Search

Item does not contain fulltext BACKGROUND: Immune checkpoint inhibitors and targeted therapies have dramatically improved outcomes in patients with advanced melanoma, but approximately half these patients will not have a durable benefit. Phase 1-2 trials of adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) have shown promising responses, but data from phase 3 trials are lacking to determine the role of TILs in treating advanced melanoma. METHODS: In this phase 3, multicenter, open-label trial, we randomly assigned patients with unresectable stage IIIC or IV melanoma in a 1:1 ratio to receive TIL or anti-cytotoxic T-lymphocyte antigen 4 therapy (ipilimumab at 3 mg per kilogram of body weight). Infusion of at least 5×10(9) TILs was preceded by nonmyeloablative, lymphodepleting chemotherapy (cyclophosphamide plus fludarabine) and followed by high-dose interleukin-2. The primary end point was progression-free survival. RESULTS: A total of 168 patients (86% with disease refractory to anti-programmed death 1 treatment) were assigned to receive TILs (84 patients) or ipilimumab (84 patients). In the intention-to-treat population, median progression-free survival was 7.2 months (95% confidence interval [CI], 4.2 to 13.1) in the TIL group and 3.1 months (95% CI, 3.0 to 4.3) in the ipilimumab group (hazard ratio for progression or death, 0.50; 95% CI, 0.35 to 0.72; P

The inv(16) acute myeloid leukemia-associated CBFβ-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFβ-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFβ-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFβ-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.

Peripheral bloodmononuclear cellswere isolated and cultured to a pure pro-EBL population and reprogrammed using episomal plasmids. The pluripotency of transgene-free induced pluripotent stem cell (iPSC) line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. Peripheral blood is a non-invasive easy accessible cell source and combined with EBL outgrowth in vitro, a routine process obtaining sufficient amount of homogenous cells can be obtained within a week. Using episomal delivery, pro-EBLs can be reprogrammed in a transgene-free, cost effective system. (c) 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8-10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryoid and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications.HighlightsEfficient hematopoietic differentiation from single cell-derived iPSC coloniesReproducible feeder-free, monolayer differentiation system independent of iPSC lineProduction of erythroid, megakaryoid and myeloid cells with high-purityPlatform for hematopoietic developmental research and future clinical application

Hematopoietic differentiation of human induced pluripotent stem cells (iPSCs) provide opportunities not only for fundamental research and disease modelling/drug testing but also for large-scale production of blood effector cells for future clinical application. Although there are multiple ways to differentiate human iPSCs towards hematopoietic lineages, there is a need to develop reproducible and robust protocols. Here we introduce an efficient way to produce three major blood cell types using a standardized differentiation protocol that starts with a single hematopoietic initiation step. This system is feeder-free, avoids EB-formation, starts with a hematopoietic initiation step based on a novel single cell-derived iPSC colony differentiation and produces multi-potential progenitors within 8–10 days. Followed by lineage-specific growth factor supplementation these cells can be matured into well characterized erythroid, megakaryocytic and myeloid cells with high-purity, without transcription factor overexpression or any kind of pre-purification step. This standardized differentiation system provides a simple platform to produce specific blood cells in a reproducible manner for hematopoietic development studies, disease modelling, drug testing and the potential for future therapeutic applications. Keywords: iPSC, Differentiation, Erythroid, Hematopoietic, Megakaryocytic, Myeloid

Mobilized peripheral blood (MPB) CD34+ cells were cultured to CD41+/CD34+ megakaryoblasts. Cells were sorted to obtain a pure megakaryoblast population that was reprogramed by a hOKSM self-silencing polycistronic vector using lentiviral delivery. The generated induced pluripotent stem cell (iPSC) lines were tested for silencing of the reprogramming construct by flow cytometry. Pluripotency of MML-6838-Cl2 iPSC line was confirmed by expression of associated markers and by in vivo spontaneous differentiation towards the 3 germ layers. The genomic integrity of iPSC line was shown by karyotyping. The MML-6838-Cl2 iPSC is, to our knowledge, the first to be generated from megakaryoblasts.

