ALBERT

Description: Introduction: Rearrangement of ZNF384, a transcription factor of poorly characterized function, defines a subtype of acute leukemia that may manifest as either B-ALL with aberrant myeloid marker expression or B/myeloid mixed phenotype acute leukemia (MPAL). Such leukemias are characterized by chromosomal rearrangements that result in the fusion of a diverse group of partners, often transcription factors or epigenetic modifiers, to ZNF384. Our prior studies have shown that ZNF384 B-ALL and B/myeloid MPAL are genomically indistinguishable, and that the fusion may be identified in a subset of hematopoietic stem cells, suggesting that the acquisition of a ZNF384 fusion in a primitive progenitor directly perturbs hematopoietic differentiation. The goals of this study were to determine the effect of expression of ZNF384 rearrangements on human hematopoietic stem and progenitor cell differentiation in vitro and in vivo, using TCF3-ZNF384 as a commonly observed exemplar of this form of leukemia. Methods: For in vitro experiments, human CD34+ cord blood cells were sorted into stem and progenitor populations (hematopoietic stem cell (HSC), multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP)) and lentivirally infected with wild type ZNF384, TCF3-ZNF384, or vector control. Single cells were sorted onto an MS-5 stromal layer and the immunophenotype of colonies was determined 15 days later by flow cytometry. In vivo studies were performed by sorting human CD34+ cord blood cells into stem-enriched (CD34+CD38-) or progenitor-enriched (CD34+CD38+) populations and lentivirally infecting with wild type ZNF384, TCF3-ZNF384, or vector control. Cells were transplanted into sub-lethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-SGM3) mice. Results: Single-cell MS-5 stromal experiments revealed that expression of the fusion protein perturbed hematopoietic differentiation. In all stem and progenitor populations, cells expressing TCF3-ZNF384 lost the ability to differentiate into erythroid colonies. HSC, MPP, and CMP cells expressing the fusion most commonly form undifferentiated, CD45+, CD33+ colonies. Additionally, GMP and MEP cells expressing the fusion lost their ability to form colonies. Human CD34+ cells expressing TCF3-ZNF384 successfully initiate leukemia in NSG-SGM3 mice with a median latency of 123 days. Mice presented with anemia and pathological analysis using hematoxylin and eosin staining showed infiltration of leukemic cells into the bone marrow, spleen, liver, central nervous system, and ovary. Additionally, CD33, myeloperoxidase, and major basic protein staining confirmed myeloid leukemia with a subset of eosinophil differentiation. Conclusion: Our results demonstrate that hematopoietic lineage determination is altered by the expression of TCF3-ZNF384 in human stem and progenitor cell populations. Additionally, we have created the first model of TCF3-ZNF384 leukemia which mimics the complexity of lineage deregulation in ZNF384-rearranged leukemia. Disclosures Mullighan: Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Abbvie: Research Funding.

Description: The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM−Sca-1+ cells, osteoblast-enriched ALCAM+Sca-1−, and ALCAM–Sca-1− cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM+Sca-1− cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion–related genes in HSCs. Microarray analysis showed that ALCAM−Sca-1+ fraction highly expressed cytokine-related genes, whereas the ALCAM+Sca-1− fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic markerlow/− subpopulation in ALCAM+Sca-1− fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow.

