107 results on '"A. Barakat"'
Search Results
2. Impact of soluble BCMA and non–T-cell factors on refractoriness to BCMA targeting T-cell engagers in multiple myeloma
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Lee, Holly, Durante, Michael, Skerget, Sheri, Vishwamitra, Deeksha, Benaoudia, Sacha, Ahn, Sungwoo, Poorebrahim, Mansour, Barakat, Elie, Jung, David, Leblay, Noemie, Ziccheddu, Bachisio, Diamond, Benjamin, Papadimitriou, Marios, Cohen, Adam D., Landgren, Ola, Neri, Paola, Maura, Francesco, and Bahlis, Nizar J.
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- 2024
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3. CRBN Structural Changes, Copy Number Changes and COP9 Signalosome Subunits Gene Expression Mediate Sensitivity to New Celmod Compound CC-92480 in Multiple Myeloma Patients
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Tilmont, Remi, primary, Maity, Ranjan, additional, Leblay, Noemie, additional, Lee, Holly, additional, Barakat, Elie, additional, Neri, Paola, additional, and Bahlis, Nizar J., additional
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- 2022
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4. Dysfunctional Hyper-Expanded Clonotypes and Lack of TCR Clonal Replacement Predict Resistance to T Cell Engagers in Multiple Myeloma
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Neri, Paola, primary, Ahn, Sungwoo, additional, Lee, Holly, additional, Leblay, Noemie, additional, Friedrich, Mirco, additional, Maity, Ranjan, additional, Tilmont, Rémi, additional, Barakat, Elie, additional, Raab, Marc S, additional, and Bahlis, Nizar Jacques, additional
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- 2022
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5. The Economic Impact of Treatment Sequencing in the Management of Chronic Lymphocytic Leukemia in Canada Using Venetoclax Plus Obinutuzumab
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Guinan, Kimberly, primary, Lachaine, Jean, additional, Paul Roc, Nancy, additional, Bull, Sarah-Jane, additional, Tankala, Dipti, additional, Barakat, Stéphane, additional, Hillis, Christopher M., additional, and Banerji, Versha, additional
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- 2022
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6. Barriers to Transition from Pediatric to Adult Care in Adolescents and Young Adults with Sickle Cell Disease: A Mixed-Method Study on the Impact of Systems of Inequity
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Prussien, Kemar V., primary, Faust, Haley L., additional, Smith-Whitley, Kim, additional, Barakat, Lamia P., additional, and Schwartz, Lisa A., additional
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- 2022
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7. Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)
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Lee, Holly, primary, Neri, Paola, additional, Ahn, Sungwoo, additional, Maity, Ranjan, additional, Leblay, Noemie, additional, Ziccheddu, Bachisio, additional, Chojnacka, Monika, additional, Tilmont, Rémi, additional, Barakat, Elie, additional, Landgren, Ola, additional, Maura, Francesco, additional, and Bahlis, Nizar Jacques, additional
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- 2022
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8. A Link between Blood Flow and the Inflammatory Networks That Govern Hematopoietic Stem Cell Specification
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Campbell, Clyde Allen, primary, Barakat, Radwa, additional, Calderon, Rodolfo, additional, and Espin Palazon, Raquel, additional
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- 2022
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9. Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)
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Holly Lee, Paola Neri, Sungwoo Ahn, Ranjan Maity, Noemie Leblay, Bachisio Ziccheddu, Monika Chojnacka, Rémi Tilmont, Elie Barakat, Ola Landgren, Francesco Maura, and Nizar Jacques Bahlis
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
10. CRBN Structural Changes, Copy Number Changes and COP9 Signalosome Subunits Gene Expression Mediate Sensitivity to New Celmod Compound CC-92480 in Multiple Myeloma Patients
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Remi Tilmont, Ranjan Maity, Noemie Leblay, Holly Lee, Elie Barakat, Paola Neri, and Nizar J. Bahlis
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
11. Barriers to Transition from Pediatric to Adult Care in Adolescents and Young Adults with Sickle Cell Disease: A Mixed-Method Study on the Impact of Systems of Inequity
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Kemar V. Prussien, Haley L. Faust, Kim Smith-Whitley, Lamia P. Barakat, and Lisa A. Schwartz
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
12. The Economic Impact of Treatment Sequencing in the Management of Chronic Lymphocytic Leukemia in Canada Using Venetoclax Plus Obinutuzumab
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Kimberly Guinan, Jean Lachaine, Nancy Paul Roc, Sarah-Jane Bull, Dipti Tankala, Stéphane Barakat, Christopher M. Hillis, and Versha Banerji
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
13. Dysfunctional Hyper-Expanded Clonotypes and Lack of TCR Clonal Replacement Predict Resistance to T Cell Engagers in Multiple Myeloma
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Paola Neri, Sungwoo Ahn, Holly Lee, Noemie Leblay, Mirco Friedrich, Ranjan Maity, Rémi Tilmont, Elie Barakat, Marc S Raab, and Nizar Jacques Bahlis
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
14. Mapping the Multiple Myeloma T Cell Landscape By Immunotherapeutic Perturbation Reveals Mechanism and Determinants of Response to Bispecific T Cell Engagers
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Friedrich, Mirco, primary, Neri, Paola, additional, Leblay, Noemie, additional, Kehl, Niklas, additional, Michel, Julius, additional, Lee, Holly, additional, Barakat, Elie, additional, Ahn, Sungwoo, additional, Goldschmidt, Hartmut, additional, Platten, Michael, additional, Weinhold, Niels, additional, Raab, Marc-Steffen, additional, and Bahlis, Nizar J., additional
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- 2021
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15. A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma
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Maity, Ranjan, primary, Benaoudia, Sacha, additional, Zemp, Franz, additional, Lee, Holly, additional, Barakat, Elie, additional, Leblay, Noemie, additional, Ahn, Sungwoo, additional, Mahoney, Douglas, additional, Neri, Paola, additional, and Bahlis, Nizar J., additional
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- 2021
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16. Integrated Epigenetic and Transcriptional Single Cell Analysis of t(11;14) Myeloma and Its BCL2 Dependency
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Leblay, Noemie, primary, Ahn, Sungwoo, additional, Maity, Ranjan, additional, Lee, Holly, additional, Barakat, Elie, additional, Bahlis, Nizar J., additional, and Neri, Paola, additional
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- 2021
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17. Scl regulates the quiescence and the long-term competence of hematopoietic stem cells
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Lacombe, Julie, Herblot, Sabine, Rojas-Sutterlin, Shanti, Haman, André, Barakat, Stéphane, Iscove, Norman N., Sauvageau, Guy, and Hoang, Trang
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- 2010
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18. Mapping the Multiple Myeloma T Cell Landscape By Immunotherapeutic Perturbation Reveals Mechanism and Determinants of Response to Bispecific T Cell Engagers
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Marc-Steffen Raab, Niels Weinhold, Julius Michel, Paola Neri, Nizar J. Bahlis, Michael Platten, Elie Barakat, Niklas Kehl, Sungwoo Ahn, Hartmut Goldschmidt, Mirco Friedrich, Noemie Leblay, and Holly Lee
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medicine.anatomical_structure ,Mechanism (biology) ,Chemistry ,T cell ,Immunology ,medicine ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Multiple myeloma ,Cell biology - Abstract
