Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Simultaneous Detection ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3620-3620
Author(s):  
Yang Liu ◽  
Min Ni ◽  
Aldo M. Roccaro ◽  
Xavier Leleu ◽  
Yong Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Binding Sites ◽  
Research Funding ◽  
Enrichment Analysis ◽  
Promoter Regions ◽  
Advisory Committees ◽  
Pol Ii ◽  
Normal Controls

Abstract Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Dynamic Changes in Clonal Clearance with Decitabine Therapy in AML and MDS Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 689-689
Author(s):  
John S. Welch ◽  
Allegra Petti ◽  
Christopher A. Miller ◽  
Daniel C. Link ◽  
Matthew J. Walter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Complete Remission ◽  
Clinical Response ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Advisory Committees ◽  
Clonal Hematopoiesis ◽  
Clinical Responses

Abstract To determine how AML subclonal architecture changes during decitabine treatment, and whether specific mutations might correlate with sensitivity vs. resistance to decitabine, we performed exome sequencing at multiple time points during single agent decitabine therapy. We enrolled 69 patients with either AML (age ≥ 60, or with relapsed/refractory disease, N = 45) or MDS (N = 24) on a phase I clinical trial. All subjects were treated with decitabine 20 mg/m2 on days 1-10 of 28 day cycles. With a median follow-up of 13.7 months, the intention to treat clinical response (complete remission with or without complete count recovery: CR/CRi) is 40%, with survival correlating with response (median survival - CR/CRi: 583 days; partial response/stable disease (PR/SD): 260 days; progressive disease (PD) or failure to complete cycle 1: 36 days, p < 0.0001). We performed exome sequencing on unfractionated bone marrow cells at diagnosis (day 0), cycle 1 day 10, cycle 1 day 28, cycle 2 day 28, and, when possible, during remission and at clinical relapse/progression. We have completed sequencing analysis for the first 34 cases (outcomes: 5 CR, 15 CRi, 3 PR, 8 SD, and 3 PD). Several important themes have emerged, as follows: 1) We correlated mutation status at diagnosis with clinical response. All six patients with TP53 mutations obtained clinical CR or CRi, and exome analysis demonstrated near complete elimination of the TP53- associated founding clones by the end of cycle 2 (p < 0.03). Long-term outcomes were similar in these patients compared with other patients who achieved CR/CRi: four patients relapsed after 8, 9, 10, or 17 cycles; 1 patient is doing well post-transplant; and one patient died of an infectious complication after cycle 2. No other mutations were significantly associated with clinical response or with consistent mutation clearance. 2) We observed a reduction in blast counts, which preceded mutation elimination in fourteen cases with CR, CRi or PR. This suggests that decitabine may induce morphological blast differentiation in vivo prior to mutation elimination. 3) In eight of nine cases with a clinical response followed by relapse, clinical progression was associated with expansion of a pre-existing subclone. We have not yet observed any recurrent mutations that reliably predict whether a subclone will contribute to relapse. Intriguingly, in two of these cases, the relapse-associated subclone was detectable at diagnosis and was eliminated more slowly than the founding clone mutations, suggesting that this subclone harbored intrinsic decitabine-resistance. 4) Complete remission can occur with concomitant non-malignant, clonal hematopoiesis. In three cases with a CR, a new clonal population was selected for during the remission. In two of these cases, there were no shared mutations between the founding clone and the emergent, non-malignant, clonal hematopoiesis, suggesting that these clones were unrelated. 5) Mutational architecture is generally stable, but differential chemo-sensitivity can be detected even between subclones in the same patient. In ten cases with PR or SD, we observed minimal shifts within the mutational burden over the course of eight weeks, suggesting that "clonal drift" is a relatively slow process. However, in four cases with SD, what appeared clinically to be simple persistent disease was in fact a dynamic elimination of one subclone, and its replacement by a different subclone. Similarly, in three cases with CRi, we observed rapid clearance of a subclone with slower clearance of the founding clone, again suggesting differential chemo-sensitivity among subclones. 6) Finally, we correlated pharmacologic markers with clinical outcomes. We observed no correlation between steady-state plasma decitabine levels and clinical responses. Using Illumina 450k methylation arrays, we observed a correlation between response and the extent of decitabine-induced hypomethylation in total bone marrow cells that persisted on cycle 1 day 28 (p < 0.01), but not on cycle 1 day 10 (p < 0.1). In summary, these data reveal that response to decitabine is associated with morphologic blast clearance before mutations are eliminated, that relapse is associated with subclonal outgrowth that may be identified early in the treatment course, and that TP53 mutations may be predictive of rapid clinical responses, although, like most responses to decitabine, these are not necessarily durable. Disclosures Off Label Use: Decitabine treatment of AML.. Uy:Novartis: Research Funding. Oh:CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Abboud:Novartis: Research Funding; Gerson Lehman Group: Consultancy; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Merck: Research Funding; Teva Pharmaceuticals: Research Funding. Cashen:Celgene: Speakers Bureau. Schroeder:Celgene: Other: Azacitidine provided for this trial by Celgene; Incyte: Consultancy. Jacoby:Sunesis: Research Funding; Novo Nordisk: Consultancy.