Description: Currently, the niche for long-term hematopoietic stem cells (HSCs) is thought to consist conceptually of two parts: the endosteal surface (the osteoblastic niche) and a sinusoidal endothelium (the vascular niche), and a subset of osteoblasts functions as a key component of the hematopoietic stem cell niche. However, it is still unclear that the precise cellular and molecular contribution of osteoblastic cells on the HSC supportive microenvironment. In this study, we try to characterize the osteoblastic cells and investigate the property of osteoblastic niche cells. For isolation of osteoblastic cells, we treated the bone fragments of femur and tibiae with collagenase following flush-out of the bone marrow (BM). Non-hematopoietic and non-endothelial cells were then enriched by magnetic cell sorting of the CD45-CD31-Ter119- population, and expression of Sca-1 and platelet derived growth factor receptor α (PDGFRα) was analyzed. FACS analysis showed that CD45-CD31-TER119- cells were subdivided into three fractions: Sca-1+PDGFRα+, Sca-1-PDGFRα-, Sca-1-PDGFRα+. First we examined the multilineage differentiation potential of three populations. Although Sca-1- fractions efficiently differentiated into the osteoblastic lineage and showed calcium deposition, these cells hardly differentiated into adipocytes. In contrast to the Sca-1- cells, we found that Sca-1+PDGFRα+ cells can differentiate into osteoblastic and adipocytic lineages, suggesting that Sca-1+ cells have multi-potency. Next we examined the expression of osteoblastic marker expression by quantitative RT-PCR analysis, and found that Sca-1- populations expressed Runx2 and OB-cadherin. Alkaline phosphatase (ALP) staining of freshly isolated cells showed that Sca-1- fractions expressed ALP, while Sca-1+ cells did not express ALP. These data suggest that Sca-1- populations were the cell fractions, which were already committed to osteoblastic lineage. In addition, osteocalcin was expressed in PDGFRα+ fraction in Sca-1- cells, indicating that Sca-1-PDGFRα+ cells are more mature osteoblastic cells than Sca-1-PDGFRα-cells. Furthermore, N-cadherin was specifically expressed in Sca-1-PDGFRα+ cells, suggesting that N-cadherin was up-regulated with the maturation of osteoblastic cells. In addition, N-cadherin expression was up-regulated in Sca-1-PDGFRα+ cells with the postnatal development of BM. Interestingly, in the freshly isolated cells, we found that Sca-1+PDGFRα+ cells showed higher expression of Angiopoietin-1 (Ang-1), compared to Sca-1- fractions. Ang-1 expression was up-regulated in Sca-1-PDGFRα+ cells after over night incubation. Next we investigated the ability of these fractionated cells to support hematopoiesis. We examined the capacity of these fractionated cells on maintenance of colony formation ability of BM linage-Sca-1+c-Kit+ cells after 5 days of co-culture. Although CFU-C formation was supported Sca-1+PDGFRα+ cells, Sca-1-PDGFRα+ cells maintained CFU-Mix formation compared to the Sca-1+PDGFRα+ and Sca-1-PDGFRα-cells. From these data above, we hypothesize that multiple osteoblastic populations form a “niche complex” and collaborate with other supporting cells, such as CXCL12-abundant reticular (CAR) cells, to support HSCs, and that N-cadherin+ osteoblastic cells provide a foothold for anchoring of quiescent HSCs. Now we are investigating the gene expression profiles of these three populations and are tying to clarify the changes of characteristics of osteoblastic cells during postnatal BM development.

Description: Introduction “Ph-like acute lymphoblastic leukemia (ALL)” or “BCR-ABL1-like ALL” is a recently identified subtype of high-risk B-cell precursor (BCP)-ALL with poor outcome and high frequency of IKZF1 deletion. They exhibit a gene expression profile similar to BCR-ABL1-positive (Ph1-)ALL but do not have BCR-ABL1 and other common chimeric genes. Recent study from others has identified rearrangements involving tyrosine kinase genes, such as ABL1, PDGFRB, JAK2, and CRLF2, by employing transcriptome sequencing using next generation sequencer. In the present study, we intended to explore the biologic characteristics and clinical outcomes of Ph-like ALL cases in Japanese patients. Method We investigated gene expression of 235 BCP-ALL cases enrolled on Tokyo Children Cancer Study Group (TCCSG) L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using both clustering analysis and Gene Set Enrichment Analysis (GSEA), a computational method that ascertains whether a given gene set is significantly enriched in a list of genes ranked by their correlation with a phenotype of interest. Four-year event-free survival (EFS) rate of the entire cases was 83.2%. Results Real-time PCR analysis revealed that 155 cases did not have common chimeric genes (“chimera-”), including BCR-ABL1, ETV6-RUNX1, E2A-PBX1, MLL-AF4, MLL-AF9, and MLL-ENL. Among “chimera-” cases, 11 (4.7% of the entire BCP-ALL cases) patients were identified as ALL with gene expression profile similar to that of Ph1-ALL by clustering analysis. Their 4-year EFS rate and the frequency of IKZF1 deletion were 66.7% and 80.0%, respectively. On the other hand, GSEA detected 26 ALL cases with Ph-like signature (11.1%), including 9 cases those identified to have Ph-like phenotype by clustering analysis, and they exhibited 4-year EFS rate of 58.3% and IKZF1 deletion frequency of 38.1%. Interestingly, GSEA also revealed that 7 cases simultaneously possess ETV6-RUNX1-like signature. When we excluded 7 cases having ETV6-RUNX1-like signature and 5 cases with higher NOM p-value than 0.05, 14 cases were remained and they represented a highly unfavorable outcome (event-free survival 38.1%), while the frequency of IKZF1 deletion was 41.7%. We performed transcriptome sequencing of ALL cases with Ph-like phenotype using next generation sequencer and identified rare and novel chimeric fusions involving tyrosine kinases, such as ABL1, as well as CRLF2-related chimeric fusions. Conclusions We identified the presence of ALL subset having Ph-like phenotype in Japanese patients. They represent unfavorable outcome. However, the diagnosis of “Ph-like ALL” varies according to the method to identify Ph-like phenotype. Therefore, the definition of “Ph-like ALL” needs more consideration. Disclosures: No relevant conflicts of interest to declare.

