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Rapid proliferation of cancer cells assisted by endothelial cell-mediated angiogenesis and acquired inflammation at the tumor microenvironment (TME) lowers the success rate of chemotherapeutic regimens. Therefore, targeting these processes using localized delivery of a minimally toxic drug combination may be a promising strategy. Here, we present engineering of a biocompatible self-assembled lithocholic acid-dipeptide derived hydrogel (TRI-Gel) that can maintain sustained delivery of antiproliferating doxorubicin, antiangiogenic combretastatin-A4 and anti-inflammatory dexamethasone. Application of TRI-Gel therapy to a murine tumor model promotes enhanced apoptosis with a concurrent reduction in angiogenesis and inflammation, leading to effective abrogation of tumor proliferation and increased median survival with reduced drug resistance. In-depth RNA-sequencing analysis showed that TRI-Gel therapy induced transcriptome-wide alternative splicing of many genes responsible for oncogenic transformation including sphingolipid genes. We demonstrate that TRI-Gel therapy targets the reversal of a unique intron retention event in β-glucocerebrosidase 1 (Gba1), thereby increasing the availability of functional Gba1 protein. An enhanced Gba1 activity elevates ceramide levels responsible for apoptosis and decreases glucosylceramides to overcome drug resistance. Therefore, TRI-Gel therapy provides a unique system that affects the TME via post-transcriptional modulations of sphingolipid metabolic genes, thereby opening a new and rational approach to cancer therapy.

Introduction: Acquired mutations, most commonly in RNA splicing factors and epigenetic regulators are thought to be disease drivers of clonal myeloid disorders such as MDS and AML. Both MDS and AML are amenable to epigenetic therapies regardless of underlying mutational subtypes. This prompted our hypothesis that alterations to epigenetic and chromatin landscape is important in pathobiology of clonal myeloid disorders driven by splicing factor mutations. In this study, we demonstrate that SF3B1 mutations induce distinct changes to epigenetic landscape and chromatin organization by altering RNA Polymerase II (Pol2) transcription kinetics. Critically, such epigenetic changes are also targetable - inhibiting components in the H3K4me/Sin3/HDAC pathway reverses Pol2 transcription defects and downstream effects on chromatin organization and genomic integrity.

Introduction: Recurrent mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are common in clonal myeloid disorders. These mutations are typically hemizygous, exclusive to each other, and non-synonymous, pointing to neomorphic functions. Molecular mechanisms connecting the mutations to disease pathogenesis remain unclear. Previous studies predominantly focused on the well-established role of splicing factors in pre-mRNA splicing, seeking to explain how mutations in these SFs leading to changes in the abundance of key regulatory genes through their effects on alternative splicing. These studies are limited by inconsistent and relatively low isoform changes across mutations and independent datasets. They also don't explain the mutual exclusivity of these mutations. In this study, we explored a novel mechanism that link SF mutations to disease biology: dysregulation of RNA Polymerase II (Pol2) transcriptional kinetics arising from impaired spliceosome assembly.

Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway leads to R-loop accumulation that contribute to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.

Beam-column joint is considered as a crucial zone in moment resisting frames. It is subjected to large forces during an earthquake, due to ground shaking and the response of the building depends on the behavior of the beam-column joint. During analysis, the joints are usually treated as rigid and this fails to consider the effects of various shear forces developed within the joint. So there emerges the need of seismic upgrading owing to structural deterioration, change in functions or increased performance requirements. Damping is one of the commonly adopted methods proposed for achieving optimal performance of the building subjected to seismic actions. In the present study, an economical approach towards the use of dampers in buildings to reduce the seismic effect is studied. A hybrid combination of dampers with steel bracings for retrofitting is studied in this paper. A cost effective hybrid configuration is presented which can simultaneously reduce the seismic effect and the overall cost for retrofitting.

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. StemCells2014;32:662–673

Solid oxide fuel cells (SOFCs) with anode-side supports, along with -stabilized (YSZ) anode, YSZ electrolyte, and LSM-YSZ cathode, were prepared. Button cells yielded a power density up to in humidified and air at . The cells showed much-improved stability against coking in methane compared with conventional Ni-YSZ anode-supported SOFCs. A detailed model was used to fit the SOFC electrical results and to show that the good stability in methane was a result of the high O-to-C ratio in the Ni-YSZ anode due to the support.

