Monitoring of Phosphorylated CRKL in Imatinib Resistant Patients with BCR-ABL Positive Leukemias Expressing BCR-ABL Mutants Treated with AMN107.

Abstract

AMN107 is a new, highly potent and selective BCR-ABL inhibitor currently in clinical development for the treatment of imatinib-resistant chronic myelogenous leukemia (CML) or Philadelphia positive acute lymphoblastic leukemia ALL (Ph+ALL). Pre-clinical testing has revealed AMN107 to inhibit all but one (T315I) BCR-ABL mutants which have been associated with imatinib resistance. We sought to determine the pharmacodynamic activity of AMN107 by measuring the proportion of phosphorylated CrkL (CrkL-P) as a surrogate of BCR-ABL activity in vivo. Assay validation revealed a CV-value of 13%, which was defined as cut-off value for significant modulation of the Crkl-P/CrkL ratio. A total of 34 patients (median age 61 years, range 35–80) diagnosed with imatinib resistant Ph+ ALL (n=10), CML in chronic phase (n=1), accelerated phase (n=13), myeloid (n=7), or lymphoid blast crisis (n=3) were investigated in a phase I study permitting individual dose escalation (50–1200 mg/day). Proportion of CrkL-P (Crkl-P/total Crkl) was determined by Western blot, ratio BCR-ABL/ABL by quantitative RT-PCR, and mutation status by direct sequencing in 73 peripheral blood or bone marrow samples from baseline and during treatment with AMN107. Median follow up was 89 days (range 13–386). Patients expressed e1a2 (n=7), b2a2 (n=12), b3a2 (n=14), and b2a2&b3a2 (n=1) BCR-ABL transcripts. At baseline, 18 pts exhibited BCR-ABL mutations (P-loop, n=4; T315I, n=3; others, n=11), in 4 pts two different mutations were found in parallel. Prior to treatment with AMN107, the median proportion of CrkL-P indicating BCR-ABL activity was 47% (range 0–69%). Significant reductions of the proportion of CrkL-P were observed from a dose level of 200 mg AMN107/day. CRKL-P (0%) became undetectable during treatment with AMN107 indicating complete suppression of BCR-ABL in 16 pts starting at AMN107 dose levels of 200 (n=1), 400 (n=2), 600 (n=4), 800 (n=8) or 1200 mg/d (n=1). At baseline, patients had unmutated BCR-ABL (n=8), M244V, Y253H, E255K, T315I, M351T, L384M/H396P, A217V/F311L, L324Q/A350V (n=1 each). Undetectability of CrkL-P, correlated with a good molecular response (ratio BCR-ABL/ABL <2%) in 3 pts. We conclude that a minimum of 200 mg of AMN107 is required to induce effective BCR-ABL inhibition in patients. Effectively repressed CrkL phosphorylation in patients lacking molecular response indicates multifactorial resistance mechanisms. Even in patients with BCR-ABL mutations, BCR-ABL may be inactive suggesting alternative signaling pathways that stimulate proliferation. However, treatment with AMN107 is associated with a reduction of the proportion of CrkL-P indicating suppression of BCR-ABL activity in a significant proportion of patients after imatinib resistance. The CrkL phosphorylation status may help to determine alternative treatment strategies including dose optimization in phase I studies.