ALBERT

Description: Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1–infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1–infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV–infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1–infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1–infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1–infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1–infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Description: Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remains unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, while ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. ASNS CpG island is largely unmethylated in normal hematopoietic cells but is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knock-in mice. In three childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL with favorable karyotypes but is mostly unmethylated in BCP-ALL with poor prognostic karyotypes. Higher ASNS methylation is associated with higher l-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene due to aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.

Description: Lindsey Montefiori, Sonja Seliger, and Zhaohui Gu contributed equally. Acute leukemias of ambiguous lineage (ALAL), including those that express combinations of myeloid, T-lineage and stem cell markers such as T/myeloid mixed phenotype acute leukemia (MPAL) and early T cell precursor acute lymphoblastic leukemia (ETP-ALL), remain challenging to diagnose, classify and treat. To define the genomic basis of these leukemias, we conducted a large pan-acute leukemia analysis of 2,573 samples, including 774 T-ALL, 126 MPAL, 262 AML, and 1,411 B-ALL cases, with transcriptomic sequencing of all cases and whole genome sequencing of a subset. tSNE and hierarchical clustering analyses of RNA-seq data identified a new subtype of 60 samples with a distinct gene expression profile (Figure 1A) and immunophenotype (typically cCD3+ CD7+ CD1a- CD2+ CD5- CD8- cMPO+/- and myeloid/stem cell marker positive); of 55 cases with data, 25 (45.5%) were T/myeloid MPAL, 20 (36.4%) ETP-ALL, 8 (14.5%) AML and 2 (3.6%) undifferentiated leukemia; 80% of cases harbored FLT3 alterations. These cases exhibited monoallelic expression of BCL11B which encodes a T-lineage transcription factor that is repressed in hematopoietic stem and progenitor cells (HSPCs), a putative cell of origin for ALAL. WGS/RNA-seq identified recurrent BCL11B-deregulating structural variants (SVs) in 56/60 (93.3%) cases, including BCL11B fusion to RUNX1 or ZEB2 in 6 (10%) cases. Most SVs were noncoding and included rearrangement of BCL11B to a gene desert upstream of ARID1B on chromosome 6 (23 cases; 41%); rearrangement to the BENC enhancer at CCDC26, distal to MYC on chromosome 8 (9; 16%); rearrangement to an intronic region of CDK6 on chromosome 7 (4; 7%); a novel high-copy (~20x) tandem amplification of a 2.5 kb noncoding region 700 kb downstream of BCL11B on chromosome 14 which we term BCL11B Enhancer Tandem Amplification (BETA) in 12 cases (21%); and rearrangement to noncoding regions at the SATB1 and ETV6 loci, each observed in a single case (Figure 1B). BCL11B-deregulating SVs were otherwise not identified in WGS analysis of 5,550 pediatric and adult hematological malignancies, 344 pediatric brain tumors and 797 pediatric solid tumors. We hypothesized that these SVs result in enhancer hijacking and ectopic activation of BCL11B in a CD34+ HSPC. Accordingly, the ARID1B, CCDC26, CDK6, and ETV6 loci all harbor CD34+ super enhancers which are absent (ARID1B, CCDC26, ETV6) or diminished (CDK6) in committed T cell precursors (Figure 1C,D). The BETA region is nominally active in HSPCs; however, tandem amplification generates a ~50 kb chromatin domain which may transform this region into a potent transcriptional activator. To investigate this, we performed histone H3 lysine 27 acetyl (H3K27ac) chromatin conformation capture followed by high-throughput sequencing (HiChIP) on 5 primary samples (1 ARID1B, 1 CCDC26, 1 CDK6 and 2 BETA cases), normal cord blood CD34+ cells, and 2 T-ALL cell lines (Figure 1E,F). In each primary sample, HiChIP confirmed that the rearranged CD34+ enhancers are active and interact with BCL11B, supporting an enhancer hijacking mechanism. Moreover, in addition to looping to BCL11B, BETA also activates the T cell-specific ThymoD enhancer 1 Mb distal of BCL11B (Figure 1E). Thus, the tandem amplification of a short, inconspicuous noncoding region generates a powerful de novo enhancer that ectopically activates BCL11B, 700kb downstream, and co-opts a dormant T cell enhancer 300 kb in the opposite direction. These activation events likely collaborate to drive oncogenic BCL11B expression in HSPCs. In conclusion, this large-scale analysis has not only identified a new subtype-defining lesion in leukemia and a new mechanism of enhancer generation in cancer (BETA) but has also resolved two controversies. First, genotypic alterations transcend immunophenotype in the classification of lineage ambiguous leukemias, with BCL11B rearrangements unifying a subgroup of T/myeloid MPAL, ETP-ALL and poorly differentiated AML that often differ only by cMPO expression. This recapitulates prior observation of ZNF384-rearrangement defining a subtype of B-ALL and B/myeloid MPAL. Second, chromatin topology analysis demonstrates enhancer hijacking of BCL11B in a primitive stem/progenitor cell, and thus, at least for a subset of cases, a hematopoietic stem cell is the cell of origin for T/myeloid antigen-expressing lineage ambiguous leukemias. Disclosures Iacobucci: Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; AbbVie, Inc.: Research Funding.

