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Mutations of Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITDs) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in approximately 7% of AML patients. Patients carrying the FLT3-ITD but not the FLT3-TKD mutation have a significantly worse prognosis. Therefore, both FLT3 mutations seem to exert different biologic functions. FLT3-ITD but not FLT3-TKD has beenshown to induce robust activation of the STAT5 signaling pathway. In the present study, we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD uses SRC to activate STAT5. Coimmunoprecipitation and pull-down experiments revealed an exclusive interaction between SRC but not other Src family kinases and FLT3-ITD, which is mediated by the SRC SH2 domain. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for SRC binding and subsequent STAT5 activation. Using sitespecific Abs, we found that both residues were significantly more strongly phosphorylated in FLT3-ITD compared with FLT3-TKD. SRC inhibition and knockdown blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. We conclude that SRC might be a therapeutic target in FLT3-ITD ؉ AML. IntroductionThe Fms-like tyrosine kinase-3 (FLT3) receptor tyrosine kinase is expressed on blast cells in most patients with acute myeloid leukemia (AML). 1 Activating mutations of FLT3 have been detected in approximately 30% of AML patients. 2,3 Two distinct groups of FLT3 mutations have been described: (1) internal tandem duplications (FLT3-ITDs), which are most common in the juxtamembrane or the beta1-sheet of the tyrosine kinase domain-1 coding sequence in approximately 20%-27% of patients with AML; and (2) point mutations within the second tyrosine kinase domain (FLT3-TKDs) in approximately 7% of AML patients. 2,4,5 Both types of mutations activate the FLT3 receptor constitutively, leading to activation of downstream signaling proteins and resulting in factor-independent proliferation of murine lymphoid and myeloid cells. [6][7][8][9][10] Studies have identified FLT3-ITD as a prognostic factor because patients harboring this type of mutation have elevated peripheral blood and BM blast counts and an increased chance of relapse and inferior overall survival. 2,11,12 For FLT3-TKD mutations, a clear association with an inferior outcome could not be demonstrated in all studies and the relevance of FLT3-TKD for clinical prognosis is being investigated further. 13 Because FLT3 mutations can be found in approximately 30% of all AML patients and FLT3-ITD is associated with an inferior clinical prognosis, FLT3 is considered as an attractive therapeutic target. 14 Therefore, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been developed and tested in AML patients. 15,16 These studies showed that a single application of the inhibitor led to a short-term clinical response in some pat...

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R e s e a R c h a R t i c l e5. Group 2 correlated with high STAT3 expression (i.e., STAT3 activation). (G) GSEA showed a significantly different distribution of the STAT3 activation-associated genes, with group 1 being negatively correlated (NES, -1.97; P < 0.001). Figure 1A; supplemental material available online with this article; doi:10.1172/JCI69094DS1). Gene set enrichment analysis (GSEA) showed a significantly different distribution of the STAT3 activation-associated genes among the hierarchical clustering-defined gene expression groups (normalized enrichment score [NES], -1.97; nominal P < 0.001; Figure 1G). In addition, mining the public repository Oncomine (www.oncomine. org) showed that STAT3 target genes were elevated in MM versus control tissue (Supplemental Figure 1B). Furthermore, we found the STAT3 signaling gene expression pattern to be associated with low bone disease, the MMSET groups, and the presence of a gain of the 1q21 locus, whereas we identified an inverse correlation with hyperdiploid disease (Supplemental Figure 1, C-E). Thus, STAT3 phosphorylation and target gene activation seems to be a major hallmark of a large subgroup of human MM. Constitutive GP130 signaling induces myeloma formation in a murine BM transduction-transplantation model. To test whether constitutive activation of GP130 signaling enables B cells to proliferate independently of cytokine stimulation, the IL-3-dependent Analysis of the malignant plasma cells revealed a t(12;15) translocation involving c-Myc (16). While Eμ-Myc transgenic mice (in which MYC expression is under control of the Eμ enhancer) develop aggressive pro-/pre-B cell lymphomas early in life (17), activationinduced deaminase-dependent expression of MYC in germinal center B cells leads to a high incidence of MM with a median survival of approximately 2 years (4). Transgenic mice expressing abnormally high levels of XBP-1, a protein involved in the terminal differentiation of B cells, develop monoclonal gammopathy of undetermined significance (MGUS), and some develop MM later in life (18). The Journal of Clinical Investigation R e s e a R c h a R t i c l e5In the present study, we showed that constitutive activation of GP130/JAK/STAT3 signal transduction in a retroviral murine BM transduction-transplantation model was sufficient to induce or facilitate MM development in mice. This model was characterized by very high penetrance and relatively short latency. Importantly, constitutive GP130 activation efficiently cooperated with MYC overexpression by driving cell growth and differentiation of malignant plasma cells. Our data indicate that constitutive GP130 signal transduction is a critical early step in myelomagenesis. Results STAT3 phosphorylation and target gene activation is a hallmark of human MM.Activation of the IL-6/IL-6R/GP130 complex is crucial for survival and proliferation of human myeloma (6,11,13), and the JAK/STAT3 pathway is a major target downstream of IL-6R/GP130 signaling (8,14). We therefore evaluated a series of BM biopsies from pati...

