Background: The Growth factor receptor bound protein-2 (Grb2) is essential to cancer cell signaling. Grb2 binds to numerous tyrosine kinases, including the Bcr-Abl chimeric oncogene found in Philadelphia chromosome positive chronic myelogenous leukemia (CML) cells. Upon binding to tyrosine kinases, Grb2 transduces the kinases signals to the activation of Ras and its downstream protein, Extracellular signals regulated protein kinases-1,2 (Erk1,2). The direct binding of Grb2 to Bcr-Abl is required for Bcr-Abl to transform fibroblasts and induce CML-like diseases in mice. We hypothesize that Grb2 is a potential therapeutic target against CML. BP1001, a liposome-incorporated antisense oligodeoxynucleotide targeted against the Grb2 mRNA, was developed to inhibit Grb2 expression. Previously we demonstrated that BP1001 suppressed the growth of CML cell lines and CML xenografts.
Aim: Since the clinical activity of BP1001 will be investigated in gleevec-resistant CML patients, preclinical studies were conducted to determine whether BP1001 could suppress the proliferation of gleevec-resistant CML cells. Additionally, whether BP1001 could enhance the inhibitory effects of dasatinib in CML cells was determined. The clinical activity of BP1001 was studied in CML blast patients, who were part of a larger Phase I clinical study.
Methods: The alamarBlue dye incorporation method and the CellTiter Glow method were used to determine cell viability. Flow cytometry was utilized to analyze cell cycling and Grb2 expression. The clinical study was a standard 3+3 dose-finding study in which BP1001 was given twice weekly, IV over 2-3 hours for 28 days. The clinical activity of BP1001 was evaluated in its ability to reduce target Grb2 protein in CD33-expressing cells and to reduce leukemia blast counts.
Results: BP1001 decreased the proliferation of gleevec-resistant CML cells in a dose-dependent manner. Furthermore, BP1001 pretreatment enhanced the inhibitory effects of dasatinib in CML cells, by about 2-6 fold. The major inhibition mechanism of the BP1001 + dasatinib combination was cell death. A total of39 patients with refractory/relapsed hematologic malignancies was enrolled in the clinical trial. Five were CML blast phase patients; they were enrolled in the first cohort (5 mg/m2BP1001). Two CML patients, who had T315I mutation, showed significant reductions in circulating blasts during treatment. One patient's blasts were reduced from 89% to 9%, while another patient's blasts were reduced from 17% to 8%. BP1001 decreased >25% Grb2 levels in 10 of 12 samples (average decrease: 49%) and >25% phosphorylated Erk1,2 levels in 7 of 12 samples (average decrease: 52%).
Conclusions: BP1001 was active against gleevec-resistant CML cells and enhanced dasatinib efficacy in preclinical studies. BP1001 may have therapeutic activity against CML. We are planning a Phase I/II trial to study the safety and efficacy of BP1001 in combination with dasatinib in accelerated or blast phase CML patients.
Tari Ashizawa:Bio-Path Holdings, Inc.: Employment, Equity Ownership.
Asterisk with author names denotes non-ASH members.