scholarly journals Single-Cell Transcriptomics Study of Human Hematopoietic Progenitors Reveals Alterations Associated with Aging and Myeloid Malignancies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1082-1082
Author(s):  
Marina Ainciburu ◽  
Teresa Ezponda ◽  
Nerea Berastegui ◽  
Ana Alfonso Pierola ◽  
Amaia Vilas-Zornoza ◽  
...  
Keyword(s):  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Healthy Aging ◽  
The Elderly ◽  
Advisory Committees ◽  
Myeloid Malignancies ◽  
Age Related ◽  
Healthy Donors ◽  
Transcriptional Alterations

Abstract Hematopoietic stem and progenitor cells (HSPCs) comprise a continuum of cells with varying differentiation potential and priming toward specific lineages. During both healthy aging and myeloid malignancies, changes occur in the composition and regulation of HSPCs. In this study, we evaluated human HSPCs obtained from young and elderly healthy donors using single-cell RNA sequencing to identify the transcriptional and regulatory alterations associated with aging at single cell resolution. We then applied this knowledge to the study of specific perturbations associated with the development of myeloid pathologies. We isolated >90,000 bone marrow CD34+ cells from 5 young (18-20 y/o), 3 elderly (>65 y/o) healthy donors, 1 patient with myelodysplastic syndrome (MDS) and 1 patient with acute myeloid leukemia (AML), using fluorescence-activated cell sorting. scRNA libraries were prepared with the 10X chromium platform and sequenced. Finally, bioinformatic analysis was performed using available R and Python algorithms such as Seurat, Palantir and Scenic. First, we characterized HSPC subpopulations in young donors by unsupervised clustering and manual annotation. Taking the previous findings as reference, we then classified the elderly and pathological HSPC using elastic-net regularization prediction models (Figure 1A). Comparison of subpopulations in young and elderly donors confirmed the age-related increase in HSC, as well as reduction of lymphoid progenitors and myelomonocytic compartments. Next, we performed differential expression and pathways analysis to uncover age-associated alterations in the transcriptional profile of cells with the same identity. We found a generalized enrichment in elderly HSPC of pathways activated upon stress and inflammation, such as p53, hypoxia and TNF alpha response. This suggests an age-related increased response to the more inflammatory microenvironment of elderly individuals. On the other hand, young HSPC were enriched for cell cycle activation and proliferation pathways, as well as metabolic processes (Figure 1B). Using trajectory analysis, we recovered 6 differentiation paths present in our young donor's data. When compared to the elderly, the greatest changes occurred along the monocytic trajectory. For some genes, expression differed through the whole trajectory, indicating the existence of original transcriptional alterations already at the HSC compartment. On the other hand, expression of myelomonocytic differentiation markers, such as MPO and CD74, reached lower levels in our elderly HSPC data, pointing towards a loss of capacity for monocytic differentiation in progenitors from elderly individuals. Finally, to identify key transcription factors regulating the progression of differentiation routes, we built gene regulatory networks. Overall, we found lower activation levels for transcriptional programs in the early progenitors from elderly donors. In addition, gene ontology enrichment analysis showed that the active networks in the young were enriched for differentiation-related terms, while networks from the elderly were not. These results also indicate an age-associated loss of differentiation capability. We then applied the same computational tools to analyze aberrant hematopoiesis in samples from 2 patients suffering from myeloid malignancies (MDS and AML). On one hand, we subjected the MDS sample to trajectory analysis, focusing on the erythroid lineage. We observed perturbations in the expression dynamics of genes playing a role in erythropoiesis. In the AML sample, we encountered a significant expansion of the most immature cell compartments (HSC, LMPP and MEP). In addition, GRN reconstruction showed up the specific activity of transcription programs activated by factors deregulated during leukemia, such as ZSCAN18 and GFI1. In conclusion, our work described the transcriptional alterations that occur in early hematopoiesis, both during healthy aging and myeloid pathology. We used multiple approaches, such as the study cellular proportions, differentiation trajectories and GRNs. The inclusion of samples from patients with myeloid pathology provided insights into the potential role of single-cell technologies for understanding and treating hematological malignancies. Figure 1 Figure 1. Disclosures Sanchez-Guijo: Gilead: Consultancy, Honoraria; Celgene/Bristol-Myers-Squibb,: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Diez-Campelo: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Valcarcel: BMS: Consultancy, Honoraria, Speakers Bureau; CELGENE: Consultancy, Honoraria, Speakers Bureau; ASTELLAS: Consultancy, Honoraria, Speakers Bureau; AMGEN: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; TAKEDA: Consultancy, Honoraria, Speakers Bureau; JAZZ: Consultancy, Honoraria, Speakers Bureau; SOBI: Consultancy, Honoraria, Speakers Bureau; SANOFI: Consultancy, Honoraria, Speakers Bureau. Romero: 10X Genomics: Current Employment. Prosper: Janssen: Honoraria; Oryzon: Honoraria; BMS-Celgene: Honoraria, Research Funding.