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Background: NIPA is involved in timing mitotic entry and is inactivated by phosphorylation. The kinase responsible for NIPA phosphorylation at G 2 /M is unknown. Results: We show that cell cycle-dependent phosphorylation of NIPA is mediated by ERK2. Conclusion: NIPA is identified as one of the very few ERK2-specific substrates at the G 2 /M transition. Significance: This demonstrates divergent functions of ERK1 and ERK2 in cell cycle regulation.

The SH2-containing adaptor protein Grb10 was first identified in a yeast screen as a new binding partner for BCR-ABL and associates with BCR-ABL in a tyrosine-dependent manner. However, its function in BCR-ABL-mediated leukemogenesis in vivo is still unknown. Here we describe an important role of Grb10 in BCR-ABL-induced leukemia by using a versatile system for efficient oncogene expression and simultaneous Grb10 knockdown from a single vector. Primary bone marrow (BM) cells coexpressing Grb10-miR/BCR-ABL showed a significant decrease in colony formation and cell cycle progression. Transplantation of Grb10miR/BCR-ABL- or control-miR/BCR-ABL- transduced BM leads to a CML/B-ALL-like phenotype with significantly delayed disease onset and progression resulting in prolonged overall survival in Grb10-miR-transplanted mice. Methylcellulose experiments exhibit additive effects of imatinib treatment and Grb10 knockdown. Cell cycle analysis suggests an anti-proliferative effect of Grb10 knockdown in BCR-ABL(+) primary BM cells. However, Grb10 abrogation was not capable of completely abolishing the BCR-ABL-induced disease. Our findings were confirmed in the human BCR-ABL(+) cell line K562, where we demonstrate reduced viability, cell cycle progression and induction of apoptosis by stable Grb10 microRNA expression. Taken together, our results suggest that Grb10 knockdown in vivo leads to impaired proliferation, longer survival and reduced colony formation, suggesting an important role of Grb10 in BCR-ABL-mediated leukemogenesis.

Mature donor-derived T cells in allogeneic bone marrow (BM) transplants mediate the graft-versus-tumor (GVT) effect by recognizing alloantigens on leukemic cells. However, alloantigen reactivity towards non-malignant tissues also induces graft-versus-host disease (GVHD). Defining T-cell subpopulations that mediate the GVT effect in the absence of GVHD induction remains a major challenge in allogeneic BM transplantation. In this study, we show that in vitro-generated alloantigen-specific CD8 þ cytotoxic T cells (CTLs) established by weekly stimulation with alloantigen-expressing antigenpresenting cells did not induce GVHD in two major histocompatibility complex-mismatched BM transplantation models, where induction of lethal GVHD is dependent on the presence of either CD4 þ or CD8 þ T cells. Despite their strong alloantigen specificity, transplantation of CTLs did not induce the expression of GVHD-associated cytokines IFN-c and TNF-a or clinical or histological signs of GVHD, and lead to a survival rate of above 90%. However, transplantation of unstimulated CD8 þ T cells, which were not primed by the alloantigen in vitro, induced GVHD in both the transplantation models. Although CTLs were impaired in GVHD induction, they efficiently eradicated Bcr-Abl-transformed B-cell leukemias or mastocytomas. Thus, in vitro-derived CTLs might be useful for optimizing anti-tumor therapy in the absence of GVHD induction.