30 results on '"Pankaj GUPTA"'
Search Results
2. Characteristics of Environmental Exposure-Associated Versus Unexposed High-Grade B-Cell Lymphomas in Veterans: A Single Center, Matched Case-Control Study
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Yi Ouyang, Helen Ma, and Pankaj Gupta
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Case-control study ,Cell Biology ,Hematology ,Environmental exposure ,Single Center ,Biochemistry ,medicine.anatomical_structure ,Internal medicine ,medicine ,business ,B cell - Abstract
Introduction: Environmental exposures, such as Agent Orange and other chemicals, are associated with the development of non-Hodgkin lymphomas (NHL). Military personnel and civilians in theaters of conflict are especially at high risk. The mechanism is still not well understood. We have initiated studies to examine the association of dioxin exposure, changes in DNA methylation, and the development of high-grade B-cell NHL. Methods: Patients diagnosed with high grade B-cell NHL were identified from the VA Long Beach medical record system for database abstraction and tissue analysis. Subjects with sufficient clinical data including diagnosis, date of diagnosis, age of diagnosis, exposure to toxins, treatments, date of last follow up, date of death, cause of death were included in the study. Patients were matched by exposure vs no exposure and international prognostic index (IPI), as well as duration in military and tour of duty when possible. Formalin-fixed paraffin-embedded specimens from a subset of these patients (where sufficient tissue was available for analysis) with and without known exposures were analyzed for differences in DNA methylation by enzymatic methyl sequencing. Descriptive statistics were used to summarize patient characteristics. Two sample t- and chi-square tests were used to compare the exposed and unexposed lymphoma groups. Overall survival was calculated from time of diagnosis to death from any cause and plotted on a Kaplan-Meier curve and compared using the Log rank test. The Fisher's exact test was used to compare the causes of death between the two groups using SAS 9. Results: Fifty-two patients were included in the study. Patient characteristics were well-balanced between the two groups. The duration of active duty, interval from military enlistment to diagnosis of NHL, and first-line therapy (generally anthracycline-based) were similar. There was a trend toward higher proportion of patients with advanced (stage III or IV) disease and younger age of diagnosis in patients with exposure-associated NHL (p=0.07). The median duration of response to first-line therapy (DOR1) trended towards being shorter (median, 0.8 years; range 0.03-25 years) in patients with exposure related NHL compared to median DOR1 of 2.7 (range, 0.02-25 years) in those without exposure, p=0.056. Median overall survival was 11.5 years in the exposure group and 10 years in the unexposed group, p=0.31. A higher portion of patients in the exposed group died from NHL compared to unexposed group (38% vs 15%), p=0.057. Other competing causes of death, which were more likely in the unexposed group, included other cancers, cardiac disease, infection, end stage kidney disease, dementia and unknown. Six exposed and six unexposed samples were sequenced with over 10,000 statistically significant differentially methylated regions (DMRs) out of 40,605 DMRs tested. Details of differences in methylation of specific genes and their potential pathophysiological and therapeutic implications will be discussed in the presentation. Conclusions: Patients with exposure-related high-grade B cell NHL who were matched by IPI with unexposed lymphomas did not differ significantly in baseline characteristics including time from enlistment to diagnosis and overall survival. In the exposure-related NHL group, certain findings trended toward significance, including younger age at diagnosis, higher proportion diagnosed at an advanced stage, shorter DOR1, and higher proportion died of NHL. This single center cohort, however, was small and heterogeneous. We are therefore currently expanding this study to examine a larger, multi-center cohort to confirm and extend our clinical and translational findings. Disclosures No relevant conflicts of interest to declare.
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- 2021
3. Relapse-Initiating Clones Preexisting at Diagnosis in B- Cell Acute Lymphoblastic Leukemia Help Predict Molecular Pathways of Relapse
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Debbie Payne-Turner, Michael Rusch, Xiaotu Ma, John E. Dick, Alex Murison, Ying Shao, Mark D. Minden, Jessica McLeod, Jeffery Wintersinger, John Easton, Mathieu Lupien, Yiping Fan, Charles G. Mullighan, Laura Garcia Prat, Michelle Chan-Seng-Yue, Michael N. Edmonson, Zhaohui Gu, Pankaj Gupta, Jinghui Zhang, Stephanie M. Dobson, Esmé Waanders, Quaid Morris, Robert Vanner, and Olga I. Gan
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Immunology ,Clone (cell biology) ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Somatic evolution in cancer ,Transplantation ,Leukemia ,Acute lymphocytic leukemia ,medicine ,Cancer research ,Epigenetics ,Gene ,Exome sequencing - Abstract
Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring. Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL. Disclosures Mullighan: Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.
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- 2018
4. Dual-targeting immunotherapy of lymphoma: potent cytotoxicity of anti-CD20/CD74 bispecific antibodies in mantle cell and other lymphomas
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Richard R. Furman, Chien-Hsing Chang, Edmund A. Rossi, John C. Byrd, Natarajan Muthusamy, Pankaj Gupta, Thomas M. Cardillo, and David M. Goldenberg
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MAP Kinase Signaling System ,medicine.drug_class ,medicine.medical_treatment ,Immunology ,Apoptosis ,Lymphoma, Mantle-Cell ,Mice, SCID ,Antibodies, Monoclonal, Humanized ,Monoclonal antibody ,Biochemistry ,Mice ,chemistry.chemical_compound ,Cancer immunotherapy ,Cell Line, Tumor ,hemic and lymphatic diseases ,Antibodies, Bispecific ,medicine ,Animals ,Humans ,Cytotoxicity ,CD20 ,B-Lymphocytes ,biology ,Cytotoxins ,Chemistry ,Histocompatibility Antigens Class II ,Cell Biology ,Hematology ,Immunotherapy ,Antigens, CD20 ,Veltuzumab ,medicine.disease ,Xenograft Model Antitumor Assays ,Molecular biology ,Milatuzumab ,Antigens, Differentiation, B-Lymphocyte ,Actin Cytoskeleton ,Drug Design ,Immunoglobulin G ,biology.protein ,Female ,Mantle cell lymphoma ,Lysosomes - Abstract
We describe the use of novel bispecific hexavalent Abs (HexAbs) to enhance anticancer immunotherapy. Two bispecific HexAbs [IgG-(Fab)4 constructed from veltuzumab (anti-CD20 IgG) and milatuzumab (anti-CD74 IgG)] show enhanced cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as patient tumor samples, without a crosslinking Ab, compared with their parental mAb counterparts, alone or in combination. The bispecific HexAbs have different properties from and are more potent than their parental mAbs in vitro. The juxtaposition of CD20 and CD74 on MCL cells by the HexAbs resulted in homotypic adhesion and triggered intracellular changes that include loss of mitochondrial transmembrane potential, production of reactive oxygen species, rapid and sustained phosphorylation of ERKs and JNK, down-regulation of pAkt and Bcl-xL, actin reorganization, and lysosomal membrane permeabilization, culminating in cell death. They also displayed different potencies in depleting lymphoma cells and normal B cells from whole blood ex vivo and significantly extended the survival of nude mice bearing MCL xenografts in a dose-dependent manner, thus indicating stability and antitumor activity in vivo. Such bispecific HexAbs may constitute a new class of therapeutic agents for improved cancer immunotherapy, as shown here for MCL and other CD20+/CD74+ malignancies.
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- 2012
5. Therapy of B-cell malignancies by anti–HLA-DR humanized monoclonal antibody, IMMU-114, is mediated through hyperactivation of ERK and JNK MAP kinase signaling pathways
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Richard R. Furman, Thomas M. Cardillo, Chien-Hsing Chang, Susan Chen, Xiaochuan Chen, Pankaj Gupta, Rhona Stein, and David M. Goldenberg
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MAPK/ERK pathway ,MAP Kinase Kinase 4 ,MAP Kinase Signaling System ,medicine.drug_class ,Chronic lymphocytic leukemia ,Immunology ,Mice, SCID ,Humanized antibody ,Monoclonal antibody ,Biochemistry ,Mice ,Antigens, CD ,immune system diseases ,Cell Line, Tumor ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,Hairy cell leukemia ,Extracellular Signal-Regulated MAP Kinases ,B cell ,CD20 ,B-Lymphocytes ,biology ,Cytotoxins ,Antibodies, Monoclonal ,HLA-DR Antigens ,Cell Biology ,Hematology ,medicine.disease ,Molecular biology ,Enzyme Activation ,Leukemia ,medicine.anatomical_structure ,Hematologic Neoplasms ,Cancer research ,biology.protein ,Female ,Drug Screening Assays, Antitumor - Abstract
A humanized IgG4 anti–HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required.
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- 2010
6. Functional abnormalities of heparan sulfate in mucopolysaccharidosis-I are associated with defective biologic activity of FGF-2 on human multipotent progenitor cells
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Lisa Koodie, Scott B. Selleck, Charles Peters, Morayma Reyes, Sally E. Stringer, Robert C.H. Zhao, Joseph J. Brazil, Chendong Pan, Pankaj Gupta, Elliot J. Stephenson, and Matthew S. Nelson
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medicine.medical_specialty ,Cell Survival ,Mucopolysaccharidosis I ,Immunology ,Biology ,Fibroblast growth factor ,Biochemistry ,Dermatan sulfate ,chemistry.chemical_compound ,Internal medicine ,medicine ,Humans ,Receptor, Fibroblast Growth Factor, Type 1 ,Progenitor cell ,Hurler syndrome ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Cell Proliferation ,Multipotent Stem Cells ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Hematology ,Heparan sulfate ,medicine.disease ,Receptors, Fibroblast Growth Factor ,Hematopoiesis ,Cell biology ,Endocrinology ,chemistry ,Multipotent Stem Cell ,Case-Control Studies ,Fibroblast Growth Factor 2 ,Heparitin Sulfate ,Stem cell - Abstract
In mucopolysaccharidosis-I (MPS-I), α-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2–FGFR1-HS interactions, resulting in defective FGF-2–induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS–cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.