scholarly journals Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 74-74
Author(s):  
Yusuke Kamihara ◽  
Edouard Forcade ◽  
John Koreth ◽  
Hongye Liu ◽  
Tomohiro Kubo ◽  
...  
Keyword(s):  
B Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Low Dose ◽  
Cell Mass ◽  
Mass Cytometry ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD. Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. Disclosures Forcade: Neovii: Other: Travel grant. Koreth: Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow: Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand: Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler: Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

scholarly journals Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 42-43
Author(s):  
Prajish Iyer ◽  
Lu Yang ◽  
Zhi-Zhang Yang ◽  
Charla R. Secreto ◽  
Sutapa Sinha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Gene Signature ◽  
T Cell Subsets ◽  
Rna Seq ◽  
Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

A Comprehensive Analysis of Single-Cell Chromatin Accessibility and Gene Expression Identifies Intra-Tumor Heterogeneity and Molecular Treatment Responses in Relapsed/Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 575-575
Author(s):  
Alexandra M Poos ◽  
Jan-Philipp Mallm ◽  
Stephan M Tirier ◽  
Nicola Casiraghi ◽  
Hana Susak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Tumor Heterogeneity ◽  
Mek Inhibitor ◽  
Chromatin Accessibility ◽  
University Research ◽  
Sample Processing ◽  
Advisory Committees

Introduction: Multiple myeloma (MM) is a heterogeneous malignancy of clonal plasma cells that accumulate in the bone marrow (BM). Despite new treatment approaches, in most patients resistant subclones are selected by therapy, resulting in the development of refractory disease. While the subclonal architecture in newly diagnosed patients has been investigated in great detail, intra-tumor heterogeneity in relapsed/refractory (RR) MM is poorly characterized. Recent technological and computational advances provide the opportunity to systematically analyze tumor samples at single-cell (sc) level with high accuracy and througput. Here, we present a pilot study for an integrative analysis of sc Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) and scRNA-seq with the aim to comprehensively study the regulatory landscape, gene expression, and evolution of individual subclones in RRMM patients. Methods: We have included 20 RRMM patients with longitudinally collected paired BM samples. scATAC- and scRNA-seq data were generated using the 10X Genomics platform. Pre-processing of the sc-seq data was performed with the CellRanger software (reference genome GRCh38). For downstream analyses the R-packages Seurat and Signac (Satija Lab) as well as Cicero (Trapnell Lab) were used. For all patients bulk whole genome sequencing (WGS) data was available, which we used for confirmatory studies of intra-tumor heterogeneity. Results: A comprehensive study at the sc level requires extensive quality controls (QC). All scATAC-seq files passed the QC, including the detected number of cells, number of fragments in peaks or the ratio of mononucleosomal to nucleosome-free fragments. Yet, unsupervised clustering of the differentially accessible regions resulted in two main clusters, strongly associated with sample processing time. Delay of sample processing by 1-2 days, e.g. due to shipment from participating centers, resulted in global change of chromatin accessibility with more than 10,000 regions showing differences compared to directly processed samples. The corresponding scRNA-seq files also consistently failed QC, including detectable genes per cell and the percentage of mitochondrial RNA. We excluded these samples from the study. Analysing scATAC-seq data, we observed distinct clusters before and after treatment of RRMM, indicating clonal adaptation or selection in all samples. Treatment with carfilzomib resulted in highly increased co-accessibility and >100 genes were differentially accessible upon treatment. These genes are related to the activation of immune cells (including T-, and B-cells), cell-cell adhesion, apoptosis and signaling pathways (e.g. NFκB) and include several chaperone proteins (e.g. HSPH1) which were upregulated in the scRNA-seq data upon proteasome inhibition. The power of our comprehensive approach for detection of individual subclones and their evolution is exemplarily illustrated in a patient who was treated with a MEK inhibitor and achieved complete remission. This patient showed two main clusters in the scATAC-seq data before treatment, suggesting presence of two subclones. Using copy number profiles based on WGS and scRNA-seq data and performing a trajectory analysis based on scATAC-seq data, we could confirm two different subclones. At relapse, a seemingly independent dominant clone emerged. Upon comprehensive integration of the datasets, one of the initial subclones could be identified as the precursor of this dominant clone. We observed increased accessibility for 108 regions (e.g. JUND, HSPA5, EGR1, FOSB, ETS1, FOXP2) upon MEK inhibition. The most significant differentially accessible region in this clone and its precursor included the gene coding for krüppel-like factor 2 (KLF2). scRNA-seq data showed overexpression of KLF2 in the MEK-inhibitor resistant clone, confirming KLF2 scATAC-seq data. KLF2 has been reported to play an essential role together with KDM3A and IRF1 for MM cell survival and adhesion to stromal cells in the BM. Conclusions: Our data strongly suggest to use only immediately processed samples for single cell technologies. Integrating scATAC- and scRNA-seq together with bulk WGS data showed that detection of individual clones and longitudinal changes in the activity of cis-regulatory regions and gene expression is feasible and informative in RRMM. Disclosures Goldschmidt: John-Hopkins University: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Molecular Partners: Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees.

