Abstract
Overview: Bcr-AblT315I is detected in the majority of CML patients who relapse after dasatinib- or nilotinib-based second-line Bcr-Abl kinase inhibitor therapy. SGX70393, an azapyridine-based Abl kinase inhibitor, is effective against Bcr-Abl and Bcr-AblT315I at low nanomolar concentrations in vitro and in cell lines. Here, we comprehensively profiled SGX70393 against native and mutant Bcr-Abl in vitro and in vivo. We also used a cell-based mutagenesis screen to evaluate the resistance profile of SGX70393 alone and in combination with imatinib, nilotinib, or dasatinib.
Methods: We assessed colony formation in the presence of SGX70393 by murine bone marrow infected with retroviruses for expression of Bcr-Abl, Bcr-AblT315I, or a variety of other mutants. Toxicity was tested in clonogenic assays of normal bone marrow. SGX70393 effects on cellular tyrosine phosphorylation were measured by immunoblot and FACS in primary Bcr-AblT315I cells isolated from patients with CML or Ph+ B-ALL. In vivo activity was evaluated in a xenograft model using Ba/F3 cells expressing Bcr-AblT315I. Lastly, the resistance profile of SGX70393 was evaluated alone and in dual combinations with imatinib, nilotinib, or dasatinib in a cell-based mutagenesis assay.
Results: Colony formation by murine bone marrow cells expressing Bcr-AblT315I (IC50: 180 nM) was reduced by SGX70393 in a dose dependent manner, while no toxicity was observed in colony forming assays of normal human or murine mononuclear cells at concentrations up to 2 μM. Ex vivo exposure of human Bcr-AblT315I mononuclear cells to SGX70393 decreased CrkL phosphorylation, while imatinib, nilotinib, or dasatinib had no effect. SGX70393 inhibited Bcr-AblT315I-driven tumor growth in mice and this was correlated with reduced levels of pCrkL in tumor tissue, while imatinib was ineffective. A cell-based mutagenesis screen revealed a profile of resistant clones confined to four p-loop residues and position 317. SGX70393 in combination with imatinib contracted the spectrum of resistant mutations relative to either single agent, though outgrowth could not be completely suppressed. Combining SGX70393 with low concentrations of nilotinib or dasatinib narrowed the resistance profile still further (residues 248 and 255 for nilotinib; 317 for dasatinib) and, with clinically achievable doses of either second drug, completely abrogated emergence of resistant subclones.
Conclusions: SGX70393, a potent inhibitor of Bcr-AblT315I, exhibits a resistance profile centered around the p-loop and residue 317 of Bcr-Abl. Remarkably, in combination with nilotinib or dasatinib, outgrowth of resistant clones is completely suppressed. Single-agent therapy with an effective T315I inhibitor may provide a viable option for patients who relapse with Bcr-AblT315I. However, as a broader spectrum of mutations accounts for imatinib resistance, patients with acquired dasatinib or nilotinib resistance may continue to harbor residual mutant clones other than T315I. Thus, the full clinical potential of SGX70393 may be realized in combinations with a second Abl kinase inhibitor. Our findings provide the first demonstration that Abl kinase inhibitor combinations that include a T315I-targeted component such as SGX70393 have the potential to pre-empt Bcr-Abl-dependent resistance.
In Vivo and In Vitro Characterization of Insulin-Producing Cells Obtained From Murine Bone Marrow