Direct internal reforming of hydrocarbon fuels such as gasoline and diesel has been problematic due to coking on Ni-based SOFC anodes. This paper describes stable direct internal reforming SOFCs (Ni-YSZ/YSZ/LSM-YSZ) using anode reforming catalysts. Button cells were tested with a separate catalyst piece placed against the anode. Button cell power densities of ~ 0.5W/cm2 at ~ 800oC have been demonstrated with n-dodecane and n-tridecane mixed with H2O-CO2-air. Life tests with n-dodecane showed stable operation for > 140 h. Flattened-tube segmented-in- series modules with 14 cells on each side were tested with a catalyst layer coating on the fuel side of the zirconia support tube. For iso-octane mixed with either air or H2O-CO2-air, a stable maximum power density of ~ 0.6W/cm2 was obtained at ~ 800oC.

Regulatory molecules produced by stromal cells are often membrane bound until cleaved by matrix metalloproteinases (MMPs); cleavage can either activate or inactivate regulatory functions. We report here that marrow stromal cells induce the expression of MMP-9 in monocytes. Induction was contact independent and could be reproduced with recombinant MCP-1/CCL2, whereas IL-6, M-CSF, G-CSF, GM-CSF, IL-8/CXCL8, SDF-1/CXCL12, and MGSA/CXCL1 did not have this effect. Stroma-induced levels of MMP-9 in the monocyte population from healthy donors were relatively consistent, whereas induced levels varied significantly (P < .001) in the CD14+ population from 27 patients with myelodysplastic syndrome (MDS). In patients with a clonal chromosomal marker, the level of inducible MMP-9 expression in the monocyte population was inversely correlated with the percentage of marker-positive cells (n = 11, P = .01), suggesting that the ability to induce MMP-9 may be compromised in clonally derived monocytes. The inducible levels of MMP-9 were also inversely correlated with marrow cellularity observed in biopsies from MDS patients (P < .001). We conclude that monocytes can express MMP-9 in response to stromal factors and that this response may be significantly decreased in MDS-derived monocytes.

Regulatory molecules produced by stromal cells are often membrane bound until cleaved by matrix metalloproteinases (MMPs); cleavage can either activate or inactivate regulatory functions. We report here that marrow stromal cells induce the expression of MMP-9 in monocytes. Induction was contact independent and could be reproduced with recombinant MCP-1/CCL2, whereas IL-6, M-CSF, G-CSF, GM-CSF, IL-8/CXCL8, SDF-1/CXCL12, and MGSA/CXCL1 did not have this effect. Stroma-induced levels of MMP-9 in the monocyte population from healthy donors were relatively consistent, whereas induced levels varied significantly (P< .001) in the CD14+population from 27 patients with myelodysplastic syndrome (MDS). In patients with a clonal chromosomal marker, the level of inducible MMP-9 expression in the monocyte population was inversely correlated with the percentage of marker-positive cells (n = 11, P= .01), suggesting that the ability to induce MMP-9 may be compromised in clonally derived monocytes. The inducible levels of MMP-9 were also inversely correlated with marrow cellularity observed in biopsies from MDS patients (P< .001). We conclude that monocytes can express MMP-9 in response to stromal factors and that this response may be significantly decreased in MDS-derived monocytes.

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The re-combinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The re-combinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

The 2,4,6-triphenylthiapyrylium ion has been obtained imprisoned inside the supercages of the tridirectional, large pore zeolites Y and beta viaship-in-a-bottle synthesis from chalcone and acetophenone in the presence of hydrogen sulfide. The resulting solids are efficient and robust photocatalysts that are able to degrade phenol and aniline in water with a higher efficiency than the P-25 TiO2standard. Preliminary tests have shown that these encapsulated dye materials are also efficient photocatalysts for the oxidative degradation of malodorous sulfurcontaining molecules.

The 2,4,6-triphenylthiapyrylium ion has been obtained imprisoned inside the supercages of the tridirectional, large pore zeolites Y and beta viaship-in-a-bottle synthesis from chalcone and acetophenone in the presence of hydrogen sulfide. The resulting solids are efficient and robust photocatalysts that are able to degrade phenol and aniline in water with a higher efficiency than the P-25 TiO2standard. Preliminary tests have shown that these encapsulated dye materials are also efficient photocatalysts for the oxidative degradation of malodorous sulfur-containing molecules.