A Phase 1 Study of Sea-CD70 in Myeloid Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Ahmed Aribi ◽  
Anjali S Advani ◽  
William Donnellan ◽  
Amir T. Fathi ◽  
Marcello Rotta ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Immune Evasion ◽  
Dose Escalation ◽  
Research Funding ◽  
Phase 1 ◽  
Steering Committee ◽  
Advisory Board ◽  
Open Label ◽  
Advisory Committees ◽  
Myeloid Malignancies

Background SEA-CD70 is being developed in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Current treatment options are limited for patients (pts) with relapsed or refractory (r/r) MDS or r/r AML and outcomes remain poor. SEA-CD70 is an investigational humanized, non-fucosylated monoclonal antibody targeting CD70. Expression of CD70 is limited in normal tissue, but is aberrantly expressed on malignant myeloid blasts while absent from healthy hematopoietic progenitor cells. CD70 and its ligand, CD27, may play a role in malignant blast cell survival and/or tumor immune evasion. SEA-CD70 uses a novel sugar-engineered antibody (SEA) platform to produce a non-fucosylated antibody with enhanced effector function. The proposed mechanism of action of SEA-CD70 includes elimination of CD70 positive cells via enhanced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and mediation of complement-dependent cytoxicity (CDC). Additionally, SEA-CD70 has the potential to block the interaction of CD70 with CD27, which may disrupt signals that enhance blast proliferation and survival and may modulate the immune system to limit immune evasion and increase antigen specific T cell responses. Methods SGNS70-101 is a phase 1, open-label, multicenter, dose-escalation, and cohort expansion study designed to establish the safety, tolerability, and preliminary activity of SEA-CD70 in pts with myeloid malignancies (NCT04227847). Dose escalation is ongoing. In dose escalation, pts must have r/r MDS with 5-20% blasts which has failed prior treatment with a hypomethylating agent (HMA), and have no other therapeutic options known to provide clinical benefit for MDS. After conclusion of dose escalation, monotherapy expansion cohorts will be opened for pts with MDS and for pts with AML. Primary objectives are to evaluate the safety and tolerability, and to determine the maximum tolerated dose (MTD) or recommended expansion dose of SEA-CD70. Secondary objectives are to assess antitumor activity, PK, and immunogenicity of SEA-CD70. Once dose escalation is complete and the recommended monotherapy dose is identified, combination cohorts will be considered in AML and MDS. The study is currently enrolling with sites opening in the US and EU. Disclosures Aribi: Seattle Genetics: Consultancy. Advani:OBI: Research Funding; Takeda: Research Funding; Novartis: Consultancy, Other: advisory board; Pfizer: Honoraria, Research Funding; Kite: Other: Advisory board/ honoraria; Amgen: Consultancy, Other: steering committee/ honoraria, Research Funding; Seattle Genetics: Other: Advisory board/ honoraria, Research Funding; Immunogen: Research Funding; Glycomimetics: Consultancy, Other: Steering committee/ honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Donnellan:Kite Pharma/Gilead: Research Funding; Janssen: Research Funding; Karyopharm Therapeutics: Research Funding; AstraZeneca: Research Funding; Astex Pharmaceuticals: Research Funding; Incyte: Research Funding; MedImmune: Research Funding; TCR2 Therapeutics: Research Funding; Genentech: Research Funding; PTC Therapeutics: Consultancy, Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Consultancy; Abbvie: Consultancy, Research Funding; Bellicum Pharmaceuticals: Research Funding; CTI Biopharma: Research Funding; Celgene: Research Funding; Celularity: Research Funding; Forma Therapeutics: Research Funding; Forty Seven: Research Funding; Takeda: Research Funding; H3 Biomedicine: Research Funding; Ryvu Therapeutics: Research Funding; Seattle Genetics: Consultancy, Research Funding. Fathi:Astellas: Consultancy; Agios: Consultancy, Research Funding; Amphivena: Consultancy, Honoraria; AbbVie: Consultancy; Pfizer: Consultancy; Daiichi Sankyo: Consultancy; Celgene: Consultancy, Research Funding; Forty Seven: Consultancy; Jazz: Consultancy, Honoraria; Kite: Consultancy, Honoraria; NewLink Genetics: Consultancy, Honoraria; Novartis: Consultancy; PTC Therapeutics: Consultancy; Takeda: Consultancy; TrovaGene: Consultancy; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Blue Print Oncology: Consultancy; Boston Biomedical: Consultancy; Kura: Consultancy; Trillium: Consultancy; Seattle Genetics: Consultancy, Research Funding. Rotta:Merck: Speakers Bureau; Jazz Pharma: Speakers Bureau. Vachani:Blueprint: Consultancy; CTI Biopharma: Consultancy; Daiichi Sankyo: Consultancy; Incyte: Consultancy, Research Funding; Jazz: Consultancy; Astellas: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy; Abbvie: Consultancy. Yang:AROG: Research Funding; Protagonist: Research Funding; Jannsen: Research Funding; AstraZeneca: Research Funding. Ho:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Garcia-Manero:Novartis: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Jazz Pharmaceuticals: Consultancy; Onconova: Research Funding; Amphivena Therapeutics: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; AbbVie: Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; H3 Biomedicine: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clinicopathological Features of Nodular Sclerosis-Type Classical Hodgkin Lymphoma In the Elderly: Multicenter Study of Hodgkin Lymphoma In Japan

