Transcriptome Profiling of Pediatric Myeloproliferative Neoplasms Demonstrates Dysregulation of Platelet-Relevant Genes and Enrichment of Inflammatory and JAK2 Mediated Complement Pathways

Introduction : Myeloproliferative neoplasms (MPNs) are rare clonal bone marrow disorders in children characterized by high blood counts, predisposition to clotting events, and the potential to transform to myelofibrosis or acute myeloid leukemia (AML). Children with MPNs have lower rates of the known driver mutations (in JAK2, MPL, and CALR) than adult patients, and the underlying pathways and molecular derangements in young patients remain unknown. Given the lack of knowledge about pediatric MPNs, it is critical that we gain a better understanding of the dysregulated pathways in these diseases, which is necessary for improving disease understanding and broadening treatment options in children. Therefore, the objective of this work was to identify differentially expressed genes and pathways between children with MPNs and healthy controls, as well as children with AML, to guide further study. Methods : Mononuclear cells were extracted from peripheral blood of pediatric MPN patients (n=20) and pediatric and young adult AML patients (n=1410), and bone marrow of normal controls (NC, n=68). AML patient samples were being evaluated as part of a Children's Oncology Group planned analysis. To identify an expression profile unique to MPNs, transcriptome data from MPN patients was contrasted against NC and AML patients. All samples were ribodepleted and underwent Illumina RNA-Seq to generate transcriptome expression data. All analyses were performed in R. Differentially expressed genes were identified using the voom function from the limma package (v. 3.38.3), and enriched pathways were identified using the pathfindR package (v. 1.3.1). Unsupervised hierarchical clustering and heatmap generation was performed using the ComplexHeatmap package (v. 1.20.0). Results : MPN patient samples showed a unique expression signature, distinct from both AML patients and normal controls. Unsupervised PCA plot (Figure 1A) and heatmaps (Figure 1B) show that MPN samples cluster together. There were 4,012 differentially expressed (DE) genes in MPNs compared to NC and 6,743 DE genes in MPNs compared to AML patients. There were 2,493 shared genes between the 2 groups (Figure 1C.) Significantly DE genes between MPNs and other groups included multiple platelet-relevant genes including PF4 (CXCL4), PF4V1, P2RY12, and PPBP (CXCL7). Interestingly, PF4V1 was the most DE gene in MPNs compared to AML, and third highest versus NC. Dysregulation of some of these genes has been seen in adult MPNs, as well as thrombosis. Further comparison of transcriptome profiles between children with (n=13) and without (n=7)JAK2 mutations showed upregulation of three genes, CFB, C2, and SERPING1, which are all known complement genes, implicating complement activation in JAK2-mutated MPN patients. Complement activation has previously been reported in adult MPNs. Pathway enrichment analysis shows a number of immune and inflammatory pathways as enriched in MPN patients compared to both AML and NC. There were 179 enriched pathways in MPNs compared to AML and 142 compared to NC, with 134 common pathways (Figure 1D.) The systemic lupus erythematosus pathway was the most heavily enriched pathway in MPNs compared to both AML and NC. Additional pathways with significant enrichment include hematopoietic cell lineage, cytokine-cytokine interactions, DNA replication, and various infection-relevant pathways. The JAK-STAT signaling pathway was also enriched in MPNs compared to both AML and NC, as was the platelet activation pathway. Conclusion: Transcriptome evaluation of childhood MPNs shows enrichment of numerous inflammatory and immune pathways, highlighting that, as in adult MPNs, inflammation is implicated in pediatric MPNs. Furthermore, specific complement genes were upregulated in JAK2-mutant MPN. Upregulation of platelet-specific genes implies potential insights into disease mechanisms and warrants more study. Variations in the cell populations may account for some of the differences seen, however all samples were largely mononuclear cells, making their comparisons reasonable. Further analysis of this early data is needed to better assess inflammatory changes and platelet activation in pediatric MPNs, as are larger sample sizes. Individual cells may have differential expression of various genes, and future experiments with single-cell RNA-seq would be helpful to further elucidate differences. Disclosures Levine: Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Roche: Consultancy, Research Funding; Lilly: Honoraria; Amgen: Honoraria; Qiagen: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees.

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Phase 1/2 Study of Elotuzumab in Combination with Bortezomib in Patients with Multiple Myeloma with One to Three Prior Therapies: Interim Results.