scholarly journals RON Kinase Is a Novel Therapeutic Target for Philadelphia-Negative Myeloproliferative Neoplasms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1462-1462
Author(s):  
Lindsay Meg Gurska ◽  
Rachel Okabe ◽  
Meng Maxine Tong ◽  
Daniel Choi ◽  
Kristina Ames ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Marrow Transplant ◽  
Jak Inhibitor ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Stat Signaling ◽  
Genetic Deletion

Abstract The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown. We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D). To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target. To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. Figure 1 Figure 1. Disclosures Halmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

scholarly journals Baseline and on-Treatment Bone Marrow Microenvironments Predict Myeloma Patient Outcomes and Inform Potential Intervention Strategies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1882-1882 ◽  
Author(s):  
Samuel A Danziger ◽  
Mark McConnell ◽  
Jake Gockley ◽  
Mary Young ◽  
Adam Rosenthal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Profiles ◽  
Cell Types ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

scholarly journals DNMT3A and TET2 mutant Clonal Hematopoiesis May Drive a Proinflammatory State and Predict Enhanced Response to Immune Checkpoint Inhibitors

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4295-4295
Author(s):  
Abhay Singh Singh ◽  
Nuria Mencia-Trinchant ◽  
Elizabeth A. Griffiths ◽  
Mahesh Swaminathan ◽  
Matthew Gravina ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Checkpoint Inhibitors ◽  
Advisory Board ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Serial Sample ◽  
Serial Samples

