Phospho-Specific Flow Cytometry of Fixed Whole Blood Demonstrates In Vivo FLT3 Inhibition in Circulating Leukemic Blasts During AC220 Therapy and Accurately Detects the Development of Therapeutic Resistance,

AC220 (quizartinib) is a potent, highly selective inhibitor of FLT3, c-KIT, and c-FMS tyrosine kinases with promising clinical activity in AML, particularly among patients with FLT3 internal tandem duplication mutations (FLT3-ITD). However, direct confirmation of biochemical FLT3 inhibition in AML blasts in vivo by AC220 has not been previously described. We report separately that phospho-ribosomal protein S6 (pS6) by flow cytometry provides a sensitive and dynamic readout of FLT3 kinase activity during FLT3 inhibitor therapy. S6 is a downstream target of the PI3 kinase/mammalian target of rapamycin (mTOR) pathway, is constitutively phosphorylated in nearly all FLT3-ITD+ samples, and its phosphorylation shows dynamic changes in response to FLT3 ligand (FL) or FLT3 inhibitors. We hypothesized that reductions in S6 phosphorylation by flow could serve as a biomarker for FLT3 inhibition and predict response and/or resistance to AC220.

Serial peripheral blood samples were collected during a phase II AC220 clinical trial (Cortes, et al. EHA 2011, abstract #1019). Samples were aliquoted into FACS tubes within four hours of collection and a subset was exposed to signaling inhibitors (ex vivo AC220, rapamycin × 30 min.) or activators (phorbol ester/PMA or FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed and red cells were lysed with the permeabilizing agent triton X-100. Samples were stored at −20C in glycerol medium. Samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single antibody cocktail. Blasts were identified by using CD45 and side scatter (SSC) and confirmed by expression of multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive and negative pS6 gates were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls.

6 subjects provided blood samples (5 FLT3-ITD, 1 FLT3-WT) and had evaluable peripheral blasts (>100/microliter) for pS6 monitoring. As previously described by our group and others using intracellular flow, pS6 is heterogeneous in primary AML samples and, at a basal state, frequently only demonstrable in a subset of blasts. The mean percentage of blasts demonstrating S6 phosphorylation prior to AC220 therapy was 29% (median 9.8%, range 2–97%). To quantify FLT3 kinase inhibition in vivo, we monitored pS6 prior to and following subjects' initial dose of AC220. Samples obtained two hours following the initial dose consistently reduced the percentage of blasts with pS6 to a mean of 2.8% (median 1, range 0.02 to 11). Consistent with its long half-life, pS6 following a single dose of AC220 remained consistently and potently suppressed at trough concentration on day 2. Comparing clinical response and biochemical inhibition by AC220, all 5 subjects with marked reduction in pS6 to AC220 cleared peripheral blasts by day 29 and 3 subjects achieved marrow blasts <5%. By contrast, a FLT3-WT subject with no WBC reduction during AC220 therapy showed persistent constitutive S6 phosphorylation in FLT3-expressing cells. Re-activation of S6 phosphorylation also occurred in 2/2 FLT3-ITD+ subjects who relapsed during AC220. In both cases, we detected treatment-emergent FLT3-D835 mutations, which have been shown to confer potent in vitro resistance to AC220 (Smith, et al, AACR 2011, abstract #4737). Although S6 phosphorylation in relapsed patients' blasts was not suppressed by additional ex vivo AC220, plasma obtained at relapse potently inhibited S6 phosphorylation in the FLT3-ITD+ cell line Molm14. Taken together, these data confirm that these subjects' blasts show intrinsic resistance to AC220 and that drug failure was not secondary to pharmacokinetic changes over time.

We demonstrate the feasibility and utility of intracellular flow cytometry for phospho-S6 to monitor the biochemical efficacy of FLT3 inhibitors. The potential of these methods to predict clinical response/resistance paired with the rapid turnaround time of flow cytometry suggests potential future application of this technology in the screening evaluation of patients offered novel signal transduction inhibitors. Studies correlating the magnitude of signal inhibition with IWG responses are ongoing in a larger cohort treated with AC220 and will be reported once clinical data mature.

Carroll:TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding; Agios Pharmaceuticals: Research Funding.