Heterogeneity of clonal expansion and maturation-linked mutation acquisition in hematopoietic progenitors in human acute myeloid leukemia.

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML), but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34(+)/CD33(-) cells revealed polyclonal growth in highly curable AMLs, suggesting that mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34(+)/CD33(-) cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications.

Advances in the management of patients with non-Hodgkin's lymphoma.

Non-Hodgkin's lymphoma is the fifth most common cause of death due to cancer and has been rising at a rate of 4% per year for the last four decades. Although 'traditional' chemotherapy and radiotherapy have had important contributions to improving outcomes, new tools in the treatment of non-Hodgkin's lymphoma are needed. This review describes therapeutic modalities that are currently being used or are in the process of being developed and which are based on concepts divergent from 'traditional' approaches to managing non-Hodgkin's lymphoma.

Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck.

Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.

The c-Fes protein-tyrosine kinase suppresses cytokine-independent outgrowth of myeloid leukemia cells induced by Bcr-Abl.

The c-Fes protein-tyrosine kinase exhibits strong expression in myeloid hematopoietic cells. Previous studies have shown that Fes induces differentiation in the chronic myelogenous leukemia-derived cell line K-562, suggesting that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in these cells. In addition, Fes has been shown to associate with and phosphorylate Bcr on NH2-terminal sequences retained within Bcr-Abl. To determine whether Fes interacts directly with Bcr-Abl, kinase-inactive Bcr-Abl was coexpressed with Fes in 293T cells, and phosphorylation was assessed by anti-phosphotyrosine immunoblotting. Bcr-Abl was strongly phosphorylated by Fes under these conditions, suggestive of direct interaction. Similarly, tyrosine phosphorylation of kinase-inactive Fes was observed after coexpression with active Bcr-Abl. To test for the interaction of Fes with Bcr-Abl under physiological conditions, wild-type and kinase-defective Fes were stably expressed in the cytokine-dependent myeloid leukemia cell line, DAGM. Expression of either form of Fes alone did not affect the proliferation or interleukin 3 dependence of these cells. The DAGM/Fes cells were then infected with Bcr-Abl retroviruses, and their rates of cytokine-independent outgrowth were compared. Fes dramatically suppressed Bcr-Abl-induced DAGM cell outgrowth relative to a cell line expressing beta-galactosidase as a negative control. This effect required Fes tyrosine kinase activity, because the kinase-inactive form of Fes did not affect Bcr-Abl-induced cell outgrowth. The phosphotyrosine content of both wild-type and kinase-inactive Fes was strongly enhanced after coexpression with Bcr-Abl in DAGM cells, similar to the 293T result. Phosphorylation of wild-type Fes correlated with stimulation of Fes tyrosine kinase activity in the presence of Bcr-Abl. These results show that Fes and Bcr-Abl interact in myeloid cells, leading to Fes activation and suppression of Bcr-Abl-induced conversion to cytokine independence.

The c-Fes family of protein-tyrosine kinases.

The human c-fes protooncogene encodes a protein-tyrosine kinase (c-Fes) distinct from c-Src, c-Abl and other nonreceptor tyrosine kinases. Although originally identified as the cellular homolog of several transforming retroviral oncoproteins, Fes was later found to exhibit strong expression in myeloid hematopoietic cells and to play a direct role in their differentiation. Recent work has shown that Fes exhibits a more widespread expression pattern in both developing and adult tissues, suggesting a general physiological function for this kinase and its closely related homolog, Fer. This review highlights the unique aspects of Fes structure, regulation, and function that set it apart from other tyrosine kinase families.

Oligomerization of the Fes tyrosine kinase. Evidence for a coiled-coil domain in the unique N-terminal region.

The c-fes proto-oncogene encodes a non-receptor tyrosine kinase (Fes) that has been implicated in cytokine receptor signal transduction and myeloid differentiation. Previous work from our laboratory has shown that Fes autophosphorylates via an intermolecular mechanism more commonly associated with growth factor receptor tyrosine kinases. Analysis of the Fes amino acid sequence with the COILS algorithm indicates that the N-terminal region of the protein has a very high probability of forming coiled-coil structures often associated with oligomeric proteins. These findings suggest that oligomerization may be a prerequisite for trans-autophosphorylation and activation of Fes. To establish whether the active form of Fes is oligomeric, we performed gel-filtration experiments with recombinant Fes and found that it eluted as a single symmetrical peak of approximately 500 kDa. No evidence of the monomeric, 93-kDa form of the protein was observed. Deletion of the unique N-terminal domain (amino acids 1-450, including the coiled-coil homology region) completely abolished the formation of oligomers. Furthermore, co-precipitation assays demonstrated that an immobilized glutathione S-transferase fusion protein containing the Fes N-terminal region bound to full-length Fes but not to a mutant lacking the N-terminal region. Similarly, a recombinant Fes N-terminal domain protein was readily cross-linked in vitro, whereas the SH2 and kinase domains were refractory to cross-linking. Incubation of wild-type Fes with a kinase-inactive Fes mutant or with the isolated N-terminal region suppressed Fes autophosphorylation in vitro, suggesting that oligomerization may be essential for autophosphorylation of full-length Fes. The presence of an oligomerization function in the Fes family of tyrosine kinases suggests a novel mechanism for non-receptor protein-tyrosine kinase regulation.