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- 2005
7. Activating Mutations Are Potent Pro-Leukemic Mediators in Murine MLL-MLLT3 Leukemia That Cause Distinct Transcriptional Profiles
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Tanja A. Gruber, Anna Andersson, James R. Downing, Helena Sturesson, Jing Ma, Jenny Hansson, Julhash U. Kazi, Axel Hyrenius-Wittsten, Michael P. Walsh, Pankaj Gupta, Mattias Pilheden, Stephanie Nance, Jinghui Zhang, and Guangchun Song
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Mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Chromatin ,Transplantation ,Leukemia ,hemic and lymphatic diseases ,Acute lymphocytic leukemia ,medicine ,Cancer research - Abstract
Acquired activating mutations in kinase/PI3K/RAS signaling pathways occur in about half of pediatric acute leukemia cases with Mixed Lineage Leukemiagene rearrangements (MLL-R; MLL, also known as KMT2A). Mutations resulting in activated signaling cooperate with the MLL-R in mouse leukemia models, however, detailed insight on the effect on the transcriptional and proteomic landscape is lacking. In infant MLL-R acute lymphoblastic leukemia (ALL), a subtype with a very poor prognosis, a majority of the activating mutations are subclonal, but the biological mechanisms by which subclonal mutations affect leukemogenesis remain unclear. Here we show that NRASG12D, FLT3internal tandem duplication(ITD)and FLT3N676K,cooperates with MLL-MLLT3 in myeloid leukemogenesis using a competitive murine retroviral bone marrow transplantation model. The addition of an activating mutation remodels the gene expression patterns of the MLL-R leukemia with distinct profiles for each mutation as determined by RNA-sequencing. Gene set enrichment analysis revealed enrichment of genes involved in chromatin assembly, transcription and stemness in leukemia induced by MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Leukemia induced by only MLL-MLLT3 displayed upregulation of genes involved in signal transduction suggesting activation of such pathways by alternative mechanisms. Upon secondary transplantation, mice receiving MLL-MLLT3-only leukemic cells succumbed to disease at a similar latency to those receiving MLL-MLLT3 and an activating mutation, supporting that fully transformed MLL-MLLT3 leukemias have sustained active signaling via high expression of genes involved in signal transduction. Using the same model as above but with significantly reduced numbers of transplanted cells containing an activating mutation, we could further show that a subclonal mutation, exemplified by FLT3N676K, causes significantly reduced disease latency as compared to mice receiving MLL-MLLT3 only (34 vs 50 days). The size of the double positive (MLL-MLLT3+FLT3N676K) and single positive (MLL-MLLT3) clones within each mouse was determined by flow cytometry revealing that 8/24 mice had a double positive subclone (≤50%). The clonal evolution of the double positive cells from 22/24 mice was assessed in secondary recipients showing three distinct patterns 1) increase in size, 19/22 mice 2) maintained, 2/22 mice or 3) decreased in size, 1/22 mice. Targeted gene re-sequencing of the latter leukemia that lost its MLLT3+FLT3N676K subclone, identified a de-novo CblA308T in the SH2-like domain, in the MLL-MLLT3 onlycells that had gained clonal dominance in the secondary recipient. The decreased disease latency in mice with subclonal activating mutations raises the possibility that cells with an activating mutation support the growth of other leukemic cells by direct cell-cell contact or through secreted factors. Transcriptome and proteomic analyses identified a high expression of the macrophage inhibitory factor (MIF), a pro-inflammatory cytokine, in mice with MLL-MLLT3 and NRASG12D, FLT3ITD or FLT3N676K. Addition of rMIF increased the survival of MLL-MLLT3 murine leukemic cells in vitro. Although additional factors likely mediate pro-leukemic effects in vivo, our data suggest that MIF could be one of these factors. In summary, our data demonstrate that activating mutations cooperate with the MLL-R in murine leukemogenesis and cause widespread changes in the transcriptional landscape. In addition, our results suggest that cells containing an activating mutation in addition to an MLL-fusion positively influence the survival and likely also the growth of other leukemic cells, suggesting a pro-leukemic effect mediated by interclonal cooperation between clones carrying distinct mutational set-ups in leukemogenesis. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
8. Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche
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Joseph J. Brazil, Theodore R. Oegema, Pankaj Gupta, Arkadiusz Z. Dudek, Arne Slungaard, and Catherine M. Verfaillie
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Stromal cell ,Cellular differentiation ,Immunology ,Antigens, CD34 ,Bone Marrow Cells ,Biology ,Biochemistry ,Extracellular matrix ,Cell Adhesion ,Humans ,Progenitor cell ,Cells, Cultured ,Glycosaminoglycans ,Interleukin 3 ,Extracellular Matrix Proteins ,Molecular Structure ,Sulfates ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Hematopoiesis ,Cell biology ,Molecular Weight ,Haematopoiesis ,Cell culture ,Culture Media, Conditioned ,Cytokines ,Proteoglycans ,Heparitin Sulfate ,Stem cell ,Heparan Sulfate Proteoglycans ,Protein Binding - Abstract
Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P
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- 1998
9. Human CD34+ Bone Marrow Cells Regulate Stromal Production of Interleukin-6 and Granulocyte Colony-Stimulating Factor and Increase the Colony-Stimulating Activity of Stroma
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Catherine M. Verfaillie, Pankaj Gupta, Kalpna Gupta, and Bruce R. Blazar
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Stromal cell ,medicine.medical_treatment ,Immunology ,CD34 ,Antigens, CD34 ,Bone Marrow Cells ,Cell Count ,Biology ,Biochemistry ,Feedback ,Colony-Forming Units Assay ,Granulocyte Colony-Stimulating Factor ,medicine ,Humans ,Erythropoietin ,Cells, Cultured ,Interleukin-6 ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Molecular biology ,Coculture Techniques ,Hematopoiesis ,Granulocyte colony-stimulating factor ,Haematopoiesis ,medicine.anatomical_structure ,Cytokine ,Gene Expression Regulation ,Cytokines ,Tumor necrosis factor alpha ,Bone marrow ,Stem cell ,Cell Division - Abstract
Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34(+) cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34(+) cells for 3 to 5 weeks. Coculture of more mature CD15(+)/CD14(-) myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34(+) nor CD15(+)/CD14(-) cells produced IL-6, G-CSF, IL-1beta, or tumor necrosis factor alpha. When CD34(+) cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34(+) cells or when CD34(+) cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit-granulocyte-macrophage-stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34(+) progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant. ispartof: Blood vol:91 issue:10 pages:3724-33 ispartof: location:United States status: published
- Published
- 1998
10. Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells
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Pankaj Gupta, Catherine M. Verfaillie, and James B. McCarthy
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Stromal cell ,medicine.medical_treatment ,Immunology ,Cell Culture Techniques ,Bone Marrow Cells ,Stem cell factor ,Biology ,Hematopoietic Cell Growth Factors ,Biochemistry ,Biological Factors ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Cell Differentiation ,Cell Biology ,Hematology ,Heparan sulfate ,Fibroblasts ,Hematopoietic Stem Cells ,Molecular biology ,Recombinant Proteins ,Haematopoiesis ,Cytokine ,chemistry ,Connective Tissue ,Cell culture ,Culture Media, Conditioned ,Cattle ,Proteoglycans ,Heparitin Sulfate ,Stem cell ,Leukemia inhibitory factor - Abstract
We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2-10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2-10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2-10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2-10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors. ispartof: Blood vol:87 issue:8 pages:3229-36 ispartof: location:United States status: published
- Published
- 1996
11. Identification of an MIP-1α–binding heparan sulfate oligosaccharide that supports long-term in vitro maintenance of human LTC-ICs
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Sally E. Stringer, Matthew S. Nelson, and Pankaj Gupta
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Chemokine ,medicine.medical_treatment ,Immunology ,Oligosaccharides ,Antigens, CD34 ,Bone Marrow Cells ,Plasma protein binding ,Polysaccharide ,Biochemistry ,chemistry.chemical_compound ,Antigen ,medicine ,Humans ,Chemokine CCL4 ,Cells, Cultured ,Chemokine CCL3 ,chemistry.chemical_classification ,biology ,HLA-DR Antigens ,Cell Biology ,Hematology ,Heparan sulfate ,Macrophage Inflammatory Proteins ,Oligosaccharide ,Hematopoietic Stem Cells ,In vitro ,Cytokine ,chemistry ,biology.protein ,Heparitin Sulfate ,Protein Binding - Abstract
We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1alpha binding ability. A specific MIP-1alpha-binding HS oligosaccharide preparation of M(r) 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1alpha binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.
- Published
- 2003
12. Incidence of Unprovoked Venous Thromboembolic Events in Patients with Chronic Lymphocytic Leukemia (CLL)
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Mark A. Klein, Lauren Katkish, Gerhard J. Johnson, Pankaj Gupta, Thomas S. Rector, and Sravanti Rangaraju
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education.field_of_study ,Pediatrics ,medicine.medical_specialty ,business.industry ,Incidence (epidemiology) ,Deep vein ,Immunology ,Population ,Cell Biology ,Hematology ,Thrombophilia ,medicine.disease ,Biochemistry ,Chemotherapy regimen ,Thrombosis ,Pulmonary embolism ,medicine.anatomical_structure ,medicine ,Risk factor ,business ,education - Abstract
While lymphoma is a known risk factor for venous thromboembolism (VTE), the thrombogenicity of chronic lymphocytic leukemia (CLL) has not been investigated. One study reported a ten-fold increased risk in VTE compared to the general population, but this included both provoked and unprovoked events, making it difficult to discern the independent contribution of CLL to the thrombogenic predisposition (Whittle A et al. Leuk Res 2011; 35:419-21). Since we found no studies examining only unprovoked VTE in CLL, we sought to estimate the risk of spontaneous VTE occurring in patients with CLL in the absence of known thrombogenic risk factors. The study was approved by the institutional human subjects committee. We examined patients diagnosed with CLL between 1997-2014 by the persistent presence of ≥ 5,000 circulating small, mature, monoclonal, CD5+, CD23+ B lymphocytes (per diagnostic criteria in: Halleck M et al. Blood 2008;111:5446-56) and listed in the Minneapolis VA Tumor Registry. Clinical and laboratory data were obtained from the electronic medical record. Each patient's chart was reviewed individually for VTE, either pulmonary embolism (PE) or deep vein thrombosis (DVT), and the report of a confirmatory imaging study was examined to confirm the diagnosis. We followed patients (n = 308, 99% males, mean age 70 years) starting at the date of CLL diagnosis or the date after CLL diagnosis when the patient began follow-up at the VA and ending at last patient contact or death (122/308 [39.6%] patients died during the observation period). Each patient made at least annual contact with the VA between the start and end dates, with a mean VA follow-up time of 4.6 +/- 2.9 years; 90% patients were followed continuously at the VA since after the diagnosis of CLL. Patients were excluded if there was a preceding period of immobility, serious illness requiring hospitalization, diagnosed hypercoagulable state, or recent chemotherapy known to be associated with increased thrombotic risk. The total follow-up time was 1408 patient-years, during which 10 unprovoked VTE occurred, for an estimated incidence rate of unprovoked VTE of 0.71 per 100 patient-years, 95% CI [0.38, 1.32]. Eight VTE were originally diagnosed at our institution. Confirmatory imaging was available for the 2 VTE diagnosed elsewhere. All 10 patients were males between the ages of 55 to 95 years at the time of CLL diagnosis. Six had DVT, three had PE, and one had both DVT and bilateral PE. Nine patients had symptoms that prompted diagnostic imaging, while one PE was diagnosed incidentally on a chest CT scan. Nine patients had bulky lymphadenopathy at the time of VTE, but these did not compress the relevant vein in any patient. Small, stable monoclonal paraproteinemia was identified in 2/5 VTE patients tested (one patient at 0.39 and 0.35 g/dL, one patient at 0.32 and 0.27 g/dL). At time of development of VTE, 4, 1, 1 and 3 patients had Rai stage I, II, III or IV CLL, respectively. Thus, while there were relatively few events, there did not appear to be any clear relationship of VTE with advanced CLL stage. Recurrence of unprovoked VTE occurred in only 1 of 10 (10%) patients. In a study of the general population in our region of the US (Olmsted County, MN), the overall incidence of VTE in 70-74 year old males was 0.65 per 100 patient-years (Silverstein M et al. Arch Internal Med 1998;158:585-93). Since 17-47% of all VTEs were unprovoked in various population series (Cushman M et al. Thromb Haemost 2001;86[suppl 1]:OC2349, Heit JA et al. Arch Intern Med 2002;162:1245-8, Spencer FA et al. J Thromb Thrombolysis 2009;28:401-9, White RH Circulation 2003;107:I-4-8), the incidence rate of unprovoked VTEs observed in CLL patients in the current series (0.71; 95% CI: 0.38, 1.32) approximates the expected incidence of unprovoked VTEs in the general population. Further, the development of unprovoked VTE would have been expected to be skewed towards patients with advanced stage (Rai stages III/IV), and/or have been associated with more frequent recurrences, if CLL was an independently thrombogenic condition. Conclusion: This is the first long-term study of unprovoked VTE in patients with CLL, with >1400 patient-years of follow-up. The low incidence of unprovoked VTEs observed in our study suggests that primary thromboprophylaxis may not be required in patients with CLL who do not have additional risk factors for thromboembolism. Disclosures No relevant conflicts of interest to declare.