Description: Introduction: Myeloid/Natural killer (NK) cell precursor acute leukemia (MNKPL) is a rare hematologic malignancy prevalent in East Asia. MNKPL is characterized by extramedullary involvement, immature lymphoblastoid morphology without myeloperoxidase (MPO) reactivity, the CD7+/CD33+/CD34+/CD56+/HLA−DR+ phenotype. MNKPL is classified as mixed phenotype acute leukemia, and not otherwise specified rare types (MPAL NOS rare types) in WHO classification. However, its characteristic clinical feature and undetermined genetic feature suggests that MNPKL leaves open the possibility of a new independent disease concept. Here, we report clinical features and genetic alterations in patients with MNKPL. Methods: The Leukemia and Lymphoma Committee of the Japanese Society of Pediatric Hematology and Oncology (JSPHO) sent out questionnaires to 110 JSPHO affiliated hospitals and collected cases of MNPKL diagnosed during the period 2000-2013. Besides, the cases published as literature were recruited. The data of clinical features, cell surface antigen profiling, overall survival (OS), and event-free survival (EFS) defined as relapse or death were also collected as a secondary survey. The protocol of this retrospective study was approved by the review boards of JSPHO and Ehime Prefectural Central Hospital. Comprehensive genetic analysis including 13 whole-exome sequences (WES), 2 target sequence, 6 RNA sequence (RNA-seq), and 8 DNA methylation analysis was performed. We also performed single-cell RNA-seq using 1 sample of MNKPL patients and a normal bone marrow sample as the reference. The research protocol was approved by the review board of TMDU. Results: Sixteen children or young adults (〈 39 years old) and 2 older adults with MNKPL were identified. The median age of MNKPL patients was 11 (0.5-75) years old. There are 12 males and 6 females. The extramedullary involvement was observed in 7 patients. Complete remission after induction therapy was achieved in 8/14 (57%) patients treated with acute myeloid leukemia (AML) type chemotherapy and 2/4 (50%) patients treated with acute lymphoblastic leukemia (ALL)/non-Hodgkin lymphoma type chemotherapy, respectively. Fifteen patients underwent hematopoietic cell transplantation (HCT). The median follow-up period was 3.8 (0.1-16.0) years. 5-year OS and 5-year EFS was 49.5% and 40.7%, respectively. In genetic analysis, median 388 somatic mutations in MNKPL were identified by WES. The recurrent mutations were observed in NOTCH1 (n=5), MAML3 (n=4), NRAS, MAP3K4, RECQL4, CREBBP, ASXL2, and KMT2D (n=3, respectively), and MAML2, MAP3K1, FLT3, CARD11, MSH4, FANCI, WT1, ZNF384, and ERG (n=2, respectively). The distinct expression pattern, higher expression of RUNX3 and NOTCH1, and lower expression of BCL11B were identified in MNKPL samples which were compared to MPAL, AML, and T cell ALL in RNA-seq. The distinct methylation profile, hypomethylation of RUNX3 regulatory region, and hypermethylation of BCL11B regulatory region were identified in DNA methylation analysis. Single-cell RNA-seq analysis also showed distinct 4 subsets of MNKPL. Discussion and Conclusions: NK cells are the founding member of a family of innate lymphoid cells (ILC). Genetic abnormality of NOTCH1 pathway is a hallmark of MNPKL. RUNX3 is required for NK cell survival and proliferation in response to IL-15 signaling. RUNX3 high expression and hypomethylation of RUNX3 regulatory region also characterize MNKPL. Currently, MNKPL is classified as MPAL NOS, our genetic analysis revealed that MNKPL is a distinct group from MPAL. The prognosis of MNKPL was not satisfactory even though HCT was performed. The development of new therapeutic approaches based on these genetic analyses is highly expected. Disclosures Saito: Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.

