Potent activity of ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies

Abstract

Ponatinib (AP24534) is a novel multitargeted kinase inhibitor that potently inhibits native and mutant BCR-ABL at clinically achievable drug levels. Ponatinib also has in vitro inhibitory activity against a discrete set of kinases implicated in the pathogenesis of other hematologic malignancies, including FLT3, KIT, fibroblast growth factor receptor 1 (FGFR1), and platelet derived growth factor receptor α (PDGFRα). Here, using leukemic cell lines containing activated forms of each of these receptors, we show that ponatinib potently inhibits receptor phosphorylation and cellular proliferation with IC50 values comparable to those required for inhibition of BCR-ABL (0.3 to 20 nmol/L). The activity of ponatinib against the FLT3-ITD mutant, found in up to 30% of acute myeloid leukemia (AML) patients, was particularly notable. In MV4-11 (FLT3-ITD(+/+)) but not RS4;11 (FLT3-ITD(-/-)) AML cells, ponatinib inhibited FLT3 signaling and induced apoptosis at concentrations of less than 10 nmol/L. In an MV4-11 mouse xenograft model, once daily oral dosing of ponatinib led to a dose-dependent inhibition of signaling and tumor regression. Ponatinib inhibited viability of primary leukemic blasts from a FLT3-ITD positive AML patient (IC50 4 nmol/L) but not those isolated from 3 patients with AML expressing native FLT3. Overall, these results support the investigation of ponatinib in patients with FLT3-ITD-driven AML and other hematologic malignancies driven by KIT, FGFR1, or PDGFRα.

Figure 1

Chemical structure of ponatinib (A), sorafenib (B), and sunitinib (C).

Figure 2

Ponatinib inhibits phosphorylation of activated RTKs and cell viability in AML cell lines. A, MV4-11, Kasumi-1, KG1, and EOL1 cells were incubated with the indicated concentrations of ponatinib for 1 hour. Lysates were prepared and immunoblotted for phosphorylated FLT3, KIT, FGFR, or PDGFRα, respectively. Blots were subsequently stripped and reprobed for total levels of the relevant kinase. Similar results were obtained in 2 independent experiments. B, AML cells were incubated with increasing concentrations of compound for 72 hours, and cell viability was assessed using an MTS assay. Data are presented as mean ± SD from 3 experiments.

Figure 3

Ponatinib induces apoptosis in MV4-11 cells. A, MV4-11 cells were seeded in 96-well plates, treated with increasing concentrations of ponatinib, and caspase-3/7 activity measured at the indicated times. Data are expressed as fold induction of caspase activity relative to vehicle-treated cells and are presented as mean ± SD from 3 individual experiments. B, MV4-11 cells were treated with the indicated range of ponatinib concentrations for 24 hours. Cells were harvested and immunoblotted for phosphorylated and total STAT5, as well as total and cleaved (Cl)-PARP. GAPDH was included as a loading control.

Figure 4

Ponatinib showed dose-dependent efficacy, tumor regression, and target inhibition in MV4-11 xenografts. A, daily oral administration of vehicle or ponatinib for 4 weeks at doses of 1, 2.5, 5, 10, and 25 mg/kg/d was initiated when MV4-11 flank xenograft tumors reached approximately 200 mm³ (10 mice per group). Mean tumor volumes (± SEM) are plotted. Three of ten animals in the vehicle control group were sacrificed before the last treatment on day 28 due to tumor burden. Therefore, TGI was calculated from days 0 to 24 (as indicated by the *), the next to last time-point for tumor measurement during the dosing phase. B, mice bearing established MV4-11 tumor xenografts were administered a single oral dose of ponatinib (4 mice per group) at the level indicated; control animals received vehicle alone (5 mice). Tumors were harvested 6 hours later and analyzed for levels of phosphorylated and total FLT3 and STAT5 by immunoblotting. GAPDH was examined as a control. Each lane represents a separate animal. C, relative phosphorylation levels of FLT3 and STAT5 are shown as mean (± SEM) from 2 independent experiments (one of which is shown in B). GAPDH was used as a loading control. Plasma ponatinib levels are shown as mean (± SEM) from the same 2 experiments.

Figure 5

Ponatinib selectively inhibits cell viability of FLT3-ITD primary AML blasts ex vivo. Primary leukemic blast cells were isolated from peripheral blood from 4 individual AML patients. FLT3-ITD status was determined by PCR and sequencing. Primary cell cultures were treated with the indicated concentrations of ponatinib for 72 hours, at which time viability was assessed using an MTS assay. All values are normalized to the viability of cells incubated in the absence of drug.