Molecular studies have identified a number of signal transduction pathways that are deregulated in human cancers. A major priority for the cancer research community is the translation of this growing body of knowledge into therapeutics targeted specifically to these pathways. Imatinib (Gleevec, formerly STI571), a small-molecule, orally administered inhibitor of the pathogenetic tyrosine kinases in chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GISTs), has validated this approach.
Imatinib was identified at Novartis Pharmaceuticals in a high-throughput screen for tyrosine kinase inhibitors and showed a high degree of specificity for the ABL and PDGFR (platelet-derived growth factor receptor) tyrosine kinases. Subsequent studies from my laboratory showed that it also inhibits the c-KIT tyrosine kinase, but no other kinases tested. We also demonstrated that imatinib specifically inhibits the proliferation of BCR-ABL–expressing hematopoietic cells or c-KIT–dependent cells, with no obvious effects on normal cells or cells transformed by other tyrosine kinase oncogenes. Based on antileukemic activity in several preclinical models, we designed a phase I clinical trial for patients with CML who had failed other treatment options. This clinical trial was conducted in collaboration with Charles Sawyers (HHMI, University of California, Los Angeles) and Moshe Talpaz (M. D. Anderson Cancer Center).
CML is a disorder marked by increased numbers of myeloid cells with full maturation. All patients with CML express the constitutively activated BCR-ABL tyrosine kinase, formed as a consequence of a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22). In the early stages of CML (known as the chronic phase), BCR-ABL is likely to be the sole molecular abnormality. Indeed, studies in cellular and animal models have conclusively established that BCR-ABL alone is sufficient to cause CML, and mutational analysis has established that tyrosine kinase activity is required for oncogenic activity. Over time, the capacity for differentiation of the malignant clone is lost as the disease progresses to an acute leukemia (blast crisis).
In the phase I clinical trials of imatinib in chronic-phase patients with CML, once therapeutic doses of imatinib were reached, virtually all patients had their blood counts returned to normal. A majority of patients had a cytogenetic response, that is, a significant decrease in the number of marrow metaphases positive for t(9;22). Remarkably, no significant imatinib-related toxicities have been observed; therefore, we have been unable to define a maximum tolerable dose. In subsequent studies, imatinib has been established as the standard treatment for newly diagnosed patients with CML. With more than 5 years of follow-up, the overall survival rate is 89 percent; the relapse rate has been approximately 4 percent per year, but has been declining in the past few years. Despite achieving high rates of durable remissions, almost all patients have detectable disease as measured by polymerase chain reaction (PCR) technologies; thus, patients are required to remain on therapy. From our preclinical data that showed that imatinib also inhibits the c-KIT tyrosine kinase, we hypothesized that imatinib would have clinical activity in patients with GIST, as this malignancy is driven by KIT mutations. As predicted, imatinib has remarkable single-agent activity in this highly chemotherapy refractory tumor.
Our current program is focused on determining the mechanisms of disease resistance and persistence, with the goal of translating these findings into improved outcomes for CML patients and identifying similar targets in other leukemias. Our studies on the molecular mechanisms of resistance began with an evaluation of BCR-ABL kinase activity in patients. We had identified CRKL as the major tyrosine-phosphorylated protein in CML patient samples, and we used this information to develop an assay for CRKL phosphorylation in patients being treated with imatinib. In addition, we have analyzed the necessity of the CRKL protein for BCR-ABL–mediated transformation. These studies demonstrated that an SH3 domain of CRKL interacts directly with a proline-rich region in the carboxyl terminus of the ABL protein. Deletion of this direct binding site had no consequences on the transforming potency of BCR-ABL. However, CRKL remains bound to BCR-ABL and heavily tyrosine phosphorylated, presumably because of indirect interactions. Therefore, in collaboration with Akira Imamoto (University of Chicago), we generated a CRKL null animal. These animals have a variety of developmental defects that recapitulate some of the features seen in DiGeorge syndrome. This syndrome is characterized by cranial, cardiac, and facial abnormalities, which in humans appear to be modified by deletion of the CRKL locus. In cells obtained from CRKL null animals, we showed the transformation by BCR-ABL is impaired. We are evaluating other signaling pathways that may compensate for this defect.
Our studies of imatinib resistance were aided greatly by the crystal structure of imatinib in complex with the ABL kinase from John Kuriyan's laboratory (HHMI, University of California, Berkeley). Amie Corbin from my laboratory made mutations of all of the predicted contact points between ABL and imatinib. As expected, many of these contact points were required for ATP binding, and mutations resulted in a kinase-inactive protein. Mutations at amino acids 253 and 315 resulted, however, in a kinase that was much less sensitive to imatinib. These residues were subsequently identified as mutations in patients who relapsed on therapy with imatinib. We have now characterized numerous mutations and have found that these BCR-ABL mutants have a wide range of sensitivity to imatinib. From this knowledge, we were able to identify a class of compounds that is capable of inhibiting most, but not all, of the imatinib-resistant mutants. Based on this work, similar compounds are now in clinical trials for imatinib-resistant patients.
The bigger problem faced by most patients with CML, however, is the inability of imatinib to eradicate the disease, which we term molecular persistence. We have begun to investigate this phenomenon by purifying leukemic cells from patients who are responding to imatinib, yet have disease persistence. Our goal is to determine why these cells are incapable of being killed by imatinib.
Several elements are required to recapitulate the success with imatinib. The first is the identification of a molecular pathogenetic event in a cancer and validation of this target. Once a target has been validated as a pathogenetic event, a drug must be developed to restore the function of this target to normal, to inhibit its function, or to delete the target. Thus, this final goal of our laboratory is to identify other molecular targets in leukemia and to develop agents that target these events. For this project, we screened a panel of 30 acute myeloid leukemia cell lines for STAT5 tyrosine phosphorylation. This screen, for a marker of tyrosine kinase activity, identified eight cell lines with constitutive STAT5 tyrosine phosphorylation. We used a phosphoproteomic approach to analyze these cell lines and generate a list of tyrosine kinases that are phosphorylated. Expression of individual tyrosine kinases was down-regulated with RNAi (RNA interference) to determine whether a specific tyrosine kinase is critical to the growth and survival of the cells. Using this approach, we determined that two cell lines expressed known leukemic oncogenes, BCR-ABL and TEL-ARG. We also identified a JAK2 mutation in an erythroleukemia cell line.
More recently, we identified a novel JAK3 mutation in a megakaryoblastic cell line. In collaboration with D. Gary Gilliland (HHMI, Brigham and Women's Hospital, Boston), we determined that expression of this JAK3 mutant in mice induces a dual myelo-lymphoproliferative disorder. Additionally, leukemic blasts from several patients with acute megakaryoblastic leukemia express related JAK3 mutations. JAK3 kinase inhibitors are already in clinical trials as immunosuppressive agents and could be tried in patients with JAK3 mutations.