Immunotherapies have transformed the clinical care of patients with cancer. Bispecific T cell engagers (TCEs) have recently entered early-phase clinical trials of multiple myeloma (MM) and shown remarkable response rates even in heavily pretreated patients. However, T cells are heterogeneous with respect to phenotype, function and specificity for tumor antigens and currently we have limited understanding how to identify and monitor tumor specific T cells in hematological malignancies. It is furthermore unclear why individual patients fail to elicit an antitumor immune response upon treatment with TCEs and whether a persistent T cell response to TCEs relies on reinvigoration of pre-existing tumor-infiltrating lymphocytes or on recruitment of novel T cells. Here we performed longitudinal paired single-cell RNA and T cell receptor (TCR) sequencing on >100,000 immune cells from patients with MM before, during and after TCE therapy. We defined transcriptional gradients of MM-infiltrating immune cells between n=5 healthy bone marrow donors, n=10 newly diagnosed MM patients and n=11 refractory MM patients undergoing immunotherapy with bispecific BCMA-targeting antibodies. By tracking T cell clones over time using their TCR as individual barcode, we further integrated these longitudinal in vivo data with protein-level analysis and functional validation in MM bone-marrow cultures exposed to TCEs. Refractory MM patients exhibited a highly individual bone-marrow immune composition, that was significantly perturbed compared to healthy or diseased, but therapy-naïve bone marrow. We observed that the inter-patient heterogeneity in the T cell landscape composition is superimposed by conserved TCR repertoire dynamics forming a trajectory between early anti-tumor effector states and exhaustion. In all patients, we observed a dichotomy of TCE-responsive versus TCE-refractory T cell clones. Longitudinal tracking of TCE-responsive T cell clones and their transcriptional phenotypes revealed coupling of tumor recognition, clonal expansion and T cell dysfunction marked by expression of cytotoxicity (GZMB, GNLY) and terminal exhaustion markers, such as TOX and CD39. Significant clonal replacement of T cells was evident in n=5 clinically responding patients with MM throughout continued TCE therapy and driven by a subset of non-exhausted, naïve-like CD8 + T cells. The top 1% TCE-responsive clones were fate-determined and either followed a memory-exhaustion or cytotoxicity trajectory. Patients who did not respond to TCE therapy exhibited a dysfunctional T cell landscape before therapy that limited clonal expansion and TCR persistence. As proof-of-concept, we matched single-cell profiling data of n=10 individual patients with protein-level analysis and functional validation of TCE-driven T cell expansion in vitro, providing the first signals of preferential expansion of specific fate- and avidity-determined clones upon TCE-mediated stimulation. We propose the mode of action of TCE therapy in MM to be driven by pre-existing T cell fate commitments that determine clonotype diversification and persistence, and ultimately, clinical response. Our results further demonstrate that clinical TCE response derives from a distinct repertoire of pre-existing T cell clones, whereas other clonotypes are functionally excluded from the repertoire and subsequently lost during therapy. We define the determinants of response to TCE treatment to be inherent to the individual's T cell repertoire before therapy. Our results provide the rationale for response prediction and monitoring of future immunotherapy approaches in MM patients beyond TCE therapy. Figure 1 Figure 1. Disclosures Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Goldschmidt: Amgen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Adaptive Biotechnology: Consultancy; Celgene: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; BMS: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Chugai: Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; GSK: Honoraria; Incyte: Research Funding; Janssen: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Johns Hopkins University: Other: Grant; Molecular Partners: Research Funding; MSD: Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Dietmar-Hopp-Foundation: Other: Grant; Sanofi: Consultancy, Honoraria, Other: Grants and/or Provision of Investigational Medicinal Product, Research Funding; Takeda: Consultancy, Research Funding. Weinhold: Sanofi: Honoraria. Raab: Abbvie: Consultancy, Honoraria; Roche: Consultancy; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Bahlis: Amgen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.
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- 2021
19. Integrated Epigenetic and Transcriptional Single Cell Analysis of t(11;14) Myeloma and Its BCL2 Dependency
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Paola Neri, Nizar J. Bahlis, Elie Barakat, Holly Lee, Sungwoo Ahn, Ranjan Maity, and Noemie Leblay
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Dependency (UML) ,Single-cell analysis ,Immunology ,Cell Biology ,Hematology ,Epigenetics ,Computational biology ,Biology ,Biochemistry - Abstract
Multiple myeloma is characterized by recurrent chromosomal translocations that involve the immunoglobulin gene enhancers and partners such as the cyclin D genes (CCND1, CCND2 or CCND3) or other genes like WHSC1 and MAF. t(11;14) results in upregulation of CCND1 with unique morphological, phenotypic markers, and drug sensitivity profiles with exquisite sensitivity to BCL2 inhibitors. It is evident now that this unique sensitivity profile is driven by the BH3-proapoptotic protein priming of BCL2 with high BCL2/MCL1 or BCL2/BCL2L1 ratios. However, the epigenetic mechanisms associated with t(11;14) and their impact on genes regulation and clinical response to venetoclax remains elusive. In the present study we compared the transcriptomics (scRNA-seq) and chromatin accessibility (scATAC-seq) of single plasma cells of MM patients with and without t(11;14) as well as pre- and post-venetoclax exposure in order to establish the epigenomic signature of t(11;14) and/or BCL2-sensitivity in myeloma. Serial BM aspirates (n=24) were collected from 15 relapsed or refractory myeloma patients (RRMM); harboring t(11;14) (n=6 pairs) and 9 without this translocation prior to initiation of salvage therapy and at time of relapse. All t(11;14) MM patients were treated with venetoclax. Unbiased chromatin accessibility and mRNA profiling of CD138 pos cells were performed using the chromium single cell ATAC and RNA-Seq 3' solution (10x Genomics), respectively. Cell Ranger, Seurat and ArchR were used for sample de-multiplexing, barcode processing, single-cell 3' gene, peaks counting, and data analysis. We first compared the scATAC-seq and scRNA-seq profiles found in CD138 pos MM cell isolated from patients harboring t(11;14) with the one obtained in patients without this translocation. As expected, t(11:14) patients had high chromatin accessibility at the CCND1 locus and high mRNA expression. Differentially accessible chromatin analysis identified 147518 peaks that were specific to t(11;14) patients. Of interest, motifs enrichment analysis of accessible peaks identified a "B cell-like" motifs signature with enriched TFs motifs such as TCF4 and PAX5 in t(11;14) patients compared to non t(11:14) enriched for IRF and STAT family of motifs. The integration of the scATAC-seq and scRNA-seq data confirmed the B cell signature of t(11;14) patients with upregulation of B cell markers such as MS4A1, VPREB3, CD79A, CD19, and down-regulation of plasma cell markers such as TDO2, EFEMP1, CD28, SLAMF7, and IL6R. Additionally, we found PAX1, PAX5, TCF3, TCF5, and SPI1 transcription factors to be highly expressed in t(11;14) while the non t(11:14) were enriched for IRF1-9 transcription factors. Of interest, the clustering analysis performed on scATAC-seq data identified 3 non t(11;14) patients with a chromatin accessibility profile similar to that of t(11;14) patients. They expressed B cell markers (PAX5, VPREB3 or FCRLA), overexpressed BCL2 and we are currently examining whether this B cell-like epigenetic signature determines sensitivity to venetoclax. In order to define the epigenetic contribution to the acquired resistance to venetoclax in t(11;14) myeloma, we compared the chromatin accessibility profiles of t(11;14) patients pre- vs. post-venetoclax treatment. Enriched motifs within accessible peaks differed significantly between pre- and post-venetoclax with RELA, REL, RELB and EGR1 motifs predominantly enrichmed in the pre-samples in contrast to JUN, JUNB, JUND and FOSL1/L2 motifs enrichment in the post-samples. Of note, integration analysis of scRNAseq (differentially expressed genes) and ATACseq data (differentially accessible peaks) identified MCL1 and ENSA (a gene 60 Kb centromeric to MCL1 on chr1q) as the top enriched genes and peaks in resistant samples suggesting that copy number gain at the MCL1 locus (which we confirmed by single cell CNV analysis) rather than epigenetic modifications is likely the main determinant of acquired resistant to venetoclax in t(11;14) MM. In the current study we have defined the epigenetic regulome and transcriptome associated with t(11;14) myeloma and its relatedness to B cell rather than plasma cell biology. Our studies also suggest that acquired resistance to venetoclax is largely driven by copy number gain at the MCL1 locus. Disclosures Bahlis: Karyopharm: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria. Neri: BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.
