MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors.

Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila melanogaster , loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling.

FGFR1 is fused with a novel zinc-finger gene, ZNF198, in the t(8;13) leukaemia/lymphoma syndrome.

Various histological subtypes of leukaemia and lymphoma are associated with diagnostic chromosome translocations, and substantial strides have been made in determining the specific oncogenes targetted by those translocations. We report the cloning of a novel fusion oncogene associated with a unique leukaemia/lymphoma syndrome. Patients afflicted with this syndrome present with lymphoblastic lymphoma and a myeloproliferative disorder, often accompanied by pronounced peripheral eosinophilia and/or prominent eosinophilic infiltrates in the affected bone marrow, which generally progress to full-blown acute myelogenous leukaemia within a year of diagnosis. A specific chromosome translocation, t(8;13)(p11;q11-12), is found in both lymphoma and myeloid leukaemia cells from these patients, supporting bi-lineage differentiation from a transformed stem cell. We find that the 8p11 translocation breakpoints, in each of four patients, interrupt intron 8 of the fibroblast growth factor receptor 1 gene (FGFR1). These translocations are associated with aberrant transcripts in which four predicted zinc-finger domains, contributed by a novel and widely expressed chromosome-13 gene (ZNF198), are fused to the FGFR1 tyrosine-kinase domain. Transient expression studies show that the ZNF198-FGFR1 fusion transcript directs the synthesis of an approximately 87-kD polypeptide, localizing predominantly to the cytoplasm. Our studies demonstrate an FGFR1 oncogenic role and suggest a tumorigenic mechanism in which ZNF198-FGFR1 activation results from ZNF198 zinc-finger-mediated homodimerization.

Interallelic V(D)J trans-rearrangement within the beta T cell receptor gene is infrequent and occurs preferentially during attempted D beta to J beta joining.

Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1.

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.

Calmodulin binds to chick lens gap junction protein in a calcium-independent manner.

A biochemically active conjugate of calmodulin and tetramethylrhodamine isothiocyanate (CaM-RITC) was synthesized. When incubated with sections of chick lens, this conjugate bound to the surface membranes of lens fiber cells in the presence of absence of calcium. Incubation of lens sections with antibodies to gap junction protein of lens completely blocked the binding of the conjugate to cell membranes, whereas serum from nonimmunized animals or antibodies to others lens proteins reduced the binding only slightly. By means of a gel overlay procedure, 125I-labeled calmodulin was found to bind to the gap junction protein of lens, also in a calcium-independent manner. These results support the concept that calmodulin may interact with and regulate gap junctions in living cells.

NOTCH1-induced T-cell leukemia in transgenic zebrafish.

Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of human NOTCH1-induced T-cell leukemia was generated. Seven of sixteen mosaic fish developed a T-cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. However, stable transgenic fish exhibited a longer latency for leukemia onset. When the stable transgenic line was crossed with another line overexpressing the zebrafish bcl2 gene, the leukemia onset was dramatically accelerated, indicating synergy between the Notch pathway and the bcl2-mediated antiapoptotic pathway. Reverse transcription-polymerase chain reaction analysis showed that Notch target genes such as her6 and her9 were highly expressed in NOTCH1-induced leukemias. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.

Fatal myeloproliferation, induced in mice by TEL/PDGFbetaR expression, depends on PDGFbetaR tyrosines 579/581.

The t(5;12)(q33;p13) translocation associated with chronic myelomonocytic leukemia (CMML) generates a TEL/PDGFbetaR fusion gene. Here, we used a murine bone marrow transplant (BMT) assay to test the transforming properties of TEL/PDGFbetaR in vivo. TEL/PDGFbetaR, introduced into whole bone marrow by retroviral transduction, caused a rapidly fatal myeloproliferative disease that closely recapitulated human CMML. TEL/PDGFbetaR transplanted mice developed leukocytosis with Gr-1(+) granulocytes, splenomegaly, evidence of extramedullary hematopoiesis, and bone marrow fibrosis, but no lymphoproliferative disease. We assayed mutant forms of the TEL/PDGFbetaR fusion protein - including 8 tyrosine to phenylalanine substitutions at phosphorylated PDGFbetaR sites to which various SH2 domain-containing signaling intermediates bind - for ability to transform hematopoietic cells. All of the phenylalanine (F-) mutants tested conferred IL-3-independence to a cultured murine hematopoietic cell line, but, in the BMT assay, different F-mutants displayed distinct transforming properties. In transplanted animals, tyrosines 579/581 proved critical for the development of myeloproliferative phenotype. F-mutants with these residues mutated showed no sign of myeloproliferation but instead developed T-cell lymphomas. In summary, TEL/PDGFbetaR is necessary and sufficient to induce a myeloproliferative disease in a murine BMT model, and PDGFbetaR residues Y579/581 are required for this phenotype.

Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.

Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.

Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1.

Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types, including lymphoid cells. Signaling is normally initiated through one or more ligand-mediated proteolytic cleavages that permit nuclear translocation of the intracellular portion of the Notch receptor (ICN), which then binds and activates transcription factors of the Su(H)/CBF1 family. Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, a gene initially identified based on its involvement in a (7;9) chromosomal translocation found in sporadic T-cell lymphoblastic leukemias and lymphomas (T-ALL). To investigate which portions of ICN1 contribute to transformation, we performed a structure-transformation analysis using a robust murine bone marrow reconstitution assay. Both the ankyrin repeat and C-terminal transactivation domains were required for T-cell leukemogenesis, whereas the N-terminal RAM domain and a C-terminal domain that includes a PEST sequence were nonessential. Induction of T-ALL correlated with the transactivation activity of each Notch1 polypeptide when fused to the DNA-binding domain of GAL4, with the exception of polypeptides deleted of the ankyrin repeats, which lacked transforming activity while retaining strong transactivation activity. Transforming polypeptides also demonstrated moderate to strong activation of the Su(H)/CBF1-sensitive HES-1 promoter, while polypeptides with weak or absent activity on this promoter failed to cause leukemia. These experiments define a minimal transforming region for Notch1 in T-cell progenitors and suggest that leukemogenic signaling involves recruitment of transcriptional coactivators to ICN1 nuclear complexes.

Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

A new recurrent 9q34 duplication in pediatric T-cell acute lymphoblastic leukemia.

Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses approximately 4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17-39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

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 (proline, glutamate, serine, threonine) 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 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 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.

Molecular biology of Burkitt's lymphoma.

The diagnostic category of Burkitt's lymphoma encompasses a closely related group of aggressive B-cell tumors that includes sporadic, endemic, and human immunodeficiency virus-associated subtypes. All subtypes are characterized by chromosomal rearrangements involving the c-myc proto-oncogene that lead to its inappropriate expression. This review focuses on the roles of c-myc dysregulation and Epstein-Barr virus infection in Burkitt's lymphoma. Although the normal function of c-Myc remains enigmatic, recent data indicate that it has a central role in several fundamental aspects of cellular biology, including proliferation, differentiation, metabolism, apoptosis, and telomere maintenance. We discuss new insights into the molecular mechanisms of these c-Myc activities and their potential relevance to the pathogenesis of Burkitt's lymphoma and speculate on the role of Epstein-Barr virus.

Myelodysplastic syndrome as a late complication following autologous bone marrow transplantation for non-Hodgkin's lymphoma.

PURPOSE: To determine the incidence, natural history, and risk factors associated with myelodysplastic syndrome (MDS) occurring as a late complication following autologous bone marrow transplantation for patients with non-Hodgkin's lymphoma. METHODS: We retrospectively reviewed the charts of all 262 patients who underwent autologous bone marrow transplantation for non-Hodgkin's lymphoma at the Dana-Farber Cancer Institute from 1982 through 1991. Although patients received a variety of treatments before they were eligible for transplant, identical myeloablative therapy (cyclophosphamide 60 mg/kg/d for 2 days plus total-body irradiation twice daily for 3 days) was administered in each case. By collecting data on pretransplant and early posttransplant variables, we attempted to identify risk factors for the development of MDS. RESULTS: The crude overall incidence of posttransplant MDS or acute myeloid leukemia (AML) was 7.6%. The actuarial risk at 6 years was 18% +/- 9%. The median time of onset was 31 months (range, 10 to 101) after transplant or 69 months (range, 27 to 141) after initial treatment for lymphoma. Pretreatment variables predictive for the development of MDS (univariate analysis) included prolonged interval between initial treatment and the transplant procedure (P = .003), increased duration of exposure to chemotherapy (P = .019) or to alkylating agents (P = .045), and use of radiation therapy (P = .032) or pelvic radiation (P = .003) before transplant. CONCLUSION: MDS is a potential complication of autologous bone marrow transplantation for non-Hodgkin's lymphoma; bone marrow stem-cell damage sustained before the transplant may be the most important risk factor.

