Ponatinib is an orally active, multitargeted kinase inhibitor that has shown potent activity against BCR-ABL, and all mutant variants tested, in preclinical models of CML (2). Viability of cells driven by native or mutant BCR-ABL, including BCR-ABLT315I
, has previously been shown to be inhibited with IC50
values between 0.5 and 36 nmol/L. Previous studies (2) have also shown potent in vitro
inhibitory activity against a discrete set of additional kinases, including several implicated in the pathogenesis of other hematologic malignancies (4
): FLT3, KIT, and members of the FGFR and PDGFR families. Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib exhibits activity against each of these kinases with potency similar to that observed for BCR-ABL: IC50
values for inhibition of target protein phosphorylation and cell viability ranged from 0.3 to 20 nmol/L and 0.5 to 17 nmol/L, respectively. Other multitargeted kinase inhibitors, such as sorafenib and sunitinib, have previously been shown to have inhibitory activity against a subset of these kinases. However we found that ponatinib was unique in its ability to inhibit activity of all 4 kinases with high potency. Importantly, preliminary results reported from an ongoing phase 1 clinical trial of ponatinib that includes patients with refractory CML show that levels of ponatinib required to functionally inhibit BCR-ABL, and mutant variants, are attainable (24
). In the models tested here, ponatinib exhibited potency against FLT3, KIT, FGFR1, and PDGFRα comparable to that observed previously in BCR-ABL–driven models of CML (2), suggesting that inhibition of these additional targets is clinically achievable. Overall these results provide support for clinical testing of ponatinib in diseases in which these kinases play a role.
Myeloproliferative neoplasms with genetic rearrangements of FGFR1 and PDGFRα are considered to be rare; however, it has been shown that the resulting fusion proteins play a major role in the pathogenesis of these diseases (6
). The 8p11 myeloproliferative syndrome is an aggressive disease that can rapidly transform to AML in the absence of treatment. We have shown here that ponatinib potently inhibits viability of the AML KG1 cell line, which is driven by an FGFR1OP2-FGFR1 fusion protein, suggesting that ponatinib may have clinical activity in this disease type. HEL/CEL patients with a PDGFRα fusion achieve dramatic hematological responses when treated with the PDGFR inhibitor imatinib (6
) and we have shown that ponatinib has potent activity against the FIP1L1-PDGFRα fusion protein as shown in the leukemic EOL cell line. However, the T674I mutant of PDGFRα, which is mutated at the position analogous to the T315I gatekeeper residue in BCR-ABL, has been shown to confer resistance to imatinib in patients (6
). Importantly, ponatinib has potent activity against the PDGFRα T674I mutant kinase, with an IC50
of 3 nmol/L (2), suggesting that ponatinib may be effective in treating patients who carry this fusion protein. More generally, the unique linker of ponatinib is specifically designed to accommodate mutated gatekeeper residues, suggesting that the ability to inhibit such mutations may also apply to other targets (2, 3). Indeed ponatinib potently inhibits the FGFR1 gatekeeper mutant FGFR1V561M
with an IC50
of 7 nmol/L (2). The fact that the same isoleucine side chain is shared by BCR-ABLT315I
, and FLT3F691I
suggests that ponatinib should also be active against these KIT and FLT3 gatekeeper mutants, based on the molecular interactions observed in the crystal structure of T315I ABL bound with ponatinib (2, 3).
Both the incidence and prognostic significance of FLT3-ITD alterations in AML suggest that this kinase plays a critical role in the pathogenesis of the disease (25
) and, as such, represents a major target for therapeutic intervention. In the studies reported here, using the FLT3-ITD expressing cell line MV4-11, we show a close relationship between inhibition of FLT3 activity, both in vitro
and in vivo
, and inhibition of tumor cell viability. In vitro
, low nmol/L concentrations of ponatinib (i.e., <10 nmol/L) led to a decrease in FLT3 phosphorylation, a decrease in viability, and an increase in markers of apoptosis. In an in vivo
xenograft model, a daily oral dose of 1 mg/kg ponatinib led to significant inhibition of tumor growth and a dose of 5 mg/kg or greater led to tumor regression. Consistent with the effects on tumor growth being due to inhibition of FLT3, a single dose of 1 mg/kg ponatinib led to a partial inhibition of FLT3-ITD and STAT5 phosphorylation, while doses of 5 and 10 mg/kg led to substantial inhibition. Finally, ponatinib potently inhibited viability of primary blasts isolated from a FLT3-ITD positive AML patient (IC50
of 4 nmol/L), but not those isolated from 3 FLT3 wild-type patients (IC50
> 100 nmol/L).
Multiple compounds with FLT3 activity have been described and several have already been evaluated in patients. Relatively modest clinical activity has been reported to date (11
), although AC220 has begun to show promise (16
). Based on preclinical studies that show that FLT3 inhibition needs to be sustained to effect killing of FLT3-dependent AML cells (26
), a view has emerged that to achieve maximum therapeutic benefit, continuous and near-complete inhibition of FLT3 kinase may be required (26
). Our in vitro
studies show that complete inhibition of FLT3 phosphorylation and function can be obtained at 10 nmol/L or more concentrations. Importantly, preliminary analysis of the pharmacokinetic and pharmacodynamic properties of ponatinib show that well-tolerated oral daily doses lead to trough plasma drug levels exceeding 40 nmol/L, and sustained inhibition of BCR-ABL activity in circulating leukemic cells (24
). These data suggest that the potency and pharmacologic properties of ponatinib may allow continuous and near-complete inhibition of FLT3 in patients.
In summary, ponatinib is a multitargeted kinase inhibitor that displays potent inhibition of FLT3 and is cytotoxic to AML cells harboring the FLT3-ITD mutation. Importantly, this agent exhibits activity against additional RTKs, FGFR1, KIT, and PDGFRα, which have also been shown to play roles in the pathogenesis of hematologic malignancies. Notably, the potency of ponatinib against these RTKs in vitro and plasma levels of ponatinib observed in humans suggest that ponatinib may have clinical activity against these targets. Taken together, these observations provide strong preclinical support for the evaluation of ponatinib as a novel therapy for AML and other hematologic malignancies.