- Published
- 2021
20. A BCL2L1 Armoured BCMA Targeting CAR T Cell to Overcome Exhaustion and Enhance Persistence in Multiple Myeloma
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Ranjan Maity, Sacha Benaoudia, Franz Zemp, Holly Lee, Elie Barakat, Noemie Leblay, Sungwoo Ahn, Douglas Mahoney, Paola Neri, and Nizar J. Bahlis
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Chimeric antigen receptor (CAR) T cells targeting the B-cell maturation antigen (BCMA) have resulted in deep responses in patients with relapsed MM however most remissions are not sustained. While cellular and molecular mediators of relapse post CAR T therapy in MM are not fully delineated, current data suggest three possible mechanisms including the lack of persistence of the CAR T cell product, acquired exhaustion and less commonly loss of BCMA expression. Using CITE-seq we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in serial blood and marrow samples (n=10) collected from patients treated with BCMA targeting CAR T cells. CAR T cells were identified by the expression of the chimeric CAR T cell transcript. With the exception of one patient where biallelic loss of BCMA was identified at relapse, CAR T cells of resistant patients were enriched with terminally exhausted CD45RA+ cells with loss of CD28, low BCL2L1 (gene encoding BCL-XL) expression, high CD57 with co-expression of checkpoint inhibitors (LAG3, TIGIT and PD1). The lack of persistence of the CAR T cells product was notable in all relapsing patients consistent with an activation induced cells death (AICD) specially in the setting of chronic antigenic stimulation. Cognizant of the role BCL-XL plays in T cells survival in response to CD28 co-stimulatory signaling, we postulated that increasing BCL-XL expression is a feasible strategy to enhance CAR T cell resistant to AICD, improve their persistence and anti-BCMA reactivity. To this goal, we designed a 2nd generation lentiviral CAR construct where the anti-BCAM scFV-41BBz CAR and the BCL2L1 cDNA were linked with self-cleaving 2A sequence. The efficiency in eradicating MM cells of this BCL-XL armored CAR (BCMA_BCL2L1_CAR) was compared to that of non-unarmored CAR (BCMA_CAR) in vitro and i n vivo studies. While BCMA_BCL2L1_CAR and BCMA_CAR were equally cytotoxic to OPM2 MM cells, in MM cell lines expressing the FAS death receptor ligand FASLG (MM1S, OCMY5 and H929) BCMA_BCL2L1_CAR viability and cytolytic activity was significantly superior to that of unarmored BCMA_CAR. Of note, the expression of FASLG, a known interferon response gene, was upregulated in H929 cells when co-cultured with CAR T cells. Importantly, under chronic antigenic stimulation conditions (FIG 1A), where CAR T cells were stimulated every 6 days over a 28 days period with irradiated OPM2 cells, we found no phenotypic difference between BCMA_BCL2L1_CAR and BCMA_CAR with respect to the composition of effector memory T cells (Tem: CCR7− CD45RO+ CD45RA−) or central memory T cells (Tcm: CCR7+CD45RO+CD45RA−) or terminal effector / exhausted T cells. However, under these chronic antigenic stimulation conditions, the CAR T cells viability, proliferation (FIG 1B) and anti-MM cytotoxic activities (FIG 1C) of the BCMA_CAR were dramatically reduced compared to that of the BCL2L1 armored CAR. Furthermore, in initial animal studies where NOD-SCID mice were tail vein injected with 2e6 OPM2 MM cells transduced with a luciferin reporter gene, followed 10 days later by control T cells, BCMA_CAR or BCMA_BCL2L1_CAR T cells IV injection, and despite a skewing to a larger initial disease burden in the BCMA-BCL2L1-CAR group, BCL2L1 armored CAR T cells resulted in more prolonged disease control and animal survival compared to the BCMA_CAR treated mice (FIG 1D). Our studies indicate that BCL2L1 blockade of AICD not only enhanced the viability and proliferation of BCMA targeting CAR T cells but surprisingly also reduced their functional exhaustion. Our findings provide an novel approach for CAR T optimization and overcoming disease relapse resulting from lack of persistence and/or T cells exhaustion. Figure 1 Figure 1. Disclosures Neri: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.
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- 2021
21. Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia
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Knee, Kelly M., primary, Barakat, Amey, additional, Tomlinson, Lindsay, additional, Ramaiah, Lila, additional, Wenzel, Zane, additional, Kapinos, Brendon, additional, Ahn, Youngwook, additional, Lintner, Nathanael G., additional, Field, S. Denise, additional, Jasuja, Reema, additional, and Pettersen, Betty A., additional
- Published
- 2020
- Full Text
- View/download PDF
22. Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy
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Leblay, Noemie, primary, Maity, Ranjan, additional, Barakat, Elie, additional, McCulloch, Sylvia, additional, Duggan, Peter, additional, Jimenez-Zepeda, Victor, additional, Bahlis, Nizar J., additional, and Neri, Paola, additional
- Published
- 2020
- Full Text
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23. Cite-Seq Profiling of T Cells in Multiple Myeloma Patients Undergoing BCMA Targeting CAR-T or Bites Immunotherapy
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Paola Neri, Nizar J. Bahlis, Noemie Leblay, Ranjan Maity, Peter Duggan, Victor H Jimenez-Zepeda, Elie Barakat, and Sylvia McCulloch
- Subjects
medicine.medical_treatment ,T cell ,Immunology ,GATA3 ,CD28 ,Cell Biology ,Hematology ,Immunotherapy ,Biology ,Biochemistry ,Chimeric antigen receptor ,Immune system ,medicine.anatomical_structure ,TIGIT ,medicine ,Cancer research ,CD8 - Abstract
Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) have demonstrated encouraging responses in heavily pre-treated multiple myeloma (MM) patients. However, the cellular and molecular predictors of clinical response are not fully understood as well as the mediators of acquired resistance remain elusive. Local immune suppression and T cell exhaustion are important mediators of responses therefore, it is plausible to speculate that a tolerant tumor microenvironment and the expansion of specific T cell populations may dictate clinical responses. In this study, we performed at the single cell level a broad immunophenotypic and transcriptomic characterization of the blood and bone marrow (BM) T cells of sensitive and resistant MM patients treated with adaptive T cell therapies. Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) we measured the expansion of variable T cell subsets, T cell specific activation and inhibitor markers and their functional states in order to identify cellular mediators of resistance to these adoptive immune therapies. Serial blood samples and BM aspirates (n=12) were collected from patients treated with anti-BCMA CAR-T or BCMA-CD3 BiTEs at variable time points, prior and post initiation of therapy and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD3pos T cells. Unbiased mRNA profiling coupled with feature barcoding technology for cell surface protein (TotalSeq-B) of BM CD3pos T cells was then performed by using the chromium single cell (10x Genomics). Paired-end sequencing was performed on Illumina platform. Cell Ranger and Seurat pipeline were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting, cell surface protein expression and data analysis. CAR-T cells were identified by the expression of the chimeric CAR-T cell transcript. The parallel measurement of transcripts and cell surface protein phenotypes of CD3pos T cells using a panel of 19 immune surface markers underlined the T cell repertoire diversity and identified different T cell subsets among the CD8pos and CD4pos T cells. Notably, the cell surface protein information overlaid on the transcript-generated UMA allowed accurate identification of all main immune clusters, in particular for the CD45RA and CD45RO positive cells. Comparison of CITE-Seq features revealed that the T cells composition of the blood and BM niches differed significantly between sensitive and resistant patients. As such an enrichment of CD4pos T cells with a higher CD4:CD8 ratio was noted in responding patients. Phenotypic (CD45RA, CD45RO, CD95, CCR7, CD62L, CD28, CD27) and transcriptional signatures (TCF7, LEF1, GATA3, EOMES, TBX21, PRDM1) also identified a higher proportion of memory like T cells (Tscm, Tcm) in responding patients. In contrast, T cells of resistant patients were enriched with terminally exhausted (Tex) and senescent cells with loss of CD28, high GMZHand GMZB, CD57pos, CD69pos and CD160pos as well as upregulation of TBX21. Expression of T cell checkpoint inhibitors such as LAG3, TIGIT and PD1 was high in these Tex cells as well as in some Tem. Of note, ex vivo T cell activation studies with TIGIT blockade demonstrated T cell activation in an autologous MM and T cell co-culture system with enhanced MM cells death. An expanded cluster of regulatory T cells (Treg) FOXP3pos,CD25pos was also observed in two resistant patients. Of note, no loss of BCMA transcript or surface expression was noted in MM cells at the time of acquired resistance. Single cell transcriptome of primary MM cells and chromatin accessibility (ATAC-seq) analyses of T cells of these patients are ongoing to investigate the transcriptional programs and epigenetic factors underlying the immune escape. Combined single cell features profiling of the transcriptome and surface protein expression of T cells from MM patients receiving BCMA targeted CAR-T or BiTEs therapies revealed potential mediators of resistance. In particular, T cells composition (low CD4:CD8 ratio and reduced population of Tscm, Tcm) along with an enrichment of terminally exhausted T cells are the main features observed in resistant patients. Delineating these mechanisms will guide future T cells engineering studies to enhance the efficacy and response durability of adoptive immunotherapy in MM. Disclosures McCulloch: Amgen: Honoraria; Sanofi: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Duggan:Jannsen: Consultancy; Amgen: Consultancy; Novartis: Honoraria; Celgene: Consultancy; Astra Zeneca: Consultancy. Jimenez-Zepeda:Janssen, Celgene, Amgen, Takeda: Honoraria. Bahlis:AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Karyopharm Therapeutics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria. Neri:Celgene/BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.