Blood ◽

10.1182/blood.v114.22.3876.3876 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3876-3876 ◽

Cited By ~ 1

Author(s):

Andrzej J Jakubowiak ◽

William Bensinger ◽

David Siegel ◽

Todd M. Zimmerman ◽

Jan M. Van Tornout ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Phase 1 ◽

Surface Glycoprotein ◽

Advisory Committees ◽

Cell Surface Glycoprotein

Abstract Abstract 3876 Poster Board III-812 Background Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein, which is highly and uniformly expressed in multiple myeloma (MM). In mouse xenograft models of MM, elotuzumab demonstrated significantly enhanced anti-tumor activity when combined with bortezomib compared to bortezomib alone (Van Rhee et al., Mol. Cancer Ther., in press, 2009). This phase 1/2 trial will determine the maximum tolerated dose (MTD), overall safety, pharmacokinetics (PK) and clinical response of elotuzumab in combination with bortezomib in patients with relapsed MM following 1-3 prior therapies. Methods The study consists of 4 escalating cohorts of elotuzumab (2.5 mg/kg to 20 mg/kg) administered on Days 1 and 11 and bortezomib (1.3 mg/m2) administered on Days 1, 4, 8 and 11 of a 21-day cycle. Patients with progressive disease at the end of Cycle 2 or 3 also receive oral dexamethasone (20 mg) on Days 1, 2, 4, 5, 8, 9, 11 and 12 of each subsequent cycle. Patients with stable disease or better at the end of 4 cycles will continue treatment for 6 or more cycles unless withdrawn earlier due to unexpected toxicity or disease progression. Key entry criteria: age ≥ 18 years; confirmed diagnosis of MM and documentation of 1 to 3 prior therapies; measurable disease M-protein component in serum and/or in urine; and no prior bortezomib treatment within 2 weeks of first dose. Results To date, a total of 16 MM patients with a median age of 64 years have been enrolled in the study. The median time from initial diagnosis of MM was 3.5 years and patients had received a median of 2 prior MM treatments. Patients have been treated in four cohorts; 3 each in 2.5, 5 and 10 mg/kg elotuzumab cohorts, and 7 in the 20 mg/kg elotuzumab cohort. No dose limiting toxicity (DLT) was observed during the first cycle of the study and the MTD was not established. Five SAEs have been reported in four patients in later treatment cycles; two events, chest pain and gastroenteritis, occurring in one patient, were considered elotuzumab-related. Other SAEs include grade 3 sepsis, vomiting, pneumonia and grade 2 dehydration. The most common AEs reported include Grade 1-3 diarrhea, constipation, nausea, fatigue, thrombocytopenia, neutropenia, anemia and peripheral neuropathy. The best clinical response (EBMT criteria) for the 16 patients who have received at least two cycles of treatment is shown in the table below. Preliminary PK analysis suggests a serum half-life of 10-11 days at higher doses (10 and 20 mg/kg). Preliminary analysis of peripheral blood mononuclear cells and bone marrow of patients on study indicates that objective responses in the study correlate well with complete saturation of CS1 sites by elotuzumab on bone marrow plasma and NK cells. Conclusions The combination of elotuzumab with bortezomib has a manageable adverse event profile and shows promising preliminary efficacy with ≥PR in 44% and ≥MR in 75% of all enrolled patients. Accrual is ongoing in the expanded 20 mg/kg cohort. Updated safety, efficacy, and PK data will be presented at the meeting. Disclosures: Jakubowiak: Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Centocor Ortho Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Bortezomib in combination with elotuzumab for the treatment of relapsed/refractory multiple myeloma. Bensinger:Millennium: Membership on an entity's Board of Directors or advisory committees. Siegel:Millennium: Speakers Bureau; Celgene: Speakers Bureau. Zimmerman:Millennium: Speakers Bureau; Centecor: Speakers Bureau. Van Tornout:BMS: Employment. Zhao:Facet Biotech: Employment. Singhal:Facet Biotech: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Promoter-Wide Transcriptional Deregulation In Waldenstrom Macroglobulinemia

Blood ◽

10.1182/blood.v116.21.3620.3620 ◽

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.

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

Blood ◽

10.1182/blood-2021-148192 ◽

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.

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Significance of Abnormalities of PPM1D in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2019-129565 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4207-4207

Author(s):

Bridget Marcellino ◽

Joseph Tripodi ◽

Michal Bar-Natan ◽

Harry Fruchtman ◽

Noushin Farnoud ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Myeloproliferative Neoplasms ◽