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2677-2677
Author(s):  
Naoko Asano ◽  
Tomohiro Kinoshita ◽  
Koichi Ohshima ◽  
Tadashi Yoshino ◽  
Nozomi Niitsu ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Hodgkin Lymphoma ◽  
Research Funding ◽  
The Elderly ◽  
Clinicopathological Features ◽  
Line Treatment ◽  
Classical Hodgkin Lymphoma ◽  
First Line ◽  
Advisory Committees ◽  
First Line Treatment

Abstract Abstract 2677 Background: Classical Hodgkin lymphoma (CHL), which is characterized by the presence of Hodgkin and Reed Sternberg (H-RS) cells in a background of non-neoplastic inflammatory cells, is divided into four histological subgroups, nodular sclerosis (NSCHL), mixed cellularity (MCCHL), lymphocyte-rich, and lymphocyte depletion. While NSCHL in young adults is characterized by a mediastinal mass and good prognosis, the clinicopathological characteristics of NSCHL in the elderly (NSCHL-e) remain uncertain. Patients and methods: Enrolled patients were diagnosed with CHL between 1986 and 2006 as part of the Hodgkin Lymphoma's Multicenter Study Group. To better characterize NSCHL-e, we compared the clinicopathological profiles of 84 NSCHL-e patients aged 50 or over with 237 NSCHL-y patients aged 49 or younger and 302 with MCCHL. Results: The total of 743 CHL patients consisted of 496 men and 247 women with a median age of 48 years (range, 15– 89 years). The pathological diagnoses were NSCHL in 324 patients (43%) and MCCHL in 303 (41%). NSCHL patients showed a bimodal age distribution, with an initial peak in their 20s and a second small peak in their 60s. We categorized the former as NSCHL-y (49 or younger) and the latter as NSCHL-e (50 and over). NSCHL-e patients were characterized by male predominance and a more advanced clinical stage (53%) than NSCHL-y. Immunophenotypically, H-RS cells had the prototypic immunophenotype of CD15+ CD30+ and Pax5+. NSCHL-e cases showed a significantly higher rate of CD20 (24%) than NSCHL-y (8%, P = 0.001). Furthermore, H-RS cells in 29 of 75 (39%) patients with NSCHL-e were positive for EBV RNA transcripts by in situ hybridization, whereas only 7% of NSCHL-y cases were EBER-positive (P < 0.0001) (Table). Regarding NSCHL-e and MCCHL, no significant difference between these patients was seen in clinical characteristics. Immunophenotypically, NSCHL-e patients showed significantly higher rates for CD3 and TIA-1, while MCCHL patients showed higher EBV positivity (75%). Fifty-five of 63 patients received systemic multi-agent chemotherapy as first-line treatment, consisting of doxorubicin, bleomycin, vinblastine, and dacarbacin (ABVD) in 38 patients; CHOP in 8; C-MOPP in 8; and BEACOPP in 1. Overall, 51 patients responded to first-line treatment, 39 with complete response and 12 with partial response. Disease-specific survival of NSCHL-e was poorer than that of NSCHL-y (P < 0.001) but similar to that of MCCHL (P = 0.43) (Figure). Conclusion: NSCHL-e is characterized by an unfavorable prognosis and different clinicopathological features to NSCHL-y, which is considered as typical NSCHL. A number of cases of NSCHL-e might have been associated with MCCHL, with most being EBV-positive. These results suggest the limitations of current histological subgroupings for CHL. Disclosures: Matsushita: Pfizer CO.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter Co.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Clonotype-Immunophenotype Relationships in TET2 and IDH-Mutant Myeloid Transformation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 373-373
Author(s):  
Linde A. Miles ◽  
Robert L. Bowman ◽  
Nicole Delgaudio ◽  
Troy Robinson ◽  
Martin P. Carroll ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Patterns ◽  
Clonal Expansion ◽  
Mutant Mice ◽  
Triple Mutant ◽  
Advisory Committees ◽  
Disease Evolution

