Detection of Phosphorylated and Total Flt3 and STAT5 in Whole Blood: Modulation by AC220 From Phase I and II Trials in AML

Abstract 2607

Fms-like tyrosine kinase 3 (Flt3) plays a critical role in the leukemogenesis of a significant percentage of acute myeloid leukemias (AML) and acute lymphoblastic leukemias making it an attractive target for therapeutic intervention with small molecule inhibitors. To evaluate the pharmacodynamic (PD) effects of AC220, a highly selective and potent Flt3 inhibitor, in phase I and II clinical trials, we developed Flt3 immunoassays using the Meso Scale Discovery (MSD) 96-well platform. Several antibodies were screened to identify capture and detection pairs for both total (t) and phospho (p) Flt3 that were optimized for selectivity, sensitivity, and reproducibility. To validate the assays, cell lysates from SEM-K2 cells that express constitutively phosphorylated Flt3, the MSD assays reproducibly detected tFlt3 with high signal to background ratios, and consistently tracked changes in Flt3 phosphorylation when cells were subjected to Flt3 inhibitors. The functionality of these assays was minimally affected in the context of a whole blood matrix, as p/tFlt3 levels were similarly detected when SEM-K2 cells were spiked into human blood before lysis.

To determine the PD effects of AC220 in humans, we tested whole blood samples from a phase I trial in predominantly relapsed or refractory AML patients (pts). AC220 was dosed once daily with escalations from 12 to 450 mg/day. Blood samples were initially examined in 6 pts taken pre-drug administration, at 2, 9, and 24 hrs, and 8 and 15 days post-administration. AC220 markedly reduced pFlt3 levels in the blood lysates at the 2 hr time point and this reduction was maintained over the 5 subsequent time points. Interestingly, the tFlt3 levels increased in all 6 pts at 2 and 9 hrs after treatment, began decreasing by 24 hrs (albeit still elevated from pre-dose levels), and was severely reduced by day 8 and day 15, consistent with elimination of Flt3-expressing peripheral blasts. Expanding the analysis to 45 patients from this phase I study, statistically significant reductions were observed in pFlt3 levels at 2 hrs, 24 hrs, and 15 days after dosing. Furthermore, pts receiving >90 mg demonstrated significantly greater reductions in pFlt3 compared to those receiving <90 mg. There was also significantly higher inhibition of pFlt3 in pts with Flt3 harboring the internal tandem duplication (ITD) activating mutation compared to those lacking this mutation. Importantly, absolute levels, as well as reductions in tFlt3 from day 1 to day 15 showed a statistically significant (p<0.001) rank correlation with the absolute levels and reductions in peripheral blast counts over the same time period. These results demonstrate that the Flt3 levels detected by the MSD assay effectively parallels the peripheral disease burden in AML pts.

More recently, the PD effects of AC220 were assessed for pts from the exploratory part of an ongoing phase II trial in relapsed/refractory Flt3 ITD+ AML pts. Blood was taken at day 1 just before the first dose, and 24 hrs and 8 days following the initial dose. Phosphorylated STAT5, a marker closely associated with the Flt3 ITD mutation, was also monitored using a commercially available MSD assay. Blood samples from 52 pts were tested in both MSD assays. For Flt3, 13 pts were excluded from the analysis due to signals below the level of quantitation. Of the 39 pts remaining, a median pFlt3 inhibition of 75% (mean 70%) was observed at 24 hrs following the first dose, consistent with a direct effect of AC220 on its intended target. Similar to what was observed in the phase I trial, 52% of the pts had an increase in tFlt3 levels at 24 hrs. Importantly, 47% of pts had >90% reduction in tFlt3 levels after one week on AC220, signifying a major inhibitory effect on peripheral blast counts. Consistent with the strong linkage between Flt3 ITD autoactivation and STAT5 phosphorylation, there was a significant (p<0.006) correlation between changes in pFlt3 and pSTAT5 levels 24 hrs after dosing. These newly developed assays allow for the rapid assessment of Flt3 autophosphorylation and correlation with downstream signaling and peripheral disease burden.