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- 2015
13. Genomic Landscape of Relapsed Acute Lymphoblastic Leukemia
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Scott R. Olsen, Sima Jeha, Jinghui Zhang, Yiping Fan, Stephanie M. Dobson, William E. Evans, Pankaj Gupta, Gang Wu, Esmé Waanders, Xiaotu Ma, Geoffrey Neale, Colin Bailey, John E. Dick, Ying Shao, Ilaria Iacobucci, Kathryn G. Roberts, Kelly McCastlain, Jun J. Yang, Ching-Hon Pui, Charles G. Mullighan, Ji Wen, Jing Ma, James R. Downing, Mary V. Relling, John Easton, Matthew Lear, Deanna Naeve, Guangchun Song, Michael Rusch, Shann-Ching Chen, Zhaohui Gu, and Debbie Payne-Turner
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Oncology ,Neuroblastoma RAS viral oncogene homolog ,medicine.medical_specialty ,business.industry ,Immunology ,Cancer ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Somatic evolution in cancer ,Leukemia ,Internal medicine ,medicine ,KRAS ,business ,EP300 ,Exome sequencing - Abstract
Introduction: Despite risk stratification according to presenting clinical and genetic features, 10-25% of children with acute lymphoblastic leukemia (ALL) relapse, which is associated with a poor prognosis. Here, we sought to provide a comprehensive overview of the genetic alterations associated with relapse in ALL. Methods: We studied 93 children (27 female, 43 male) diagnosed with ALL (62 B-progenitor, 25 T-lineage) between 1987 and 2008 and treated on total therapy studies XI-XVI who experienced relapse and/or a second tumor. Age at diagnosis ranged from 3 months to 18 years. Median time to relapse was 3 years (range 3 months to 10 years). Seventy patients had a single relapse, 15 cases had 2 relapses, and 8 cases developed a second tumor of different lineage (B-cell lymphoma, chronic myeloid leukemia (n=1 each) and acute myeloid leukemia (n=6)). Diagnosis, relapse and matched normal samples (n=299) were studied using Affymetrix SNP 6.0 microarrays and whole genome or whole exome sequencing. Results: We found 2692 copy number aberrations (CNAs) with a median of 9 (range 0-109) in the diagnosis samples (n=91) compared to a median of 10 (range 0-112) in the relapse samples (n=89) and 12 (range 0-70) in subsequent samples (n=20). The number of CNAs did not differ significantly between diagnosis, relapse or subsequent samples. We identified a 7286 non-silent single nucleotide variants (SNVs) and small insertions or deletions (indels) in 5002 genes, 1392 of which were recurrent. The median number of variants was 12 (range 0-70) at diagnosis (n=91), 21 (range 0-858) at relapse (n=91; P=0.0029 v. diagnosis) and 60 (range 10-650) in subsequent samples (n=20; P The most frequently mutated genes were NOTCH1 (n=33), NRAS (n=24), CREBBP (n=20) and KRAS (n=16). Of the recurrently altered genes, only 87 genes were known to be affected in cancer (Cancer Gene Census, COSMIC database), of which 59 were affected in leukemia and lymphoma tissues, indicating that we have identified 1306 novel recurrently affected genes, most commonly C13orf40 and MKI67. Mutations in epigenetic regulators were particularly frequent, with genes mutated in at least 3 cases altered in over 60% of the cohort (e.g. CREBBP, EP300, MLL2, MLL3, KDM6A/B, CTCF, SETD2, TET2/3, and EZH2). Clonal evolution analyses showed multiple patterns of evolution, with relapses sharing either few or many variants with the diagnosis sample in a frequency that reflects both predominant clones and minor subclones propagating relapse. Variants in NOTCH1, NRAS, and CREBBP were preserved from a major clone at diagnosis in 4, 6, and 5 cases respectively, but acquired at relapse or grown out from a minor subclone at diagnosis in 3, 5, and 8 cases respectively. In contrast, variants in USH2A (n=4), FOXA1 (n=3), and purine/pyrimidine synthesis pathway genes NT5C2 (n=3), PRPS1 (n=3) and NT5C1B (n=1) were exclusively found in relapse samples. Notably, the NT5C2 mutations, which are thought to confer resistance to thiopurines, were subclonal at relapse in the majority of cases. We identified 13 cases (10 B-lineage, 3 T-lineage) in which the diagnosis and relapse were fully discordant for all CNAs and sequence mutations, only 4 of which showed a prolonged remission time (>5 years). This suggests that these patients developed a second primary malignancy and may be predisposed to leukemia development. Indeed, one case revealed focal amplifications on chromosome 1q21.1 encompassing the neuroblastoma breakpoint family genes, which are implicated in cancer development. Comprehensive germline analyses are underway. Conclusion: This study has provided detailed insight into the genetic basis of relapse, implicating multiple new genes and pathways involved in treatment resistance, demonstrating multiple patterns of clonal evolution, and revealing an unexpectedly high frequency of genetically discordant second malignancy in relapse in ALL. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Science Institute: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria; Loxo Oncology: Research Funding.
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- 2015
14. Multiple signaling pathways induced by hexavalent, monospecific, anti-CD20 and hexavalent, bispecific, anti-CD20/CD22 humanized antibodies correlate with enhanced toxicity to B-cell lymphomas and leukemias
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David M. Goldenberg, Chien-Hsing Chang, Edmund A. Rossi, and Pankaj Gupta
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Lymphoma, B-Cell ,MAP Kinase Signaling System ,Chronic lymphocytic leukemia ,Sialic Acid Binding Ig-like Lectin 2 ,Immunology ,Antineoplastic Agents ,Apoptosis ,Humanized antibody ,Antibodies, Monoclonal, Humanized ,Biochemistry ,chemistry.chemical_compound ,Antibodies, Monoclonal, Murine-Derived ,Cell Line, Tumor ,medicine ,Humans ,B cell ,Cell Proliferation ,Leukemia ,Lymphoid Neoplasia ,biology ,CD22 ,NF-kappa B ,PTEN Phosphohydrolase ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,Veltuzumab ,medicine.disease ,Antigens, CD20 ,Lymphoma ,Gene Expression Regulation, Neoplastic ,medicine.anatomical_structure ,chemistry ,Proto-Oncogene Proteins c-bcl-2 ,Mitochondrial Membranes ,biology.protein ,Cancer research ,Antibody ,Rituximab ,Epratuzumab ,medicine.drug ,Signal Transduction - Abstract
We have generated hexavalent antibodies (HexAbs) comprising 6 Fabs tethered to one Fc of human IgG1. Three such constructs, 20-20, a monospecific HexAb comprising 6 Fabs of veltuzumab (humanized anti-CD20 immunoglobulin G1κ [IgG1κ]), 20-22, a bispecific HexAb comprising veltuzumab and 4 Fabs of epratuzumab (humanized anti-CD22 IgG1κ), and 22-20, a bispecific HexAb comprising epratuzumab and 4 Fabs of veltuzumab, were previously shown to inhibit pro-liferation of several lymphoma cell lines at nanomolar concentrations in the absence of a crosslinking antibody. We now report an in-depth analysis of the apoptotic and survival signals induced by the 3 HexAbs in Burkitt lymphomas and provide in vitro cytotoxicity data for additional lymphoma cell lines and also chronic lymphocytic leukemia patient specimens. Among the key findings are the significant increase in the levels of phosphorylated p38 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by all 3 HexAbs and the notable differences in the signaling events triggered by the HexAbs from those incurred by crosslinking veltuzumab or rituximab with a secondary antibody. Thus, the greatly enhanced direct toxicity of these HexAbs correlates with their ability to alter the basal expression of various intracellular proteins involved in regulating cell growth, survival, and apoptosis, with the net outcome leading to cell death.
- Published
- 2010
15. Genomic Characterization and Experimental Modeling Of BCR-ABL1-Like Acute Lymphoblastic Leukemia
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Kathryn G. Roberts, Deqing Pei, Jing Ma, Andrew J. Carroll, Sima Jeha, I-Ming Chen, Charles G. Mullighan, William L. Carroll, Cheng Cheng, Debbie Payne-Turner, Yongjin Li, Guido Marcucci, Jinghui Zhang, Richard C. Harvey, Daniela S. Gerhard, Julie M. Gastier-Foster, Eric Larsen, Natalia Santiago-Morales, Elizabeth A. Raetz, Yung-Li Yang, Mignon L. Loh, James R. Downing, Naomi J. Winick, Shann-Ching Chen, Nyla A. Heerema, Clara D. Bloomfield, Stephen P. Hunger, Michael Rusch, Meenakshi Devidas, Steven M. Kornblau, Elisabeth Paietta, Jinjun Cheng, Pankaj Gupta, Guangchun Song, Cheryl L. Willman, and Ching-Hon Pui
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Oncology ,medicine.medical_specialty ,ABL ,Immunology ,Imatinib ,PDGFRB ,Cell Biology ,Hematology ,Biology ,Bioinformatics ,medicine.disease ,Biochemistry ,Erythropoietin receptor ,Dasatinib ,hemic and lymphatic diseases ,Internal medicine ,Acute lymphocytic leukemia ,medicine ,Interleukin-7 receptor ,Exome sequencing ,medicine.drug - Abstract
BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (B-ALL) accounts for 10-15% of childhood B-ALL and is characterized by alteration of IKZFI, a gene expression profile similar to BCR-ABL1 ALL and poor outcome. Using next-generation sequencing, we have shown that BCR-ABL1-like ALL patients harbor genetic alterations activating kinase pathways that are sensitive to tyrosine kinase inhibitors (TKIs), and have shown that refractory BCR-ABL1-like ALL is responsive to TKIs in vivo (Weston et al., J. Clin. Oncol 2013). Furthermore, the outcome of ALL in adolescent and young adult (AYA) patients is inferior to children, yet the genetic basis underlying treatment failure is poorly understood. To define the frequency and genomic landscape of BCR-ABL1-like ALL in children, adolescents, and young adults we have extended our studies to include 665 high-risk childhood ( To characterize the full spectrum of kinase lesions in the remaining BCR-ABL1-like ALL cases we performed mRNA-seq on pediatric (n=39), adolescent (n=21) and young adult (n=22) cases, and whole genome (WGS; n=18) or exome sequencing (n=10) on cases with matched tumor and normal material. Fusion transcripts were identified using deFuse and CICERO, a novel assembly-based structural variation detection method specifically designed for mRNA-seq analysis. We identified 23 different kinase rearrangements involving 7 tyrosine kinase or cytokine receptor genes. These consist of 5 ABL1, 2 PDGFRB, 8 JAK2 fusions and 2 EPOR translocations to IGH@ and IGK@ loci, along with new fusions involving the tyrosine kinases ABL2 (n=3), CSF1R (n=1), AKT2 (n=1) and STAT5B (n=1). We performed frequency testing for 15 of these fusions on 555 cases from the COG AALL0232 trial of high-risk B-ALL. Several alterations were recurrent in BCR-ABL1-like ALL, including NUP214-ABL1, RCSD1-ABL2, SSBP2-CSF1R, PAX5-JAK2 and EPOR translocations. Notably, we did not identify any of these fusions in non BCR-ABL1-like cases. The frequency of ABL1/ABL2 and EPOR translocations was consistent across all age groups (∼16% and 7% of BCR-ABL1-like cases, respectively), while JAK2 rearrangements were more common in young adult than in pediatric and adolescent ALL (12%). Importantly, ∼10% of BCR-ABL1-like ALL cases lacked a kinase-activating alteration on analysis of mRNA-seq data. Notably, we identified two additional cases with IL7R or SH2B3 sequence mutations, indicating the requirement for complementary approaches such as WGS to fully define the genomic landscape of BCR-ABL1-like ALL. Current functional studies include the development of experimental models using the Ba/F3 hematopoietic progenitor cell line, primary mouse pre-B cultures and the generation of xenografts to determine the role of these alterations in leukemogenesis, and to enable testing of targeted therapies. For example, we show that RCSD1-ABL1 and SSBP2-CSF1R confer factor-independent growth and constitutive activation of JAK/STAT pathways in Ba/F3 cells. Furthermore, RCSD1-ABL1 and SSBP2-CSF1R are both sensitive to the TKIs, imatinib (IC50 378nM and 327nM, respectively) and dasatinib (IC50 2.1nM and 2.5nM, respectively). Together, these complementary approaches will further define the genetic landscape of both pediatric and AYA ALL, and facilitate the development of diagnostic and therapeutic strategies to improve the treatment outcome for high-risk BCR-ABL1-like ALL patients. Disclosures: Hunger: Bristol Myers Squibb: Consultancy.