Abstract Background. DNA methylation is a key epigenetic process involved in development, aging, and cancer. Mutations in DNMT3A and TET2 in the hematopoietic stem cell compartment lead to increased self-renewal. In addition to mutations in ASXL1, collectively, these DTA mutations are recognized as an aging phenomenon, known as the most common Clonal hematopoiesis of Indeterminate Potential (CHIP) mutations and alone are not predictive of increased risk for hematopoietic malignancy. Recently, DNMT3A mutations in donor hematopoietic cells were suggested to be associated with enhanced T-cell activity in allografted patients. Additionally, role of DNMT3A mutations in creating a proinflammatory state in cardiovascular disease setting and associated elevation of T-cell markers in the myocardium have been recently explored (Sano S et al. Circ Res. 2018). Since an inflamed tumor microenvironment is associated with improved immune checkpoint inhibitors (CPI) activity, we sought to determine the impact of CHIP (a proinflammatory state) on response to CPI and CPI's effects on clonal dynamics. Additionally, while classical chemotherapy (CTX) can create selective external pressure providing survival advantage to mutant stem cells, the selective pressure of T-cell activating therapies on hematopoietic stem cells is unclear. Methods. To study the relationship between CHIP and CPI, we used paired peripheral-blood samples taken before and after treatment with CPI therapy in patients (pts) with melanoma (MEL; n= 32) and non-small cell lung cancer (NSCLC; n=109). Serial samples (or post CPI samples) were evaluable in 5 MEL pts and 6 NSCLC pts. Error-corrected sequencing of a targeted panel of genes recurrently mutated in clonal hematopoiesis (CH) was performed on peripheral blood genomic DNA. Statistical comparisons between baseline and serial sample VAFs were performed using two-sided fisher's exact test, with a p &lt; 0.05 considered significant. Results. In both the MEL and NSCLC cohort, baseline samples were collected before extensive therapy exposure. 90% (29/32) of the MEL cohort had no CTX or targeted therapy prior to the baseline sample; 28% (9/32) had prior radiotherapy (RT). 10% (11/109) of the NSCLC cohort samples had prior CTX, but only 2 of these were treated for more than 1 month before sample collection. CH was frequent in these minimally pre-treated patient samples; 28.1% (9/32) and 37.6% (41/109) of the baseline MEL and NSCLC samples, respectively. As expected, DTA mutations were the most common events in these cohorts. Samples with CH were from patients of older age, but had normal hematological parameters with exception of increased RDW (p=0.022). Primary tumor responses in this cohort were defined as durable (receipt of ≥12 CPI cycles) or not durable (&lt;12 cycles). DNMT3Amut patients (VAF ≥1%, n=5) had more durable responses, i.e. higher median number of CPI cycles (21 cycles, range:10-40) compared to non-DNMT3Amut pts (7 cycles, range:1-13; p= NS). Additionally, pts with larger DNMT3Amut clones (figure 1- MEL cohort) tended to receive higher numbers of CPI cycles. In the serial sample analysis, we observed that mutations in DNMT3A and TET2 increased in size with longer CPI exposures (Figure 2, MEL cohort); pts 2, 3 and 5 received 13, 15 and 18 CPI cycles respectively, while pt 4 with the most notable clonal expansion in DNMT3A received 40 CPI cycles. All serial samples in MEL cohort showed a statistically significant change in VAF from baseline. In the serial sample analysis of NSCLC pts, we observed that those with ≥ 3 months of CPI exposure demonstrated decreases in clone size for non-DTA gene mutations such as SRCAP, STK11 and TPM1 (Table 1), but increases or stability in DNMT3A and TET2 mutations (Table 1). However, this VAF increase in DNMT3A and TET2 mutations in NSCLC cohort was not statistically significant. Conclusions. In this small cohort of pts with MEL and NSCLC, the presence of DNMT3A/TET2 CH was associated with longer checkpoint inhibitor exposure and increased allelic frequency over time. These findings need further validation in larger cohorts and delineation of the relationship between DTA mutations such as DNMT3A and enhanced immune activity. Acknowledgement: Data and samples for this study were provided by the Data Bank and BioRepository (DBBR), which is funded by the National Cancer Institute (P30 CA016056) and is a Roswell Park Cancer Institute Cancer Center Support Grant shared resource. Figure 1 Figure 1. Disclosures Griffiths: Taiho Oncology: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Research Funding; Novartis: Honoraria; Boston Biomedical: Consultancy; Astex Pharmaceuticals: Honoraria, Research Funding; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Apellis Pharmaceuticals: Research Funding; Genentech: Research Funding; Takeda Oncology: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Hassane: Tempus Labs, Inc: Current Employment. Guzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Wang: Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Genentech: Membership on an entity's Board of Directors or advisory committees; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

scholarly journals Gene Expression Profiling of CD93-Selected CP-CML Stem Cells Confirms Their Quiescent Character and Biomarker Potential

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4231-4231
Author(s):  
Gillian A. Horne ◽  
Chinmay Rajiv Munje ◽  
Ross Kinstrie ◽  
Eduardo Gómez-Castañeda ◽  
Helen Wheadon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Single Cells ◽  
Gene Clustering ◽  
Single Cell Level ◽  
Cell Level ◽  
Advisory Committees ◽  
Two Populations