- Published
- 2020
24. Sickle Cell Disease Model Mice Lacking 2,3-Dpg Show Reduced RBC Sickling and Improvements in Markers of Hemolytic Anemia
- Author
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Betty Pettersen, Lila Ramaiah, Nathanael G. Lintner, Kelly M. Knee, S. Denise Field, Reema Jasuja, Youngwook Ahn, Lindsay Tomlinson, Amey Barakat, Brendon Kapinos, and Zane Wenzel
- Subjects
Hemolytic anemia ,medicine.anatomical_structure ,business.industry ,Immunology ,Cell ,Medicine ,Cell Biology ,Hematology ,Disease ,business ,medicine.disease ,Biochemistry - Abstract
Sickle cell disease (SCD) is a severe genetic disorder caused by a mutation in hemoglobin (b6Glu-Val), which allows the mutant hemoglobin to assemble into long polymers when deoxygenated. Over time, these polymers build up and deform red blood cells, leading to hemolytic anemia, vaso-occlusion, and end organ damage. A number of recent therapies for SCD have focused on modulating the mutant hemoglobin directly, however, reduction or elimination of 2,3-DPG to reduce Hb S polymerization and RBC sickling has recently been proposed as a therapeutic strategy for SCD. Current clinical studies focus on activation of pyruvate kinase to reduce 2,3-DPG, however, direct targeting of the enzyme which produces 2,3-DPG; Bisphosphoglycerate Mutase (BPGM) may also be possible. In this study we evaluate the impact of elimination of 2,3-DPG on SCD pathology by complete knockout of BPGM in Townes model mice. Animals with complete knockout of BPGM (BPGM -/-) have no detectable 2,3-DPG, while animals that are heterozygous for BPGM (BPGM -/+) have 2,3-DPG levels comparable to Townes mice. Western Blot analysis confirms that BPGM -/- animals completely lack BPGM, while BPGM -/+ animals have BPGM levels that are nearly equivalent to Townes mice. As expected from the lack of 2,3-DPG, BPGM -/- animals have increased oxygen affinity, observed as a 39% decrease in p50 relative to Townes mice. Complete elimination of 2,3-DPG has significant effects on markers of hemolytic anemia in BPGM -/- mice. Mice lacking 2,3-DPG have a 60% increase in hemoglobin (3.7 g/dL), a 53% increase in red blood cell count, and a 29% increase in hematocrit relative to Townes mice. The BPGM -/- mice also have a 57% decrease in reticulocytes, and a 61% decrease in spleen weight relative to Townes animals, consistent with decreased extramedullary hematopoiesis. Consistent with the reduction in hemolysis, BPGM -/- animals had a 59% reduction in red blood cell sickling under robust hypoxic conditions. BPGM -/+ animals had hemoglobin, RBC, and hematocrit levels that were similar to Townes animals, and a similar degree of RBC sickling to Townes mice. Liver phenotype was similar across all variants, with areas of random necrosis observed in BPGM -/-, BPGM -/+ and Townes mice. Higher percentages of microcytic and/or hyperchromic RBCs were observed in BPGM -/- animals relative to BPGM -/+ or Townes animals. These results suggest that modulation of 2,3-DPG has a positive effect on RBC sickling and hemolytic anemia, which may have therapeutic benefits for SCD patients. However, the lack of improvement in organ damage suggests that modulation of 2,3-DPG alone may not be sufficient for complete elimination of SCD phenotypes, and further investigation of this therapeutic avenue may be necessary. Disclosures No relevant conflicts of interest to declare.
- Published
- 2020
25. A Novel Non-Covalent Modulator of Hemoglobin Improves Anemia and Reduces Sickling in a Mouse Model of Sickle Cell Disease
- Author
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Knee, Kelly M., primary, Jasuja, Reema, primary, Barakat, Amey, primary, Rao, Dharani, primary, Wenzel, Zane, primary, Sahasrabudhe, Parag, primary, Narula, Jatin, primary, Jasti, Jay, primary, Chang, Jeanne S., primary, Beaumont, Kevin, primary, Piotrowski, David W., primary, and Janz, Jay M., primary
- Published
- 2019
- Full Text
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26. A Novel Non-Covalent Modulator of Hemoglobin Improves Anemia and Reduces Sickling in a Mouse Model of Sickle Cell Disease
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Kevin Beaumont, Dharani Rao, Amey Barakat, David W. Piotrowski, Jay Jasti, Parag Sahasrabudhe, Jeanne S. Chang, Jay M. Janz, Zane Wenzel, Reema Jasuja, Kelly M. Knee, and Jatin Narula
- Subjects
Hemolytic anemia ,medicine.diagnostic_test ,Anemia ,Chemistry ,Immunology ,Cell ,Cell Biology ,Hematology ,Hypoxia (medical) ,Hematocrit ,medicine.disease ,Biochemistry ,Sickle cell anemia ,medicine.anatomical_structure ,medicine ,Cancer research ,Hemoglobin ,medicine.symptom ,Vaso-occlusive crisis - Abstract
Sickle cell disease (SCD) is a severe genetic disorder caused by a single point mutation on the β-chain of adult hemoglobin (Hb A), β6 Glu→Val (Hb S). In the deoxygenated state Hb S polymerizes, leading to RBC sickling and precipitating all downstream consequences, including vaso-occlusion (pain crisis), hemolytic anemia, and stroke. Over time, these features cause significant organ damage and eventual organ failure, dramatically impacting both quality of life and expected lifespan. Numerous small molecules which covalently bind to Hb S have been evaluated clinically, however, the molecules that have demonstrated clinical efficacy all carry a reactive aldehyde group. The reactive aldehyde, a moiety that has the potential to react with any free amine, forms a covalent Schiff base with the N-terminal amine of the α1-Val. At least one member of this class of molecules, Tucaresol, showed a significant safety signal attributed to off-target Schiff base formation. An early investigation of covalent hemoglobin modification, extracorporeal carbamylation, both improved anemia and decreased the frequency of vaso-occlusive events by 80%, when there was a sufficiently high level of modification (30-50%). These results suggest that a molecule that binds Hb S and stabilizes the oxygenated state can impact both hemolytic anemia and vaso-occlusive crisis, if the molecule can achieve the necessary degree of hemoglobin modification. PFE-001 is a non-covalent molecule which binds selectively to Hb S and stabilizes the oxygenated state. Biochemical and biophysical studies show that PFE-001 binds specifically to Hb with double digit nanomolar potency and exhibits strong in vivo partitioning into RBCs. In a two-week multiple dose study using Townes SCD model animals (200 mg/kg, twice daily), PFE-001 significantly improved markers of hemolytic anemia, increased oxygen affinity, and reduced RBC sickling. Following 15 days of treatment blood drawn from PFE-001 treated animals and exposed to intense hypoxic conditions (4% O2, 4 hr) showed a 37.8% reduction in sickling compared to vehicle treated mice. Oxygen affinity was increased, demonstrated by a 53.7% reduction in p50 and an 84.4% reduction in p20 in the PFE-001 treated group. Hemoglobin levels in mice treated with PFE-001 increased by 42%, a mean increase of 5 g/dL. Hematocrit in the PFE-001 treated group increased to 42%, in contrast to 29% in the vehicle group. Reticulocyte percentages were reduced from 53% in vehicle treated animals to 24% in PFE-001 treated animals. In addition to the significant impact PFE-001 had on hemolytic anemia, a 10% reduction in sVCAM-1 levels in the PFE-001 treated group indicates a small but statistically significant improvement in vasculopathy following 15 days of treatment. This improvement in vasculopathy suggests that PFE-001 has the potential to address vaso-occlusive crisis in addition to anemia. In total, the in vitro and in vivo data suggest that PFE-001 is a potent, selective, and effective inhibitor of Hb S polymerization and RBC sickling. PFE-001 can reduce hemolytic anemia, improve vasculopathy, increase oxygen affinity, and reduce RBC sickling under hypoxic conditions. Plans for advancement of PFE-001 to clinical trials are in progress. Disclosures Knee: Pfizer Inc: Employment. Jasuja:Pfizer Inc.: Employment. Barakat:Pfizer Inc.: Employment. Rao:Pfizer Inc.: Employment. Wenzel:Pfizer Inc.: Employment. Sahasrabudhe:Pfizer Inc.: Employment. Narula:Pfizer Inc.: Employment. Jasti:Pfizer Inc.: Employment. Chang:Pfizer Inc.: Employment. Beaumont:Pfizer Inc.: Employment. Piotrowski:Pfizer Inc.: Employment. Janz:Pfizer Inc.: Employment.