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- 2013
16. Genomic- and Transcriptomic Profiling Of Acute Lymphoblastic Leukemia With Dicentric Chromosomes
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Mignon L. Loh, Nyla A. Heerema, Jing Ma, Pankaj Gupta, Debbie Payne-Turner, Guangchun Song, Andrew J. Carroll, Susana C. Raimondi, Linda Holmfeldt, Charles G. Mullighan, Paolo Vignali, Jinghui Zhang, Michael Rusch, and Stephen P. Hunger
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Genetics ,Candidate gene ,Immunology ,Aneuploidy ,Cell Biology ,Hematology ,Chimeric gene ,Biology ,medicine.disease ,Biochemistry ,Molecular biology ,Gene expression profiling ,Dicentric chromosome ,CDKN2A ,medicine ,Exome ,Exome sequencing - Abstract
Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, comprising multiple subtypes characterized by aneuploidy, recurring submicroscopic gains and losses of DNA, and inter- and intra-chromosomal rearrangements. A common feature in ALL with 44-45 chromosomes (from here on referred to as near-diploid ALL) is the presence of a dicentric chromosome, which may lead to the formation of gene fusions and the expression of chimeric proteins. At present, however, we have a limited understanding of the nature of these chimeric proteins, but some are known or predicted to dysregulate the function of oncoproteins or hematopoietic growth factors. We recently performed genome-wide profiling of over 120 pediatric cases with hypodiploid ALL, including 68 near-haploid cases (harboring 24-31 chromosomes), 34 low-hypodiploid cases (32-39 chromosomes) and 22 near-diploid cases, 20 of which harbored a dicentric chromosome. Near-haploid ALL is characterized by a high frequency of activating mutations of Ras signaling (mainly NF1 alterations) and IKZF3 deletions, and low-hypodiploid ALL by IKZF2, TP53 and RB1alterations. In contrast, these alterations are infrequent in dicentric ALL. To gain insight into the genetic basis of near-diploid ALL with dicentric chromosomes, we examined the 20 cases including 6 with dic(9;20), 3 with dic(7;12) and 2 with dic(9;17), while the remaining 9 cases had dicentric chromosomes involving various chromosomal arms. Affymetrix SNP 6.0 microarrays, gene expression profiling and candidate gene resequencing for 19 genes were performed for all cases, and transcriptome sequencing (mRNA Seq) on tumor and whole exome sequencing on tumor and matched normal genomic DNA have to date been carried out for 10 cases. The most common lesion was focal deletion of CDKN2A/CDKN2B, encoding the tumor suppressors INK4/ARF (77%). Approximately one third of near-diploid cases harbored mutations targeting Ras and receptor tyrosine kinase signaling, including mutations in NRAS (18%), KRAS (9%), PTPN11 (9%) and NF1 (5%). The B-lymphoid transcription factor PAX5 was altered by deletion, sequence mutation or amplification in 59% of near-diploid ALL. PAX5 has previously been reported to be involved in gene fusions created by dicentric chromosomes in ALL, including PAX5-ETV6 in some cases with dic(9;12)(p13;p13). PAX5 has previously also been shown to be targeted by dic(9;20)(p11-13;q11), which creates several non-productive fusion transcripts. The burden of non-synonymous single nucleotide variations (SNVs) and insertion/deletion (indel) mutations in coding- and splice regions in mRNA- and exome sequenced dicentric ALL varied with 8-25 SNVs and 4-15 indels per case. Except for the recurrent lesions stated above, the only recurrently sequence mutated gene identified in these 10 cases was NOTCH1, with mutations found in two cases, both of T-lineage. Singleton mutations were identified in e.g. IL7R, JAK3, STAT3, MYCN, PTEN and DOT1L. Fusion detection in mRNA Seq data identified 1-4 head-to-tail gene fusions per case, including the previously characterized fusions DDX3X-MLLT10, PICALM-MLLT10, MLL-AFF1 and P2RY8-CRLF2, none of which are involved in the dicentric chromosomes. The P2RY8-CRLF2 fusion was by RT-PCR shown to be present in two additional dicentric ALL cases. Further, novel in-frame fusions including ETV6-AMPH and PAX5-FBRSL1 created by dic(7;12)(p11.2;p11.2) and dic(9;12), respectively, were identified, the function of which in tumorigenesis is still unknown. An extended mRNA Seq study is currently ongoing, investigating an additional 30 near-diploid ALL cases with a focus on the dicentric chromosomes dic(7;9), dic(9;12) and dic(9;20). Further, recurrence screening will be performed on a cohort including another 45 cases, and functional evaluation is on an ongoing basis carried out on identified fusions. Altogether, this study is anticipated to provide critical new insights into the genetic basis of near-diploid ALL harboring a dicentric chromosome, and to shed more light on the putative chimeric genes produced by the formation of dicentric chromosomes. Further, we hope to identify new targets for therapy. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
17. Combination Therapy with FTY720 (Fingolimod) and Bispecific Anti-CD20/CD74 Antibodies in Mantle Cell Lymphoma
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Chien-Hsing Chang, David M. Goldenberg, Rosana B. Michel, and Pankaj Gupta
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CD20 ,biology ,medicine.drug_class ,business.industry ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,Monoclonal antibody ,medicine.disease ,Veltuzumab ,Humanized antibody ,Biochemistry ,Milatuzumab ,Transplantation ,chemistry.chemical_compound ,chemistry ,hemic and lymphatic diseases ,medicine ,Cancer research ,biology.protein ,Annexin A5 ,business - Abstract
Abstract 3736 Introduction: Mantle cell lymphoma (MCL) is one of the most challenging B-cell lymphomas to treat. Although the response rates with first-line conventional or high-dose chemotherapy, with or without stem-cell transplantation, are high, most patients relapse, after which their prognosis is considered poor. Thus, new therapeutic interventions in MCL are needed, among which targeted therapy with a variety of monoclonal antibodies (mAbs), either alone or in combination with other biological agents or drugs, is a major focus of ongoing clinical studies. FTY720 (fingolimod), an immunosuppressive agent approved by the FDA as a frontline treatment for relapsing multiple sclerosis, shows promising pre-clinical activity in chronic lymphocytic leukemia and MCL. More recently, FTY720 was also found to increase CD74 expression and sensitize MCL cells to milatuzumab (anti-CD74 humanized antibody)-mediated cell death (Alinari L, et al. ASH 2011 Abstract submitted). Here we report that the in vitro cytotoxicity of two novel bispecific anti-CD20-/CD74 antibodies to MCL lines can be enhanced significantly by combining with FTY720. Methods: The two bispecific, anti-CD20/CD74, hexavalent antibodies (HexAbs), designated 20-(74)-(74) and 74-(20)-(20), were generated from veltuzumab (anti-CD20 humanized mAb) and milatuzumab with the Dock-and-Lock (DNL) method. The effect of FTY720 on cell-surface expression of CD20, CD22, CD74, and HLA-DR was assessed by flow cytometry after treating JeKo-1, Granta-519 and Mino MCL cell lines with FTY720 for 18 h followed by labeling the cells with primary antibody and FITC-conjugated secondary antibody at 4°C. The in vitro cytotoxicity of the combination treatment was determined by Annexin V /PI binding assays and the data analyzed by the Chou-Talalay equation to calculate the combination index (CI). Results: In the three MCL lines tested, FTY720 at 12.5 μM induced almost 9-fold increase of CD74, a modest decrease of CD20, and no apparent change in CD22 and HLA-DR, when compared to untreated controls. Further studies in JeKo-1 incubated with varying doses of FTY720 revealed a 5-fold enhanced expression of CD74 could be achieved at 4 μM, but no notable changes were observed with FTY720 at 2.5 μM, or below. The viability of JeKo-1 cells treated with a single agent for 16 h was determined by the Annexin V/PI staining assay to be 45% for FTY720 at 4 μM and 65–70 % for either 20-(74)-(74) or 74-(20)-(20) at 33 nM. Combined treatments with FTY720 and 20-(74)-(74) at 4 μM and 33 nM, respectively, resulted in about 15% live cells (Annexin V-negative/PI-negative) with statistically significant P values ( Disclosures: Gupta: Immunomedics, Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics, Inc.: Consultancy, Employment, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Chang:Immunomedcis, Inc.: Employment, Patents & Royalties, Stock Options.