Abstract Large scale molecular profiling studies in AML patients have suggested that stepwise acquisition of somatic mutations is crucial in driving leukemic development. High variant allele frequency (VAF) mutations in epigenetic modifier genes, such as TET2 and IDH1/2, are thought to occur early in AML pathogenesis while oncogenic mutations with typically lower VAF mutations, including FLT3 and NRAS, are suggested to occur late in disease evolution. While bulk DNA sequencing has catalogued co-mutations found in individual AMLs, it cannot unveil the heterogeneity and composition of clones that makes up the disease. Elucidating the architecture and clone-specific molecular profiles at the single cell resolution will be key to understanding how sequential and/or parallel mutation acquisition drives myeloid transformation. To assess the clonal architecture of AML, we previously performed single cell DNA sequencing (scDNA seq) in 146 patients with myeloid malignancies. We have further identified specific mutational combinations driving clonal expansion in TET2- or IDH1/2- mutant AML samples. These studies suggest TET2 and IDH1/2 can cooperate to promote clonal expansion with DNMT3A and NPM1 (Figure 1A). However, TET2 or IDH1/2 mutant clones that acquired KRAS mutations underwent minimal clonal expansion, suggesting mutant-pair specific fitness alterations (Figure 1B). To further identify how co-mutational pairing impacted clonal fitness and differentiation, we integrated the scDNA platform with immunophenotypic profiling of 45 cell surface markers and analyzed new TET2- and IDH1/2- mutant AML samples (Figure 1C). We identified clone-specific differences in lineage markers depending on co-mutational partners. NPM1 co-mutant clones were enriched for more primitive markers (CD33), whereas NRAS co-mutant clones possessed high expression of myeloid differentiation markers (CD14/CD11b), suggestive of clone-specific fitness landscapes across hematopoietic differentiation. We also identified divergent clonotype-immunophenotype patterns in TET2- and IDH2-mutant clones harboring NPM1/RAS mutations, suggesting that initiating mutations may prime mutant clones for very different evolutionary trajectories as they acquire similar mutations in leukemogenesis (Figure 1D). To deterministically delineate the relationship between clonal evolution and myeloid transformation, we generated Cre-inducible single (Tet2 -/-), double (Tet2 -/-/Nras G12Dand Tet2 -/-/Npm1 cA/wt), and triple (Tet2 -/-/Npm1 cA/wt/Nras G12D) mutant mice and evaluated differences in chimerism, immunophenotype, and survival. We observed a shortened survival for double and triple mutant mice, compared to Tet2 -/- only mice (Figure 1E). As previously reported, Tet2 -/-/Nras G12D mice developed a CMML-like phenotype. Critically, the addition of Npm1 resulted in a more rapid disease onset and transformation to AML (Figure 1F). Moreover, triple mutant WBM transplanted to form a fully penetrant disease into secondary recipients, while double mutant Tet2 -/-/Nras G12D WBM failed to form disease within 3 months of transplant, suggesting a difference in the cell population responsible for disease propagation. Immunophenotypic alterations were evident with Tet2 -/-/ Nras G12D displaying an increase in Mac1 +Gr1 + cells compared to Tet2 -/-/Npm1 cA/wt/Nras G12D mice which possessed increased Mac1 +Gr1 - cells and expansion of lineage negative cells (Figure 1G). These findings align with the clonotype specific expression patterns observed in clinical specimen and suggest that myeloid transformation and maturation biases are influenced by specific mutational combinations. Figure 1 Figure 1. Disclosures Miles: Mission Bio: Honoraria, Speakers Bureau. Bowman: Mission Bio: Honoraria, Speakers Bureau. Carroll: Janssen Pharmaceutical: Consultancy; Incyte Pharmaceuticals: Research Funding. Levine: Astellas: Consultancy; Janssen: Consultancy; Auron: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; QIAGEN: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Incyte: Consultancy; Imago: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Prelude: Membership on an entity's Board of Directors or advisory committees; Ajax: Membership on an entity's Board of Directors or advisory committees; Zentalis: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Morphosys: Consultancy.

scholarly journals Racial and Age-Related Differences in Impacts of High-Risk Cytogenetic Abnormalities on Survival in Multiple Myeloma in a Nationwide Electronic Health Record-Derived Database

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4121-4121
Author(s):  
Gregory S Calip ◽  
Mustafa S Ascha ◽  
Xiaoliang Wang ◽  
Amy E Pierre ◽  
Kathleen Maignan ◽  
...  
Keyword(s):  
High Risk ◽  
Board Of Directors ◽  
Research Funding ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Cytogenetic Abnormalities ◽  
Age Related ◽  
Black Patients ◽  
Double Hit ◽  
White Patients