Description: Background: During chemotherapy for hematological malignancies, febrile neutropenia (FN) is a frequent and serious complication. The causative organisms of FN vary from era to era and depending on bacterial resistance in each country. Therefore, epidemiological data are required for each era and country from prospective large clinical trials. We previously reported a prospective clinical trial indicating the importance of the D-index, a measure of neutropenia severity (CEDMIC trial, J Clin Oncol. 2020 10; 38: 815). We hypothesized that analyzing these data would provide accurate contemporary epidemiological data for hematological malignancies. Furthermore, as encountered in practice, we suspected that gram-negative (GN) bloodstream infections (BSIs) are more common at high body temperatures (BTs) than at low BTs and examined the relationship between causative bacteria and BT. Patients and Methods: Between June 2013 and April 2017, we enrolled patients with hematological malignancies aged 16-79 years post-chemotherapy or -stem cell transplantation (SCT) with expected neutropenia durations 〉7 days. Neutrophil counts

Description: Hematopoiesis is extrinsically controlled by cells of the bone marrow microenvironment, including skeletal lineage cells. The identification and subsequent studies of distinct subpopulations of maturing skeletal cells is currently limited due to a lack of methods to isolate these cells. We found that murine Lineage-CD31-Sca-1-CD51+ cells can be divided into four subpopulations using flow cytometry, based on their expression of the platelet derived growth factor receptors ⍺ and β (PDGFR⍺ and PDGFRβ). The use of different skeletal lineage reporters confirmed the skeletal origin of the four populations. Multiplex immunohistochemistry studies revealed that all four populations were localized near the growth plate and trabecular bone and were rarely found near cortical bone regions or in central bone marrow. Functional studies revealed differences in their abundance, colony-forming unit-fibroblast capacity and potential to differentiate into mineralized osteoblasts or adipocytes in vitro. Furthermore, the four populations had distinct gene expression profiles and differential cell surface expression of leptin receptor (LEPR) and vascular cell adhesion molecule 1 (VCAM-1). Interestingly, we discovered that one of these four different skeletal populations showed the highest expression of genes involved in the extrinsic regulation of B lymphopoiesis. This cell population varied in abundance between distinct hematopoietically active skeletal sites, and significant differences in the proportions of B lymphocyte precursors were also observed in these distinct skeletal sites. It also supported pre-B lymphopoiesis in culture. Our method to isolate four distinct maturing skeletal populations will assist in elucidating the roles of distinct skeletal niche cells in regulating hematopoiesis and bone.

Description: Background: The pathogenesis of chronic lymphoproliferative disorder of NK-cells (CLPD-NK) is poorly understood. Mutations in the JAK/STAT pathway (especially STAT3) are found in 30% of patients, but the genetic or exogenous drivers responsible for other cases are unknown. Here we comprehensively define the genetic drivers of CLPD-NK by integrated genome and transcriptome sequencing (WGS/WTS) of a large cohort of cases and complementary functional analyses. Methods: We studied 63 CLPD-NK patients (M/F: 42/21, median age: 71 years [35-89 y]) by WGS, WTS and flow cytometry. A validation cohort of 67 patients (M/F: 43/24, median age: 64 years [7-91]) was analyzed by targeted sequencing. To study the role of CCL22 in CLPD-NK pathogenesis, we examined internalization of the CCL22 receptor, CCR4, and cell chemotaxis in response to exogenous wild type (wtCCL22) or mutant CCL22 (mtCCL22: L45R, P79R, IL87_88NF) in CCR4-expressing Ba/F3 cells (Ba/F3-CCR4). To examine potential autocrine/paracrine activity, we exchanged supernatants of Ba/F3-CCR4-wtCCL22 and -mtCCL22 cells and examined CCR4 expression. To examine the in vivo effects of the mutations on proliferation and phenotype, GFP-tagged empty vector, wtCCL22, or mtCCL22-transduced NK-92 cells were engrafted into IL15-transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(IL15)1Sz/SzJ (NSG) mice. Human NK-92 cells isolated from spleens of moribund mice were analyzed by WTS and immunophenotyping. Results: WGS of 63 CLPD-NK identified STAT3 mutations in 18 (29%) cases, with mutually exclusive CCL22 mutations (mtCCL22) in 14 (22%) patients. WGS of 4500 hematological malignancies showed that mtCCL22 were only found in CLPD-NK. Recurrent co-mutations in both groups were found in ATM (n=3), FAS (n=2) and TET2 (n=5). Of the remaining patients, 23/31 had one or more mutation including epigenetic regulators (n=12), signaling components (n=7) or TP53 (n=4). Our findings of CCL22 mutations were confirmed in an independent validation cohort with STAT3 mutations in 19/67 (28%) and mtCCL22 in 13/67 (19%). CCL22 mutations were clustered at the conserved leucine 45 and proline 79 residues (Fig. 1A). Sequencing of purified CD3+ T and CD56+ NK cells showed that the mtCCL22 were somatic mutations acquired by the CD56+ NK population. mtCCL22 defined a subgroup of CLPD-NK, with high NCAM1 (CD56) positivity by flow cytometry (p