A specific need for CRKL in p210BCR-ABL-induced transformation of mouse hematopoietic progenitors.

CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation.

A BCR-ABL mutant lacking direct binding sites for the GRB2, CBL and CRKL adapter proteins fails to induce leukemia in mice.

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62(DOK), and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62(DOK) and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.

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

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.

In vitro and in vivo activity of ATP-based kinase inhibitors AP23464 and AP23848 against activation-loop mutants of Kit.

Oncogenic mutations of the Kit receptor tyrosine kinase occur in several types of malignancy. Juxtamembrane domain mutations are common in gastrointestinal stromal tumors, whereas mutations in the kinase activation loop, most commonly D816V, are seen in systemic mastocytosis and acute myelogenous leukemia. Kit activation-loop mutants are insensitive to imatinib mesylate and have been largely resistant to targeted inhibition. We determined the sensitivities of both Kit mutant classes to the adenosine triphosphate (ATP)-based inhibitors AP23464 and AP23848. In cell lines expressing activation-loop mutants, low-nM concentrations of AP23464 inhibited phosphorylation of Kit and its downstream targets Akt and signal transducer and activator of transcription 3 (STAT3). This was associated with cell-cycle arrest and apoptosis. Wild-type Kit-and juxtamembrane-mutant-expressing cell lines required considerably higher concentrations for equivalent inhibition, suggesting a therapeutic window in which cells harboring D816V Kit could be eliminated without interfering with normal cellular function. Additionally, AP23464 did not disrupt normal hematopoietic progenitor-cell growth at concentrations that inhibited activation-loop mutants of Kit. In a murine model, AP23848 inhibited activation-loop mutant Kit phosphorylation and tumor growth. Thus, AP23464 and AP23848 potently and selectively target activation-loop mutants of Kit in vitro and in vivo and could have therapeutic potential against D816V-expressing malignancies.

Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970.

Oncogenic mutations of the receptor tyrosine kinase KIT occur in gastrointestinal stromal tumors (GISTs), some cases of acute myelogenous leukemia (AML), and systemic mastocytosis (SM). GISTs commonly contain mutations of the KIT juxtamembrane region while SM and AML harbor active site KIT mutations. Imatinib, which potently inhibits juxtamembrane mutants, is effective for the treatment of GISTs but has no activity against active site mutants. We analyzed the inhibitory potential of 2 small molecule inhibitors, MLN518 and PD180970, against different classes of KIT mutants. Both compounds inhibit the growth of cell lines expressing juxtamembrane mutant KIT. MLN518 additionally targets active site mutant cell lines, inhibiting cell proliferation, KIT, and signal transducer and activator of transcription-3 (Stat3) phosphorylation and inducing apoptosis at concentrations that may be clinically achievable. As phase 1 clinical trials of MLN518 in AML have shown little toxicity, our data suggest MLN518 is a promising candidate for the treatment of SM or AML with KIT mutations.

Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis.

Internal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant, implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3, KIT, and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells, we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions, after chemotherapy-induced myelosuppression, and during bone marrow transplantation. In these assays, we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML, at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL-positive acute leukemia, MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols.

The dynamic behavior of CheW from Thermotoga maritima in solution, as determined by nuclear magnetic resonance: implications for potential protein-protein interaction sites.

Measurements of the 15N relaxation parameters have been used to characterize the backbone dynamics of CheW from Thermotoga maritima. The dynamic nature of residues that appeared disordered in our recent solution structure of CheW is confirmed by these dynamics measurements. We have interpreted the data in terms of the Lipari and Szabo 'model-free' approach. The derived order parameter, S(2), the [1H]-(15)N heteronuclear nuclear Overhauser effect (NOE) values, the chemical exchange rate, R(ex), and the internal correlation time, tau(e), show that CheW exhibits considerable motional freedom from the picosecond to millisecond time scales. These regions of the protein cluster within the framework of the three-dimensional structure and may indicate possible binding sites for other protein components of the bacterial chemotaxis receptor-signaling complex. The structure of CheW consists of two five-stranded beta-barrel domains that pack together with an extensive hydrophobic core between the domains. Regions highlighted by dynamics measurements co-localize to specific regions of the three-dimensional structure of CheW previously implicated in the formation of bacterial chemotaxis receptor signaling complex. The motional properties of domain 2 of CheW suggest that this domain may be able to experience structural rearrangements that allow the exposure of a hydrophobic surface area that could be used as a binding surface for the other members of the receptor complex. Residues within domain 2 have been implicated in binding interactions for two chemotaxis proteins, CheA and the receptor. We further propose that domain 1 interacts with other components of the chemotaxis machinery, such as CheZ, or in the formation of clusters of signaling components.

The solution structure and interactions of CheW from Thermotoga maritima.

Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored by nuclear magnetic resonance (NMR) techniques. The chemical shift perturbation data show the probable contacts that CheW makes with CheA. In combination with previous genetic data, the structure also suggests a possible binding site for the chemotaxis receptor. These results provide a structural basis for a model in which CheW acts as a molecular bridge between CheA and the cytoplasmic tails of the receptor.