Amie S. Corbin

AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity

Imatinib therapy, which targets the oncogene product BCR-ABL, has transformed chronic myeloid leukemia (CML) from a life-threatening disease into a chronic condition. Most patients, however, harbor residual leukemia cells, and disease recurrence usually occurs when imatinib is discontinued. Although various mechanisms to explain leukemia cell persistence have been proposed, the critical question from a therapeutic standpoint--whether disease persistence is BCR-ABL dependent or independent--has not been answered. Here, we report that human CML stem cells do not depend on BCR-ABL activity for survival and are thus not eliminated by imatinib therapy. Imatinib inhibited BCR-ABL activity to the same degree in all stem (CD34+CD38-, CD133+) and progenitor (CD34+CD38+) cells and in quiescent and cycling progenitors from newly diagnosed CML patients. Although short-term in vitro imatinib treatment reduced the expansion of CML stem/progenitors, cytokine support permitted growth and survival in the absence of BCR-ABL activity that was comparable to that of normal stem/progenitor counterparts. Our findings suggest that primitive CML cells are not oncogene addicted and that therapies that biochemically target BCR-ABL will not eliminate CML stem cells.

Dasatinib (BMS-354825), a Dual SRC/ABL Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane, and Activation Loop Mutant KIT Isoforms Associated with Human Malignancies

Activating mutations of the activation loop of KIT are associated with certain human neoplasms, including the majority of patients with systemic mast cell disorders, as well as cases of seminoma, acute myelogenous leukemia (AML), and gastrointestinal stromal tumors (GISTs). The small-molecule tyrosine kinase inhibitor imatinib mesylate is a potent inhibitor of wild-type (WT) KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GIST. However, KIT activation loop mutations involving codon D816 that are typically found in AML, systemic mastocytosis, and seminoma are insensitive to imatinib mesylate (IC50 > 5-10 micromol/L), and acquired KIT activation loop mutations can be associated with imatinib mesylate resistance in GIST. Dasatinib (formerly BMS-354825) is a small-molecule, ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomolar range. Some small-molecule SRC/ABL inhibitors also have potency against WT KIT kinase. Therefore, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. We report herein that dasatinib potently inhibits WT KIT and juxtamembrane domain mutant KIT autophosphorylation and KIT-dependent activation of downstream pathways important for cell viability and cell survival, such as Ras/mitogen-activated protein kinase, phosphoinositide 3-kinase/Akt, and Janus-activated kinase/signal transducers and activators of transcription. Furthermore, dasatinib is a potent inhibitor of imatinib-resistant KIT activation loop mutants and induces apoptosis in mast cell and leukemic cell lines expressing these mutations (potency against KIT D816Y >> D816F > D816V). Our studies suggest that dasatinib may have clinical efficacy against human neoplasms that are associated with gain-of-function KIT mutations.

Characterization of Murine JAK2V617F-Positive Myeloproliferative Disease

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib.

ABSTRACT Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

Analysis of the Structural Basis of Specificity of Inhibition of the Abl Kinase by STI571

STI571, a selective inhibitor of Bcr-Abl, has been a successful therapeutic agent in clinical trials for chronic myelogenous leukemia. Chronic phase chronic myelogenous leukemia patients treated with STI571 have durable responses; however, most responding blast phase patients relapse despite continued therapy. Co-crystallization studies of Abl kinase and an STI571-related compound identify specific amino acid residues as critical to STI571 binding, one of which, T315, has been characterized as an acquired Thr to Ile mutation in relapsed patients. Other studies, however, suggest that mutations other than these predicted contact points are capable of conferring STI571 resistance in relapsed patients. Using a variety of models of STI571 binding to the Abl kinase, we have performed an extensive mutational analysis of sites that might alter the sensitivity of the Abl kinase to STI571. Although mutation of many of the predicted contact points between Abl and STI571 result in a kinase-inactive protein, additional mutations that render the Abl kinase less sensitive to STI571 demonstrate a broad range of possibilities for clinical resistance that are now becoming evident.

SGX393 inhibits the CML mutant Bcr-AblT315I and preempts in vitro resistance when combined with nilotinib or dasatinib

Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-AblT315I, however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-AblT315I, with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-AblT315I. These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

Absence of SKP2 expression attenuates BCR-ABL–induced myeloproliferative disease

BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF(SKP2) (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G(1) arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2(V617F), and TEL-PDGFRbeta, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2(-/-) marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2(+/+) marrow. SKP2(-/-) leukemic cells demonstrated higher levels of nuclear p27 than SKP2(+/+) counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF(SKP2) may be therapeutically useful.

Zoledronate inhibits proliferation and induces apoptosis of imatinib-resistant chronic myeloid leukaemia cells

Although imatinib mesylate has revolutionized the treatment of chronic myeloid leukaemia (CML), resistance to the drug, manifesting as relapse after an initial response or persistence of disease, remains a therapeutic challenge. In order to overcome this, alternative or additional targeting of signaling pathways downstream of Bcr-Abl may provide the best option for improving clinical response. Bisphosphonates, such as zoledronate, have been shown to inhibit the oncogenicity of Ras, an important downstream effector of Bcr-Abl. In this study, we show that zoledronate is equally effective in inhibiting the proliferation and clonogenicity of both imatinib-sensitive and -resistant CML cells, regardless of their mechanism of resistance. This is achieved by the induction of S-phase cell cycle arrest and apoptosis, through the inhibition of prenylation of Ras and Ras-related proteins by zoledronate. The combination of imatinib and zoledronate also augmented the activity of either drug alone and this occurred in imatinib-resistant CML cells as well. Since zoledronate is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.

Interactions of p62 dok with p210 bcr-abl and Bcr-Abl-associated Proteins

A 62-kDa Ras GTPase-activating protein (RasGAP)-associated protein is tyrosine-phosphorylated under a variety of circumstances including growth factor stimulation and in cells transformed by activated tyrosine kinases. A cDNA for p62 dok , reported to be the RasGAP-associated 62-kDa protein, was recently cloned from Abl-transformed cells. In this study, the interactions of p62 dok with Bcr-Abl and associated proteins were examined. In 32D myeloid cells and Rat-1 fibroblasts transformed by p210 bcr-abl , p62 dok is tyrosine-phosphorylated and co-immunoprecipitates with Bcr-Abl, RasGAP, and CrkL, a Src homology 2 (SH2) and SH3 domain-containing adaptor protein. Tyrosine-phosphorylated p62 dok from cells expressing p210 bcr-abl bound directly to the SH2 domains of Abl and CrkL in a gel overlay assay. Previous work has shown that an SH2 domain deletion mutant of Bcr-Abl is defective in transforming fibroblasts but remains capable of inducing myeloid growth factor independence. In both fibroblasts and myeloid cells expressing this mutant, p62 dok is underphosphorylated as compared with cells expressing full-length p210 bcr-abl but remains capable of associating with Bcr-Abl. However, in a gel overlay assay, p62 dok from cells expressing the SH2 domain deletion was incapable of associating directly with SH2 domains of Abl and CrkL. Interestingly, no direct binding between Bcr-Abl and p62 dok could be demonstrated in a yeast two-hybrid assay. These data suggest that indirect interactions mediate the interaction between Bcr-Abl and p62 dok and that the SH2 domain of Bcr-Abl is required for hyperphosphorylation of p62 dok . Further, hyperphosphorylation of p62 dok correlates with the ability of Bcr-Abl to transform fibroblasts but not with the induction of growth factor independence in myeloid cells.