Fold Increase ◽

Advisory Committees ◽

Transcript Levels ◽

Activating Mutations ◽

Cytogenetic Aberrations ◽

Tp53 Pathway

The TP53 pathway has been shown to be dysregulated in myeloproliferative neoplasms (MPN) through several different mechanisms, including TP53 mutations, TP53 deletions and Murine Double Minute 2 (MDM2) overexpression. Downregulation of the TP53 pathway likely plays a role in MPN progression as evidenced by the association of TP53 loss of heterozygosity with transformation to acute leukemia and the presence of inactivating mutations of TP53 found in a proportion of MPN-blast phase (MPN-BP) cases (Kubesova et al, Leukemia. 2018;32(2):450-61). Furthermore, MDM2 inhibition induces TP53 pathway upregulation and consequent targeting of hematopoietic stem and progenitor cells in polycythemia vera (PV) and myelofibrosis (MF). The MDM2 inhibitor, idasanutlin, has shown activity in a Phase I trial of high-risk PV patients (Mascarenhas et al. Blood. 2019 Jun 5). An additional regulator of TP53 is protein phosphatase, Mg2+/Mn2+ dependent 1D (PPM1D) which by dephosphorylating TP53 and stabilizing MDM2 regulates the DNA damage response pathway and apoptosis. Somatic activating mutations of PPM1D are present in both solid and hematologic malignancies and are specifically associated with therapy-related myeloid disorders (Hsu et al. Cell Stem Cell. 2018 Nov 1;23(5):700-13). Grinfeld et al. (N Engl J Med. 2018 Oct 11;379(15):1416-30) recently reported that in their genomic analysis of 2035 MPN patients, PPM1D was the eighth most common mutated gene in MPNs at 1.9% frequency. To determine whether there was an association of PPM1D mutations with MPNs we analyzed the genomic data of patients who participated in several clinical trials executed by the Myeloproliferative Neoplasm Research Consortium (MPN-RC). Of 89 MPN-BP patients, 5 patients harbored a PPM1D mutation with a median variant allele frequency (VAF) of 17%. In comparison, 4 out of 135 high risk PV and ET patients harbored a PPM1D mutation with a median VAF of 24%. All mutations were truncating which is consistent with previous reports of PPMID mutations in malignancies. The PPM1D gene is located on the long arm of chromosome 17 (17q23.2) and we hypothesized that cytogenetic aberrations involving this gene locus might also contribute to abnormalities of PPM1D function. Through analysis of our cytogenetic database, 16/1,124 (1.4%) MPN patients were found to have cytogenomic abnormalities involving the region containing the PPM1D gene. Eight of these patients had karyotypic abnormalities, including 3 pts with isochromosome 17q resulting in a gain of 17q and a loss of 17p, including the TP53 gene. The other 4 patients had structural variations of 17q which might lead to aberrant expression of PPM1D. One patient by FISH analysis had gain of 17q. Ten patients had cytogenomic aberrations of 17q detected through analysis of array comparative genomic hybridization and single nucleotide polymorphism (aCGH+SNP), 2 of which had concurrent karyotypic abnormalities of 17q. All patients had a gain or copy neutral loss of heterozygosity (cnLOH) of the 17q22-24.2 region. CnLOH of this region could lead to aberrant overexpression of the PPM1D gene. One of the 8 patients with cnLOH of 17q harbored a PPM1D mutation. Of the 16 patients with 17q cytogenomic abnormalities, 7 (44%) had MPN-BP and 7 (44%) patients had ET or ET that progressed to myelofibrosis (MF) or MPN-BP indicating an association of these abnormalities with advanced disease. By quantitative real time-PCR we determined the PPM1D transcript levels in mononuclear cells from 31 MPN patients without known PPM1D mutations (7 polycythemia vera (PV), 6 ET, 14 MF, 4 MPN-BP) and compared the transcript levels to mononuclear cells from healthy control patients. Forty-two percent (13/31) patients had overexpression of PPM1D (>1.5 fold increase range). Fold increase ranged from 1.5-8 and overexpression was present in the following diagnoses: 4/7 PV, 1/6 ET, 8/14 MF and 0/4 MPN-BP. We have provided evidence that a number of abnormalities of PPM1D including activating mutations, cytogenetic aberrations and transcript overexpression are present in a significant proportion of MPN patients. These abnormalities in PPM1D can be additional events that lead to the downregulation of TP53 and contribute to MPN disease progression. We propose that inhibitors of PPM1D may be used in combination with other drugs that upregulate TP53 to treat MPN patients. Disclosures Rampal: Constellation, Incyte, and Stemline Therapeutics: Research Funding; Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy. Mascarenhas:CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Promedior: Research Funding; Merus: Research Funding. Hoffman:Merus: Research Funding.

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GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Blood ◽

10.1182/blood-2020-134071 ◽

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.

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Platelet Transcriptome Yields Progressive Markers in Chronic Myeloproliferative Neoplasms and Identifies Putative Targets of Therapy

Blood ◽

10.1182/blood-2021-153323 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1469-1469

Author(s):

Zhu Shen ◽

Wenfei Du ◽

Cecelia Perkins ◽

Lenn Fechter ◽

Vanita Natu ◽

...

