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Purpose: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. Experimental Design: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. Results: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMSlike tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sitesY589 and Y599 and colocalized in vivo. Expression of CBLDexon8 and CBLDexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor^independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand^dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). Conclusion: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.CBL, a known negative regulator of activated receptor tyrosine kinases (RTK), is localized on human chromosome 11q23, a region frequently associated with chromosomal aberrations. Translocations t(4;11) and t(11;14), and mixed-lineage leukemia fusion genes involving CBL have been described in patients with leukemia and lymphoma (1 -3). CBL oncogenes were initially identified in the murine system. CBL-70Z, carrying an internal deletion of 17 amino acids, was isolated from the 70Z/ 3 mouse pre-B-cell lymphoma cell line (4). CBL-70Z deregulates the cellular tyrosine kinase machinery, as NIH3T3 serumstarved cells expressing CBL-70Z showed significantly increased endothelial growth factor receptor (EGFR) kinase activity after EGF stimulation (5). p95CBL, expressed in the murine reticulum sarcoma cell line J-774, lacks internal 111 amino acids, comprising whole exons 8 and 9 (6). CBL70Z and p95CBL mutations both target the linker and RING finger domain, which points to a mutation-sensitive region within the CBL protein. Recently the first human CBL mutation has been reported in a patient...

IntroductionIn recent years, substantial progress has been made in understanding the biology of acute myeloid leukemia (AML). One of the pathogenetic hallmarks of AML are chromosomal translocations generating leukemogenic fusion genes that often act as aberrant transcription factors. 1 The second key genetic characteristics in AML are mutations, particularly those found in patients with normal karyotype and affecting the receptor tyrosine kinase FLT3 or the nucleophosmin protein (NPM1). Beside these structural genetic changes, large-scale gene expression analyses of cDNA samples from patients with AML have demonstrated that deregulated expression of nonaltered genes characterizes many AML cases. The most prominent example for this is the deregulated expression of homeobox genes in AML. [2][3][4] Homeobox genes form a highly conserved family of transcription factors known to be key regulators of normal hematopoietic stem cell and progenitor development. 5 Several studies have demonstrated that aberrant HOX gene expression profoundly perturbs normal murine and human hematopoietic development and causes leukemia in mice. [5][6][7][8][9] The aberrant expression of homeobox genes such as HOXA9 and HOXA10 is strongly associated with certain AML subtypes characterized by MLL fusion genes, NPM1 mutations (NPMc ϩ ), and by more rare translocations such as the translocation t(10;11)(p13q14) generating the CALM-AF10 fusion gene. 4,[10][11][12][13] All together, deregulated homeobox gene expression characterizes more than every third case of AML. So far, it is largely unknown how the aberrant expression of homeobox genes is initiated in the malignant clone. In cases with 11q23 chromosomal translocations, it is thought that aberrant function of the MLL gene, a known positive upstream regulator of HOX gene expression, is responsible for the perturbed expression of these key regulatory genes of early hematopoietic development. 14 In contrast, the aberrant HOX gene expression in patients with AML with normal karyotype and NPM1 mutation is not well understood. 15 In particular, the patients with NPMc ϩ AML demonstrate that aberrant HOX gene expression cannot be just explained by the stage of differentiation at which the leukemic clone is arrested: NPMc ϩ patients are CD34 Ϫ in more than 95% of patients, and represent therefore a cell stage in which HOX genes are normally silenced. 8,16 Another gene family critically involved in Hox gene regulation is the family of the so-called ParaHox genes, comprising the different "caudal-related homeobox genes" such as CDX1, CDX2, and CDX4, and the GSH2 homeobox gene. 17 Several experimental systems have demonstrated that loss of Cdx2 causes homeotic alterations and posterior shifts in Hox expression domains, 18 and that consensus-binding sites for the 3 Cdx homologs are present in the promoters of multiple Hox genes. [19][20][21][22] Expression of Cdx2 is tightly restricted to intestinal development in the adult. 23 Aberrant expression of CDX2 is associated with intestinal metaplasia, 24,25...