Abstract Background: The incidence of multiple myeloma (MM) and enrichment of cytogenetic abnormalities differ significantly between racial/ethnic groups in the US, and their significance in determining myeloma progression and survival is not well understood. Whole genome sequencing has identified unique mutational signatures in MM, including an age-related process common in hyperdiploid myeloma. Our purpose was to describe racial and age-related differences in the impact of high-risk cytogenetic abnormalities (HRCAs) on survival in MM. Methods: We conducted a retrospective cohort study of adult MM patients starting first-line therapy between January 2011 and May 2021 using the nationwide Flatiron Health electronic health record-derived de-identified database. Patient-level demographic and clinical characteristics were ascertained using structured and unstructured data, curated via technology-enabled abstraction. Patients who had documented fluorescence in situ hybridization testing within 30 days prior to or 90 days following the start of first-line treatment were included. HRCAs, including gain or amplification 1q21, deletion 17p, t(4;14), t(14;16) and t(14;20), were identified and categorized as 0, 1, or 2+ HRCAs. Our outcomes of interest were real world progression free survival (rwPFS) and overall survival (rwOS). Cox proportional hazards models were used to calculate adjusted hazard ratios (HR) and 95% confidence intervals (CI), adjusted for demographic and clinical characteristics and treatment including time-dependent receipt of autologous stem cell transplantation. Results: From a cohort of 4889 MM patients, there were 790 (16%) Black and 2995 (61%) White patients with median ages at diagnosis of 68 and 70 years, respectively. Compared to White patients, a higher proportion of Black patients had IgG M-protein (61% vs 55%) and a lower proportion had 1+ HRCAs identified (31% vs 34%). Among all racial groups, compared to patients aged &lt;65 years (N=1771), a higher proportion of patients aged 65+ years (N=3118) had IgA M-protein (21% vs 17%) and 1+ HRCAs identified (35% vs 33%). Multivariable models showed evidence of significant statistical interaction between age and prevalence of HRCA for rwPFS (P-int: 0.02). Among White patients, having 2+ HRCAs ("double-hit MM") compared to no HRCAs was associated with worse rwPFS in both younger and older patients (&lt;65 years: HR 2.88, 95% CI 1.93-4.32, P&lt;0.01; 65+ years: HR 1.51, 95% CI 1.18-1.94, P&lt;0.01). Among Black patients, associations between double-hit MM and rwPFS were attenuated and not statistically significant regardless of age (&lt;65 years: HR 1.81, 95% CI 0.69-4.74, P=0.23; 65+ years: HR 1.61, 95% CI 0.92-2.81, P=0.09). Similarly, we also found evidence of statistical interaction between age and prevalence of HRCA for rwOS (P-int: 0.02). Among White patients, double-hit MM was significantly associated with worse rwOS but the magnitude of increased risk differed for younger (HR 3.39, 95% CI 2.24-5.14, P&lt;0.01) and older (HR 1.61, 95% CI 1.27-2.05, P&lt;0.01) patients. Double-hit MM was significantly associated with worse rwOS among older Black patients (HR 1.78, 95% CI 1.03-3.06, P=0.04), but not younger Black patients (HR 1.60, 95% CI 0.58-4.40, P=0.36). Conclusions: In this cohort of newly diagnosed MM patients treated in routine practice, having double-hit MM was differentially predictive of poor survival across age groups. Double-hit MM was associated with worse rwPFS and rwOS among White patients, but these trends were less consistent among Black patients. Our current understanding of cytogenetic risk stratification of MM requires further study and additional data for identifying low- and high-risk subsets of patients across different ages and racial groups. Figure. Kaplan-Meier survivor functions for rwPFS in White (Panel A) and Black (Panel B) patients by age group and number of HRCAs Figure 1 Figure 1. Disclosures Calip: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company; Pfizer: Research Funding. Ascha: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wang: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Pierre: Flatiron Health, Inc: Current Employment; Roche: Current holder of stock options in a privately-held company. Maignan: Flatiron Health: Current Employment; Roche: Current equity holder in publicly-traded company. Wadé: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Leng: Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment. Seymour: Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Current equity holder in publicly-traded company; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Flatiron Health Inc: Current Employment. Patel: Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Neparidze: Eidos Therapeutics: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Research Funding; Janssen: Research Funding.

scholarly journals Molecular Spectrum of CSF3R variants Correlate with Specific Myeloid Malignancies and Secondary Mutations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4389-4389
Author(s):  
Vera Adema ◽  
Cassandra M. Hirsch ◽  
Bartlomiej Przychodzen ◽  
Yasunobu Nagata ◽  
Tomas Radivoyevitch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Board Of Directors ◽  
Research Funding ◽  
Cytoplasmic Domain ◽  
Tier 2 ◽  
Juxtamembrane Domain ◽  
Advisory Committees ◽  
Myeloid Malignancies ◽  
Tier 1 ◽  
Allelic Burden