Keyword(s):

Machine Learning ◽

Growth Factor ◽

Risk Stratification ◽

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Rna Seq ◽

Advisory Committees ◽

Chronic Myeloproliferative Neoplasms ◽

Healthy Donors

Abstract Predicting disease progression remains a particularly challenging endeavor in chronic degenerative disorders and cancer, thus limiting early detection, risk stratification, and preventive interventions. Here, profiling the spectrum of chronic myeloproliferative neoplasms (MPNs), as a model, we identify the blood platelet transcriptome as a proxy strategy for highly sensitive progression biomarkers that also enables prediction of advanced disease via machine learning algorithms. Using RNA sequencing (RNA-seq), we derive disease-relevant gene expression in purified platelets from 120 peripheral blood samples constituting two time-separated cohorts of patients diagnosed with one of three MPN subtypes at sample acquisition - essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or post ET/PV secondary myelofibrosis, MF (n=42), and healthy donors (n=21). The MPN platelet transcriptome reveals an incremental molecular reprogramming that is independent of patient driver mutation status or therapy and discriminates each clinical phenotype. Differential markers in each of ET, PV and MF also highlight candidate genes as potential mediators of the pro-thrombotic and pro-fibrotic phenotypes in MPNs. In ET and PV, a strong thromboinflammatory profile is revealed by the upregulation of several interferon inducible transmembrane genes (IFITM2, IFITM3, IFITM10, IFIT3, IFI6, IFI27L1, IFI27L2), interleukin receptor accessory kinases/proteins (IRAK1, IL15, IL1RAP, IL17RC) and several solute carrier family genes (SLC16A1, SLC25A1, SLC26A8, SLC2A9) as glucose and other metabolic transport proteins, and coagulation factor V (F5). In MF, fibrosis-specific markers were identified by an additional focused comparison of MF patients versus ET and PV, showing increased expression of several pro-fibrotic growth factors (FGFR1, FGFR3, FGFRL1), matrix metalloproteinases (MMP8, MMP14), vascular endothelial growth factor A (VEGFA), insulin growth factor binding protein (IGFBP7), and cell cycle regulators (CCND1, CCNA2, CCNB2, CCNF). Also, focusing on the JAK-inhibitor ruxolitinib/RUX-specific signatures, we not only confirm previous observations on its anti-inflammatory and immunosuppressive effects (e.g. downregulation in our RUX-treated cohort of IL1RAP, CXCR5, CPNE3, ILF3) but also identify new gene clusters responsive to RUX - e.g. inhibition of type I interferon (e.g. IFIT1, IFIT2, IFI6), chromatin regulation (HIST2H3A/C, HIST1H2BK, H2AFY, SMARCA4, SMARCC2), epigenetic methylation in mitochondrial genes (ATP6, ATP8, ND1-6 and NDUFA5), and other proliferation, and proteostasis-associated markers as putative targets for MPN-directed therapy. Mechanistic insights from our data highlight impaired protein homeostasis as a prominent driver of MPN evolution, with a persistent integrated stress response. Preliminary ex vivo data on MPN patient bone-marrow-derived CD34+ cells and cultured megakaryocytes validate a proteostasis-focused subset of our peripheral platelet RNA-seq signatures. Further leveraging this substantive dataset, and in particular a progressive expression gradient across MPN, we develop a machine learning model (Lasso-penalized regression) predictive of the advanced subtype MF at high accuracy and under two conditions of validation: i) temporal Stanford internal (AUC-ROC of 0.96) and ii) geographic external cohorts (AUC-ROC of 0.97, using independently published data of an additional n=25 MF and n=46 healthy donors). Lasso-derived signatures offer a robust core set of < 5 MPN progression markers. Together, our platelet transcriptome snapshot of chronic MPNs demonstrates a methodological avenue for disease risk stratification and progression beyond genetic data alone, with potential utility in a wide range of age-related disorders. Part of the work contributing to this abstract has been posted as a preprint at this link: https://www.biorxiv.org/content/10.1101/2021.03.12.435190v2 Figure 1 Figure 1. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; Allakos: Consultancy.

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Immunogenomic Exploration of the Acute Myeloid Leukemia Microenvironment Identifies Determinants of T-Cell Fitness

Blood ◽

10.1182/blood-2018-99-118424 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2750-2750

Author(s):

Lauren K. Brady ◽

David Soong ◽

Evan F. Lind ◽

Yoko Kosaka ◽

Adam J. Lamble ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Immune Modulation ◽

