Aravind Ramakrishnan

Five-group cytogenetic risk classification, monosomal karyotype, and outcome after hematopoietic cell transplantation for MDS or acute leukemia evolving from MDS

Clonal cytogenetic abnormalities are a major risk factor for relapse after hematopoietic cell transplantation (HCT) for myelodysplastic syndrome (MDS). We determined the impact of the recently established 5-group cytogenetic classification of MDS on outcome after HCT. Results were compared with the impact of the International Prognostic Scoring System (IPSS) 3 cytogenetic risk groups, and the additional effect of a monosomal karyotype was assessed. The study included data on 1007 patients, 1-75 years old (median 45 years), transplanted from related (n = 547) or unrelated (n = 460) donors. Various conditioning regimens were used, and marrow, peripheral blood, or cord blood served as stem cell source. Both IPSS and 5-group cytogenetic risk classifications were significantly associated with post-HCT relapse and mortality, but the 5-group classification discriminated more clearly among the lowest- and highest-risk patients. A monosomal karyotype tended to further increase the rates of relapse and mortality, even after considering the IPSS or 5-group classifications. In addition, the pathologic disease category correlated with both relapse and mortality. Mortality was also impacted by patient age, donor type, conditioning regimen, platelet count, and etiology of MDS. Although mortality declined significantly in recent years, novel strategies are needed to overcome the barrier of high-risk cytogenetics.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells

Canine embryonic stem (cES) cell lines were generated to establish a large‐animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell‐derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12–16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13–14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)‐7, has been maintained through 34 passages and is presented here. FHDO‐7 cells are alkaline phosphatase‐positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency‐associated microRNAs (miRs) (miR‐302b, miR‐302c, and miR‐367) characteristic of human and mouse ES cells. The FHDO‐7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm‐derived neuronal cells expressing γ‐enolase and β3‐tubulin; mesoderm‐derived cells producing collagen IIA1, cartilage, and bone; and endoderm‐derived cells expressing α‐fetoprotein or Clara cell‐specific protein.

Genome-Wide Analysis of miRNA-mRNA Interactions in Marrow Stromal Cells

Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell‐surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA‐mRNA interactions are indirect and have high false‐positive and negative rates. In this report, a genome‐wide biochemical technique (high‐throughput sequencing of RNA isolated by cross‐linking immunoprecipitation or HITS‐CLIP) was used to generate unbiased genome‐wide maps of miRNA‐mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome‐wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. Stem Cells 2014;32:662–673

Primary Marrow-Derived Stromal Cells: Isolation and Manipulation

Marrow stromal cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give rise to several mesenchymal lineages. MSC also refers to mesenchymal stem cells which implies that all cells in the population are multipotent. It is generally agreed that while there may be a few multipotent stem cells in an MSC population the majority are not stem cells. In either case MSCs do not produce hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone marrow is their most common source for research and clinical use. Primary MSC populations can be derived from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular heterogeneity within a culture and how this may vary from donor to donor. In this chapter, we describe methodology to derive primary MSCs from bone marrow screens, an otherwise discarded by-product of bone marrow harvests used for clinical transplantation. We also describe some useful techniques to characterize and manipulate MSCs-both primary and immortalized cell lines.

Efficient iPS cell generation from blood using episomes and HDAC inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Allogeneic Hematopoietic Cell Transplantation for Patients with Myelodysplastic Syndrome and Myeloproliferative Disorders

Myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD) are clonal diseases of hematopoietic precursors/stem cells. While the course may be protracted, these diseases are progressive in nature and, unless treated effectively, generally prove fatal. MDS represents a complex, heterogeneous group of disorders that are characterized by dysplastic marrow morphology, various cytogenetic abnormalities, peripheral blood cytopenias due to clonally dysregulated hematopoiesis and an increased risk for developing acute myelogenous leukemia (AML) [1]. In many patients with MPD, proliferative features with increased blood cell counts are prominent early in the disease, while cytopenias may develop eventually. Following an initial cellular marrow phase, severe marrow fibrosis, generally associated with splenomegaly, may develop [2]. The recently identified JAK2 (V617F) kinase mutation, which results in constitutive cell activation, may lead to a new classification of the disorders that we currently refer to as Polycythemia vera (PV), essential thrombocythemia (ET) or chronic idiopathic myelofibrosis (CIMF) [3, 4]. Some patients present with clinical and histological features of both MDS and MPD [1, 2]. In fact, chronic myelomonocytic leukemia (CMML), listed under MDS in the original French American British (FAB) classification [5], has been re-classified as a separate (overlap) entity by the World Health Organization (WHO) [6]. MDS and MPD are predominantly, but not exclusively, diseases of the elderly. This is one reason why, until very recently, the standard of care was supportive therapy. Recently, some therapeutic agents, including DNA methyltransferase inhibitors and lenalidomide, have become available. However, as of now, the only treatment modality that has been shown to have curative potential is hematopoietic cell transplantation (HCT) [7]. Chapter 9