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that two of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, two of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

Purpose: Mutations in the receptor tyrosine kinase FLT3 are found in up to 30% of acute myelogenous leukemia patients and are associated with an inferior prognosis. In this study, we characterized critical tyrosine residues responsible for the transforming potential of active FLT3-receptor mutants and ligand-dependent activation of FLT3-WT. Experimental Design: We performed a detailed structure-function analysis of putative autophosphorylation tyrosine residues in the FLT3-D835Y tyrosine kinase domain (TKD) mutant. All tyrosine residues in the juxtamembrane domain (Y566, Y572, Y589, Y591, Y597, and Y599), interkinase domain (Y726 andY768), and COOH-terminal domain (Y955 andY969) of the FLT3-D835Y construct were successively mutated to phenylalanine and the transforming activity of these mutants was analyzed in interleukin-3-dependent Ba/F3 cells. Tyrosine residues critical for the transforming potential of FLT3-D835Y were also analyzed in FLT3 internal tandem duplication mutants (FLT3-ITD)and the FLT3 wild-type (FLT3-WT) receptor. Result: The substitution of the tyrosine residues by phenylalanine in the juxtamembrane, interkinase, and COOH-terminal domains resulted in a complete loss of the transforming potential of FLT3-D835Y-expressing cells which can be attributed to a significant reduction of signal tranducer and activator of transcription 5 (STAT5) phosphorylation at the molecular level. Reintroduction of single tyrosine residues revealed the critical role of Y589 and Y591in reconstituting interleukin-3-independent growth of FLT3-TKD-expressing cells. Combined mutation of Y589 and Y591 to phenylalanine also abrogated ligand-dependent proliferation of FLT3-WT and the transforming potential of FLT3-ITD-with a subsequent abrogation of STAT5 phosphorylation. Conclusion: We identified two tyrosine residues,Y589 and Y591, in the juxtamembrane domain that are critical for the ligand-dependent activation of FLT3-WTand the transforming potential of oncogenic FLT3 mutants.FLT3 is a member of the class III protein receptor tyrosine kinase family (RTK) that is characterized by five extracellular immunoglobulin-like domains, a juxtamembrane domain (JM), and two protein tyrosine kinase domains (TKD) split by an interkinase domain (IK; ref. 1). The class III receptors also include KIT, FMS, platelet-derived growth factor receptor-a (PDGFRA), and platelet-derived growth factor receptor-h (PDGFRB). Binding of FLT3 ligand (FL) to its receptor induces dimerization, phosphorylation, and subsequent activation of downstream signaling pathways such as signal tranducer and activator of transcription 5 (STAT5), Ras/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/AKT (2 -6). FLT3 has been shown to play an important role in normal hematopoiesis and is highly expressed in CD34 + hematopoietic progenitor cells (2, 7 -9).Activating mutations of FLT3 are found in 30% of patients with acute myelogenous leukemia (AML) and are associated with an inferior clinical outcome (10 -12). FLT3 internal tandem dup...

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Phage display has emerged as one of the leading technologies for the selection and generation of highly specific antibodies, offering a number of advantages over traditional ways of antibody generation such as mouse hybridoma techniques. While there are various possibilities to conduct phage display, selection of antibodies via solution panning is an elegant way to circumvent conformation changes of antigen, which may arise when performing panning with antigen immobilized on a solid surface. Here, a standard solution panning procedure using a Fab based antibody library including primary screening for selectivity is described.

<div>Abstract<p><b>Purpose:</b> CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL.</p><p><b>Experimental Design:</b> The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells.</p><p><b>Results:</b> We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMS-like tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sites Y589 and Y599 and colocalized <i>in vivo</i>. Expression of CBLΔexon8 and CBLΔexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor–independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand–dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin).</p><p><b>Conclusion:</b> CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.</p></div>