Immune Cell ◽

T Cell Proliferation ◽

Rna Seq ◽

Advisory Committees ◽

Equity Ownership

Abstract Advances in Acute Myeloid Leukemia (AML) research have shown that the bone marrow microenvironment may distinctly vary across disease subtypes, and that this variation is associated with disease risk and response to conventional therapies. Novel therapies aimed at altering the tumor microenvironment, such as T-cell redirection, CAR-T and checkpoint inhibition, are emerging as promising treatment options for AML patients; however, there remains a critical need to determine how response to immune modulation may vary within different subsets of AML. Thus, in collaboration with the Beat AML Consortium, we carried out comprehensive mass cytometry profiling of patient bone marrow samples of nearly 100 Beat AML subjects and characterized their ex vivo response to several immune modulators. As a complement to this study, we leveraged the Beat AML Consortium dataset (including next-generation sequencing, functional cell-based assays, small molecule screening and clinical information) to investigate connections between disease subtype, immune function and clinical outcome. The mass cytometry time of flight (CyTOF) immune profiling, combined with matched genomic, cytogenetic, and outcome data from the same subjects, provided a unique opportunity to investigate features of the immune environment at single-cell resolution and test for their association with clinical covariates in a large treatment-naïve cohort. Interestingly, flow cytometry analysis of T-cells isolated from patient bone marrow showed a distinct subset of AML subjects with highly proliferative T-cells and a group of AML subjects with non-proliferative T-cells. To characterize molecular determinants of T-cell function in the AML microenvironment, we compared the transcriptional profiles of tumor specimens from subjects within these two groups. The data revealed a distinct set of differentially expressed genes associated with T-cell proliferation; pathway enrichment analysis indicated that these genes were involved in leukocyte migration, inflammation and response to hypoxia. Genes related to immune function were also enriched, likely due to the presence of immune cell infiltrates and stromal cells in addition to tumor cells from the AML specimens used for RNA-Seq. To estimate the extent of immune and stromal cells in the AML bone marrow, we next computed the approximate cellularity of the RNA-Seq samples using the xCell algorithm. The results of this analysis indicated enrichment of several types of immune cells in the RNA-Seq specimens from the proliferator group, including monocytes, neutrophils and activated dendritic cells. These observations were validated by preliminary results of the CyTOF immune cell profiling of the same subjects. Ongoing work is focused on the biological interpretation of CyTOF data collected for these subjects, including evaluating the association of functional marker expression on T-cell and myeloid cell populations with T-cell proliferation. Furthermore, we are exploring the functional impact of variation in T-cell fitness and immune cell composition on response to several immune modulators in a series of ex vivo experiments using Beat AML patient samples. Initial findings suggest that for a subset of patients, low baseline levels of T-cell proliferation did not prevent response to immune modulation. We are interrogating the Beat AML dataset for common molecular features of patients in this responder group. Overall, this study evaluates determinants of immune function and variation within the tumor microenvironment of AML patients to advance current knowledge of AML disease biology and to assess the impact of immune fitness on response to immune modulation. These results will contribute to early target identification and development, and importantly shed light on features of the AML bone marrow environment associated with response to therapy. Disclosures Brady: Janssen R&D: Employment. Soong:Janssen R&D: Employment. Lind:Celgene: Research Funding; Monojul: Research Funding; Amgen: Research Funding; Janssen Pharmaceutical R&D: Research Funding; Fluidigm: Honoraria. Schaffer:Janssen Research & Development: Employment, Equity Ownership. Hodkinson:Janssen R&D: Employment. Adams:Janssen Pharmaceutical R&D: Employment. Abraham:Janssen R&D: Employment. Safabakhsh:Janssen R&D: Employment. Tyner:AstraZeneca: Research Funding; Aptose: Research Funding; Array: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Druker:Aileron Therapeutics: Consultancy; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, 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; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: 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, Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Celgene: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Monojul: Consultancy; Henry Stewart Talks: Patents & Royalties. Huang:Janssen R&D: Employment, Equity Ownership.

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Human MICL Is An Early Marker In Myeloid Differentiation and Identifies a Subgroup Of CML Patients With Expanded Granulocyte-Macrophage Progenitor Populations At Diagnosis

Blood ◽

10.1182/blood.v122.21.2704.2704 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2704-2704

Author(s):

Peter Buur van Kooten Niekerk ◽

Anne Stidsholt Roug ◽

Charlotte Christie Petersen ◽

Line Nederby ◽

Charlotte Guldborg Nyvold ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Mononuclear Cells ◽