Modulation of Hematopoietic Lineage Specification Impacts TREM2 Expression in Microglia-Like Cells Derived From Human Stem Cells:

Microglia are the primary innate immune cell type in the brain, and their dysfunction has been linked to a variety of central nervous system disorders. Human microglia are extraordinarily difficult to obtain for experimental investigation, limiting our ability to study the impact of human genetic variants on microglia functions. Previous studies have reported that microglia-like cells can be derived from human monocytes or pluripotent stem cells. Here, we describe a reproducible relatively simple method for generating microglia-like cells by first deriving embryoid body mesoderm followed by exposure to microglia relevant cytokines. Our approach is based on recent studies demonstrating that microglia originate from primitive yolk sac mesoderm distinct from peripheral macrophages that arise during definitive hematopoiesis. We hypothesized that functional microglia could be derived from human stem cells by employing BMP-4 mesodermal specification followed by exposure to microglia-relevant cytokines, M-CSF, GM-CSF, IL-34, and TGF-β. Using immunofluorescence microscopy, flow cytometry, and reverse transcription polymerase chain reaction, we observed cells with microglia morphology expressing a repertoire of markers associated with microglia: Iba1, CX3CR1, CD11b, TREM2, HexB, and P2RY12. These microglia-like cells maintain myeloid functional phenotypes including Aβ peptide phagocytosis and induction of pro-inflammatory gene expression in response to lipopolysaccharide stimulation. Addition of small molecules BIO and SB431542, previously demonstrated to drive definitive hematopoiesis, resulted in decreased surface expression of TREM2. Together, these data suggest that mesodermal lineage specification followed by cytokine exposure produces microglia-like cells in vitro from human pluripotent stem cells and that this phenotype can be modulated by factors influencing hematopoietic lineage in vitro.

Abstract IA36: SRSF2 mutations impair hematopoietic differentiation by altering exonic splicing enhancer preference.

