agents including conventional chemotherapeutics are probably necessary to optimize clinical efficacy. Results from our combination study suggest a sequence-dependent synergistic effect between AKN-028 and cytarabine or daunorubicin, with better antileukemic activity when cells were exposed to chemotherapy simultaneously or 24
h before adding AKN-028, whereas antagonism was observed when cells were pre-treated with AKN-028. The finding of possible synergy between cytotoxic drugs and targeted therapy is in line with the results reported from other TKIs.51, 52
Sequence-dependent antagonism after pre-treatment with TKIs has also been reported, possibly because of the fact that (some) TKIs may cause cell cycle arrest.52
Further studies of the cell cycle effect of AKN-028 are underway.
Pharmacokinetic studies show that anticipated effective plasma exposure can be obtained in mice after oral administration. If the IC90
value is divided with fraction unbound of the compound, taking into account the plasma protein binding of 96,5%, the plasma target concentration should be in the range of 1–3
. Given no strain difference and linear pharmacokinetics, a similar exposure in NMRI mice as in the C57 black mice would be expected. The predicted plasma profile in the hollow-fiber experiment corresponds to 1
h of exposure above the plasma target concentration of 1–3
twice daily. Thus, continuous exposure was not necessary to obtain an antitumoral effect, which is in accordance with some other TKIs, for example, dasatinib, being clinically effective despite a relatively short plasma half-life.53
In conclusion, AKN-028 is a novel TKI with potent in vitro and in vivo activity in AML, irrespective of FLT3 status. In vitro synergy with the standard antileukemic agents cytarabine and daunorubicin, along with good oral bioavailability, make AKN-028 a candidate drug for clinical trials. An international two-part multi-center phase-I study of AKN-028 in patients with AML started in January 2012 (ClinicalTrials.gov NCT01573247). Additional studies on its mechanism of action are underway.