- Published
- 2019
27. Quality of Life in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group
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Nagarajan, Rajaram, primary, Alonzo, Todd A, additional, Gerbing, Robert B, additional, Johnston, Donna, additional, Aplenc, Richard, additional, Kolb, E. Anders, additional, Meshinchi, Soheil, additional, Barakat, Lamia, additional, and Sung, Lillian, additional
- Published
- 2018
- Full Text
- View/download PDF
28. Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model
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Barakat, Amey, primary, Jasuja, Reema, additional, Murphy, John E., additional, and Pittman, Debra D, additional
- Published
- 2018
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29. Quality of Life in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group
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Richard Aplenc, Lillian Sung, Lamia P. Barakat, Soheil Meshinchi, Todd A. Alonzo, Rajaram Nagarajan, Donna L. Johnston, E. Anders Kolb, and Robert B. Gerbing
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0301 basic medicine ,Sorafenib ,Oncology ,medicine.medical_specialty ,medicine.medical_treatment ,Immunology ,Neutropenia ,Biochemistry ,law.invention ,03 medical and health sciences ,Randomized controlled trial ,law ,Internal medicine ,medicine ,Adverse effect ,Chemotherapy ,business.industry ,Repeated measures design ,Common Terminology Criteria for Adverse Events ,Cell Biology ,Hematology ,medicine.disease ,Chemotherapy regimen ,030104 developmental biology ,business ,medicine.drug - Abstract
Background: Objectives were to describe guardian proxy-reported quality of life (QoL) during chemotherapy for pediatric acute myeloid leukemia (AML) and to identify treatment and demographic factors associated with worse QoL. Methods: Children's Oncology Group phase 3 AAML1031 study was a randomized trial for de novo AML patients age 0-30 to receive standard AML therapy with and without bortezomib. Patients with high risk FLT3-internal tandem duplication high allelic ratio (ITD HAR) were allocated to receive sorafenib in addition to the standard chemotherapy. Patients enrolled on the AAML1031 study who were 2-18 years of age at diagnosis with English or Spanish-speaking guardians were eligible to participate in the QoL portion of the study which included the PedsQL 4.0 Generic Core Scales, PedsQL 3.0 Acute Cancer Module and PedsQL Multidimensional Fatigue Scale. QoL assessments were obtained at four timepoints - at diagnosis and following completion of second, third and fourth (final) course of therapy. Guardians provided proxy assessments for all patients, while self-report for patients 5 years of age or older who could understand English was optional. This analysis focused on guardian proxy-reported QoL for patients who did not have FLT3-ITD HAR. In addition to demographic and treatment related factors, the total number of non-hematological grade 3-4 CTCAE (Common Terminology Criteria for Adverse Events) toxicities occurring during the time frame of QoL assessments was examined as a potential predictor of QoL. Results: There were a total of 4105 QoL submissions and there were 3513 non-hematological grade 3-4 CTCAE toxicities reported: 1339 submissions at diagnosis with 1088 toxicities reported, 1112 submissions following the second course with 721 toxicities, 929 submissions following third course with 911 toxicities, and 725 submissions following the fourth course with 793 toxicities. In repeated measures linear regression the number of submitted CTCAE toxicities was significantly associated with worse physical health (β±standard error (SE) -3.00±0.69; P Conclusions: The number of CTCAE toxicities was an important factor influencing physical QoL and fatigue among children on treatment for AML. Identifying novel approaches for reducing toxicities should be a priority to potentially improve QoL. Disclosures No relevant conflicts of interest to declare.
- Published
- 2018
30. Efficacy of Anti-TFPI Antibody PF-06741086 Alone and in Combination with Activated Prothrombin Complex Concentrates in Mouse Hemophilia a Bleeding Model
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Amey Barakat, Reema Jasuja, John E. Murphy, and Debra D. Pittman
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biology ,business.industry ,Protein C inhibitor ,Immunology ,Cell Biology ,Hematology ,Pharmacology ,medicine.disease ,Biochemistry ,Hemostatics ,Bleeding diathesis ,Tissue factor pathway inhibitor ,Anti-Inhibitor Coagulant Complex ,medicine ,biology.protein ,Thromboplastin ,Antibody ,business ,Factor IX ,medicine.drug - Abstract
BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.
- Published
- 2018
31. An Antibody to Tissue Factor Pathway Inhibitor (TFPI) Restores Hemostasis after the Onset of Bleeding in Hemophilic a Mouse Injury Models
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Jasuja, Reema, primary, Barakat, Amey, additional, Murphy, John E., additional, and Pittman, Debra D, additional
- Published
- 2016
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32. Translation from SCD Mouse to Man: Chronic Administration of Tucaresol Reduces RBC Sickling and Improves Hematological Parameters, but Does Not Impact Inflammatory Markers in SCD Model Mice
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Knee, Kelly Marie, primary, Jasuja, Reema, additional, Barakat, Amey, additional, Kelly, John J., additional, Nneji, Joseph C., additional, Singh, Pratap, additional, and Janz, Jay M., additional
- Published
- 2016
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33. Immune and Genome Profiling of Myeloma Patients Treated with Sequential Immunotherapies Reveal Differential Non-Overlapping Mechanisms of Resistance
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Lee, Holly, Ahn, Sungwoo, Maity, Ranjan, Leblay, Noémie, Durante, Michael, Ziccheddu, Bachisio, Brake, Alexis, Tilmont, Remi, Barakat, Elie, Poorebrahim, Mansour, Landgren, Ola, Diamond, Benjamin, Maura, Francesco, Neri, Paola, and Bahlis, Nizar J
- Abstract
Chimeric antigen receptor T cells (CAR T) or bispecific T cell engagers (TCE) targeting multiple myeloma (MM) antigens result in deep responses in relapsed patients. Nevertheless, resistance invariably emerges requiring salvage treatments. Sequential therapies with CAR T or TCE, in patients with or without prior anti-BCMA therapy exposure, reported durable responses. However the genomic and molecular determinants of response to sequential immunotherapies are poorly defined.
- Published
- 2023
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34. A High-Risk Subgroup Multiple Myeloma Classification Based on the Detection of PR Minor Subclones
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Leblay, Noemie, Ahn, Sungwoo, Skerget, Sheri, Tilmont, Rémi, Lee, Holly, Poorebrahim, Mansour, Penaherrera, Daniel, Barakat, Elie, Jung, David, Bahlis, Nizar J, Keats, Jonathan, and Neri, Paola
- Abstract
In the last two decades several classifications of Multiple Myeloma (MM) have been proposed based on gene expression signatures. These include the supervised translocation and cyclin D (TC) molecular subgroup approach or unsupervised methods to elucidate the molecular heterogeneity of MM and identify high risk subgroups. In particular, two RNA based gene expression studies have identified 8 to 10 unique subtypes, including the MS, MAF, CD1, CD2, and PR subtypes. The PR subgroup represents a highly proliferative gene signature that is associated with very poor survival outcomes. However, assigning this PR subgroup based on bulk transcriptome signatures can be challenging giving the mixture of genetic backgrounds that can be observed within this subtype. Furthermore, the presence of a minor PR subclone can often be missed with bulk genomic studies leading to the misclassification of a patient as standard risk. We here postulated that minor PR subclones expand under selective therapeutic pressures to become the predominant clones at subsequent relapses and the presence of PR subclones can impact any of the molecular subgroups pertaining poor survival outcomes.
- Published
- 2023
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35. Bclxl Prevents Progressive Exhaustion in BCMA CAR T Cells with Maintenance of High TCF1 Expressing T Cells
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Poorebrahim, Mansour, Lee, Holly, Benaoudia, Sacha, Maity, Ranjan, Ahn, Sungwoo, Leblay, Noémie, Barakat, Elie, Tilmont, Remi, Mahoney, Douglas, Boise, Lawrence H., Neri, Paola, and Bahlis, Nizar J
- Abstract
Although BCMA targeted CAR T cell therapies are highly effective in the treatment of multiple myeloma (MM), patients with high disease burden have not fully benefited from this therapy with shorter disease remissions despite initial responses. It is postulated that high disease burden results in chronic antigenic stimulation of CAR T cells with the induction of exhaustion-associated gene signatures and CAR T cells contraction, limiting their in vivopotency. We have previously reported that BCL2L1(gene encoding for BCLxL) armoring of BCMA CAR T cells enhanced CAR T cells functional fitness and improved proliferation and cytotoxicity after chronic antigenic stimulation in vitro, as well as superior tumor clearance in xenograft models of MM. In the current study we aimed to define the molecular mechanisms that mediates BCL2L1-armored CAR T cell and examine the effect of BCLxL variants on CAR T cells fitness.