Spliceosomal mutations account for the most frequent class of mutations in patients with myelodysplastic syndromes, yet the mechanism by which these mutations perform their driver function is not well understood. Given the genetic heterogeneity of primary patient samples, we generated a model for conditional expression of the commonly occurring SRSF2P95H mutation from the endogenous murine locus of Srsf2 and compared expression of the Srsf2P95H mutation with genetic inactivation of 0, 1 or 2 copies of Srsf2. Mx1-cre Srsf2P95H/wildtype mice exhibited significant morphologic dysplasia, leukopenia, macrocytic anemia, and preserved bone marrow (BM) cellularity as early as 2 weeks after mutation expression. Moreover, Mx1-cre Srsf2P95H/wildtype mice exhibited an increase in hematopoietic stem/progenitor cells (HSPCs) with an increase in lineage-negative Sca1+ c-Kit+ cells (LSK cells) in S-phase and early apoptosis. In competitive transplantation, Srsf2P95H mice HPSCs were expanded in the BM at 16 weeks post-transplantation despite having a reduced contribution to peripheral blood chimerism. In contrast, mice with homozygous deletion of Srsf2 exhibited anemia and leukopenia due to BM aplasia with striking loss of HSPCs. Collectively, these data show that Srsf2 is required for hematopoiesis, while mutations in Srsf2 provide a competitive advantage at the level of HSPCs but impair differentiation into mature circulating blood elements. Next, to identify transcriptional alterations caused by SRSF2 mutations, we performed deep RNA-seq on sorted HSPC populations from wildtype and Srsf2P95H mice, stable K562 cell lines ectopically expressing an empty vector or a single allele of SRSF2 (WT, P95H, P95L, P95R), as well as primary CMML and AML patient samples. We quantified global changes in splicing of ~125,000 alternative splicing events and ~160,000 constitutive splice junctions associated with SRSF2 mutations in these datasets. Intersection of differentially spliced genes in primary murine HSPC, CMML, and AML samples identified 75 genes that were differentially spliced in association with SRSF2 mutations in murine HSPCs and at least one primary patient cohort. Many of the genes that were differentially spliced in SRSF2 mutant cells participate in biological processes of known importance in myeloid malignancies. For example, a cassette exon of EZH2 that alters the reading frame inducing nonsense-mediated decay was promoted by SRSF2 mutations. We next sought to determine how SRSF2 mutations alter SRSF29s normal role in RNA splicing. As SRSF2 recognizes exonic splicing enhancer (ESE) elements within the pre-mRNA to promote exon recognition, we hypothesized that SRSF2 mutations might alter its normal sequence-specific activity. To test this, we performed an ab initio motif identification screen to identify motifs that were enriched or depleted in cassette exons promoted versus repressed in primary Srsf2P95H cells relative to wildtype. This analysis identified CCAG and GGTG as the most enriched and depleted consensus motifs, respectively. A recent solution structure of SRSF2 in complex with RNA revealed that SRSF2 normally recognizes the motifs CCNG and GGNG equally well. Analysis of the spatial distribution of CCNG and GGNG motifs across genomic loci containing cassette exons that were promoted or repressed in association with SRSF2 mutations revealed that CCNG and GGNG were respectively enriched and depleted specifically over the cassette exons, that were differentially spliced in association with SRSF2 mutations. Together, our data indicate that mutant SRSF2 drives widespread changes in splicing due to alterations in its sequence-specific recognition of exonic splicing enhancers. The biological as well as molecular data here identify an effect of the SRSF2P95H mutation distinct from haploinsufficient or complete loss of SRSF2 and reveal that mutations in SRSF2 alter ESE preference to contribute to key aspects of MDS. Citation Format: Eunhee Kim, Janine O. Ilagan, Stanley Lee, Aravind Ramakrishnan, Young Rock Chung, Jean-Baptiste Micol, Michele E. Murphy, Min-Kyung Kim, Ahmad S. Zebari, Silvia Buonamici, Peter Smith, H. Joachim Deeg, Camille Lobry, Iannis Aifantis, Robert K. Bradley, Omar Abdel-Wahab. SRSF2 mutations impair hematopoietic differentiation by altering exonic splicing enhancer preference. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA36.

Anti-thymocyte globulin plus etanercept as therapy for myelodysplastic syndromes (MDS): a phase II study: ATG and Etanercept Treatment in MDS

Immunosuppressive therapies have proven valuable in treating patients with myelodysplastic syndromes (MDS). We evaluated the combination of equine anti‐thymocyte globulin (ATGAM®) and the soluble tumour necrosis factor receptor, etanercept (Enbrel®), in a phase II trial. Twenty‐five patients with MDS [4‐refractory anaemia (RA), 2‐RA with ring sideroblasts, 15‐refractory cytopenia with multilineage dysplasia (RCMD), 3‐RCMD and ring sideroblasts, 1‐RA with excess blasts type 1] in International Prognostic Staging System risk groups low (nu2003=u200311) or intermediate‐1 (nu2003=u200314) were enrolled. All patients were platelet or red cell transfusion‐dependent. Nineteen patients completed therapy with ATG at 40u2003mg/kg per day for four consecutive days, followed by etanercept, 25u2003mg subcutaneous twice a week for 2u2003weeks, every month for 4u2003months. Thirteen patients had haematological improvement (HI)‐erythroid, 2 HI‐neutrophil, and 6 HI‐platelet. One patient with a co‐existing diagnosis of multiple sclerosis and rheumatoid arthritis had a complete remission. The overall response by intent to treat analysis among the 25 patients was 56% (95% confidence interval 35–56%). Four patients did not complete their first course of therapy and one patient did not survive to the 8‐week post‐treatment assessment. Among patients who completed treatment and survived to the 8‐week assessment, 70% had at least haematological responses lasting for at least 5 to more than 36u2003months. Thus, combination therapy with ATG and etanercept was active and safe in patients with MDS.