CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation.

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyedMBNL1expression in matched tumor/normal pairs across The Cancer Genome Atlas and found thatMBNL1was down-regulated in several common cancers. Down-regulation ofMBNL1predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared betweenMBNL1-low cancers and embryonic stem cells including aMAP2K7∆exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversingMAP2K7∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

Key Points• Lineage-inappropriate expression of the B-cell master regulator PAX5 in t(8;21) AML depends on aberrant MAP kinase signaling.• MAP kinase signaling by a mutated growth factor receptor leads to the dissociation of polycombrepressive complexes from PAX5 chromatin.The activation of B-cell-specific genes, such as CD19 and PAX5, is a hallmark of t(8;21) acute myeloid leukemia (AML) which expresses the translocation product RUNX1/ETO. PAX5 is an important regulator of B-lymphoid development and blocks myeloid differentiation when ectopically expressed. To understand the molecular mechanism of PAX5 deregulation, we examined its chromatin structure and regulation in t(8;21) AML cells, non-t(8;21) myeloid precursor control cells, and pre-B cells. In non-t(8;21) myeloid precursors, PAX5 is poised for transcription, but is repressed by polycomb complexes. In t(8;21) AML, PAX5 is not directly activated by RUNX1/ETO, but expression requires constitutive mitogen-activated protein (MAP) kinase signaling. Using a model of t(8;21) carrying an activating KIT mutation, we demonstrate that deregulated MAP kinase signaling in t(8;21) AML abrogates the association of polycomb complexes to PAX5 and leads to aberrant gene activation. Our findings therefore suggest a novel role of activating tyrosine kinase mutations in lineage-inappropriate gene expression in AML. (Blood. 2013;122(5):759-769) IntroductionThe t(8;21) is one of the most widely studied chromosomal translocations associated with the core-binding factor (CBF) complex in human myeloid leukemia. 1 The CBF complex consists of a member of the RUNX family of transcription factors along with its non-DNA-binding partner CBFb. 2 In t(8;21) acute myeloid leukemia (AML), the N-terminal domain of RUNX1 is fused to the transcriptional repressor ETO (also called RUNX1T1 or MTG8), 3 generating the fusion protein RUNX1/ETO. RUNX1/ ETO expression initiates extensive reprogramming of the epigenome and deregulates the normal myeloid gene expression program. 4,5 Heterozygous RUNX1/ETO knock-in mice fail to establish hematopoiesis and die at embryonic day 11.5, 6 but conditional expression of the full-length RUNX1/ETO alone in precursor cells is unable to establish AML.7-10 t(8;21) AML patients present additional mutations, such as activating mutations in growth factor receptors. This includes the KIT N822K or FLT3-length mutations [FLT3-LM]) which, in cooperation with RUNX1/ETO, cause fatal leukemia in mice. 11,12 Activating growth factor receptor mutations provide a proliferative advantage to leukemic cells; however, little is known of how aberrant signaling impacts on gene expression in t(8;21) AML.One of the defining features of t(8;21) AML is a mixed lineage phenotype indicated by the coexpression of myeloid and B-cell-specific genes such as CD19 and PAX5, an expression pattern that is only rarely seen in other types of AML. [13][14][15] Within the hematopoietic system, PAX5 is normally expressed exclusively in the B-lymphoid lineage 16 where it regulates B-cell commitment by ...