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- 2023
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36. The Impact of Soluble BCMA and BCMA Gain on Anti-BCMA Immunotherapies in Multiple Myeloma
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Lee, Holly, Durante, Michael, Ahn, Sungwoo, Leblay, Noemie, Poorebrahim, Mansour, Maity, Ranjan, Tilmont, Rémi, Barakat, Elie, Jung, David, Ziccheddu, Bachisio, Brake, Alexis, Landgren, Ola, Diamond, Benjamin, Maura, Francesco, Neri, Paola, and Bahlis, Nizar J
- Abstract
B cell maturation antigen (BCMA) targeting chimeric antigen receptor T cells (CAR T) and bispecific T cell engagers (TCE) have remarkable efficacy in multiple myeloma (MM). While BCMA antigen escape, driven by TNFRSF17deletions or mutations, is a mediator of anti-BCMA CAR T/ TCE resistance, the majority of patients retain BCMA surface expression at progression. Furthermore, patients with high disease burden and extramedullary disease associated with poorer response to anti-BCMA therapies, have high levels of soluble BCMA (sBCMA). The mechanisms mediating this resistance, and in particular the contribution of sBCMA, have not been fully elucidated.
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- 2023
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37. Translation from SCD Mouse to Man: Chronic Administration of Tucaresol Reduces RBC Sickling and Improves Hematological Parameters, but Does Not Impact Inflammatory Markers in SCD Model Mice
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Reema Jasuja, Pratap Singh, Kelly M. Knee, Joseph C. Nneji, John J. Kelly, Amey Barakat, and Jay M. Janz
- Subjects
Hemolytic anemia ,medicine.medical_specialty ,Hematology ,Mean corpuscular hemoglobin concentration ,medicine.diagnostic_test ,business.industry ,Immunology ,Soluble cell adhesion molecules ,Inflammation ,Cell Biology ,Pharmacology ,medicine.disease ,Biochemistry ,Hemolysis ,In vivo ,Internal medicine ,Medicine ,Hemoglobin ,medicine.symptom ,business - Abstract
Sickle cell disease is a hematological disorder with significant unmet need that affects approximately ten million people worldwide. Currently, treatment for SCD is limited to a single approved drug, Hydroxyurea, and palliative treatments such as hydration and pain management. The limited treatment options have led to considerable interest in developing new therapies for SCD. One strategy for a disease-modifying treatment for SCD is stabilization of the oxygenated form of hemoglobin (OxyHb) by a small molecule allosteric modulator (Zaugg et. Al, JBC, 1977). One such compound, Tucaresol was advanced into clinical trials on the strength of the pre-clinical data, however a SCD mouse model was unavailable when the trial was initiated. The renewed interest in stabilizing OxyHb with a small molecule presents an opportunity to use Tucaresol as a mechanism for understanding the translation between effects seen in an animal model and clinical endpoints in patients. In this study, we treated Townes SCD mice with Tucaresol for 15 days, to evaluate its effect on hematology, oxygen affinity, sickling and markers of inflammation in vivo. These results are compared to the clinical data reported in the original trials. Tucaresol is a substituted benzaldehyde derived from a class of molecules known to preferentially bind to hemoglobin and stabilize the oxygenated state by formation of a Schiff base linkage at a site on the a-chains (Rolan et. Al, Br. J. Clin. Pharm., 1995). Earlier molecules acting by this mechanism had been shown to reduce Hb S polymerization and RBC sickling in vitro, and it was theorized that long-term treatment with such molecules would impact hemolysis and occurrence of vaso-occlusive crisis (VOC) (Beddell et. Al, Br. J. Pharmac. 1984). Preclinical studies demonstrated Tucaresol modification of Hb S delays the polymerization process and inhibits RBC sickling. In clinical trials, Tucaresol positively impacted hematological markers including hemoglobin and reticulocyte counts, and reduced the percentage of irreversibly sickled cells. However, trials of Tucaresol were halted due to a significant immune response and specific inflammatory markers were not measured during the trial. To study these effects in Townes SCD model, mice were dosed with Tucaresol at 30 mg/kg twice a day, for fifteen days. The dose was selected to be consistent with clinical dosing regimens. Blood was collected prior to study start from individual animals to obtain baseline levels for hematological parameters and soluble cell adhesion molecules in plasma. Blood was sampled at day 3 and day 10 of dosing to understand drug exposure and accumulation. At this dose, Tucaresol demonstrated a steady state achieved by day 10 and the blood concentration ranged from 0.5-1.6 mM on day 15. Following two week dosing with Tucaresol, we observed a 50% increase in hemoglobin, and 24% increase in mean corpuscular hemoglobin concentration (MCHC), relative to vehicle treated animals. Reticulocytes, a clinical indicator of hemolytic anemia, are highly elevated in SCD patients and mice, also showed a 47% decrease in Tucaresol treated relative to vehicle treated animals. Oxygen affinity measurements showed a decrease in P50 and P20 values for treated animals, indicating increased oxygen affinity, consistent with the proposed mode of action of Tucaresol- stabilization of the oxygenated state. The percentage of sickled erythrocytes was also decreased by 38% in Tucaresol treated SCD mice when compared to vehicle controls. The decrease in sickling is consistent with the clinical observation of a 52% (mean) decrease in irreversibly sickled cells. In contrast to the improvement seen with hematological parameters, inflammatory markers remained either unchanged or slightly elevated after 15 days of treatment with Tucaresol. Plasma levels of sICAM-1 and sVCAM-1 were unchanged while sE-Selectin was elevated. In summary, in this short course of treatment of SCD mice, Tucaresol had an impact on the hematological markers that was consistent with effects observed in the clinic, but did not modulate the inflammation response. All experiments were within guidelines and were reviewed and approved by Pfizer institutional animal care and use committee. Disclosures Knee: Pfizer Inc: Employment. Jasuja:Pfizer: Employment. Barakat:Pfizer: Employment. Kelly:Pfizer: Employment. Singh:Pfizer Inc: Employment. Janz:Pfizer: Employment.
- Published
- 2016
38. An Antibody to Tissue Factor Pathway Inhibitor (TFPI) Restores Hemostasis after the Onset of Bleeding in Hemophilic a Mouse Injury Models
- Author
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Debra D. Pittman, Reema Jasuja, John E. Murphy, and Amey Barakat
- Subjects
0301 basic medicine ,biology ,business.industry ,Immunology ,Cell Biology ,Hematology ,Pharmacology ,medicine.disease ,Biochemistry ,Fibrin ,Bleeding diathesis ,03 medical and health sciences ,Tissue factor ,030104 developmental biology ,Tissue factor pathway inhibitor ,Coagulation ,Hemostasis ,biology.protein ,Medicine ,business ,Hemostatic function ,Factor IX ,medicine.drug - Abstract
Hemophilia is a family of rare bleeding disorders characterized by inadequate levels of intrinsic coagulation factors, Factor VIII (FVIII) in hemophilia A and Factor IX (FIX) in hemophilia B. This leads to insufficient thrombin generation for the conversion of fibrinogen to fibrin for the development of a stable clot. Replacement factor therapies are provided as a prophylactic treatment to prevent bleeds or for on-demand treatment to treat an active bleed. Some patients develop inhibitory antibodies making them refractory to treatment. Although hemophilia patients have defects in the intrinsic pathway, the extrinsic pathway remains intact. An alternative approach to therapy would be to augment the extrinsic tissue factor pathway. TFPI is a Kunitz domain type inhibitor that negatively regulates thrombin generation within the extrinsic pathway of coagulation by rapidly inactivating the protease functions of Factor Xa and the Factor VIIa/Tissue Factor complex. PF-06741086 is a fully human monoclonal antibody directed against TFPI. Here, we investigated the activity of PF-06741086 in controlling bleeding in hemophilic mouse injury models when administered after a bleeding injury. PF-06741086 restores hemostasis prior to the onset of an injury. As a model for on demand treatment of bleeds, the effect of PF-06741086 on restoring hemostasis in hemophilia A mice following the induction of a severe bleed was monitored. PF-06741086 (1, 3, and 6 mg/kg), recombinant FVIII (200 U/mg) or vehicle were administered to mice, at 2 minutes into an active bleed after the 3 mm tail transection. A dose-dependent decrease in blood loss was observed with the infusion of PF-06741086, 51 % (3 mg/kg), and 76 % (6 mg/kg) compared to vehicle treated mice. A decrease in blood loss (81 %) was also observed for mice receiving recombinant FVIII, compared to vehicle treated mice. The magnitude of the effect in mice that received PF-06741086 (6 mg/kg) at 2 minutes post injury was similar to hemostatic effects of recombinant FVIII administered 2 minutes after the onset of bleeding. In a second model, the cremaster microvessel laser injury model was used to investigate the hemostatic effect of PF-06741086 in hemophilia A mice after the induction of a vessel injury. In each individual mouse, an injury was made without administration of PF-06741086 and data was recorded for platelet accumulation and fibrin generation. A second laser injury was made in the same mouse and a single intravenous dose of PF-06741086 was infused at 6 mg/kg immediately following the laser injury. When compared to untreated mice, enhanced platelet accumulation and fibrin deposition at the site of injury was observed when PF-06741086 was administered in hemophilia A mice during an ongoing bleed induced with laser injury. In summary, PF-06741086, an inhibitor of TFPI, restores hemostasis in on demand hemophilia mouse injury models when administered after the onset of a bleeding injury. PF 06741086 is being developed for the treatment of hemophilia A and hemophilia B, with and without inhibitors. All experiments were within guidelines and were reviewed and approved by Pfizer Institutional Animal Care and use Committee. Disclosures Jasuja: Pfizer: Employment. Barakat:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.