Abstract Different CSF3R mutations (CSF3RMT) result in aberrant G-CSF signaling pathways and are linked to a wide range of myeloid disorders. Loss-of-function mutations in its extracellular domain cause severe congenital neutropenia (SCN). Activating mutations in the juxtamembrane region have been associated with a variety of myeloid malignancies. Truncating mutations in the cytoplasmic domain are associated with SCN cases that progress to MDS or AML. In this study, we evaluate the extent to which different CSF3RMT associate with disease onset, progression to leukemia and neutrophil counts in patients (pts) diagnosed with myeloid malignancies. We identified CSF3RMT cases in a cohort of 1400 pts [median age 71 years (yrs)]. We analyzed somatic and germline mutational patterns, and cross-sectional correlation with other gene mutations in CSF3RMT. A stringent algorithm based on conserved amino acid residues and alterations of protein features was used to predict the pathogenic significance of CSF3RMT. We identified 44 CSF3RMT: 33 germline (CSF3RGL) and 11 somatic (CSF3RS) variants. Most CSF3RGL were found in pts (median age 63 yrs) with MDS or related conditions (87% of all mutant cases), conversely these mutations were present in 5% (n= 22/424) of MDS, 3% (n= 7/244) MDS/MPN and <1% (n= 3/392) of AML and in 1 out of 3 pts with aCML tested. Mutations were mostly missense and located between the cytoplasmic (58%: M696T, R698C (isoform III), D732N, P733T, S744F, Y752*, E808K), and extracellular (42%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H) domains. No mutations were detected in the juxtamembrane domain. Variants were grouped in Tier-1 (61%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H Y752*, E808K) and Tier-2 (variants with uncertain significance, 39%: S413L, M696T, R689C, D732N, P733T, S744F). E808K and R698C were the most common amino acid changes in Tier-1 (53%) and Tier-2 (44%), respectively. A total of 4/7 pts with E808K progressed to AML (but none with R698C), supporting previous observations that E808K (or E785K) represents a pathogenic variant predisposing to leukemia. A total of 46% (n=14) of pts with CSF3RGL had neutropenia [median 0.9x109/L (0.02-1.22x109/L)] at the time of sampling. Two pts diagnosed with a prior cancer manifested sustained neutropenia before the diagnosis of MDS and MDS/MPN. G-CSF was administered in 21% of pts. Alterations in -7/7q- were common (21%). Some pts also harbored other somatic mutations in NF1 (15%), DNMT3A (12%), SETBP1 (12%), or U2AF1 (12%). Of note, 1 patient carried mutations in WAS and GATA2 and another carried a mutation in VPS45, which have been previously associated with SCN/MDS. The patient with aCML harbored also a CSF3RS (T615A). Overall combined allelic burden in pts cohort was 2% vs. 1.6% expected allelic burden in control populations for the same variants (P=.02). CSF3R S were found in 11 pts (median age 71 yrs) with AML or MDS related conditions (73% of all mutant cases), conversely these mutations were present in 1.4% (n= 6/424) of AML, <1% in MDS (n= 2/244) and MDS/MPN (n= 1/392) and in 2/3 pts with aCML tested. Mutations were missense in 63% of pts, T618I being most recurrent (n=5/11; 45%). Frameshifts accounted for 36% of the mutations and were localized in the cytoplasmic domain (Q741*, Q749*, Y752*, Q768*). All mutations were heterozygous. At the time of sampling 3/11 pts had leukocytosis and 3/11 had neutropenia. Mutations were distributed between the juxtamembrane domain (55%) and the cytoplasmic domain (45%). Mutations in the extracellular domain were not detected. Pts with sAML mostly carried mutations in the juxtamembrane domain (67%), those with MDS carried only in cytoplasmic domain, and those with MDS/MPN or aCML carried mutations in both the juxtamembrane and extracellular domains. There was one somatic and one RUNX1GL mutation. The cytogenetic abnormalities -7/7q- were detected in 18% (2/11) of cases. Interestingly, T618I was found solely in pts with sAML. Focusing on associations between CSF3RMT and mutations in the class III receptor tyrosine kinases CSF1R, FLT3, and KIT we identified only FLT3 to be co-mutated with CSF3RMT. All 3 pts (2 CSF3RGL and 1 CSF3RS) with such co-mutations evolved to AML. In sum, we found that CSF3RGL do not commonly co-occur with CSF3RS, suggesting that the neutropenia observed at the sampling time most likely is causative of undetected GL variants and/or is representative of a long unrecognized disease. Disclosures Nazha: MEI: Consultancy. Carraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy. Santini:Otsuka: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Single Cell RNA Sequencing Identifies a Crucial Role for ASXL1 in Neutrophil Development

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 212-212
Author(s):  
Theodore Braun ◽  
Theresa Lusardi ◽  
Trevor Enright ◽  
Zachary Schonrock ◽  
Cody Coblentz ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Single Cell ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Crucial Role ◽  
Research Funding ◽  
Advisory Committees ◽  
Single Cell Rna Sequencing ◽  
Equity Ownership ◽  
Specific Granule

Single Cell RNA Sequencing Identifies a Crucial Role for ASXL1 in Neutrophil Development Additional sex combs-like 1 (ASXL1) is a polycomb-associated protein that is essential for normal hematopoiesis. ASXL1 is recurrently mutated across the spectrum of myeloid malignancies including myelodysplastic syndromes, myeloproliferative neoplasms and Acute Myeloid Leukemia. ASXL1 mutations are also found in the premalignant disorders clonal hematopoiesis of indeterminate potential and clonal cytopenias of indeterminate potential. In all cases, ASXL1 mutations are associated with more aggressive disease biology and resistance to treatment. Mutations in ASXL1 broadly dysregulate the hematopoietic system, opening chromatin at genes associated with differentiation and self-renewal, predisposing to malignant transformation. However, in spite of this, the specific role of ASXL1 at different phases of hematopoiesis remains unknown. Indeed, the development of therapeutic approaches for ASXL1-mutant malignancies will require a nuanced understanding of the role of ASXL1 in directing normal blood development to maximize on target effects and minimize toxicity. ASXL1 mutations are commonly identified in myeloid disorders with dysplasia. In the neutrophil lineage, morphologic dysplasia is associated with nuclear-cytoplasmic dyssynchrony, where neutrophils demonstrate differences in nuclear and cytoplasmic differentiation (i.e. hypolobated nuclei or hypogranular cytoplasm). Given its associated with dysplasia, we hypothesized that ASXL1 plays a fundamental role in neutrophil maturation. To investigate this, we performed single cell RNA sequencing (scRNA-seq) on lineage depleted bone marrow from MX-1 Cre/Asxl1FL/FL mice (Asxl1KO) or cre negative littermate controls (Asxl1WT). This analysis revealed a loss of multi-lineage differentiation potential in response to Asxl1 deletion with the most prominent effects noted in myeloid differentiation. Although the neutrophil-primed granulocyte-macrophage progenitors appeared relatively normal, a differentiation block was identified at the transition between promyelocytes and myelocytes. Specifically, Asxl1KO mice demonstrated a failure to normally upregulate specific granule constituents. Although key differentiation-associated transcription factors are present in the appropriate precursor populations, they appear to require normal Asxl1 function to effectively initiate transcription of specific granule genes. This is the first description of a crucial role for Asxl1 in terminal neutrophil differentiation. Furthermore, the failure to effectively upregulate specific granule genes in Asxl1 deficient mice may provide a mechanistic explanation for the dysplasia-associated hypogranular neutrophils present in dysplastic disorders with mutant ASXL1. Disclosures Druker: Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Monojul: Other: former consultant; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Gilead Sciences: Other: former member of Scientific Advisory Board; Celgene: Consultancy; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding.

