ALBERT

Description: The design and evaluation of therapies for the sickle cell and β-thalassemia syndromes rely on our understanding of hemoglobin accumulation during human erythropoiesis. Here we report direct measurements of hemoglobin composition and messenger RNA (mRNA) levels in cultured CD34+ cells and correlate those measurements with studies of freshly obtained bone marrow samples. Hemoglobin levels in differentiating cells were also compared with morphologic, immunophenotypic, and cell cycle assessments. A population of large preproerythroblasts was first identified within 24 hours and became the dominant population by day 5. The transition from proerythroblast to basophilic normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% ± 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts were the dominant and final cell type by day 13. High-performance liquid chromatography–based quantitation of fetal (HbF) and adult (HbA) hemoglobin and real-time polymerase chain reaction globin mRNA quantitation demonstrated a coordinate rise in the accumulation of both proteins and mRNA among these developmentally staged populations. Quantitative analyses on freshly sorted bone marrow populations demonstrated a similar rising pattern with β-globin and HbA as the dominant species at both early and late stages of differentiation. We found no evidence for HbF dominant populations or switching during differentiation in adult cells. Instead, rapid increases in both HbF (heterocellular) and HbA (pancellular) content were observed, which coincided with the apex in cell cycling and the proerythroblast-basophilic normoblast transition. Based on these measurements, we conclude that HbF and HbA content are regulated with the rate of proliferation during adult erythropoiesis.

Description: Primary mediastinal large B-cell lymphoma (MLBCL) is a clinically distinct entity that typically presents as localized, sclerotic disease in young, female patients. We previously characterized the transcriptional profiles of MLBCLs and identified important shared features with a clinically related disorder, classical Hodgkin lymphoma (cHL) (Blood 102:3871, 2003). Given the documented role of the NFkB survival pathway in Hodgkin Reed-Sternberg cells, we previously assessed NFkB activation in MLBCL by determining the subcellular location of the c-REL subunit of the NFkB heterodimer with a 2-color immunofluoresence assay. In a small pilot MLBCL series, c-REL was localized to the nucleus in the majority of examined cases, consistent with NFkB activation. In the current study, we evaluated c-REL subcellular localization in an additional series of MLBCLs and DLBCLs using a broadly applicable immunoperoxidase method. 100% of MLBCLs exhibited nuclear c-REL staining whereas DLBCL c-REL subcellular localization was more variable. Thereafter, we analyzed the transcription profiles of the 34 MLBCLs and 176 DLBCLs for coordinate expression of NFkB target genes, using literature-curated NFkB target gene lists from three independent sources and gene set enrichment analysis (GSEA). MLBCL signatures exhibited significant enrichment of 2 of the 3 NFkB target gene sets. In addition, 32 NFkB target genes from the combined set were significantly more abundant in MLBCLs than DLBCLs (〉 30% more abundant and 〉 99th percentile in permutation analysis). Similar results were obtained in an independent series of MLBCLs and DLBCLs with available gene expression profiles (J. Exp. Med. 198:851, 2003). To assess the role of c-REL amplification in NFkB activation in our lymphoma series, we compared c-REL amplification, c-REL subcellular localization and coordinate expression of the identified NFkB target genes and classified the DLBCLs according to putative cell of origin. The majority of c-REL amplifications (67%) were found in DLBCLs of germinal center (GC) subtype, consistent with the observation that c-REL is part of the described GC signature. However, most (71%) of the examined GC DLBCLs had cytoplasmic c-REL expression and the GC DLBCLs did not have increased expression of NFkB target genes. Taken together with the MLBCL analyses, these studies indicate that: 1) NFkB is consistently activated in MLBCL; 2) c-REL amplification is not closely associated with NFkB activation in large cell lymphomas (LCLs); and 3) NFkB activation in LCL subtypes does not require amplification of the c-REL locus.