Peripheral blood mononuclear cells were isolated from an individual harboring a heterozygous c.859C -> T p.Q287* mutation in GFI1B, causing an autosomal dominant bleeding disorder, platelet type, 17 (BDPLT17). PBMCs were differentiated to erythroblasts and reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. Pluripotency of iPSC line was confirmed by expression of associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Normal karyotype confirmed the genomic integrity of iPSCs and the presence of disease causingmutationwas shown by Sanger sequencing. The generated iPSCs can be used to study BDPLT17 pathophysiology and basic functions of GFI1B. (c) 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

The heterozygous Q287* mutation in Growth Factor Independence 1B (GFI1B) causes an autosomal-dominant bleeding disorder characterized by gray platelets as a result of reduced α-granule content.Affected individuals also exhibited macro-thrombocytopenia, increased megakaryocyte numbers and platelet CD34 expression. GFI1B functions as transcriptional repressor by recruiting the histone modifying enzyme LSD1/KDM1A. The C-terminally truncated GFI1B-Q287* mutant has lost its repressive function and inhibits the function of wild type GFI1B in a dominant-negative manner. To study how mutant GFI1B affects megakaryopoiesis, we expressed it in megakaryoblastic MEG01 cells by retroviral transduction. Compared to empty vector transduced cells, expression of GFI1B-Q287* caused a significant growth advantage, which is in line with the strong increase in megakaryocytes in the bone marrow of affected individuals. In contrast to GFI1B-Q287*, expression of GFI1B-WT significantly impaired MEG01 expansion (Figure 1). GFI1B recruits LSD1 through its proline and lysine amino acids at positions 2 and 8, respectively. Individual alanine mutations at these positions disturb the LSD1 interaction. We tested whether the LSD1 interaction was required for the growth phenotypes induced by GFI1B-WT and GFI1B-Q287* by separately introducing alanine mutations (P2A and K8A). This showed that both mutations nullified the growth advantage and disadvantage of GFI1B-Q287* and GFI1B-WT, respectively (Figure 1). Thus, GFI1B-WT may limit megakaryoblast expansion through LSD1 recruitment. The mutant protein may inhibit wild type GFI1B by quenching LSD1. Indeed, preliminary results show that co-expression of LSD1 and GFI1B-Q287* resulted in loss of the proliferative advantage as seen for GFI1B-Q287* alone. Figure 1: P2A and K8A mutations in GFI1B nullified the growth advantage of GFI1B-Q287* and the and growth disadvantage GFI1B-WT. GFP% of MEG01 cells was followed for 26 days. Data is normalized to day 5. To address this finding in more detail, we treated normal human CD34+ bone marrow cells with 4 µM LSD1 inhibitor GSK2879552 and induced megakaryocyte differentiation. This showed an 1.4-fold increased CD34+/CD41+ megakaryoblast expansion after 2 days of treatment. In addition, we observed that CD34 expression was retained and elevated. Thus, LSD1 inhibition of primary CD34+ cells during in vitro megakaryocyte differentiation phenocopies disease characteristics observed in individuals with the GFI1B-Q287* mutation. To further study megakaryopoiesis, we generated induced pluripotent stem (IPS) cells from two individuals with GFI1B-Q287* plus a healthy control. Hematopoietic differentiation of IPS colonies was induced using Stemline II, VEGF and BMP4 for the first 6 days and from day 6 onward in CellQuin, VEGF, BMP4, SCF, IL-1β, IL-3, IL-6 and TPO. Non-adherent hematopoietic cells where harvested and differentiated towards megakaryocytes using CellQuin, IL-1β and TPO. Upon megakaryocyte differentiation a 10-fold increase in expansion of megakaryocytes, sustained CD34 expression and increased hypolobulation was observed compared to GFI1B-WT differentiated IPS cells. Thus, GFI1B-Q287* IPS originated megakaryocytes phenocopy disease characteristics observed in individuals with the GFI1B-Q287* mutation. Our data indicate that the LSD1-GFI1B interaction is key to controlling megakaryoblast expansion and that it is important for proper megakaryocyte maturation as assessed by CD34 expression. These data also suggest that mutant GFI1B-Q287* inhibits the function of wild type GFI1B by quenching the demethylase LSD1. Induced LSD1 expression in the megakaryocyte lineage may be of therapeutic relevance for GFI1B related bleeding and platelet disorders. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.