The ''Gatekeeper'' Mutation T315I in BCR/ABL Confers Additional Oncogenic Activities to Philadelphia Chromosome Positive Leukemia

Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), having a (9;22) chromosomal translocation. The BCR/ABL fusion protein is the hallmark of Ph+ leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases, particularly by altering the natural history of chronic phase (CP) CML and preventing the previously inexorable progression to terminal blast crisis (BC). However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. The most important mechanism of resistance against TKIs is the selection of leukemic clones driven by BCR/ABL harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). The "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of Ponatinib, a multi-target kinase inhibitor. CML and Ph+ ALL, rarely present at diagnosis with a BCR/ABL harboring a resistance mutation to TKI. Resistant clones may be present and only detectable by highly sensitive methods. We have previously shown that the resistance mutations may influence the biology of BCR/ABL and its transformation potential.

We therefore hypothesized that the presence of mutations such as the T315I select for a "dormant cell population" which manifests following initial treatments with TKI inhibitors and treatment failure. The aim of this study was to determine whether the ''gatekeeper'' mutation T315I is able to confer biological features to BCR/ABL influencing its leukemogenic potential.

We investigated the influence of T315I on the biology of BCR/ABL in CML and Ph+ ALL. We used Ph+ ALL patient derived long term culture (PDLTCs), factor dependent Ba/F3 cells and syngeneic mouse model of BCR/ABL induced CML-like disease. These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. We observed significantly slower proliferation of Ba/F3 cells and PDLTCs expressing BCR/ABL-T315I compared to the native BCR/ABL. This was further confirmed by undertaking mitotic index calculations and colony formation assays on both cell types. Furthermore, the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). We undertook functional studies to determine the putative signaling pathway and found that Ras/Erk1/2 pathway was activated inT315I positive cells.

This study may assist towards therapy decisions in patients with CML/Ph+ ALL with a T315I mutation.