- Published
- 2016
39. Scl regulates the quiescence and the long-term competence of hematopoietic stem cells
- Author
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Guy Sauvageau, Sabine Herblot, Trang Hoang, Shanti Rojas-Sutterlin, Norman N. Iscove, André Haman, Stéphane Barakat, and Julie Lacombe
- Subjects
Cyclin-Dependent Kinase Inhibitor p21 ,Inhibitor of Differentiation Protein 1 ,Immunology ,Gene Expression ,Biology ,behavioral disciplines and activities ,Biochemistry ,Gene dosage ,Resting Phase, Cell Cycle ,Mice ,hemic and lymphatic diseases ,Proto-Oncogene Proteins ,Basic Helix-Loop-Helix Transcription Factors ,Animals ,Progenitor cell ,T-Cell Acute Lymphocytic Leukemia Protein 1 ,Stem Cell Factor ,Interleukin-6 ,fungi ,Graft Survival ,G1 Phase ,Hematopoietic Stem Cell Transplantation ,Gene targeting ,Cell Biology ,Hematology ,Cell cycle ,Hematopoietic Stem Cells ,Interleukin-11 ,Mice, Mutant Strains ,Cell biology ,Transplantation ,Mice, Inbred C57BL ,Haematopoiesis ,RNA Interference ,Stem cell ,Cell Division ,TAL1 - Abstract
Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
40. Cost-Effectiveness Of Arsenic Trioxide + All-Trans Retinoic Acid Compared With All-Trans Retinoic Acid + Idarubicin In The Treatment Of Newly Diagnosed Acute Promyelocytic Leukemia In Canada
- Author
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Lachaine, Jean, primary, Mathurin, Karine, additional, and Barakat, Stephane, additional
- Published
- 2013
- Full Text
- View/download PDF
41. Cost-Effectiveness Of Arsenic Trioxide In The Treatment Of Relapsed/Refractory Acute Promyelocytic Leukemia In Canada
- Author
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Lachaine, Jean, primary, Mathurin, Karine, additional, and Barakat, Stephane, additional
- Published
- 2013
- Full Text
- View/download PDF
42. Cost-Effectiveness Of Arsenic Trioxide In The Treatment Of Relapsed/Refractory Acute Promyelocytic Leukemia In Canada
- Author
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K. Mathurin, Stéphane Barakat, and Jean Lachaine
- Subjects
Acute promyelocytic leukemia ,Oncology ,medicine.medical_specialty ,Mitoxantrone ,business.industry ,Cost effectiveness ,medicine.medical_treatment ,Standard treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Surgery ,Clinical trial ,Internal medicine ,medicine ,Cytarabine ,business ,Etoposide ,medicine.drug - Abstract
Acute promyelocytic leukemia (APL) is a distinct and rare morphological, clinical and pathological variant of acute myeloid leukemia (AML). It represents approximately 10% to 15% of AML. APL is characterized by a high incidence of coagulopathy caused by disseminated intravascular coagulation and/or excessive fibrinolysis and is associated with a high early mortality. Current first-line treatments consist of all-trans retinoic acid (ATRA), anthracyclines and conventional chemotherapy (CT). Although considerable progress has been made in the first-line treatment of APL, about 20 to 30% of patients who achieved complete remission (CR) still relapse Trisenox® is a sterile injectable solution of arsenic trioxide (ATO) and has been approved in several countries, including Canada, for the induction of remission and consolidation in patients with APL who are refractory to, or have relapsed from, retinoid and anthracycline chemotherapy. At this time, ATO is recognized as the standard treatment for relapsed or refractory APL. However, it is not reimbursed yet by provincial public health care systems and was available through a special access program in Canada until product availability. The objective of this study was to assess, from a Canadian perspective, the economic impact of ATO in the treatment of patients with relapsed or refractory APL. A time-dependent Markov model was constructed to assess the cost-effectiveness of ATO compared to ATRA+CT in the treatment of relapsed/refractory APL. Because there was no head-to-head clinical trial available, data from the ATO treatment arm were taken from Soignet, 2001, while data for ATRA+CT were taken from Thomas, 2000. The comparative treatment was composed of ATRA + sequential CT including cytarabine, mitoxantrone or etoposide, followed by autologous hematopoietic stem cell transplantation (HSCT) in consolidation as described in Thomas, 2000. The Markov model comprises five health states: induction, second remission, treatment failure/relapse, post-failure, and death. The length of each Markov cycle was one month for the first 24-month study period then of one year. The model continued to run until all patients reached the absorbing state of death. All patients started in the induction state and could move to other health states thereafter. In case of treatment relapse/failure, patients were subsequently assigned to receive an allogeneic HSCT. The model also takes into account the incidence of treatment-induced grade 3-4 toxicity reported in both clinical trials (Soignet, 2001 and Thomas, 2000). Analyses were conducted from both a Canadian Ministry of Health (MoH) and a societal perspective over a lifetime horizon. In the treatment of relapsed/refractory APL, ATO is a cost-effective strategy over ATRA+CT, from both a health care system and a societal perspective. In fact, compared with ATRA+CT, ATO is associated with an incremental cost-effectiveness ratios (ICERs) of $20,443 per QALY and $22,219 per QALY, from a MoH and societal perspective respectively. Moreover, the results of the exhaustive sensitivity analysis confirm the robustness of the base-case results. In fact, according to the deterministic analysis results, ATO remained a cost-effective strategy compared with ATRA+CT from both perspectives. The ICERs vary between $9,785 and $40,732 / QALY from a MoH perspective and between $11,561 and $44,271 / QALY from a societal perspective. Results of the probabilistic sensitivity analysis indicated that, according to a willingness to pay of $50,000, ATO remains a cost-effective strategy in 99.37% and 98.98% of the simulations, from a MoH and a societal perspective respectively. In conclusion, this economic evaluation demonstrates that ATO+ATRA is a cost-effective strategy in the treatment of relapsed/refractory APL. Disclosures: Lachaine: Lundbeck Canada: Research Funding. Barakat:Lundbeck Canada: Employment.