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- 2011
18. Whole Genome Sequence Analysis of 22 MLL Rearranged Infant Acute Lymphoblastic Leukemias Reveals Remarkably Few Somatic Mutations: A Report From the St Jude Children‘s Research Hospital - Washington University Pediatric Cancer Genome Project
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Guanchun Song, Sheila A. Shurtleff, Tanja A. Gruber, Matthew Parker, Li Ding, Jing Ma, Pankaj Gupta, Xin Hong, Anna Andersson, Ching-Hon Pui, Nicola V Venn, Gang Wu, Amanda Rush, Daniel Catchpoole, James R. Downing, Charles G. Mullighan, Linda Holmfeldt, Thoas Fioretos, Albert Chetcuti, Jianmin Wang, Debbie Payne-Turner, Jared Becksfort, Jesper Heldrup, Susana C. Raimondi, Richard K. Wilson, Charles Lu, Michael Rusch, Rosemary Sutton, Jinghui Zhang, Amanda Larson Gedman, Xiang Chen, Elaine R. Mardis, John Easton, and Anatoly Ulyanov
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Neuroblastoma RAS viral oncogene homolog ,Genetics ,Mutation ,Immunology ,Cell Biology ,Hematology ,Biology ,medicine.disease_cause ,Biochemistry ,Pediatric cancer ,PTPN11 ,CDKN2A ,medicine ,KRAS ,Gene ,Exome sequencing - Abstract
Abstract 69 Infant (< 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing. Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining. An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p In summary our analysis demonstrated an exceedingly small number of mutations required to generate infant MLL rearranged leukemia. The number of detected somatic mutations may represent the lower limit of mutations required to transform a normal human cell into cancer. Disclosures: Fioretos: Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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- 2011
19. Cannabinoids as Analgesics for Pain in Sickle Cell Disease
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Donald A. Simone, Sergey G. Khasabov, Yunfang Li, Robert P. Hebbel, Kalpna Gupta, Pankaj Gupta, Divyanshoo R. Kohli, and Marna E. Ericson
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Sickle Hemoglobin ,Pathology ,medicine.medical_specialty ,business.industry ,Transgene ,Immunology ,Cell ,Cell Biology ,Hematology ,Disease ,Catalepsy ,Bioinformatics ,medicine.disease ,Biochemistry ,medicine.anatomical_structure ,Dermis ,Neuropathic pain ,Medicine ,Severe pain ,business - Abstract
Abstract 822 Neural mechanisms underlying severe pain in sickle cell disease (SCD) remain unknown. Opioids, the primary medications for pain in SCD, are frequently associated with development of tolerance and side effects. We used transgenic heterozygous BERK mice expressing human sickle hemoglobin (hBERK) and age/sex matched mice of similar genetic background expressing normal human hemoglobin (HbA-BERK) to examine changes in skin morphology and innervation that may relate to behavioral measures of pain. Since cannabinoids have shown promise in treating inflammatory and neuropathic pain, we examined the ability of cannabinoids to treat pain in hBERK (sickle) and control mice. The epidermis and dermis were thinner in hBERK mice (p< 0.01). Confocal microscopy showed that pan-neuronal marker protein gene product 9.5 (PGP) immunoreactive nerve fibers in the epidermis and dermis were disorganized and that the dermis had fewer nerve fibers in hBERK than HbA-BERK mice. Expression of Calcitonin Gene Related Peptide and Substance P, neuropeptides found in nociceptive afferent fibers, was increased in hBERK skin, suggesting neuronal sensitization. We next examined pain sensitivity in hBERK and HbA-BERK mice. Paw Withdrawal Frequency (PWF) per 10 applications of a 1.0 g von Frey monofilament and 50% paw withdrawal threshold evoked by von Frey monofilaments ranging from 0.4 to 8.0 g were used to assess mechanical sensitivity. hBERK mice showed 277.6 ± 83% higher PWF and 35.9 ± 26% lower paw withdrawal threshold than HbA-BERK (p Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
20. Therapy of B-Cell Malignancies by Anti-HLA-DR Humanized Monoclonal Antibody, IMMU-114, Is Mediated through Hyperactivation of ERK and JNK MAP Kinase Signaling Pathways
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Pankaj Gupta, Rhona Stein, Thomas M Cardillo, Richard R Furman, Susan Chen, Chien-Hsing Chang, and David M. Goldenberg
- Subjects
Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Abstract 3738 Poster Board III-674 Introduction HLA-DR is currently under investigation as a target for antibody (mAb) therapy of B-cell malignancies. IMMU-114, a humanized IgG4 form of the murine anti-HLA-DR mAb, L243, recognizes a conformational epitope in the a-chain of HLA-DR. We have previously reported that IMMU-114 lacks effector-cell functions while displaying potent antitumor activity against B-cell lymphomas in vitro and in vivo. Here, we further investigated the cytotoxic effects of IMMU-114 on leukemia, lymphoma, and multiple myeloma (MM) cell lines, as well as clinical specimens of CLL, and explored the signaling pathways involved. Methods Binding and cytotoxicity of IMMU-114 was examined on a panel of NHL, AML, MCL, ALL, and CLL cell lines. Induction of apoptosis (Annexin V binding), ROS generation (dihydoehidium staining), changes in mitochondrial membrane potential (ψM, TMRE staining) and effects on the ERK1/2 and JNK pathways (immunoblot analyses) were evaluated on selected cell lines. The effects of various inhibitors on IMMU-114-mediated apoptosis also were determined. Studies were performed in tumor-bearing SCID mice. Results High expression of HLA-DR was detected on the cell surface of all MCL, ALL, HC, CLL, and NHL cell lines tested, as well as 2/3 AML and 5/6 MM cell lines. IMMU-114 was cytotoxic to 2/2 MCL, 2/2 CLL, 2/4 ALL, 1/1 HC, 2/2 NHL and 2/2 MM cell lines without any added anti-Fc second crosslinking antibody. IMMU-114 was consistently more cytotoxic than anti-CD20 mAbs on these cell lines, as well as on rituximab-resistant variants of Raji and SU-DHL-4. Despite positive staining, AML cell lines, Kasumi-3 and GDM-1, were not killed by IMMU-114. Four of 6 CLL patient samples expressed HLA-DR at markedly higher levels than CD20, with the remaining 2 showing similar HLA-DR and CD20 expression. Approximately 60% cytotoxicity was obtained after incubation with IMMU-114 in CLL patient cells. Induction of apoptosis as well as changes in ψM and ROS correlated with sensitivity of cell lines to IMMU-114-mediated cytotoxicity. Time-course analyses demonstrated that IMMU-114 induced approximately 46% change of ψM and 24% enhancement of ROS in as little as 30 min in Raji cells. These changes were abrogated in the presence of ROS inhibitor, N-acetyl cysteine. Immunoblot analyses revealed that IMMU-114 induces phosphorylation of ERK and JNK mitogen-activated protein (MAP) kinases in all cells defined as IMMU-114-sensitive by the cytotoxicity assays, but not IMMU-114-resistant cell lines. The p38 pathway was found to be constitutively active in these cell lines. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis, although the inhibition was not complete when any single inhibitor was used. This could be due to activation of multiple pathways, since inhibition of 2 or more pathways by specific inhibitors abolishes the apoptosis induced by IMMU-114. Activation (5-fold increases of phosphorylation) of ERK1/2 and JNK signaling pathways also was observed in CLL patient samples following IMMU-114 incubation. Further studies revealed caspase-independent apoptosis and activation of apoptosis-inducing factor (AIF) mediated by IMMU-114. The therapeutic activity of IMMU-114 was evaluated in vivo in 3 NHL models, WSU-FSCCL, Namalwa, and Raji. IMMU-114 was more effective than anti-B-cell mAbs such as rituximab, in all models. For example, in WSU-FSCCL, IMMU-114 treatment yielded 100% long-term survival. Although rituximab also induced significant responses compared to untreated mice (median survival time of 87.5 vs. 36 days for untreated controls, P Conclusion IMMU-114 is cytotoxic to a variety of hematological malignancies, and its potent activity can be related to high antigen expression of target cells and hyperactivation of ERK and JNK pathways upon binding to HLA-DR. Clinical evaluation of IMMU-114 is planned. Disclosures: Gupta: Immunomedics Inc.: Employment. Stein:Garden State Cancer Center: Employment. Cardillo:Immunomedics Inc.: Employment. Chang:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
- Published
- 2009
21. Translation Regulatory Protein 4E-Binding Protein (BP)-1 Phosphorylation Identifies Diffuse Large B Cell Lymphoma Patients with Low IPI Scores Who Experience Short Survival After Chemotherapy
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Ajay Rawal, Dennis Knapp, Pankaj Gupta, Mary J. Ninan, and Hector Mesa
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Oncology ,Regulation of gene expression ,medicine.medical_specialty ,Chemotherapy ,business.industry ,medicine.medical_treatment ,Immunology ,Eukaryotic initiation factor 4E binding ,Cell Biology ,Hematology ,CHOP ,Bioinformatics ,medicine.disease ,environment and public health ,Biochemistry ,Chemotherapy regimen ,enzymes and coenzymes (carbohydrates) ,Internal medicine ,medicine ,Phosphorylation ,Rituximab ,business ,Diffuse large B-cell lymphoma ,medicine.drug - Abstract
Abstract 1925 Poster Board I-948 Eukaryotic initiation factor 4E binding protein 1 (4E-BP1) regulates the function of eukaryotic initiation factor 4E (eIF-4E) which mediates nucleo-cytoplasmic transport and cap-dependent translation of specific transcripts. eIF-4E expression itself is elevated in several carcinomas and hematopoietic malignancies. Hypophosphorylated 4E-BP1 binds eIF-4E and inhibits cap-dependent translation. Phosphorylated 4E-BP1 dissociates from eIF-4E, thereby allowing translation of several cap-dependent transcripts responsible for cell cycling, survival and angiogenesis. Hyper-phosphorylation of 4E-BP1 may thus be associated with oncogenesis and the malignant phenotype; its influence on the response to chemotherapy and prognosis of diffuse large B cell lymphoma (DLBCL) is unknown. We examined diagnostic tissues from 85 patients with DLBCL for 4E-BP1 expression and phosphorylation, and correlated these with clinical outcomes. Samples were stained for total and phosphorylated 4E-BPI using immunohistochemistry (IHC). Slides were scored for percent cells positive for the two markers (0% to 100%) and the intensity of expression (0 to 3+) by hematopathologists (A.R. and H.M.) blinded to the clinical data. An Expression Score was calculated from their product ([% cells positive × intensity]/10), thus ranging from 0-30. In parallel, expression of bcl-2 was determined by IHC on the same samples. Patients had a median age of 75 years (range, 25-91). The majority received CHOP-based chemotherapy with/without rituximab. Total 4E-BP1 was expressed uniformly (by 97% of the cases). However, there was marked heterogeneity in the level of phosphorylation of 4E-BP1 (none [Expression Score = 0] in 35%, uniformly high [Expression Score = 30] in 34%, and variable [Expression Score = 1-29] in the remainder [31%]). We therefore examined if the level of phosphorylation of 4E-BP1 varied with clinical features at diagnosis, and whether it correlated with response to chemotherapy and survival. The level of expression or phosphorylation of 4E-BP1 did not correlate with the clinical stage, IPI score or bcl-2 expression at diagnosis, and did not influence the likelihood of achieving complete remission with CHOP-based chemotherapy. However, in patients with low IPI scores (IPI 0-2), the level of phosphorylation of 4E-BP1 correlated significantly with overall survival (OS). In these patients, the OS was significantly different (P = 0.031) in patients with low levels of phosphorylation of 4E-BP1 (Expression Score = 0-15) compared to those with higher levels of 4E-BP1 phosphorylation (Expression Score = 16-30). The median survival was >146.4 months vs 20.6 months, and the 5-year survival was 73% vs 31%, in patients with low and high levels of 4E-BP1 phosphorylation, respectively. The difference in survival between the two groups was not a consequence of differences in age or IPI score at diagnosis or inclusion of rituximab in the treatment regimen. The level of phosphorylation of 4E-BP1 did not have any further impact on the survival of patients with high IPI scores (IPI 4-5). We conclude that while the large majority of DLBCL express the translation regulatory protein 4E-BP1, the level of phosphorylation (indicative of biological activity) of this protein varies widely. Importantly, the level of phosphorylation of 4E-BP1 further stratifies DLBCL patients who would be expected to have a favorable prognosis based on low IPI scores, and identifies a subset of these patients who do poorly following standard CHOP-based chemotherapy. Since hyper-phosphorylation of 4E-BP1 increases the activity of eIF-4E, this study provides the rationale for trials incorporating novel targeted agents that inhibit eIF-4E into the therapeutic regimen, specifically in DLBCL patients with low IPI scores and high levels of 4E-BP1 phosphorylation. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