Edited by Henrik G. Dohlman GNA13, the ␣ subunit of a heterotrimeric G protein, mediates signaling through G-protein-coupled receptors (GPCRs). GNA13 is up-regulated in many solid tumors, including prostate cancer, where it contributes to tumor initiation, drug resistance, and metastasis. To better understand how GNA13 contributes to tumorigenesis and tumor progression, we compared the entire transcriptome of PC3 prostate cancer cells with those cells in which GNA13 expression had been silenced. This analysis revealed that GNA13 levels affected multiple CXC-family chemokines. Further investigation in three different prostate cancer cell lines singled out pro-tumorigenic CXC motif chemokine ligand 5 (CXCL5) as a target of GNA13 signaling. Elevation of GNA13 levels consistently induced CXCL5 RNA and protein expression in all three cell lines. Analysis of the CXCL5 promoter revealed that the ؊505/؉62 region was both highly active and influenced by GNA13, and a single NF-B site within this region of the promoter was critical for GNA13-dependent promoter activity. ChIP experiments revealed that, upon induction of GNA13 expression, occupancy at the CXCL5 promoter was significantly enriched for the p65 component of NF-B. GNA13 knockdown suppressed both p65 phosphorylation and the activity of a specific NF-B reporter, and p65 silencing impaired the GNA13-enhanced expression of CXCL5. Finally, blockade of Rho GTPase activity eliminated the impact of GNA13 on NF-B transcriptional activity and CXCL5 expression. Together, these findings suggest that GNA13 drives CXCL5 expression by transactivating NF-B in a Rho-dependent manner in prostate cancer cells. Prostate cancer is the second most prevalent cancer in males and is currently ranked the 5th leading cause of cancer deaths worldwide (1). The major reasons for death from prostate cancer are metastasis, drug resistance, and tumor relapse (2). As such, identifying the cellular mechanisms that contribute to prostate tumorigenesis and cancer progression would aid in the development of novel therapeutics to improve outcomes (3). Although previous studies on prostate cancer signaling have mostly focused on androgen hormone-signaling pathways, recent investigations have implicated G-protein-coupled receptors (GPCRs) 3 and their downstream signaling molecules in prostate cancer initiation and progression (3, 4). GPCRs are the largest and most diverse group of integral membrane proteins in eukaryotes. These proteins mediate cellular responses to a wide variety of ligands and are also important for numerous physiological functions (5). GPCRs are coupled to heterotrimeric G proteins that are made up of G ␣ , G ␤ , and G ␥ subunits, from which the G ␣ class can be further categorized into the G s , G q , G i , and G 12 subfamilies (6). Heterotrimeric G proteins function as molecular switches; ligand binding to the GPCR triggers a conformational change in the transmembrane region of the receptor, resulting in a GDP-GTP exchange on the G ␣ subunit and its subsequent dissociation from the...

Comprehensive understanding of aberrant splicing in gastric cancer is lacking. We RNA-sequenced 19 gastric tumor–normal pairs and identified 118 high-confidence tumor-associated (TA) alternative splicing events (ASEs) based on high-coverage sequencing and stringent filtering, and also identified 8 differentially expressed splicing factors (SFs). The TA ASEs occurred in genes primarily involved in cytoskeletal organization. We constructed a correlative network between TA ASE splicing ratios and SF expression, replicated it in independent gastric cancer data from The Cancer Genome Atlas and experimentally validated it by knockdown of the nodal SFs (PTBP1, ESRP2 and MBNL1). Each SF knockdown drove splicing alterations in several corresponding TA ASEs and led to alterations in cellular migration consistent with the role of TA ASEs in cytoskeletal organization. We have therefore established a robust network of dysregulated splicing associated with tumor invasion in gastric cancer. Our work is a resource for identifying oncogenic splice forms, SFs and splicing-generated tumor antigens as biomarkers and therapeutic targets.