- Published
- 2013
43. Cost-Effectiveness Of Arsenic Trioxide + All-Trans Retinoic Acid Compared With All-Trans Retinoic Acid + Idarubicin In The Treatment Of Newly Diagnosed Acute Promyelocytic Leukemia In Canada
- Author
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K. Mathurin, Jean Lachaine, and Stéphane Barakat
- Subjects
Acute promyelocytic leukemia ,Oncology ,medicine.medical_specialty ,Anthracycline ,Cost effectiveness ,business.industry ,Standard treatment ,Immunology ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Chemotherapy regimen ,Surgery ,Internal medicine ,medicine ,Cytarabine ,Idarubicin ,business ,neoplasms ,medicine.drug - Abstract
Acute promyelocytic leukemia (APL) is a distinct and rare morphological, clinical and pathological subtype of acute myeloid leukemia (AML). It represents approximately 10% to 15% of AML patients. APL is characterized by a high incidence of coagulopathy caused by disseminated intravascular coagulation and/or excessive fibrinolysis and is associated with a high early mortality. Treatment can exacerbate the coagulopathy. Untreated, APL is rapidly fatal, but if promptly diagnosed and treated, it is frequently curable. Although current treatments (all-trans retinoic acid (ATRA), anthracyclines and conventional chemotherapy) are associated with high remission rates, cytotoxic effects of chemotherapy remain a concern in the management of newly diagnosed APL. Trisenox® is a sterile injectable solution of arsenic trioxide (ATO). Trisenox® has been approved in several countries, including Canada, for the induction of remission and consolidation in patients with APL who are refractory to, or have relapsed from, retinoid and anthracycline chemotherapy. Since the first approval of ATO is for the indication of relapsed/refractory APL, the safety and efficacy of ATO during induction and consolidation of newly diagnosed APL patients have been demonstrated. Specifically, Lo-Coco et al. compared the combination of ATO+ATRA to ATRA + idarubicin (IDA) and confirmed that the combination of ATO+ATRA is at least as effective as standard treatment in the first-line setting. The objective of this study was to assess, from a Canadian perspective, the economic impact of the combination of ATO+ATRA in the treatment of newly diagnosed APL. A time-dependent Markov model was constructed to assess the cost-effectiveness of ATO+ATRA compared to ATRA+IDA in the treatment of newly diagnosed APL. The Markov model comprises four health states: event-free survival (EFS), treatment failure/relapse (TF), post-failure (PF) and death. The length of each Markov cycle was one month for the 48-month period of the Lo-Coco et al. study and then of one year. The model continued to run until all patients reached the absorbing state, defined as death. All patients started in the EFS state and could then move to other health states. In the case of treatment relapse/failure, patients were subsequently treated with a salvage induction therapy composed of ATRA and conventional chemotherapy followed by autologous hematopoietic stem cell transplantation (HSCT) as consolidation treatment. The model also takes into account the incidence of treatment-induced adverse events that were significantly different between both treatment arms in the Lo-Coco et al. study(neutropenia, thrombocytopenia, fever episodes, and QTc interval prolongation). The model also allows comparison with the combination of ATRA+IDA+cytarabine, which is also used in Canada. Analyses were conducted from both a Canadian Ministry of Health (MoH) and a societal perspective over a lifetime horizon. In the treatment of newly diagnosed APL, ATRA+ATO is associated with incremental cost-effectiveness ratios (ICERs) of $50,193 per QALY and $46,367 per QALY, from a MoH and societal perspective respectively when compared with ATRA+IDA. According to the deterministic analysis Results, the ICER of ATO+ATRA compared to ATRA+IDA varied from $23,045 / QALY and $60,759 / QALY from a MoH perspective and between $21,294 / QALY and $56,933 / QALY from a societal perspective. Results of the probabilistic sensitivity analysis indicated that the ICER remains below $50,000 in 48.33% and 74.21% of the Monte Carlo simulations from a MoH and a societal perspective respectively. However, ICER remains below $100,000 in 100% of the simulations from both perspectives. The use of Trisenox in the first line therapy of patients with APL provides significant additional clinical benefits and is associated with an ICUR below the ICUR of many other oncology treatments currently in use. Disclosures: Lachaine: Lundbeck Canada: Research Funding. Off Label Use: Arsenic trioxide is not yet approved in Canada for the First-line treatment of acute promyelocytic leukemia. Barakat:Lundbeck Canada: Employment.
- Published
- 2013
44. ETO2 Controls Hematopoietic Stem Cell Expansion.
- Author
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Barakat, Stephane, primary, Lambert, Julie, additional, Sauvageau, Guy, additional, and Hoang, Trang, additional
- Published
- 2009
- Full Text
- View/download PDF
45. SCL regulates the Quiescence and the Long-Term Competence of Hematopoietic Stem Cells.
- Author
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Lacombe, Julie, primary, Herblot, Sabine, additional, Rojas-Sutterlin, Shanti, additional, Haman, André, additional, Barakat, Stephane, additional, Iscove, Norman N., additional, Sauvageau, Guy, additional, and Hoang, Trang, additional
- Published
- 2009
- Full Text
- View/download PDF
46. ETO2 Controls Hematopoietic Stem Cell Expansion
- Author
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Stephane Barakat, Guy Sauvageau, Trang Hoang, and Julie A. Lambert
- Subjects
education.field_of_study ,Cell cycle checkpoint ,Immunology ,Population ,CD34 ,Hematopoietic stem cell ,hemic and immune systems ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Stem cell ,Progenitor cell ,education - Abstract
Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
47. Sclregulates the quiescence and the long-term competence of hematopoietic stem cells
- Author
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Lacombe, Julie, Herblot, Sabine, Rojas-Sutterlin, Shanti, Haman, André, Barakat, Stéphane, Iscove, Norman N., Sauvageau, Guy, and Hoang, Trang
- Abstract
The majority of long-term reconstituting hematopoietic stem cells (LT-HSCs) in the adult is in G0, whereas a large proportion of progenitors are more cycling. We show here that the SCL/TAL1 transcription factor is highly expressed in LT-HSCs compared with short-term reconstituting HSCs and progenitors and that SCL negatively regulates the G0-G1transit of LT-HSCs. Furthermore, when SCL protein levels are decreased by gene targeting or by RNA interference, the reconstitution potential of HSCs is impaired in several transplantation assays. First, the mean stem cell activity of HSCs transplanted at approximately 1 competitive repopulating unit was 2-fold decreased when Sclgene dosage was decreased. Second, Scl+/−HSCs were at a marked competitive disadvantage with Scl+/+cells when transplanted at 4 competitive repopulating units equivalent. Third, reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was decreased compared with controls. At the molecular level, we found that SCL occupies the Cdkn1aand Id1loci in primary hematopoietic cells and that the expression levels of these 2 regulators of HSC cell cycle and long-term functions are sensitive to Sclgene dosage. Together, our observations suggest that SCL impedes G0-G1transition in HSCs and regulates their long-term competence.
- Published
- 2010
- Full Text
- View/download PDF
48. An Isotopic Method for the Determination of Vitamin B12 Levels in Blood
- Author
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Russel M. Barakat and Roger P. Ekins
- Subjects
Vitamin ,medicine.medical_specialty ,Reproducibility ,Binding protein ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Endocrinology ,chemistry ,Internal medicine ,medicine ,Specific activity ,Vitamin B12 ,Cyanocobalamin ,Isotopes of cobalt ,pernicious anemia - Abstract
Vitamin B12 is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation of protein binding sites and excess vitamin remains in free form. The ratio of free to bound fractions thus quantitatively depends upon the concentration of exogenous compound. This observation has been utilized to determine amounts of vitamin B12 extracted from the blood of normal subjects and of patients with certain pathologic conditions. The method is simple and reproducible. The sensitivity of the method is such that vitamin levels down to roughly 20 µµg./ml. may be evaluated using labeled vitamin B12 of a specific activity of about 1 µc./µg. Repeated assays on identical specimens of normal plasma have shown a reproducibility of about 5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml. with an average of 611 ± 167. Values observed in plasma taken from patients suffering from pernicious anemia were around 100 µµg./ml. or less. Results on subjects with other pathologic conditions are also presented and the limitations of the method are discussed.
- Published
- 1963
49. An Isotopic Method for the Determination of Vitamin B12 Levels in Blood
- Author
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BARAKAT, RUSSEL M. and EKINS, ROGER P.
- Abstract
Vitamin B12 is normally present in plasma mainly bound to a specific binding protein. Addition of exogenous vitamin ultimately results in saturation of protein binding sites and excess vitamin remains in free form. The ratio of free to bound fractions thus quantitatively depends upon the concentration of exogenous compound. This observation has been utilized to determine amounts of vitamin B12 extracted from the blood of normal subjects and of patients with certain pathologic conditions. The method is simple and reproducible. The sensitivity of the method is such that vitamin levels down to roughly 20 µµg./ml. may be evaluated using labeled vitamin B12 of a specific activity of about 1 µc./µg. Repeated assays on identical specimens of normal plasma have shown a reproducibility of about 5-6 per cent. Results on 39 normal subjects gave a range of 330-1070 µµg./ml. with an average of 611 ± 167. Values observed in plasma taken from patients suffering from pernicious anemia were around 100 µµg./ml. or less. Results on subjects with other pathologic conditions are also presented and the limitations of the method are discussed.
- Published
- 1963
- Full Text
- View/download PDF
50. An Isotopic Method for the Determination of Vitamin B12Levels in Blood
- Author
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Barakat, Russel M. and Ekins, Roger P.
- Published
- 1963
- Full Text
- View/download PDF
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