Description: Irradiation and high-dose chemotherapy followed by bone marrow transplantation (BMT) have been a routine treatment for hematological malignancies and solid tumors. These conditioning, however, should be avoided in the cases of BMT for non-malignant diseases since the toxicity extends to all organs generating serious side-effects for both short-term and long-term. Then, we investigated the conditioning methods to obtain the efficient engraftment of hematopoietic stem cells (HSCs) without irradiation before BMT. In the adult BM, HSCs interact with its microenvironment which supports the activity of HSCs to maintain quiescence or self-renew to provide blood cells throughout the lifetime of an individual. This particular microenvironment, called stem cell niche, is critical for the maintenance of HSCs, and osteoblastic cells are considered as a key component for sustaining slow-cycling or quiescence of HSCs (Arai et al., 2007). Then we hypothesized that manipulating the niche may provide solutions for efficient engraftment avoiding redundant side-effects of conditionings. We previously reported that thrombopoietin (THPO)/Mpl signaling pathway is one of the niche factor which maintains HSCs quiescence in the steady state (Yoshihara et al., 2007). Enhancement of THPO/Mpl signal induces quiescence of HSCs in vivo, whereas inhibition of this signaling pathway by the administration of anti-Mpl neutralizing antibody (AMM2) induces cell cycling. We have previously shown that inhibiting THPO/Mpl signal enables HSCs to engraft without lethal irradiation. Administration of AMM2 and 5-FU prior to BMT showed donor chimerism of 5.8 ± 0.7 % (Yoshihara et al., 2007). Recently, Czechowicz et al. (2007) showed strikingly efficient transplantation by administration of c-Kit neutralizing antibody (ACK2) to Rag2-deficient mice. These results imply that inhibition of SCF/c-Kit signaling pathway is a key treatment for endogenous HSCs to deplete and exogenous donor cells to engraft. Then, we performed series of transplantation with various conditions to achieve more efficient engraftment using c-Kit-deficient W/Wv mice as recipient mice. W/Wv mice were conditioned with AMM2 (1 mg/kg body weight, iv) or PBS six days, and 5-FU (150 mg/kg body weight, iv) or PBS two days prior to BMT. Lineage−Sca-1+c-Kit+ (LSK) cells were sorted from BM of donor mice and 104 LSK cells were transplanted intravenously to each recipient. Although the use of the W/Wv mice as the recipient allowed the engraftment of donor cells with the solo administration of AMM2 (6.7%) or 5-FU (9.3 %), we did not find the any reconstitution in W/Wv mice pretreated with PBS alone. Combinational administration of AMM2 and 5-FU increased the engraftment of donor cells (11.3 %) in W/Wv mice, and the engraftment rate in W/Wv mice was higher than that of wild type recipients. These data indicate that inhibition of SCF/c-Kit signaling in endogenous HSCs had synergistic effect on the engraftment of exogenous HSCs without irradiation. In addition, interestingly, increased dose of AMM2 showed the possibility of the efficient long-term engraftment of donor cells. Among high dose AMM2-treated mice (12.5mg/kg body weight iv), maximum donor chimerism was 53.8 %. These data suggest that antibody-mediated BMT is promising by the stem cell regulation in the niche. We are now focusing on the manipulation for the HSC-niche interaction in wild-type recipients. Achievement of non-irradiated BMT method will provide advancement in stem cell therapy for non-malignant diseases such as metabolic disorders or autoimmune disease overcoming serious side-effects.