Sperm forward motility is an essential parameter in mammalian fertilization. Studies from our laboratory have identified and characterized a few unique sperm motility regulatory proteins/glycoproteins from the male reproductive fluids and mammalian blood serum. The purified sperm motility-initiating protein MIP from caprine epididymal plasma as well as the forward motility-stimulating factor FMSF and motility-stimulating protein MSP from buffalo and goat serum, respectively, have high efficacy to initiate or increase motility in nonmotile or less motile sperm. "ntibody of sperm motility inhibitory factor MIF-II has the high potential to enhance sperm vertical velocity and forward motility by increasing intracellular cyclic adenosine monophosphate c"MP level. The appearance and disappearance of D-galactose-specific lectin and its receptor along the epididymis has been reported to be involved in motility regulation in spermatozoa. " novel synthetic cryopreservation method and role of lipid to protect membrane damage during cryopreservation have been demonstrated. Motility-promoting proteins may be extremely useful for improving cattle breeding and breeding of endangered species, thereby helping in enhanced production of animal products as well as in the conservation of animals. Isolated proteins and developed cryopreservation technology may also be beneficial in human infertility clinics to increase the chance of fertilization.Keywords: Spermatozoa, Epididymis, Motility regulatory proteins, Cryopreservation, Reproduction © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. . IntroductionLivestock is a very important subsector of Indian agricultural production system. The overall contribution of the livestock is almost . %. India ranks first in milk production in the world . million tons , which is mostly contributed by cattle and buffalo. The bull concentrates in itself a high economic value and thus need to be maintained on proper nutrition and management to obtain optimum performance in terms of semen production [ ]. The demand for the best males has increased considerably due to a shortage in the number of proven bulls having better semen characteristics for sustaining a successful breeding program [ ].Reproductive techniques facilitate the breeding of farm animals, which ultimately improve milk production and growth in dairy industry. "rtificial insemination "I is a first-generation reproductive biotechnology that profoundly contributed to genetic improvement, particularly in dairy cattle. Such impact would not have been possible without successfully freezing bull semen. Quality control of frozen sperm is of utmost importance for the sperm to be used in "I [ ]. Sperm cryopreservation allows prolonged preservation of semen and a wider use of a male gamete [ ]. Thi...

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS‐ALL) is characterized by high frequency of CRLF2‐rearrangements, JAK2‐mutations, or RAS‐pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub‐clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS‐ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre‐inhibition of RAS or PTPN11, but not of PI3K or JAK‐signaling, prevented TSLP‐induced RAS‐GTP boost. Cytotoxicity assays on primary clinical DS‐ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS‐inhibitor or PTPN11-inhibitor, but not PI3K/JAK‐inhibitors, suggesting a unified treatment target for up to 80% of DS‐ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein‐activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient‐stratification strategy for precision therapy in high-risk ALL.

Background VISTA is an emerging, predominantly myeloid, immune checkpoint, and its blockade has shown benefit in multiple preclinical models of cancer as both a monotherapy and in combination with other immune checkpoint inhibitors (ICI) such as anti-PD1 and anti-CTLA-4. In some murine models of cancers, such as non-small cell lung cancer, clearrenal cell carcinoma and colorectal carcinoma, VISTA is expressed on both T cells and macrophages. Understanding the cell subset specific immunomodulatory functions of VISTA is important to inform patient selection, develop effective combination strategies, and identify biomarkers of response to anti-VISTA therapy. Methods The murine colon cancer model, CT26, exhibits robust infiltrations of multiple immune cells into tumors, and has been reported to respond to VISTA blockade. This allows simultaneous investigation of VISTA-mediated immune modulation in multiple cell subsets. Here, HMBD-002, an IgG4 anti-VISTA antibody which does not deplete VISTA-expressing cells, was used to assess the functional role of VISTA blockade in the absence of Fc-mediated effects. CT26 tumor-bearing mice were treated with HMBD-002 and/or anti-PD1. Tumors were harvested and profiled via multicolor flow cytometry to determine underlying changes in the polarization and functional status of immune infiltrates associated with anti-tumor responses.Results In CT26 tumors, VISTA expression was highest on macrophages followed by MDSCs, DCs and T cells. VISTA blockade polarized macrophages to an activated pro-inflammatory anti-tumor phenotype with significant increases in TNFa and MHCII expressing macrophage subsets. VISTA blockade also resulted in significant increases in tumor antigen gp70specific CD8 T cells. A concurrent increase in CD8 T cell activation was seen with an upregulation of several cytotoxicity associated markers, including Granzyme B. However, no change in T cell exhaustion levels was observed. The combination of VISTA blockade with anti-PD1 treatment led to further increases in tumor antigen-specific CD8 T cells, significant decreases in T cell exhaustion levels and enhanced anti-tumor efficacy when compared to monotherapy anti-VISTA or anti-PD1 arms. Conclusions Reprograming of the tumor microenvironment by blockade of VISTA is associated with polarization of macrophages to an inflammatory phenotype and increases in both tumor antigen-specific CD8 T cells and their cytotoxic activity. Further, combining anti-VISTA with other immune checkpoint inhibitors that can reprogram exhausted T cells has the potential for synergistic activity.