The 2,4,6-triphenylthiapyrylium ion has been obtained imprisoned inside the supercages of the tridirectional, large pore zeolites Y and beta via ship-in-a-bottle synthesis from chalcone and acetophenone in the presence of hydrogen sulfide. The resulting solids are efficient and robust photocatalysts that are able to degrade phenol and aniline in water with a higher efficiency than the P-25 TiO₂ standard. Preliminary tests have shown that these encapsulated dye materials are also efficient photocatalysts for the oxidative degradation of malodorous sulfur-containing molecules.

A series of anils (1−6) have been incorporated in NaY zeolites and the resulting samples characterised spectroscopically. Raman spectral analysis showed that anils exist in the zeolite microcavities predominantly in a zwitterionic form. In agreement, diffuse reflectance UV/Vis spectra showed the presence of a significant concentration of the zwitterionic form, characterised by an absorption band around 400 nm. A sharp contrast is drawn between their photochemistry in solution or in the crystalline form with that in NaY zeolites. Thus, steady state irradiation at 400 nm led to persistent changes in the diffuse reflectance UV/Vis spectra with decrease of the reflectance of the band at 400 nm and the appearance of a new absorption band at longer wavelengths. No photochromism is observed and this is attributed to the isomerization of the zwitterionic form to other stereoisomers, possibly by rotation about C−C or C−N bonds with partial double bond character. This photochemical isomerization has not been reported so far. Even though the hydrogen bonding ability and polarity of NaY zeolite has been reported to be similar to that of polyfluorinated alcohols, based on the enolisation equilibrium of anils, the photochemical behaviour of these compounds incorporated within NaY is different to that reported in polyfluorinated alcohols. Specifically, no excited-state proton transfer or proton catalysed re-enolization of the zwitterionic form is observed with anils in NaY. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

The influence of the chemical composition and silylation of mesoporous MCM-41 materials on the photochromic behaviour of adsorbed spiropyran (BIPS) and 6-nitrospiropyran was studied. Upon incorporation, the spiropyrans underwent ring opening to form either zwitterionic merocyanine or its corresponding O-protonated form. In all silica MCM-41 or in the MCM-41 containing aluminium, the O-protonated merocyanine was predominantly formed. In the case of MCM-41 modified by silylation of the OH groups, a mixture of zwitterionic merocyanine and spiropyran was present. The photochromic response was studied by means of steady-state irradiation and by laser flash photolysis. Steady-state irradiation (λ > 450 nm) of the solid samples gives rise in all cases to an intensity decrease of the absorption bands corresponding to either the protonated or the unprotonated merocyanine form (reverse photochromism). In contrast, laser flash photolysis at 308 nm of spiropyrans supported on silylated MCM-41 allows observation of the photochemical ring opening of residual spiropyran to the corresponding zwitterionic form (normal photochromism).

The influence of the chemical composition and silylation of mesoporous MCM-41 materials on the photochromic behaviour of adsorbed spiropyran (BIPS) and 6-nitrospiropyran was studied. Upon incorporation, the spiropyrans underwent ring opening to form either zwitterionic merocyanine or its corresponding O-protonated form. In all silica MCM-41 or in the MCM-41 containing aluminium, the O-protonated merocyanine was predominantly formed. In the case of MCM-41 modified by silylation of the OH groups, a mixture of zwitterionic merocyanine and spiropyran was present. The photochromic response was studied by means of steady-state irradiation and by laser flash photolysis. Steady-state irradiation (?> 450 nm) of the solid samples gives rise in all cases to an intensity decrease of the absorption bands corresponding to either the protonated or the unprotonated merocyanine form (reverse photochromism). In contrast, laser flash photolysis at 308 nm of spiropyrans supported on silylated MCM-41 allows observation of the photochemical ring opening of residual spiropyran to the corresponding zwitterionic form (normal photochromism).

Background:Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA.

Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

Booher: Curis: Employment. Ramachandra:Aurigene: Employment. Samson:Curis: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Research Funding. Starczynowski:Kurome Therapeutics: Consultancy. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

SF3B1 mutations are the most frequently occurring splicing factor mutations in MDS and AML, however the misspliced genes that contribute the malignant state in SF3B mutant MDS or AML remains unclear. We determined that SF3B1 mutant cases of MDS express a longer, active isoform of interleukin 1 receptor associated kinase (IRAK4). IRAK4 is a serine/threonine kinase that is downstream of toll-like receptor (TLR) signaling and leads to activation of oncogenic signaling states, including NF-kB and MAPK. Examination of IRAK4 by RNA sequencing showed that normal cells predominantly express small IRAK4 isoforms resulting from exclusion of the part of exon 6. These isoforms are targeted for proteosomal degradation leading to diminished IRAK4 expression and activation in normal cells. In contrast, a large proportion of MDS/AML samples with SF3B1 mutation show increased expression of an IRAK4 isoform that retains full exon 6, encoding the full-length protein (IRAK4-Long). Consequently, we show that expression of mutant SF3B1-K700E in leukemic cells is associated with increased NF-kB activity, suggesting that mutations in SF3B1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Furthermore, SF3B1 mutant MDS and AML cells exhibited a block in hematopoietic differentiation in clonogenic assays. This differentiation block was ameliorated with pharmacologic inhibition of IRAK4 with CA-4948, a potent oral clinically useful small-molecule inhibitor of IRAK4. CA-4948 blocked TLR-stimulated cytokine release in various cell models and also led to decreased leukemic burden in mice xenografted with SF3B1 mutant MDS/AML cells. Finally, we determined that SF3B1 mutation induced IRAK4 activation led to TRAF6 mediated K63 ubiquitination of critical cell cycle and regulatory proteins directly implicated in oncogenesis. We had recently shown that U2AF1 mutations can lead to IRAK4 activation via retention of exon 4 (Smith et al, Nat Cell Bio, 2019). Our data now demonstrate that SF3B1 leads to overactivation of IRAK4 via retention of a different exon (exon 6), thus reinforcing that IRAK/TRAF6 activation is a common downstream oncogenic pathway in splicing factor mutated MDS/AML. Taken together, in this study, we find that mutations in SF3B1 induce expression of therapeutically targetable “active” IRAK4 isoforms and provide a genetic link between a spliceosome mutation and oncogenic innate immune signaling in MDS and AML.

Introduction:While the use of immune checkpoint inhibitors (ICI) is now an established standard of care in the management of many solid tumors, the efficacy of this approach in myeloid neoplasms (MN) is still being explored. Preliminary data regarding efficacy presented to date are modest at best. Since many leukemia physicians lack extensive experience using ICI, and MN patients experience deep thrombocytopenia and bleeding risk precluding lung or colon biopsies to assess for immune-related adverse events (irAEs), MN presents a particularly difficult space for ICI development. Particular concern may center around distinguishing progressive infections (especially fungal) from immune pneumonitis and the appropriateness of steroids in such settings We report a single-center experience on the use of ICI in MN.

Podoltsev: CTI Biopharma: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sunesis Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Jazz Pharmaceuticals: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Kartos Therapeutics: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Prebet:pfizer: Honoraria; novartis: Honoraria; pfizer: Honoraria; Agios: Consultancy, Research Funding; pfizer: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; novartis: Honoraria; Tetraphase: Consultancy; Boehringer Ingelheim: Research Funding; novartis: Honoraria; novartis: Honoraria; Genentech: Consultancy; novartis: Honoraria; Boehringer Ingelheim: Research Funding. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:miRagen: Consultancy; Eisai: Consultancy; Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Mallinckrodt: Consultancy. Huntington:Genentech: Consultancy; Pharmacyclics: Honoraria; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; DTRM Biopharm: Research Funding; AbbVie: Consultancy. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Zeidan:Otsuka: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria.

Podoltsev: Kartos Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; CTI Biopharma: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; pfizer: Honoraria; pfizer: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; novartis: Honoraria; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; novartis: Honoraria. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Spectrum: Other: fees for non-CME/CE services ; Eisai: Consultancy; Acrotech: Consultancy; miRagen: Consultancy; Mallinckrodt: Consultancy; Seattle Genetics: Consultancy, Other: fees for non-CME/CE services . Huntington:AbbVie: Consultancy; Celgene: Consultancy, Research Funding; DTRM Biopharm: Research Funding; Pharmacyclics: Honoraria; Genentech: Consultancy; Bayer: Consultancy, Honoraria. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Zeidan:Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding.