22. Diagnostic and Therapeutic Implications of Expression and Phosphorylation of the Translation Regulatory Protein 4E-Binding Protein (BP)-1 in B-Cell Lymphomas and Reactive Lymphoid Tissues
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Dhatri Kodali, Ajay Rawal, Dennis Knapp, Hector Mesa, Robert A. Kratzke, Mary J. Ninan, Manish R. Patel, and Pankaj Gupta
- Subjects
Regulation of gene expression ,Follicular dendritic cells ,Immunology ,Germinal center ,Cell Biology ,Hematology ,Biology ,Marginal zone ,environment and public health ,Biochemistry ,Molecular biology ,enzymes and coenzymes (carbohydrates) ,medicine.anatomical_structure ,Gene expression ,medicine ,Phosphorylation ,Lymph node ,B cell - Abstract
Abstract 3919 Poster Board III-855 Identifying pathogenic mechanisms that contribute to the development of lymphomas and influence clinical behavior is critical for developing targeted therapies, and selecting patients who may benefit from such drugs. An important level of control of gene expression occurs during initiation of cap-mediated mRNA translation by the eukaryotic initiation factor-4F (eIF-4F) trimolecular complex (eIF-4E, eIF-4G and eIF-4A), in which eIF-4E is rate limiting and oncogenic. eIF-4F hyperactivity plays a key role in human cancers by mediating expression of proteins critical for cell growth, transformation and tumorigenesis. eIF-4F activity is controlled by repressor eIF-4 binding proteins (BPs). 4E-BP1 activity is regulated by phosphorylation. Hypo/non-phosphorylated 4E-BP1 is active, binds eIF-4E and impedes eIF-4F formation, blocking translation and inducing apoptosis. Phosphorylation of 4E-BP1 (p4E-BP1) releases bound eIF-4E, which initiates cap-dependent translation. Because only limited information is available on the expression and phosphorylation of 4E-BP1 in lymphomas, and since agents (e.g., antisense oligonucleotides and small molecules) that target eIF-4E have been developed, we examined the frequency and level of expression of 4E-BP1 and its phosphorylation in various subtypes of mature B cell non-Hodgkin's lymphomas (BCL). Forty-six BCLs (12 follicular [FL], 13 diffuse large B-cell [DLBCL], 7 mantle cell, 5 extranodal marginal zone, and 9 small lymphocytic [SLL] lymphomas), 4 FL with incipient/partial lymph node involvement, and 11 reactive lymphoid tissues were examined using immunohistochemistry for total and phosphorylated 4E-BP1. Staining intensity was graded as from 0 to 3+. Western immunoblotting (WB) was performed on lysates of 5 mature BCLs (2 FL, 3 DLBCL) and 2 reactive lymph nodal tissues for eIF-4G (total), eIF-4E and 4E-BP1 (total and phosphorylated) expression. In reactive lymphoid tissues, there was regional and cellular specificity of expression of 4E-BP1, with either lack of, or minimal (0 to 1+) cytoplasmic expression in follicular center cells and paracortical T-cells, 2+ expression in follicular dendritic cells and paracortical zone Langerhan cells, and 3+ expression in mantle and marginal zones. p4E-BP1 expression was inverted, with 3+ cytoplasmic immunoreactivity in reactive follicular center cells and no expression in the mantle and marginal zone cells or T-cells, and 2+ or 3+ immunoreactivity in follicular dendritic cells and paracortical zone Langerhan cells. In BCLs, a consistently high level (2+ or 3+) of cytoplasmic 4E-BP1 expression was seen in neoplastic lymphocytes in 45/46 (98%) cases. In contrast, p4E-BP1 was moderately or strongly expressed in 19/46 (41%) cases of BCL, being negative in 17 (37%) cases, and only dimly expressed in the remaining 10 (22%) cases. Three of 4 cases with incipient/partial involvement by FL were easily distinguishable from reactive germinal centers by strong, diffuse staining with 4E-BP1 (and 1+ staining in the 4th case) in neoplastic follicles, distinct from negative/weak staining of adjacent reactive germinal centers. In SLL, slightly higher 4E-BP1 expression was noted in proliferation centers in comparison to surrounding small mature lymphocytes. WB confirmed that non-phosphorylated and p4E-BP1 were expressed in reactive nodes, FL and DLBCL. Other components of the eIF-4F complex including eIF-4G, total and p-eIF-4E and total 4E-BP1 were detectable in whole tissue lysates from BCL samples. We conclude that (a) while 4E-BP1 is almost uniformly expressed in various subtypes of BCL, its level of phosphorylation (indicative of activity) varies widely and has regional and cellular specificity, and (b) 4E-BP1 expression may identify minimal/early lymphomatous involvement in tissues. We speculate that 4E-BP1 phosphorylation may influence the biological behavior of BCLs, since in other investigations we found that the level of phosphorylation of 4E-BP1 correlates with survival after CHOP-based chemotherapy in DLBCL. Our findings support therapeutic trials targeting the eIF-4E pathway in many BCL subtypes, particularly in patients where immunostaining identifies high levels of 4E-BP1 phosphorylation. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
23. A Novel Ribonuclease-Based Immunotoxin Comprising Quadruple Ranpirnase (Rap) Site-Specifically Conjugated to An Anti-CD22 IgG Showing Potent Anti-Lymphoma Activity
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Edmund A. Rossi, Eric Chan, Chien-Hsing Chang, Pankaj Gupta, and David M. Goldenberg
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biology ,Chemistry ,Immunology ,Protein domain ,Cell Biology ,Hematology ,Humanized antibody ,Biochemistry ,Molecular biology ,Fusion protein ,Cell killing ,Affinity chromatography ,Immunotoxin ,Ranpirnase ,biology.protein ,sense organs ,Protein A - Abstract
Abstract 3721 Poster Board III-657 Background Rap is a single-chain ribonuclease of 104 amino acids originally isolated from the oocytes of Rana pipiens. Rap exhibits cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro, as well as antitumor activity in vivo. The amphibian ribonuclease enters cells via receptor-mediated endocytosis and once internalized into the cytosol, selectively degrades tRNA, resulting in inhibition of protein synthesis and induction of apoptosis. Rap is in advanced Phase IIIb clinical trials against malignant mesothelioma, with reversible dose-limiting renal toxicity and no reported immunogenicity. We have previously shown that effective therapy of human lymphoma xenografts could be achieved with a recombinant fusion protein comprising Rap and a rapidly internalizing anti-CD74 humanized IgG4 antibody (Chang et al., Blood, 2005; 106: 4308-14). In this study, we applied the Dock-and-Lock (DNL) method to generate another novel class of immunotoxins, each of which contains four copies of Rap site-specifically linked to a bivalent IgG. Methods The DNL platform technology exploits a pair of distinct protein domains involved in the natural binding of cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of 2 types of modules, one containing the dimerization and docking domain (DDD) of PKA and the other containing the anchoring domain (AD) of an interactive AKAP. We have combined a recombinant Rap-DDD module with a recombinant IgG-AD module derived from the internalizing anti-CD22 humanized antibody, epratuzumab, to generate 22-Rap, in which a dimer of Rap is covalently tethered to the c-terminus of each heavy chain of epratuzumab. The Rap-DDD module was expressed in E. coli as inclusion bodies, which were purified by immobilized metal affinity chromatography and refolded. The epratuzumab-AD module was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography. Results 22-Rap was prepared by mixing the epratuzumab-AD module with the Rap-DDD module under mild redox conditions and purified to near homogeneity in a single step using Protein A affinity chromatography. Size exclusion HPLC revealed the presence of a single peak of a retention time consistent with the molecular size of ∼230 kDa, indicative of an IgG and four Rap groups. In vitro transcription/translation assays showed that both the Rap-DDD2 module and 22-Rap retained the activity of Rap. Results of in vitro cytotoxicity assays using Daudi Burkitt lymphoma cells demonstrated that 22-Rap was highly cytotoxic, resulting in nearly 100% cell killing at 1 nM, with an IC50 Conclusion The DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity. Disclosures: Rossi: Immunomedics, Inc: Employment. Chan:Immunomedics, Inc: Employment. Gupta:Immunomedics Inc.: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.
- Published
- 2009
24. Does Thrombocytosis Alter the Prognosis of Myelodysplastic Syndromes (MDS) and MDS/Myeloproloferative Syndromes (MDS/MPD)? A Matched Case-Control Analysis of Outcomes
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Qing Cao, Hector Mesa, Ajay Rawal, Pankaj Gupta, and Dhatri Kodali
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medicine.medical_specialty ,Myeloid ,Thrombocytosis ,business.industry ,Proportional hazards model ,Myelodysplastic syndromes ,Immunology ,macromolecular substances ,Cell Biology ,Hematology ,Anagrelide ,medicine.disease ,Debulking ,Biochemistry ,Gastroenterology ,Surgery ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Internal medicine ,medicine ,business ,Myelofibrosis ,medicine.drug ,Cause of death - Abstract
Thrombocytosis at diagnosis is uncommon in myelodysplastic syndromes (MDS) and MDS/myeloproliferative syndromes (MDS/MPD). We conducted a retrospective analysis of 388 consecutive patients (pts) diagnosed with MDS and MDS/MPD between January 1980 and July 2006, to determine the effect of thrombocytosis on prognosis. The influence of thrombocytosis on the probabilities of progression to a higher grade of MDS, transformation to AML, and 5-year overall survival (OS) was determined by a case-matched design using the Kaplan Meier method and Cox regression analyses. A frequency summary of all 31 pts with MDS and MDS/MPD and platelet count > 400 × 106/mL at diagnosis is described. Using pre-defined matching criteria, 25 pts with thrombocytosis were case-matched with 50 control pts (1:2 matching ratio; relative risk 2, power 82%, α 0.05), for year of diagnosis, WHO classification and IPSS prognostic score. Of 388 pts with MDS or MDS/MPD, 31 (8% [95% CI, 5.3%-10.7%]) had thrombocytosis at diagnosis. The majority (N = 22, 71%) was re-classified as MDS/MPD (CMML = 12 [39%], RARS-T = 7 [23%], MDS/MPD-U = 3 [9%]) and N=9 (29%) as MDS (RARS = 5 [16%], 5q- = 2 [7%], MDS-U = 2 [6%]) according to the current WHO classification of myeloid disorders. Karyotypic abnormalities were noted in only 27% pts. The Jak-2 V617F mutation was present in 2 pts, both with RARS-T. The IPSS risk category was Low in the majority (18 pts), Int-1 in 3 pts and Int-2 in 1 pt. On > 95 patient-years of follow-up, the risks of major bleeding or thrombosis were low (0.04 and 0.01 events/patient-year, respectively). Marked thrombocytosis required cytoreduction with hydroxyurea in 6 pts and anagrelide in 1 pt, with good clinical response. Six of 31 (19%) pts progressed to a higher grade of MDS (CMML-2 or RAEB-2). Three pts (9.6%) transformed to AML, while 1 developed marked myelofibrosis. Death occurred as a consequence of AML in 3 pts, cytopenias due to MDS in 3 pts and sepsis in 1 pt. The cause of death was unrelated to MDS in 10 pts, and unknown in 3 pts. Eleven pts are currently alive. Matched case-control analyses showed that in pts with thrombocytosis, the probability of progression to a higher grade of MDS was lower (p = 0.03) but the risk of transformation to AML was equivalent to control pts. The median OS of pts with thrombocytosis was 35.4 months (range 0.3 - 60 months) compared to 27.6 months (range 0.5 - 60 months) for case-matched controls (p = 0.07). In conclusion, this study shows that the majority of pts presenting with myelodysplasia and thrombocytosis fall in the MDS/MPD category of the new WHO classification (most commonly CMML or RARS-T) and have low-risk features (IPSS category Low or Int-1). Thrombocytosis may reach levels requiring cytoreduction with hydroxyurea or anagrelide. The risks of spontaneous bleeding or thrombosis, progression of disease and myelofibrosis are low and the overall survival is comparable to that of case-matched pts without thrombocytosis. This is the only study that has examined the effect of thrombocytosis itself on prognosis by comparing pts with thrombocytosis to controls case-matched for standard prognostic factors.