Chronic Phase ◽

Colony Growth ◽

Advisory Committees ◽

Fraction Size ◽

Tki Treatment ◽

Progenitor Compartment

Abstract Although chronic myeloid leukemia (CML) originates from a primitive hematopoietic stem cell (HSC), it is the more differentiated progenitor cells that drive the expansion of the malignant clone. In addition, previous studies of chronic phase CML have shown that, despite the marked leukocytosis observed here, megakaryocyte-erythroid progenitors dominate the progenitor fraction. We sought to elucidate this by employing the new marker for leukemic stem cells, the human myeloid inhibitory C-type lectin-like receptor (hMICL), in the study of progenitor cell expansion in CML. Bone marrow or peripheral blood stem cells were acquired from 11 normal donors and 31 CML patients at diagnosis in chronic phase and/or after 3-119 months of tyrosine kinase inhibitor (TKI) treatment. Cells were stained with fluorescent monoclonal antibodies and FACS sorted into HSCs (CD34+CD38-), hMICL+ progenitors (MpP; CD34+CD38+hMICL+), and hMICL- progenitors (MnP; CD34+CD38+hMICL-). Sorted cell subsets were subjected to growth in a 14-day methylcellulose assay and analyzed quantitatively for expression of the BCR-ABL fusion transcript. In normal donors, hMICL expression reproducibly identified a well-defined subpopulation of the CD34+CD38+ cells (fig 1A). The MpPs were highly enriched for cells of granulocyte-macrophage progenitors (GMP) phenotype compared to the MnPs (p=0.012) (fig 1B). Sorted MpPs produced almost exclusively granulocyte and/or macrophage (CFU-GM) colonies (median: 92% of colonies), while colonies from MnPs were dominated by BFU-Es (91%) and, as opposed to the MpPs, also contained CFU-GEMM colonies (0.64%) (fig 1C). Thus, hMICL seems to be a useful marker for the GMP population.Figure 1Immunological and functional properties of MpPs in normal donors. (A) Identification of MpPs, MnPs, and HSCs within the CD34+ compartment. (B) Cells with GMP phenotype (CD34+CD38+CD123lowCD45RA+) in MpP and MnP subsets. (C) Colony growth of bone marrow mononuclear cells (MNC) and sorted MpPs and MnPs in a 14-day methylcellulose assay. Error bars denote SDs.Figure 1. Immunological and functional properties of MpPs in normal donors. (A) Identification of MpPs, MnPs, and HSCs within the CD34+ compartment. (B) Cells with GMP phenotype (CD34+CD38+CD123lowCD45RA+) in MpP and MnP subsets. (C) Colony growth of bone marrow mononuclear cells (MNC) and sorted MpPs and MnPs in a 14-day methylcellulose assay. Error bars denote SDs. In CML at diagnosis we found decreased numbers of MpPs (mean 23% of CD34+CD38+ cells (range: 6.3-48%)), compared to the normal donors (33% (18-48%), p=0.030) (fig 2A). Extraordinarily, the MpP fraction varied considerably in size among CML patients, and 12/23 patients had MpP fractions within the 90% reference range (RR) of normal donors (MpPHIGH patients) and thus distinctly higher than the remaining patients (MpPLOW patients) (fig 2B-C). High MpP fractions significantly correlated with high WBC (Spearman's r = 0.47, p=0.049) (fig 2D), high neutrophil counts (r = 0.55, p=0.043), large spleen size (r = 0.66, p=0.0069), and low hemoglobin at the time of diagnosis (r = -0.58, p=0.014). Within the progenitor compartment, high ratio of BCR-ABL in the MpP to BCR-ABL in the MnP significantly correlated with large MpP fractions (r = 0.54, p=0.021).Figure 2Human MICL expression in chronic phase CML patients. (A) Fraction of MpPs in normal donors and CML patients at diagnosis. (B) Typical immunological profiles of MpPLOW patients and (C) MpPHIGH patients. (D) Correlation between MpP fraction size and total white blood cell count at the time of diagnosis. (E) Development of MpP fraction size in individual patients after 3-6 months (solid lines) and after 12-119 months (dotted lines) of TKI treatment in MpPLOW patients and (F) MpPHIGH patients.Figure 2. Human MICL expression in chronic phase CML patients. (A) Fraction of MpPs in normal donors and CML patients at diagnosis. (B) Typical immunological profiles of MpPLOW patients and (C) MpPHIGH patients. (D) Correlation between MpP fraction size and total white blood cell count at the time of diagnosis. (E) Development of MpP fraction size in individual patients after 3-6 months (solid lines) and after 12-119 months (dotted lines) of TKI treatment in MpPLOW patients and (F) MpPHIGH patients. During the first 6 months of TKI treatment differing developments in MpP fraction size were observed for MpPLOW and MpPHIGH patients. While MpPLOW patients showed increasing MpP fractions during the first 6 months of treatment (fig 2E), 4/4 and 2/4 MpPHIGH patients displayed a decrease in MpPs at 3 and 6 months, respectively (fig 2F). Thus, in these patients, the majority of the Ph+ progenitor cells being cleared seemed to be GMPs. In conclusion, our data demonstrate that hMICL is an early marker of granulocyte-macrophage differentiation, and provides a readily accessible approach to assessing the GMP population during TKI therapy in CML. Using the present approach we have uncovered a higher degree of variability in the composition of the progenitor compartment at diagnosis than previously reported, and shown that a significant proportion of the patients have expanded GMP populations. Ongoing studies are aimed at determining whether these patients may represent patients with a more advanced form of disease at the time of diagnosis. Disclosures: Stentoft: Novartis: Consultancy, Financial support for relevant congress participation Other, 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; Danish Regions: Membership on an entity’s Board of Directors or advisory committees. Hokland:Novartis: Research Funding.