Description: Introduction Residence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable. Materials and methods Time-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence. Results Time-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%). Analysis of gene expression showed 138 upregulated genes (log2FC 〉2 and FDR

Description: Introduction. Prior studies have described a subset of B-progenitor ALL cases with a distinct gene expression profile and/or deletions involving ERG (encoding the ETS family member v-ets avian erythroblastosis virus E26 oncogene), however the relationship of these alterations and their role in leukemogenesis are poorly understood. We performed integrated genomic and epigenetic analyses, biochemical studies and leukemogenesis assays to define the genetic basis of this form of ALL. Methods. We studied 1674 childhood, adolescent and young adult B-progenitor ALL cases with microarray gene expression profiling and/or RNA-sequencing data to enable the identification of ERG ALL by unsupervised clustering and predictive analysis of microarrays. Detailed genomic analysis was performed for 144 ERG ALL cases, including whole genome (N=38), exome (n=46) and/or RNA-sequencing (n=57) cases, and single nucleotide polymorphism array analysis. Epigenetic profiling, including whole genome bisulfite sequencing, chromatin immunoprecipitation and sequencing for ERG and histone modifications and ATAC-sequencing were performed for a subset of 8 xenografted ERG tumors and reference cell lines. ERG transcript expression was measured by analysis of RNA-seq analysis and quantitative RT-PCR assays, and by interrogation of TCGA and PCGP RNA-seq data. The function of ERG isoforms was evaluated by EMSA and transcriptional reporter assays, immunofluoresence, colony forming assays and retroviral bone marrow transplant assays. Results. One hundred and forty four cases (8.6%) of B-ALL cases exhibited a distinct gene expression profile and lacked known chromosomal rearrangements (ERG ALL). Such cases had favorable outcome. Eighty cases (55.6%) had focal deletions of ERG with no evidence of oncogenic or chimeric ERG fusions. The deletions were most commonly heterozygous and involving exons 3-7 (n=27) or 3-9 (n=22) of 10 coding exons, and less commonly involving exon 1, or a larger region of the gene. No ERG deletions were identified in non-ERG ALL. Two cases harbored missense mutations in the ETS domain. Analysis of whole genome and exome sequencing data of 71 cases identified a high frequency of alterations of lymphoid transcription factors (46.5%; IKZF1 36.7%, PAX5 11.3%); mutation of transcription factors otherwise uncommon in ALL (21%; MYC, MYCBP2, MGA, ZEB2, GATA3); activation of signaling pathways, most commonly NRAS or KRAS (35.2%); cell cycle regulation (22.5%); and epigenetic modifiers (56.3%), most commonly KMT2D, SETD2, ARID2 and NCOR1. Notably, the five year event-free survival of ERG ALL cases with IKZF1 alterations exceeded 85% in both St Jude and Children's Oncology Group cohorts. We observed striking transcriptional deregulation at the ERG locus. Most (51/56) ERG- deleted cases expressed an ERG isoform encoded by a novel exon in intron 6 that splices in frame to distal exons, resulting in expression of a truncated C-terminal ERG protein that lacks the pointed and central regulatory domains, but retains the ETS and transactivation domain (ERGalt). ERGalt was also present in most (36/44) cases lacking an ERG deletion, and was strongly associated with presence of ERGalt protein in leukemic cells. We also identified expression of an Antisense Long non-coding RNA associated with the ERG locus (ALE) in ERG ALL. ERGalt and ALE were absent, or uncommonly expressed at very low levels in non-ERG ALL. ERGalt was absent, and ALE rarely expressed in non-ALL PCGP and TCGA samples. ERGalt and point mutant ERG were retained in the nucleus, bound DNA targets and acted as competitive inhibitors of wild type (WT) ERG in transcriptional reporter assays. Lineage-negative Arf -null bone marrow cells transduced with ERG WT induced an aggressive erythro-megakaryoblastic leukemia; in contrast ERGalt induced an immature lymphoid progenitor leukemia. Conclusions. Genomic alterations drive aberrant transcription of ERG, resulting on expression of a truncated, C-terminal oncogenic ERG protein. This represents a novel mechanism of transcription factor deregulation in leukemia. As a subset of ERG ALL cases lack ERG deletion, and as IKZF1 alterations are not associated with inferior outcome in this form of ALL, diagnostic approaches must incorporate gene expression profiling in addition to identification of ERG and IKZF1 alterations to accurately identify this form of leukemia. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Stock:Gilead: Membership on an entity's Board of Directors or advisory committees. Voorhees:Oncopeptides: Consultancy; Onyx Pharmaceuticals: Research Funding; GSK: Consultancy; Oncopeptides: Research Funding; Janssen: Research Funding; A Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy; Millennium Pharmaceuticals: Consultancy, Research Funding; Acetylon Pharmaceuticals, Inc.: Research Funding; Novartis: Consultancy; Array BioPharma: Consultancy; GSK: Research Funding; Celgene: Research Funding. Hunger:Spectrum Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Sigma Tau: Consultancy; Merck: Equity Ownership. Mullighan:Incyte: Consultancy; Amgen: Honoraria.