Subcellular localisation, secretion, and post-translational processing of normal cochlin, and of mutants causing the sensorineural deafness and vestibular disorder, DFNA9.

Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.

A microRNA-mediated regulatory loop modulates NOTCH and MYC oncogenic signals in B- and T-cell malignancies.

Growing evidence suggests that microRNAs (miRNAs) facilitate the cross-talk between transcriptional modules and signal transduction pathways. MYC and NOTCH1 contribute to the pathogenesis of lymphoid malignancies. NOTCH induces MYC, connecting two signaling programs that enhance oncogenicity. Here we show that this relationship is bidirectional and that MYC, via a miRNA intermediary, modulates NOTCH. MicroRNA-30a (miR-30a), a member of a family of miRNAs that are transcriptionally suppressed by MYC, directly binds to and inhibits NOTCH1 and NOTCH2 expression. Using a murine model and genetically modified human cell lines, we confirmed that miR-30a influences NOTCH expression in a MYC-dependent fashion. In turn, through genetic modulation, we demonstrated that intracellular NOTCH1 and NOTCH2, by inducing MYC, suppressed miR-30a. Conversely, pharmacological inhibition of NOTCH decreased MYC expression and ultimately de-repressed miR-30a. Examination of genetic models of gain and loss of miR-30a in diffuse large B-cell lymphoma (DLBCL) and T-acute lymphoblastic leukemia (T-ALL) cells suggested a tumor-suppressive role for this miRNA. Finally, the activity of the miR-30a-NOTCH-MYC loop was validated in primary DLBCL and T-ALL samples. These data define the presence of a miRNA-mediated regulatory circuitry that may modulate the oncogenic signals originating from NOTCH and MYC.

Therapy with recombinant human erythropoietin in patients with myelodysplastic syndromes.

We conducted a Phase I-II trial of recombinant human erythropoietin-beta (rhEPO) in patients with myelodysplastic syndrome (MDS). Patients with anemia and pathologically confirmed MDS were eligible for the study. Treatment consisted of rhEPO by subcutaneous injection thrice weekly for 6 weeks at one of three dose levels (100 U/kg (three patients), 200 U/kg (three patients) and 400 U/kg (14 patients)). Ferrous sulfate (325 mg po tid) was also administered if the transferrin saturation was below 30% (two patients). Patients were monitored with weekly CBC, white cell differential, and reticulocyte counts. Bone marrow examinations were performed at the conclusion of the treatment period and after a 2 week washout period. Patients who responded to therapy were continued on rhEPO at the same dose for 6 additional months. Response criteria included: 50% reduction in transfusion requirements compared with the 6 week pre-study period; doubling of reticulocyte count that was maintained on two determinations at least 1 week apart; or an increase in hemoglobin by at least 1.2 gm/dl without transfusions. Pre-treatment factors potentially predictive of response were analyzed by univariate analysis and in a multivariate fashion by classification and regression trees. Seven of the twenty patients sustained an untransfused rise in serum hemoglobin > or = 1.2 gm/dl. Four of the sixteen patients (including three of seven patients experiencing a rise in serum hemoglobin) who were transfusion-dependent prior to the study achieved a reduction or elimination of their transfusion requirements. Five of thirteen patients who received rhEPO during the extension phase had a continued response. A low baseline erythropoietin level (< 50 mU/ml) was the best predictor of hemoglobin response when controlling for other variables. rhEPO has a role in the treatment of certain patients with MDS, particularly in those whose endogenous serum erythropoietin levels are not markedly elevated.