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Chromatin Accessibility Identifies Regulatory Elements Predictive of Oncogene Expression in Multiple Myeloma

Blood ◽

10.1182/blood-2020-140717 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Benjamin G Barwick ◽

Vikas A. Gupta ◽

Shannon M Matulis ◽

Jonathan C Patton ◽

Doris R Powell ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Chromatin Accessibility ◽

Rna Seq ◽

Advisory Committees ◽

Myeloma Cells ◽

Intergenic Regions ◽

Highly Correlated

Introduction Extensive genomic characterization of multiple myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, less is known about the myeloma epigenome. One challenge that has hindered epigenetic studies are assays amenable to biobanked specimens. Here, we sought to determine whether ATAC-seq and RNA-seq of myeloma cells from cryopreserved bone marrow aspirates recapitulated those from fresh samples and used this approach to investigate enhancers of myeloma oncogenes. Methods Consent and collection of specimens followed approved Institutional Review Board protocols. Mononuclear cells were enrichment by Ficoll gradient centrifugation and were either cryopreserved in 10% DMSO and RPMI media with 10% FBS or used to isolate viable CD138+CD38+ myeloma cells. RNA-seq used the mRNA HyperPrep kit (Kapa Biosystems) with RNA from 50,000 cells. ATAC-seq used the Tn5 transposase (Illumina) on 20,000 cells. Sequencing was performed on an HiSeq 4000 (Illumina). Sequencing data were quality and adapter trimmed using Trim Galore! And mapped to the GRCh37 genome using STAR (RNA-seq) or bowtie2 (ATAC-seq). MACS2 was used to determine chromatin accessible regions and R was used for downstream analyses. H3K27ac ChIP-seq from Jin et al. (Blood, 2018) were downloaded from the European nucleotide archive (PRJEB25605). RNA-seq from CoMMpass (NCT01454297) were downloaded from dbGaP phs000748.v7.p4. Enhancer RNAs were interrogated in intergenic regions excluding 500 bp upstream of TSSs and 5 kb downstream of transcription termination sites to avoid contamination from exonic mRNAs or intronic pre-mRNAs. Results We compared RNA-seq and ATAC-seq data from myeloma cells isolated from fresh bone marrow aspirates to those cryopreserved for up to 6 months from the same aspirate. RNA-seq and ATAC-seq data from fresh and frozen samples from the same aspirate were highly correlated with each other but distinct from other samples as depicted by principal component analysis (Fig. A,B). Inspection of CCND1 showed high levels of RNA in two patients and this was consistent in both fresh and frozen specimens as well as with FISH results indicating a t(11;14) translocation in these samples (Fig. C). Similarly, fresh and frozen specimens from the same patient showed consistent expression for CCND2 and MYC and these data corresponded with chromatin accessibility found near these genes (Fig. D, see regions shaded in gray). Based on these results we expanded our analysis to include 8 biobanked specimens, which identified 91,632 regions of chromatin accessibility that were enriched around plasma cell lineage genes such as IRF4, CD38, SLAMF7, and IGH. Chromatin accessibility often predicted proximal gene expression and this was especially pronounced for regions enriched for histone 3 lysine 27 acetylation (H3K27ac) - a mark of enhancers. Active enhancers are sometimes demarcated by enhancer RNAs (eRNAs) observable in RNA-seq data, thus we queried intergenic regions marked by chromatin accessibility and H3K27ac for eRNAs using RNA-seq data on 768 myeloma specimens from the CoMMpass study. This identified transcription at 4,729 of 13,452 potential regions. eRNA expression was highly correlated with proximal gene expression. To illustrate this point, we performed t-SNE clustering based on mRNA and eRNA expression and color-coded each sample by myeloma gene expression subtype (Fig. E). Interestingly, this identified several regions highly correlated with oncogene expression between myeloma subtypes. For example, an enhancer ~154 kb upstream of CCND2 was uniquely transcribed in the MAF subtype (Fig. F) and this was highly correlated with CCND2 expression (Fig. G). Conclusions Cryopreservation of myeloma bone marrow aspirates allows isolation and analysis of biobanked samples that produce RNA-seq and ATAC-seq data that are highly congruent with those obtained from fresh samples and this represents a strategy for retrospective genomic and epigenomic studies. Chromatin accessibility analysis identified distinct enhancer elements regulating oncogenes in myeloma subtypes providing mechanistic insight into myeloma pathology. Figure 1 Disclosures Lin: Amgen: Current Employment, Current equity holder in publicly-traded company. Hofmeister:Bristol Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Nektar: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Oncopeptides: Honoraria; Oncolytics Biotech: Research Funding; Imbrium: Honoraria; Karyopharm: Honoraria, Research Funding. Nooka:Celgene: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria; Adaptive Technologies: Consultancy, Honoraria; Spectrum Pharmaceuticals: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: Personal Fees: Travel/accomodations/expenses, Research Funding; Karyopharm Therapeutics, Adaptive technologies: Consultancy, Honoraria, Research Funding; Oncopeptides: Consultancy, Honoraria. Lonial:GSK: Consultancy, Honoraria, Other: Personal fees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Takeda: Consultancy, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; Merck: Consultancy, Honoraria, Other: Personal fees; JUNO Therapeutics: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria; Onyx: Honoraria; Genentech: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Honoraria, Other: Personal fees; Sanofi: Consultancy; Abbvie: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees.