scholarly journals Single-Cell Mapping of Stress Response and Cell Death Pathways in Acute Myeloid Leukemia Reveals Stressor-Specific Alterations and Distinct Response Patterns

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 882-882
Author(s):  
Muharrem Muftuoglu ◽  
Vivian Ruvolo ◽  
Yuki Nishida ◽  
Po Yee Mak ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Mapping ◽  
Advisory Committees ◽  
Equity Ownership

Background: Cells respond to stress in various ways ranging from adaptation to environmental challenges and activation of survival pathways to induction of cell death. The initial response to stress encompasses adaptive measures to ensure survival and in the presence of irreparable damage associated with unresolved stress cell death ensues. Understanding the principles and mechanisms governing cell survival over cell death is of particular importance in the field of cancer therapy. It is intriguing that exposure of a seemingly homogenous population to death inducing stimuli, such as chemotherapeutic agents, induces fractional tumor killing in a stochastic manner while a subgroup of cells acquire a persistent state, most probably through activation of compensatory survival pathways. Fractional cell killing and, therefore, inability to completely eradicate transformed cells result in resistance to therapy. Methods/Results: To gain further insight into compensatory mechanisms and divergent responses elicited in response to death inducing stimuli we designed a multiparametric flow cytometry panel for simultaneous assessment of different forms of cell death at the single cell level, and aimed to dissect stimulus-specific death patterns and pinpoint potential compensatory mechanisms in persistent cells. We modified ( Bergamaschi et al. 2019) and utilized panels including antibodies against RIP3, LC3B, cleaved caspase 3, cleaved PARP-1, PERK, H2AX, p21, Ki-67 and dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos in response to DNA damage and as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. To test this concept, we initially utilized agents inducing DNA damage and generated two-dimensional t-SNE plots and diffusion maps to illustrate the multifaceted stress response and developmental trajectories upon challenging with DNA damaging agents. Exposure of acute myeloid leukemia (AML) cell lines to etoposide (E) and daunorubicin (DNR) dramatically altered cellular landscape and resulted in emergence of distinct stress responses characterized by differential induction of autophagy, ER stress and DNA damage response and an increase in multiple cell death subpopulations differentially expressing cleaved caspase 3, PARP-1, necrotic cell identifier (live dead aqua dye) and H2AX. We then generated diffusion maps to infer developmental trajectories of dead cells and identified H2AX+PARP+Caspase-3 co-expression as the earliest event occurring in dying cells while cells stained positive for dead cell dye only marked the latest stage. Of note, a fraction of cells exhibited increased autophagy, accompanied with high ER stress and low DNA damage. Presumably, this pattern identifies persistent cells attaining a transient state in response to E and DNR associated with higher likelihood of survival. Evidently, external stress induced a divergent multifaceted response: DNA damage followed by cell death vs. induction of adaptive mechanisms including autophagy and high ER stress. Although both E and DNR preferentially targeted proliferating cells and induced cell cycle arrest, overall stress response to E was distinct from stress to DNR in high-dimensional plane. To attain a comprehensive overview of stress response to E vs. DNR we compared t-SNE maps depicting overall stress response and observed significant segregation. Autophagy and ER stress was more pronounced in E group while DNR completely abrogated proliferation in surviving cells. To further corroborate the utility of this approach, we assessed the activity of exportin-1 (XPO1, KPT-330) and MDM2 (DS-3032b) inhibitors. KPT-330 and DS-3032b individually induced limited cell death. Combination of XPO-1 and MDM2 inhibitors resulted in enhanced apoptotic cell death with unrepaired DNA damage while surviving cells displayed an autophagy pattern. Conclusion: These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Studies to identify stress signatures of targeted agents currently developed for the treatment of AML are ongoing Figure 1 Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership.

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