Abstract The introduction of BCR-ABL tyrosine kinase inhibitors has revolutionized the treatment of chronic myeloid leukemia (CML). A major clinical aim remains the identification and elimination of low-level disease persistence, termed "minimal residual disease". Disease persistence suggests, that despite targeted therapeutic approaches, BCR-ABL-independent mechanisms exist which sustain the survival of a small population of cells, termed leukemic stem cells (LSC). We previously identified CD93 expression as a promising biomarker of LSC in chronic phase (CP)-CML. Our group has described the long term self-renewal potential of Lin-CD34+93+ CP-CML cells compared to their Lin-CD34+93- counterparts through LTCIC assays (n=3, p<0.0001) and NSG engraftment models (3.5-30-fold increased in engraftment with Lin-CD34+93+ cells, p<0.03). We hypothesized that CD93+-selected cells would represent a more immature functional phenotype compared to CD93- selected cells. The aim of this study was to characterize differences in the gene expression profile between CD93+ and CD93- CML LSC populations and determine heterogeneity of each population at a single cell level. To interrogate this, we initially identified CP-CML subpopulations with the greatest functional capability compared to normal. Normal and CP-CML samples were FACS-sorted into HSC/LSC, CMP, GMP, and MEP sub-populations. Results suggest a significant change in functional status between normal and CP-CML subpopulations within the HSC/LSC compartment (lin-CD34+CD38-CD45RA-CD90+), where CML LSC demonstrated significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture in physiological growth factors. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively (p<0.05)). Furthermore, fluorescence in situ hybridization demonstrated that >90% of lin-CD34+CD38-CD45RA-CD90+ CML LSC from all patient samples were BCR-ABL positive. Subsequent experiments were confined to the LSC population. We hypothesized that lin-CD34+CD38-CD90+CD93- CML cells would have a more mature gene expression profile compared to lin-CD34+CD38-CD90+CD93+ cells. CP-CML cells were sorted into (1) lin-CD34+, (2) lin-CD34+CD38-CD90+CD93- and (3) lin-CD34+CD38-CD90+CD93+ populations. RNA was harvested at baseline from bulk populations (1) to (3) and cDNA was generated from single cells using the Fluidigm C1 autoprep system. Using Fluidigm technology, quantitative PCR of 90 lineage-specific and cell survival genes was performed within all populations of cells (1) to (3) in 'bulk' samples (n=3), and at single cell level (n=123 CD93+, n=120 CD93-single cells; n=3 samples in total). Bulk sample analysis demonstrated a significant increase in expression of lineage commitment genes within the lin-CD34+CD38-CD90+CD93- population, as shown by increased expression of GATA1 (p=0.0007), and CBX8 (p=0.0002). The lin-CD34+CD38-CD90+CD93+ population displayed a less lineage-restricted profile with increased expression of CDK6 (p=0.05), HOXA6 (ns), CDKN1C (ns) and CKIT (p=0.0014), compared to the lin-CD34+CD38-CD90+CD93- population. Furthermore, the two populations could be segregated by differential gene expression through gene clustering. At a single cell level, differences were noted in the frequency of expression between lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations, particularly in GATA1, TPOR, and VWF. Although a statistically significant change was demonstrated in gene expression between the lin-CD34+CD38-CD90+CD93- and lin-CD34+CD38-CD90+CD93+ populations in a number of genes, we were not able to segregate the populations by differential expression using gene clustering. This highlights the heterogeneous nature of the cell populations and the inability to distinctly characterize between the two populations at a single cell level. Our results validate CD93 as a potential biomarker to separate the primitive CP-CML LSC population and highlight key lineage and cell survival pathways that are altered in CML LSC. The results demonstrate the heterogeneity seen within gene expression at the single cell level, which may allow for further insight into the CML LSC compartment with further analyses. Disclosures Wheadon: GlaxoSmithKline: Research Funding. Copland:Shire: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria.

scholarly journals GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Akira Chiba ◽  
Yosuke Masamoto ◽  
Hideaki Mizuno ◽  
Mineo Kurokawa
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Hematopoietic Progenitor Cells ◽  
Rna Seq ◽  
Hematopoietic Progenitor ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Knock Out