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Myeloproliferative Neoplasms in Patients below 25 Years Old at Diagnosis: A Retrospective International Cooperative Work

Blood ◽

10.1182/blood-2018-99-112374 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1759-1759

Author(s):

Ianotto Jean-Christophe ◽

Jean-Jacques Kiladjian ◽

Marta Sobas ◽

Parvis Sadjadian ◽

Lee-Yung Shih ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Myeloproliferative Neoplasms ◽

Young Patients ◽

Complication Rates ◽

Vascular Events ◽

Advisory Committees ◽

Disease Evolution

Abstract Myeloproliferative neoplasms (MPN) are most common in old people (>60 years) and are rarely identified in children and young adults where information about complication rates and long-term data are lacking. To improve our knowledge, we retrospectively collected cases of young patients diagnosed with MPN before 25 years of age and analysed data of their disease to date, including vascular events and disease evolution. Data were collected in 29 hospital centres from 12 countries. Between 1971 and 2018, 335 young patients were diagnosed for an MPN before the age of 25. They were mostly females (n=246; 73.4%) with a median age of 20.3 years at diagnosis (2.5 months-25 years). Essential thrombocythemia was diagnosed in 234 patients (69.8%), polycythemia vera in 60 (17.9%) and myelofibrosis or unclassified MPN in 41 (12.3%). Most of the diagnoses were made following a coincidental blood count analysis (n=75; 51%) some based on symptoms (n=57; 38.8%) or thrombotic events (n=15; 10.2%). In terms of complications before or at diagnosis, 31 (9.3%) patients experienced thrombosis, mostly venous (75%) and 13 (3.9%) had hemorrhage. At diagnosis, the median leukocyte count was 9x109/l (range: 3-22.8), median hemoglobin count 140 g/l (65-220) and median platelet count 900x109/l (99-3290). To assess the diagnosis, 158 patients (47.2%) had had bone marrow aspirates and 214 (63.9%) a bone marrow biopsy. Mutational status was available in 319 (90%) cases: 194 (60.8%) were JAK2V617F positive, 48 (15%) had a calreticulin mutation, 76 (23.8%) were triple-negative and 1 patient had MPL mutation. The median follow-up of the cohort was 7.7 years (0-46.8) with 134 patients (40%) having follow-up for more than 10 years. 81 female patients (32.9%) experienced pregnancies. During this period, 295 patients (88%) received at least one drug for their MPN: 254 patients (77.2%) received antithrombotic drug and 222 patients (66.5%) a cytoreductive drug. As first line of treatment, hydroxycarbamide was given to 111 patients (50%) whereas anagrelide was given to 56 patients (25.2%) and interferon to 50 (22.5%). During the follow-up, 97 patients (29%) experienced at least one complication. In terms of cardiovascular events, 38 (11.3%) patients experienced thromboses with 50 events in total (recurrences in 12 cases), including 33 venous events (66%) of which 15 were localized in the splanchnic territory (45.5%). Hemorrhagic events were recorded in 34 cases (10.1%). During the follow-up, 39 patients (11.6%) have evolved: 11 from ET to PV (28.2%), 26 into MF (66.7%) and 2 into AML (5.1%). All evolutions were exclusive: one event per patient. At the time of the analysis, 66 patients (19.7%) were declared as lost of follow up and 4 were dead (1.2%). This is the largest cohort of patients aged below 25 years at the time of diagnosis demonstrates that despite their youth most of them (88%) received drug(s) for the management of their MPN. There was a high incidence of complications (29%), with vascular events and disease evolution occurring at equal frequency although death was uncommon (1.2%). Rates of events were disease evolution: 1.51/100pts/y; thromboses: 1.47 and hemorrhages: 1.32. No specific national or international guidance exists for MPN patients of this age; but our data suggest that these are not benign conditions and patients need to be carefully followed and treated. Table. Table. Disclosures Kiladjian: Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding. Giraudier:Novartis: Research Funding. Griesshammer:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Harrison:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria; Gilead: Honoraria, Speakers Bureau; CTI BioPharma: Consultancy, Honoraria.

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