Podoltsev: Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AI Therapeutics: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Research Funding; Arog Pharmaceuticals: Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; Samus Therapeutics: Research Funding; Kartos Therapeutics: Research Funding. Prebet:Genentech: Consultancy; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; novartis: Honoraria; Boehringer Ingelheim: Research Funding; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Agios: Consultancy, Research Funding; novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria. Gore:Celgene Corporation: Consultancy, Research Funding. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; miRagen: Consultancy; Mallinckrodt: Consultancy; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Eisai: Consultancy. Huntington:Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; AbbVie: Consultancy; Pharmacyclics: Honoraria; Genentech: Consultancy; DTRM Biopharm: Research Funding. Neparidze:Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific Affairs, LLC: Research Funding. Zeidan:Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Seattle Genetics: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; BeyondSpring: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding.Immune checkpoint inhibitor therapy is not approved for use in patients with acute myeloid leukemia or myelodysplastic syndrome.

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects.

Introduction:TP53mutations have been associated with adverse outcomes in AML and MDS. While these mutations occur in less than 15-20% of pts, they are often associated with complex karyotypes and therapy-related (t)-myeloid neoplasms (MN). Previous studies showed poor outcomes with intensive chemotherapy (IC) and lower-intensity therapies [LIT] (e.g. hypomethylating agents [HMA]), and higher relapse rates after allogeneic hematopoietic stem cell transplant (alloSCT), leaving the question of optimal treatment unanswered.

Introduction:Isolated trisomy 11 is a rare, poorly characterized cytogenetic abnormality in myeloid neoplasms (MN) with only a few case series published. Prior reports suggested a lower response rate to standard treatments such as intensive chemotherapy (IC) and lower-intensity therapy (e.g. hypomethylating agents [HMA]), as well as higher relapse rates after allogeneic hematopoietic stem cell transplant (alloSCT) and poor overall survival (OS) [Table 1]. We sought to define the clinical and molecular characteristics of patients (pts) and their clinical outcomes.

The article describes a behavior-based process-safety program aimed at entrenching process safety in the hearts and minds of design engineers and positively influencing behaviors. The program was created before embarking on front-end engineering design (FEED) of the Al Karaana petrochemical project. It discusses nine foundations of projects process safety.