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- 2007
25. A Reliable Strategy for Validation of Real-Time Quantitative RT-PCR Amplicons from Linearly Amplified Human Hematopoietic Stem Cell mRNA
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Matthew S. Nelson, Yonghong Xie, Anil C. Asrani, and Pankaj Gupta
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Sequence analysis ,Immunology ,Cell Biology ,Hematology ,Biology ,Amplicon ,Biochemistry ,Molecular biology ,Amplicon Size ,Real-time polymerase chain reaction ,Complementary DNA ,Primer dimer ,Agarose gel electrophoresis ,Primer (molecular biology) - Abstract
Real-time quantitative RT-PCR (qRT-PCR) is a powerful tool for measuring or validating gene expression. Enrichment for the most primitive hematopoietic progenitors yields small cell numbers and thus small amounts of mRNA. Linear amplification (required for gene expression analysis) amplifies the 3′ end of mRNA and imposes limitations on qRT-PCR primer design that may require the use of sub-optimal primers that can form primer dimers (PDs). Indeed, using linear amplification and qRT-PCR to assay expression of 31 genes of the bone morphogenetic protein (BMP) signaling cascade in CD34+/CD38−/lin− human umbilical cord blood (UCB) progenitors, we found that 17/31 (55%) of products were false positives. To distinguish true target amplicons from PDs, we therefore used a five-step sequential strategy to initially generate a Primer Profile for each primer set: water blank and positive control dissociation curve analysis serial dilution gel electrophoresis and product sequencing, which we describe for one gene (BMP-2) that we found is variably expressed in UCB progenitors under different culture conditions. Total RNA was obtained from 5–20 x 103 UCB progenitors cultured in conditions that induced detectable BMP-2 expression (BMP-2+) and those that did not (BMP-2−), mRNA was linearly amplified (RiboAmp) and cDNA was synthesized using Invitrogen Superscript III. qRT-PCR was performed using Invitrogen SYBR Green qPCR SuperMix on an ABI 7900 HT Sequence Detection System. (1) Though primers were designed for the 3′ end of BMP-2 using the Primer 3 website (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi) to identify optimal primers, an amplified product was seen in the water blank and in BMP-2− cDNA. Nevertheless, the primers also amplified the correct product in the positive control (SaOS-2 human osteosarcoma cells) and in BMP-2+ cDNA. (2) Although the genuine and the false positive products appeared similar on amplification (ΔRn vs cycle) plots, analysis of the derivative dissociation curves showed that their dissociation temperatures were distinct, being lower for the false positive PD (77°C vs 81°C). (3) Positive control and BMP-2+ cDNA yielded products with the expected one-cycle increase in cycle threshold (CT) for each 2-fold serial dilution, whereas PD product CTs did not change with serial dilution of BMP-2− cDNA. Importantly, PDs also formed at higher dilutions (≥ 1:16) of BMP-2+ cDNA, possibly because of low target mRNA abundance. (4) Agarose gel electrophoresis of the amplified products from the positive control and BMP-2+ cDNA showed the expected amplicon size of 107 bp, whereas the PD product was smaller (50 bp). (5) Finally, sequence analysis of the products (at the Univ. of Minnesota DNA Sequencing and Analysis Center) confirmed that the 107 bp product was identical with the 3′ end of human BMP-2, whereas the PD product did not yield any sequence. Similar differences were seen between genuine amplicons and PDs for several other genes examined. Thus, for each primer set, the Primer Profile provided the melting temperatures of the genuine amplicon and the PDs. For subsequent experiments, we were then able to reliably predict which product was a genuine amplicon by inspection of its melting temperature. These findings demonstrate the critical importance of using such a strategy to detect false positive qRT-PCR results due to PDs when using linear amplification of mRNA from primitive hematopoietic progenitors and other rare cell subpopulations.
- Published
- 2006
26. Thrombocytosis as a Presenting Feature in Myelodysplastic Syndromes (MDS) in Adults - 40 Year Experience from a Single Institution in the USA
- Author
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Hector Mesa, Ajay Rawal, Pankaj Gupta, and Dhatri Kodali
- Subjects
medicine.medical_specialty ,Myeloid ,Thrombocytosis ,business.industry ,Myelodysplastic syndromes ,Immunology ,macromolecular substances ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Surgery ,Leukemia ,medicine.anatomical_structure ,Dysplasia ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Atypical chronic myeloid leukemia ,Refractory cytopenia with multilineage dysplasia ,Myelofibrosis ,business - Abstract
Thrombocytosis at presentation is uncommon in the myelodysplastic syndromes (MDS), and its influence on the clinical course of the disease and on prognosis is uncertain. To determine the clinical course and long-term outcomes of patients (pts) with thrombocytosis at initial diagnosis of MDS, we conducted a retrospective analysis of 503 pts diagnosed with MDS between Jan 1966 and July 2006 at the Minneapolis VA Medical Center. Original bone marrow and peripheral blood slides and reports were reviewed with a hematopathologist (H.M.) in all pts with high platelet counts (> 400 × 103/μL) and evidence of dysplasia. Clinico-pathological correlation was obtained by chart review. Patients with inadequate data, secondary causes of thrombocytosis, transient thrombocytosis, and those without evidence of dysplasia were excluded. Of 503 pts, 41 (8.2%) were found to have thrombocytosis at presentation. Their median age was 74 years. The spleen was enlarged (by imaging) in 6 pts. Peripheral blood counts (mean; range) at diagnosis were: hemoglobin (10.9 g/dL; 7.4 – 17.1), absolute neutrophils (7.9 × 103/μL; 0.8 – 30.7) and platelets (627 × 103/μL; 402 – 1231). The cases were re-classified according to the WHO classification of myeloid disorders as follows: chronic myelomonocytic leukemia-1 (CMML-1) = 17 (41%), refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) = 13 (32%) and others = 11 (27%; including 2 pts each with RA, MDS/myeloproliferative disorder-unclassified [MDS/MPD-U] and 5q- syndrome, and 1 pt each with RA with excess blasts [RAEB-1], therapy related MDS [tMDS], refractory cytopenia with multilineage dysplasia [RCMD], MDS-U and atypical chronic myeloid leukemia [Aty CML]). Bone marrow fibrosis was increased in 3 pts with CMML-1 and 1 with RARS-T, and was normal or only focally increased in all other pts. The IPSS risk category was Low in 15 pts, Int-1 in 3 pts and Int-2 in 2 pts. Cytogenetic data was not available in the other pts. Jak-2 mutation analysis was positive in 2 pts with RARS-T, negative in 1 pt each with RARS-T and MDS/MPD-U, and is pending in others. On follow-up, 2 pts with CMML-1 and 1 pt with Aty CML transformed to acute myeloid leukemia (AML) and both pts with 5q- syndrome transformed to CMML-2. Two pts with RARS-T progressed to RAEB-2 and 1 pt with CMML-1 progressed to CMML-2. One pt with CMML-1 developed marked myelofibrosis. Marked thrombocytosis required hydroxyurea treatment in 5 pts. One MDS-U pt received 5-azacytidine. Four of 41 (10%) pts experienced major bleeding events; 3 were receiving aspirin. Five pts required ongoing red cell transfusions. The median survival (MS) was 36 months in RARS-T, 60 months in CMML-1 and 27 months in others; the MS of all 41 pts was 48 months. Causes of death were AML in 3 pts, cytopenias due to MDS in 6 pts and unrelated/unknown in 21 pts. Eleven pts are currently alive. In conclusion, the majority of pts presenting with myelodysplasia and thrombocytosis fall in the MDS/MPD category of the new WHO classification (most commonly CMML or RARS-T), be older, and have low-risk features (IPSS Low or Int-1). The risks of spontaneous bleeding, transformation to AML, progression of disease or myelofibrosis are low, and the overall prognosis is relatively favorable. Platelet counts may reach levels requiring cytoreductive therapy. This study helps better understand the natural history and prognosis of this varied spectrum of MDS and overlap syndromes.