Description: It is well known that the globin genes are organized within the human genome as two gene clusters on chromosomes 16 and 11 (α cluster: ζ2-ζ1-α2-α1-? and β cluster: ε-Gγ-Aγ-δ-β). Switching in the expression patterns of those genes during human ontogeny generally follows a pattern determined by their genomic arrangement, but the genetic mechanism is not defined. In an effort to better understand the γ-to-β globin switching phenomenon, gene expression patterns from reticulocyte RNA were studied. cDNA were generated from the reticulocytes of 28 separate donors (14 cord blood; 14 adult blood) and analyzed using Affymetrix HG-U133 arrays. Quantitative PCR was used to confirm the array expression patterns. Among those cDNA identified as having expression patterns that were relatively decreased among adult reticulocytes, we were surprised by expression associated with a cluster of expressed sequence tags (EST) located within the α-globin locus in the region between the ζ1 and α2 genes. In the context of the 44,229 arrayed probes, high-level expression from that EST cluster was detected (ranked 〈 25th). Informatic analysis revealed alignment with the second and third exons previously described for the Ψα2 globin gene, but no significant similarity with the first exon of Ψα2. Based upon these comparisons, we used adult human reticulocyte RNA to amplify a cDNA clone for further study (GenBank: AY698022; named μ-globin). Sequencing of that clone revealed an insert that encodes a predicted 423 nt. open reading frame that is one amino acid residue shorter than the α2-globin gene. While the mature μ-globin mRNA results from splicing of two introns, it lacks a canonical Kozak sequence. BLASTP and CLUSTALW analyses revealed the highest level of homology with the avian αD-globin protein with a sequence identity of 55% (78/141 amino acids). In addition, the predicted heme- and globin-binding amino acids of μ-globin and avian αD-globin are largely conserved. Experimentally, we analyzed the expression level of the μ-globin transcripts using quantitative real-time PCR. Non-erythroid tissues including whole brain, white blood cells, and the Jurkat cell line demonstrated only background levels. The expression level of μ-globin (copies per ng cDNA) was 3.04x10(4) ± 1.68x10(3) in fetal liver, 1.71x10(5) ± 9.51x10(4) in cord reticulocytes, 1.15x10(4) ± 1.19x10(3) in bone marrow, and 2.17x10(4) ± 6.84x10(3) in adult reticulocytes. Based upon the consistent decrease in μ-globin expression between fetal and adult erythroid tissues, we measured the expression of γ-globin in those tissues for comparison. The expression level of γ-globin (copies per ng cDNA) was 2.49x10(6) ± 1.48x10(5) in fetal liver, 1.49x10(8) ± 7.88x10(7) in cord reticulocytes, 3.84x10(3) ± 3.83x10(2) in bone marrow, and 5.70x10(5) ± 5.80x10(5) in adult reticulocytes. To determine if μ-globin expression is regulated during erythropoiesis, we assayed primary human erythroid progenitor cells cultured in erythropoietin. The expression pattern of μ-globin was similar to the other globin genes increasing from background levels on day 0 to maximum levels in committed erythroblasts (3.56x10(5) ± 1.99x10(4) copies per ng cDNA). These results suggest that the human genome encodes a previously unrecognized α-globin that most closely resembles avian αD-globin. Expression of that gene is decreased during the fetal-to-adult transition of human ontogeny and regulated during erythropoiesis.

Description: Reccurent translocation t(4;14) associated ectopic expression of FGFR3, sometimes containing the activation mutation K650E (TDII), has been identified in 25% of human multiple myeloma (MM) patients and cell lines. However, current empirically-derived cytotoxic chemotherapy does not effectively treat this disease. One potential therapeutic strategy of treating MM is to inhibit the tyrosine kinase activity of FGFR3. In this report, we evaluated the efficacy of PKC412 (N-benzoyl-staurosporine), a small molecule tyrosine kinase inhibitor, for the treatment of FGFR3 mutants induced diseases. PKC412 effectively inhibits the tyrosine kinase activity and activation of downstream effector pathways of FGFR3 TDII or the constitutively activated TEL-FGFR3 fusion that was reported in a subtype of human peripheral T-cell lymphoma (PTCL), as well as proliferation of hematopoietic Ba/F3 cells transformed by the FGFR3 mutants. Furthermore, PKC412 drastically inhibits proliferation of four different multiple myeloma-derived primary cell lines that are associated with t(4;14) and expression of dysregulated FGFR3. Moreover, oral-gavage treatment with PKC412 resulted in statistically significant prolongation of survival in the murine bone marrow transplant (BMT) models of FGFR3 TDII-induced pre-B cell lymphoma or TEL-FGFR3 fusion-induced myeloproliferative disease, which suggests suitable pharmacokinetic and toxicity profiles of PKC412 for clinical use. Together, our data establish the small molecule inhibitor PKC412 as a molecularly targeted therapy for multiple myeloma and other human malignancies expressing activated FGFR3.