Despite progress in understanding the molecular pathogenesis in acute myeloid leukemia (AML), the disease remains highly fatal with limited therapeutic options. There is a need for novel approaches for management of AML that can overcome resistant disease. Immune checkpoint inhibitor therapy has emerged as a successful treatment strategy in several solid tumors and refractory Hodgkin lymphoma. However, preliminary data from clinical trials to date indicate that anti-PD-1 antibodies have limited single-agent activity in AML. This suggests that co-inhibitory molecules other than PD-1 may induce immune evasion in AML. Programmed Death-1 Homolog (PD-1H, VISTA) is a novel co-inhibitory molecule that induces immune evasion in solid tumors. Interestingly, The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia database both reveal that PD-1H is over-expressed in AML at the transcript level. While we found that myeloid subsets and T cells have the expression of PD-1H in bone marrow of healthy donors, we demonstrated that PD-1H protein is highly expressed in bone marrow of 8 (of 10) human AML donors using immunohistochemistry. To determine whether the high expression of PD-1H in AML bone marrow is associated with the expression of PD-1H in AML blasts, flow cytometric analyses were performed in bone marrow aspirate of human CD34+ AML donors. Mean Fluorescent Intensity (MFI) of CD34+ AML blasts was significantly higher when compared to normal CD34+ progenitor cells (MFI: 257±82 (AML CD34+ blasts) vs 10±4 (normal CD34+ progenitor cells), N=6, p<0.05). To test our hypothesis that PD-1H induces immune evasion in AML, we transplanted PD-1H-expressing murine myeloid leukemia cells (C1498) into syngeneic PD-1H knockout (KO) or wild type (WT) littermate mice. Whole body bioluminescence (unit: radiance) was used to determine the level of luciferase activity reflecting in vivogrowth of luciferase expressing C1498 cells and survival of recipient PD-1H KO mice was compared to WT controls. We observed that murine AML cell growth in vivowas diminished in PD-1H KO mice (mean radiance in one representative experiment: 2.6x106(KO) vs 4.2x107(WT) on day 26, N=5, p<0.05) and survival of PD-1H KO mice was improved compared to WT controls (median survival: 35 days (KO) vs 28 days (WT), N=5, p<0.05). In addition, PD-1H antibody (Ab) decreased AML cell growth in vivoin KO mice (mean radiance in one representative experiment: 2.4x105(KO + PD-1H Ab) vs 2.6x106(KO + Isotype control) on day 26, N=5, p<0.05) and extended survival in C1498-bearing PD-1H KO mice (median survival: 45 days (KO + PD-1H Ab) vs 35 days (KO + Isotype), N=5, p<0.05). These data suggest that PD-1H on both host cells and AML cells induces immune evasion in AML. Based on prior preclinical models, we further hypothesized that immunogenicity of AML can be enhanced by epigenetic modulation, that can potentiate anti-leukemic effect of PD-1H blockade. We found that DNA methyltransferase inhibition (DNMTi) by 5-aza-2'-deoxycytidine (decitabine) increased T cell infiltration in subcutaneously injected murine AML (C1498) tumor (CD3+ cells/tumor cells: 33±5% (DNMTi) vs 10±3% (PBS control), N=3, p<0.05) using flow cytometry and immunohistochemistry. Importantly, DNMTi potentiated the anti-leukemic effect of the PD-1H blockade (mean radiance in one representative experiment: 9.9x108(PBS + WT) vs 1.2x108(PBS + KO) vs 3.6x105(decitabine + WT) vs 6.9x104(decitabine + KO) on day 21; 5.9x109(PBS + WT) vs 3.5x108(PBS + KO) vs 1.8x106(decitabine + WT) vs 1.3x105(decitabine + KO) on day 24, N=7, p<0.05) and significantly prolonged survival in PD-1H KO mice (median survival: 28 days (WT) vs 35 days (KO) vs 39 days (DNMTi + WT) vs 60 days (DNMTi + KO), p<0.05). Together, our data suggest that PD-1H is highly expressed in human AML and can induce immune evasion in a murine model of AML (C1498). DNMTi has potential synergistic effects on PD-1H blockade and combining these two can be an important immunotherapeutic approach in AML.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate of several cell types including hematopoietic stem and progenitor cells. MSI2 was recently shown to be transcriptionally up-regulated in aggressive myeloid leukemia including chronic myelogenous leukemia in blast crisis (CML-BC) and acute myeloid leukemia (AML). Loss-of-function studies have confirmed an important role for MSI2 in the aggressive biology of these leukemia, but the precise molecular mechanisms remain undefined. MSI2 is thought to bind to the 3' untranslated region (3'UTR) of target mRNA in a sequence-specific fashion and inhibit their translation, but functional targets of MSI2 are not known. We implemented two transcriptome-wide techniques to define these targets: iCLIP (individual nucleotide-resolution cross-link and immunoprecipitation) and polysome profiling. We first defined the RNA interactome of MSI2 in a CML-BC cell line (K562) by iCLIP of FLAG-tagged MSI2. iCLIP relies on the ability of ultraviolet (UV) radiation to cross-link RBPs to interacting RNA allowing for stringent immunoprecipitation of RBP-RNA complexes and subsequent analysis by next generation sequencing. iCLIP analysis revealed direct binding of MSI2 to transcripts of 5036 genes, specifically enriched in the 3'UTR. These iCLIP peaks were also enriched in UAG, the minimal sequence motif of MSI2 previously defined through biochemical assays. Given that MSI2 is ubiquitously expressed, but has very specific cellular functions, we hypothesized that only a small fraction of the direct targets demonstrable by iCLIP are functionally relevant. To determine those target mRNA, we performed translatome profiling of K562 cells (MSI2 knock-down and control cells) with polysome profiling. Polyribosomes or polysomes are aggregates of ribosomes assembled on efficiently translated transcripts. Comparing change in abundance of polysome-associated transcripts (polysome profiling) allows determination of quantitative changes to translation. Using polysome profiling, we found that transcripts from only 413 genes underwent translational up-regulation (without a corresponding change in total transcript abundance) in response to MSI2 knock-down, of which 319 were deemed to be direct MSI2 targets from iCLIP analysis. Translational targets thus defined were highly enriched in biological pathways such as differentiation, cell-cycling, DNA damage response and apoptosis. Specific transcripts thus identified included transcription factors critical to myeloid and leukemia biology (cJUN, MYC and SP1) and cell-cycle regulators (CDK1, CDKN1B, RAD21 and RB1). Using expression profiles of 91 CML patient samples (reported in Radich JP et al PNAS 2006 Feb 21;103(8)) we tested if changes in gene-expression during disease progression (from chronic phase to blast crisis) reflect the change in translation of transcription factors as predicted by the translatome analysis. Accordingly, we found that transcripts down-regulated in CML-BC were highly enriched in targets of JUN and SP1, confirming a primary role for MSI2 targets in progression of CML. In summary, our novel approach that integrates two transcriptome-wide techniques show that while thousands of transcripts are directly bound by MSI2, only a small proportion of these undergo translational repression. Functional targets thus identified include transcription factors and cell-cycle regulators critical to progression of myeloid leukemia. Our results will form the basis of further studies to explore therapeutic targeting of MSI2 in myeloid leukemia.