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

scholarly journals Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1092-1092
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Single Cell ◽  
Research Funding ◽  
Inflammatory Responses ◽  
Inflammatory Microenvironment ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis ◽  
Otsuka Pharmaceutical ◽  
Mutant Cells

Abstract Background Clonal hematopoiesis (CH) in apparently healthy individuals has been drawing an increasing attention of recent years due to its implication in the risk of hematopoietic malignancies and also in cardiovascular complications. However, our knowledge on CH has been largely based on genetic studies, while few functional analyses have been performed using human materials with most studies being confined to artificial models using mice. The major challenge here is the difficulty of isolating CH clones from wild-type (WT) cells in human bone marrow (BM) to elucidate the effect of CH-mutations. Methods To investigate cellular phenotypes of mutated and WT cells separately in CH, we developed a Fluidigm C1-based single cell-sequencing platform for simultaneous genotyping and gene expression analysis. We analyzed a total of 10,178 hematopoietic stem/progenitors (HSPCs) derived from BM of patients with (n=11) and without (n=17) CH. Results In the analysis of HSPCs from CH(−) elderly individuals, we found a significant positive correlation between age and expression of gene sets implicated in inflammatory responses including TGFβ signalling and IL-6. We next analysed CH(+) samples, including those with mutations in TET2, DNMT3A, SF3B1 and IDH1 in which we investigated the pathways enriched in differentially expressed genes between mutated and unmutated cells. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation, while genes related to inflammatory responses were significantly downregulated. These results suggest that mutant HSPCs show an enhanced cell proliferation and an attenuated response to an inflammatory microenvironment in aged BM, compared with endogenous WT counterparts. Based on these observations, we further investigated the role of the BM microenvironment in CH, in which we compared the phenotype of WT cells in age-matched CH(+) and CH(−) cases. Compared with those from CH(−) cases, WT cells from these CH(+) cases showed an enhanced response to proinflammatory cytokines, including IL-6, interferons and TNF, suggesting a possibility that CH(+) BM might be characterized by a more enhanced inflammatory microenvironment, compared with CH(−) BM. To understand the different phenotype of WT cells between CH(+) and CH(−) cases, we next investigated a possible effect of mutant cells on endogenous WT cells. Such an effect was first documented in an IDH1-mutated CH case. IDH1-mutated cells showed a gene expression profile suggestive of an enhanced cell proliferation compared with endogenous IDH1-WT cells (WT IDH1). However, on the basis of comparison of gene expression between WT cells from CH(−) cases, it was better explained by suppressed proliferation of endogenous WT cells, rather than enhanced proliferation of mutant cells. In agreement with this, mouse BM cells treated with 2-hydroxyglutalate (2HG), an oncometabolite produced by IDH1-mutant cells, mimicked the endogenous WT cells in IDH1-mutated cases, including downregulated E2F target genes and upregulated inflammation-related genes, compared with control BM cells. A non-cell autonomous effect of mutations was also seen in cases with TET2-mutated CH, in which not only TET2-mutated cells but also endogenous WT cells exhibited a significantly different gene expression profile, compared with those from CH(−) cases. Of interest, when BM cells from WT Ly5.1/5.2 mice were co-transplanted with those from heterozygous conditional Tet2 knock-out (Ly5.2) or WT (Ly5.2) mice into lethally irradiated mice (Ly5.1) and flow-sorted Lin − WT donor competitor cells were analysed using single cell sequencing, the WT HSC-like cells co-transplanted with Tet2-mutant competitors exhibited enhanced cell proliferation and IFNα and IFNγ pathway genes, compared with those co-transplanted with WT competitors. Conclusions Taken together, these results suggest that mutant cells in CH(+) BM have non-cell autonomous effects on endogenous WT cells, which might be responsible for an accelerated inflammatory microenvironment of aged BM, favor positive selection of CH-clones, and also affect the phenotype of endogenous WT cells, contributing to the pathogenesis of CH. Disclosures Nakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Inagaki: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Yoda: Chordia Therapeutics Inc.: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

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