Description: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with recognized variability in clinical outcome, genetic features, and cells of origin. To date, transcriptional profiling has been used to highlight similarities between DLBCL tumor cells and normal B-cell subtypes and associate genes and pathways with unfavorable outcome. Given the genetic heterogeneity in DLBCL, there are likely to be subsets of tumors with different pathogenetic mechanisms and possible treatment targets. To identify DLBCL subtypes that were sufficiently robust to be captured by multiple methods, we analyzed the profiles of 176 newly diagnosed DLBCLs using three different clustering algorithms (hierarchical clustering (HC), self-organizing maps (SOM), and probabilistic clustering (PC)), the top 5% of genes with the highest reproducibility across duplicate samples and largest variation across patient tumors, and a resampling-based method (consensus clustering) that automatically selects the most stable numbers of clusters with each algorithm. Three discrete subsets of DLBCLs -- “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” and “Host Response” (HR) -- were identified, characterized using gene set enrichment analysis and confirmed in an independent series of newly diagnosed DLBCLs with available array data. There was an association between cluster membership and examined genetic abnormalities in DLBCL. BCL2 translocations were more common in the OxPhos cluster whereas BCL6 translocations were more frequent in the BCR/proliferation cluster. Translocations of either type were uncommon in the HR cluster. Patients with HR DLBCLs were also significantly younger than those with OxPhos or BCR/proliferation tumors; HR patients also had a significantly higher incidence of splenic and BM involvement. The unique characteristics of HR tumors - fewer known genetic abnormalities and prominent host immune and inflammatory cell transcripts -prompted us to assess host immune cells in study tumors using morphologic and immunohistochemical approaches. HR DLBCLs contained significantly higher numbers of morphologically distinct CD2+/CD3+ tumor-infiltrating lymphocytes and interdigitating S100+/GILT+/CD1a-/CD123- dendritic cells. The HR cluster shared features of histologically defined T-cell/histiocyte-richBCL, including fewer known genetic abnormalities, younger age at presentation and frequent splenic and bone marrow involvement. The current study identifies tumor microenvironment and host inflammatory response as defining features in DLBCL, provides insights into the nature of the host immune response in a major DLBCL subtype and suggests rational treatment targets in newly identified tumor groups.

Description: Although diffuse large B-cell lymphoma (DLBCL) is potentially curable with current therapy, a significant number of DLBCL patients still die of their disease. In a broad-based screen for genes and pathways associated with poor DLBCL outcome, we previously identified a novel risk-related gene, termed BAL (B-aggressive lymphoma). Thereafter, we cloned and characterized a major BAL partner protein, BBAP (B-aggressive lymphoma and BAL binding partner), described the nuclear trafficking patterns of both proteins and demonstrated that BAL functions as a modulator of transcription. In the current study, we characterized BAL expression in normal tonsil and primary DLBCLs and defined BAL regulation and potential functions in both cell types. Immunohistochemical staining of normal tonsil revealed that BAL was expressed by small numbers of germinal center (GC) cells and interfollicular cells with the morphologic features of centroblasts (with the GC) and immunoblasts (within the interfollicular areas). In primary DLBCLs, BAL was expressed by the malignant B cells. To gain insights regarding the regulation of BAL and BBAP expression in DLBCLs, we analyzed a series of 176 primary tumor biopsies transcriptionally profiled an U133A/B Affymetrix microarrays. BAL/BBAP high-expressing DLBCLs had evidence of a brisk host immune response, including increased expression of T/NK-cell receptor and activation pathway components, complement cascade members, macrophage/dendritic cells markers and γIFN-induced transcripts, raising the possibility that BAL was induced by γIFN. Consistent with this hypothesis, γIFN treatment of DLBCL cell lines with low basal levels of BAL markedly increased BAL expression. In silico analysis revealed that the BAL and BBAP genes are located in 3q21 in head-to-head orientation and share the same CpG-related promoter. This shared promoter contains a conserved γIFN-responsive module composed of IRF and STAT binding elements. The BAL/BBAP bidirectional promoter was cloned into a pGL3 luciferase promoterless reporter vector and found to increase luciferase activity 〉 20-fold following γIFN stimulation. To gain further insights into regulation of BAL transcription, mutant versions of BAL promoter were generated and cloned. Luciferase assays demonstrated that the IRF binding site was necessary for γIFN-induced BAL transcription, whereas the STAT1 binding site had an accessory role. Taken together, these results demonstrate that BAL and BBAP genes are transcriptionally activated by γIFN in DLBCLs with features of a brisk host immune response including γIFN signaling. Preliminary studies suggest that BAL limits the efficacy of the observed host inflammatory response.