Splicing factor 3B1 (SF3B1) is a member of the U2 snRNP complex that is a key regulator of RNA splicing. RNA splicing begins with the recognition of splice sites (ss) at the 5' and 3' ends of introns and ends with the removal of introns and joining of exons flanking them. SF3B1 plays an important role in this process by facilitating the recognition of the 3'ss. SF3B1 is frequently mutated in numerous cancers as well as the myelodysplastic syndromes (MDS). Mutations within the HEAT domain of the protein potentially contribute to disease pathogenesis. In addition to influencing splicing by binding to pre-mRNA, SF3B1 has been shown to affect splicing of exons by associating with them directly on chromatin via histone/nucleosome interactions. However, it is not understood if or how SF3B1 association with chromatin is regulated. Given that N-terminal serine and threonine residues on SF3B1 are known substrates of cyclin E-Cdk2, which phosphorylates histone subunits and other chromatin associated proteins, we hypothesized that CDK2 activity regulates SF3B1-nucleosome interactions. Although SF3B1 is phosphorylated during splicing catalysis, the function of this phosphorylation has remained unknown. We have now discovered, using nucleosome preparations and histone subunit co-immunoprecipitation assays in synchronized cells, that endogenous SF3B1 interacts with nucleosomes in a highly cell-cycle dependent manner, while total cellular abundance of SF3B1 remains invariant during cell cycle progression. In human and mouse cells, including hematopoietic cell lines, SF3B1 is excluded from chromatin during both G0 (quiescence) and G2/M phases of cell cycle. Notably, SF3B1 is enriched within chromatin maximally during S-phase. Unexpectedly, we found that the inhibition of Cdc2 (Cdk1) during G2/M enforces the SF3B1-chromatin interaction, pointing to a direct role for Cdc2 in restraining this interaction during mitosis. Further, SF3B1 loading onto chromatin during early cell cycle progression from G0 to S-phase is inhibited by Cdk2 inhibition. Thus, Cdk2 and Cdc2 appear to have antagonistic roles in controlling SF3B1-chromatin interactions during the cell cycle. Our findings suggest that Cdk activity may regulate the recruitment of the spliceosome machinery in order to coordinate splicing of particular transcripts with cell cycle progression. Current studies are focusing on how disease-associated mutations in the HEAT domain of SF3B1 affect the dynamics of its cell cycle-dependent interaction with nucleosomes and corresponding alterations to splicing outcomes.

No relevant conflicts of interest to declare.

Specific splicing Factor (SF) mutations are recurrent and mutually exclusive in hematopoietic diseases.Mutations in the splicing factor SRSF2 occur in nearly 40% of patients with CMML, 14% of MDS, and 19% of secondary AML, and portend a poor prognosis. SRSF2 binds to exonic splicing enhancers (ESEs), thereby affecting exon inclusion or exclusion. We have recently shown that SRSF2 mutations result in altered RNA binding affinity and specificity via in vitro structure-function studies, specifically isothermal titration calorimetry and nuclear magnetic resonance (NMR) modeling. While wildtype (WT) SRSF2 binds the consensus RNAs 5'-SSNG-3' equally well (S=G/C; N=A/T/C/G), mutant (MUT) SRSF2 shows increased affinity specifically for 5'-CCNG-3' and 5'-GCNG-3', due to structural changes in the N- and C-termini of the RRM (Kim et al, Cancer Cell, 2015).

Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.