- Published
- 2006
27. Heparin and Endogenous Heparan Sulfate Modulate Bone Morphogenetic Protein-4 (BMP-4) Signaling and Activity
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Shaukat Khan, Pankaj Gupta, Patrick M. Gaffney, Matthew S. Nelson, and Chendong Pan
- Subjects
animal structures ,Immunology ,Cell Biology ,Hematology ,Heparan sulfate ,Biology ,medicine.disease ,Bone morphogenetic protein ,Biochemistry ,Cell biology ,Extracellular matrix ,chemistry.chemical_compound ,Bone morphogenetic protein 4 ,chemistry ,embryonic structures ,medicine ,Signal transduction ,Chordin ,Hurler syndrome ,BMP signaling pathway - Abstract
Determining how extracellular matrix (ECM) components influence cytokine-induced growth and differentiation of cells in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel treatment strategies. Bone morphogenetic proteins (BMPs) regulate the growth, differentiation and apoptosis of cells in the brain, bone, bone marrow and diverse tissues. ECM glycosaminoglycans (GAGs) such as heparan sulfate (HS) interact with and influence the biological activity of a number of proteins including BMPs. We examined if heparin, endogenous HS in malignant cells and the structurally abnormal HS accumulated in Hurler cells influence BMP signaling and activity. First we showed using real-time quantitative RT-PCR (qRT-PCR) that the BMP signaling pathway including BMPs 2–7, BMP and activin receptors and Smad-1 and -5 are expressed by SaOS-2 human osteosarcoma cells. Western immunoblotting showed that BMP-4 induced Smad-1 phosphorylation, activation and nuclear translocation. Optimal Smad-1 activation was achieved by 25 ng/ml BMP-4 at 30–60 min, and blocked by the extracellular BMP antagonist chordin. BMP-4 also induced a concentration-dependent increase in alkaline phosphatase activity, indicative of induction of osteogenic differentiation in these malignant cells. Soluble heparin directly inhibited BMP-4 induced Smad-1 phosphorylation, and also markedly augmented the inhibitory effect of chordin. Similar effects were seen with N-desulfated, N-re-acetylated heparin but to a lesser degree than with heparin, indicating that N-sulfation of glucosamine residues in heparin/HS contributes to the effect of GAGs on BMP signaling. Inhibition of sulfation of endogenous GAGs by sodium chlorate augmented BMP-4 mediated increase in alkaline phosphatase, suggesting that endogenous sulfated GAGs themselves block BMP-4 mediated malignant cell differentiation. Because BMPs play a critical role in neurogenesis and osteogenesis, we also examined if GAGs that accumulate in Hurler syndrome impair BMP-4 signaling. Neurological dysfunction and skeletal abnormalities are among the most devastating manifestations of Hurler syndrome, an inborn metabolic disorder due to lack of lysosomal GAG-degrading α-L-iduronidase (IDUA) enzyme that leads to HS and dermatan sulfate GAG accumulation. We recently showed that HS in Hurler syndrome cells are structurally and functionally abnormal, and have impaired capability to bind and mediate FGF-2 signaling (Pan C et al. Blood2005;106:1956–64). In the present study, using Affymetrix microarrays we found that expression of the BMP signaling cascade including BMPs 1–8, -10 and -15, BMP and activin receptors, Smads 1–8, chordin and inhibitors of DNA binding (IDs) 1–4 is equivalent in normal and Hurler bone marrow derived multipotent progenitor cells. In Hurler cells, BMP-4 did induce a concentration- and time-dependent activation and nuclear translocation of Smad-1 (confocal immunofluorescent microscopy). However, BMP-4 activity was significantly enhanced following clearance of the abnormally accumulated GAGs in Hurler cells by recombinant IDUA enzyme, indicating that GAGs in Hurler cells impair BMP-4 activity. Thus, both endogenous GAGs and exogenous (soluble) heparin, via N- and O-sulfated disaccharide residues, inhibit BMP-4 activity. These findings have implications for understanding the pathobiology of diverse diseases, and for developing novel therapeutic agents that may restore BMP signaling and activity.
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- 2006
28. A Phase II Study of an Oral VEGF Receptor Tyrosine Kinase Inhibitor (PTK787/ZK222584) in Patients with Myelodysplastic Syndrome (MDS): Cancer and Leukemia Group B Study 10105
- Author
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Pankaj Gupta, Robert P. Hasserjian, Ravi Vij, Ben L. Sanford, Olatoyosi Odenike, David D. Hurd, Richard A. Larson, Clara D. Bloomfield, and Daohai Yu
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medicine.medical_specialty ,medicine.drug_class ,business.industry ,Myelodysplastic syndromes ,Immunology ,Phases of clinical research ,Common Terminology Criteria for Adverse Events ,Cell Biology ,Hematology ,Pharmacology ,medicine.disease ,Biochemistry ,Gastroenterology ,Sudden death ,Tyrosine-kinase inhibitor ,Tolerability ,Internal medicine ,Toxicity ,medicine ,Adverse effect ,business - Abstract
Angiogenesis may play a role in the pathophysiology and progression of myelodysplastic syndromes (MDS). The oral agent PTK787/ZK222584 (PTK/ZK; Novartis and Schering AG) inhibits receptor tyrosine kinases of vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) and Kit. In a phase II trial, we examined if PTK/ZK induces hematological responses and/or delays progression to acute myeloid leukemia (AML) or death in MDS. Patients (pts) previously untreated with cytotoxic agents for MDS received PTK/ZK 1,250 mg orally once daily for 28-day cycles until disease progression/unacceptable toxicity. To improve tolerability, the regimen was changed after 80 pts (cohort 1: C-I) to dose escalation: 750 mg for the first 28 d, escalated in the absence of pre-defined toxicity levels to 1,000 mg for 28 d, then 1,250 mg thereafter for cohort 2 (C-II; N=58 pts). Between 03/04 and 06/06, 138 pts were enrolled (median age, 70 yrs [range, 21–91]; 64% males) and continuous toxicity and efficacy assessments made. During the 3 months (mo) prior to study entry, 50% had received red cell and 12% received platelet transfusions. Toxicity data are available for 118/138 (85%) pts. A large proportion of toxicities occurred in the first 2 cycles, but the large majority resolved on drug discontinuation. The most common NIH Common Terminology Criteria for Adverse Events gr 2–4 non-heme toxicities at least possibly attributable to PTK/ZK were fatigue (56% in C-I; 37% in C-II), nausea (47%; 19%), vomiting (28%; 12%), dizziness (28%; 12%), ataxia (16%; 7%), anorexia (16%; 5%) and diarrhea (11%; 7%). The overall incidence of any gr 4 non-heme toxicity was 11% in C-I and 5% in C-II. We recently reported marked inter-individual variability in PTK/ZK exposure in C-I pts, but could not find an association between drug exposure and early non-heme adverse events (Gupta P et al. J Clin Oncol2006; 24[18S]:355s). Of 46 deaths, 2 (1 intra-cerebral hemorrhage, 1 sudden death) were possibly related to PTK/ZK. Heme toxicity (gr 1–4) attributable to PTK/ZK was reported in all 3 lineages. Compared with C-I, pts in C-II experienced less toxicity. However, early drug discontinuation (within 3 cycles, most often because of toxicity/intolerance, transformation to AML or lack of heme improvement) was similar (C-I=46/80 pts; C-II=31/58 pts). On central pathology review, 11 pts did not have MDS and were excluded from subsequent efficacy analyses. Of 89 MDS pts whose IPSS scores were available, 33% had high-risk (score ≥1.5; Int-2/High) and 67% had low-risk (score 0–1; Low/Int-1) MDS. At 6 and 12 mo, 16 and 2 pts respectively were still on PTK/ZK. Hematological improvement (HI; IWG criteria) occurred in 9/42 (21%) pts who received ≥3 cycles of PTK/ZK, and in no pts who received
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- 2006
29. Selective Modulation of the BMP Signaling Pathway Has Distinct Effects on Long-Term In Vitro Maintenance of Primitive Hematopoietic Progenitors in Human Umbilical Cord Blood
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Leah E. Colvin-Wanshura, Pankaj Gupta, Yonghong Xie, Matthew S. Nelson, Shaukat Khan, and Elliot J. Stephenson
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animal structures ,Immunology ,Cell Biology ,Hematology ,Biology ,Bone morphogenetic protein ,Biochemistry ,Cell biology ,BMPR2 ,Haematopoiesis ,embryonic structures ,Progenitor cell ,Noggin ,Stem cell ,Chordin ,BMP signaling pathway - Abstract
Factors responsible for long-term survival and proliferation of human hematopoietic stem cells (hHSC), and their mechanisms of action, remain to be defined. We previously showed that specific O-sulfated heparan sulfate (OS-HS) improves human long-term culture initiating cell (LTC-IC) maintenance for up to 5 wks in vitro. This is related to the ability of OS-HS to bind to and modulate the activity of heparin-binding cytokines on primitive hematopoietic progenitors (PHP). Recent studies indicate that bone morphogenetic proteins (BMPs) influence the development of embryonic hematopoiesis and also augment short-term survival and proliferation of hHSC. Since HS may modulate BMP activity, we examined how combinations of OS-HS, BMPs and specific BMP antagonists influence PHP in umbilical cord blood (UCB). First, we confirmed by real-time quantitative RT-PCR (qRT-PCR) that UCB CD34+ and/or CD34+/CD38− cells (using linear mRNA amplification) constitutively express transcripts for BMP-4 and its inhibitor Chordin, BMP receptors BMPR-IA, BMPR-IB, BMPR-II, AcvR-II and AcvR-IIB, downstream signaling proteins SMAD-1 and -5, and target genes upregulated by BMPs including Inhibitors of DNA binding (Id) proteins 1–4, and determined their relative levels of expression. BMP-4 upregulated Id2 expression 17-fold, confirming that the BMP signaling pathway is functionally active in CD34+ cells. Next, we demonstrated that OS-HS may protect BMP-4 from its inhibitors, using Western immunoblotting of immunoprecipitated proteins to show that direct binding of the antagonist Chordin to BMP-4 is inhibited by OS-HS in a dose-dependent manner. Finally, we examined the effect of exogenous supplementation with 6 BMPs, or inhibition of endogenous BMPs by 7 antagonists, on short-term (2 wk) and long-term (5 wk) LTC-IC maintenance in UCB CD34+/CD38− cells cultured in presence of OS-HS, Flt3-ligand and thrombopoietin. Long-term LTC-IC maintenance was enhanced by BMP-4 (LTC-IC maintenance: 164 +/− 11% compared to culture without BMP-4; P=0.0001) but reduced by BMP-2 or BMP-7 (P
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- 2005
30. Characterization of an Immunodeficient Mucopolysaccharidosis Type I (MPS-I) Mouse Model Suitable for Preclinical Testing of Human Stem Cell and Gene Therapy
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Leah E. Colvin-Wanshura, Walter C. Low, Pankaj Gupta, Matthew S. Nelson, Shaukat Khan, Zhenhong Nan, Mayra F. Quito-Rivera, Tyson B. Rogers, and Indrani Maitra
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Severe combined immunodeficiency ,medicine.medical_treatment ,Genetic enhancement ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Enzyme replacement therapy ,Biology ,medicine.disease ,Biochemistry ,Mucopolysaccharidosis type I ,Mucopolysaccharidosis I ,medicine ,Stem cell ,Hurler syndrome - Abstract
Mucopolysaccharidosis type I (MPS-I; Hurler syndrome) is an inborn metabolic disorder due to lack of the lysosomal glycosaminoglycan (GAG)-degrading enzyme alpha-L-iduronidase (IDUA). The resulting GAG accumulation causes progressive multi-system dysfunction and death in the first decade. We recently identified structural abnormalities in the accumulated GAGs that lead to defective heparin-binding cytokine signaling in MPS-I (Pan C et al, Blood 2005 in press: DOI 10.1182/blood-2005-02-0657), which may constitute a mechanism responsible for the diverse clinical manifestations of MPS-I and related mucopolysaccharidoses. Allogeneic hematopoietic stem cell transplantation (HSCT), if performed early in the disease, ameliorates many clinical features and extends life. However, HSCT is not available to all patients, and also does not adequately correct some of the most devastating features of MPS-I including mental retardation and skeletal deformities. Therefore, novel cellular, enzyme replacement and gene therapy approaches need to be developed for MPS-I. Such therapeutic strategies will best be tested in an immunodeficient host which is less likely to develop immune reactions to transplanted human or gene-corrected cells or their secreted IDUA enzyme. We have bred and characterized a homozygous immunodeficient MPS-I mouse (developed as a heterozygous MPS-I mouse on a NOD/SCID background by Jackson Labs) that is well suited for examining the efficacy of such therapeutic approaches. The phenotype of homozygous NOD/SCID/MPS-I mice closely mimicked the clinical features of human MPS-I including coarsening of facial features, skeletal deformities and diverse behavioral abnormalities. IDUA enzyme was completely undetectable in the liver, spleen, heart, lung, kidney and brain of homozygous animals (P
- Published
- 2005
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