Description: Juvenile Myelomonocytic Leukemia (JMML) is a clonal hematopoietic disorder of childhood that is principally characterized by proliferation of cells of the granulocytic and monocytic lineages. Previous studies showed that Ras or Nf1 mutations contribute to ~65% cases of JMML. More recently, mutations in the protein tyrosine phosphatase Shp2 (PTPN11) have been found in ~35% of sporadic JMML, and at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and childhood B-cell acute lymphoblastic leukemia (B-ALL). Interestingly, germline mutations of Shp2 cause the autosomal dominant genetic disorder Noonan Syndrome (NS) and NS patients may have an increased incidence of JMML. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and JMML mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activated. We have compared the biochemical and biological effects of NS and JMML mutations. When produced as recombinant proteins in bacteria, nearly all NS mutations studied showed increased PTP activity. However, the JMML mutations E76K and D61Y have the highest activity, resembling the fully active mutant E76A that we defined earlier. Retroviral-mediated expression of E76K or D61Y, but not wild type (WT) Shp2 in murine bone marrow (BM) cells results in cytokine-independent myeloid colony outgrowth, as well as hypersensitivity to both IL-3 and GM-CSF. Notably, NS- associated mutants (e.g., D61G or N308D) also could transform BM cells to factor-independence, but yielded far fewer colonies. Transformation by the leukemia-associated E76K mutant required the phosphatase activity and intact FLVRE motifs in the Shp2 SH2 domains. Gab2, a major Shp2 SH2 domain binding protein, also was required for transformation. When retrovirally transduced BM cells were transplanted into lethally irradiated recipients, E76K and D61Y but not WT Shp2 evoked fatal myeloproliferative disease (MPD) in 30–40% of recipients characterized by splenomegaly, leukocytosis, neutrophilia and monocytosis with occasional anemia and thrombocytopenia. Another ~20% showed less severe MPD at early stage and ultimately succumbed to T-ALL at later times. Histological analysis of mice dying MPD revealed BM and spleen packed with myeloid cells and livers with perivascular myeloid infiltrates. Flow cytometry on BM and spleen confirmed the presence of MPD. Moreover, BM from E76K and D61Y transplanted recipients exhibited enhanced factor-independent colony formation. Furthermore, mast cells derived from E76K and D61Y transplanted mice exhibit increased proliferation and enhanced activation of Erk, Akt and Stat5 in response to IL-3. In contrast, mice with a knock-in mutation of NS (D61G) displayed only a mild MPD. These data support a model in which leukemia-associated mutants of Shp2 are strong hypermorphs that enhance signaling via βc cytokines, whereas NS mutants are less severe gain of function alleles. Our results provide the first evidence that Shp2 mutants have a causal role in leukemogenesis and therefore, Shp2 is the first bona fide PTP proto-oncogene.

Description: The design and evaluation of therapies for the sickle cell and β-thalassemia syndromes rely on our understanding of hemoglobin accumulation during human erythropoiesis. Here we report direct measurements of hemoglobin composition and messenger RNA (mRNA) levels in cultured CD34+ cells and correlate those measurements with studies of freshly obtained bone marrow samples. Hemoglobin levels in differentiating cells were also compared with morphologic, immunophenotypic, and cell cycle assessments. A population of large preproerythroblasts was first identified within 24 hours and became the dominant population by day 5. The transition from proerythroblast to basophilic normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% ± 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts were the dominant and final cell type by day 13. High-performance liquid chromatography–based quantitation of fetal (HbF) and adult (HbA) hemoglobin and real-time polymerase chain reaction globin mRNA quantitation demonstrated a coordinate rise in the accumulation of both proteins and mRNA among these developmentally staged populations. Quantitative analyses on freshly sorted bone marrow populations demonstrated a similar rising pattern with β-globin and HbA as the dominant species at both early and late stages of differentiation. We found no evidence for HbF dominant populations or switching during differentiation in adult cells. Instead, rapid increases in both HbF (heterocellular) and HbA (pancellular) content were observed, which coincided with the apex in cell cycling and the proerythroblast-basophilic normoblast transition. Based on these measurements, we conclude that HbF and HbA content are regulated with the rate of proliferation during adult erythropoiesis.