Cross-regulation of the Nanog and Cdx2 promoters

The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass （ICM） and trophectoderm （TE）, is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other＇s promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.

ATP binding cassette C1 (ABCC1/MRP1)-mediated drug efflux contributes to disease progression in T-lineage acute lymphoblastic leukemia

Purpose: In acute lymphoblastic leukemia (ALL), multidrug resistance is often mediated by AT- Pase Binding Cassette (ABC) proteins, which principally involve ABCC1 (multidrug resistance protein 1, MRP1) and ABCB1 (multidrug resistance 1, MDR1). However, direct comparisons between the differential effects of ABCC1 and ABCB1 have been difficult, since identical cell lines with differential expression of these transporters have not been developed. Experimental Design: In this study, we developed and compared the biological profiles of Jurkat cell lines that selectively over-expressed ABCC1 and ABCB1. Vincristine (VCR) plays an important role in the treatment of T-lineage ALL (T-ALL), and is often the first drug given to newly-diagnosed patients. Because of its importance in treatment, we provide descalating, sub-lethal doses of VCR to Jurkat cells, and extended our observations to expression profiling of newly diagnosed patients with T-ALL. Results: We found that VCR-resistant cells over-expressed ABCC1 nearly 30-fold. The calcein AM assay confirmed that VCR-resistant cells actively extruded VCR, and that ABCC1-mediated drug resistance conferred a different spectrum of multidrug resistance than other T-ALL induction agents. siRNA experiments that blocked ABCC1 export confirmed that VCR resistance could be reversed in vitro. Analyses of T-lymphoblasts obtained from 100 newly diagnosed T-ALL patients treated on Children’s Oncology Group Phase III studies 9404 and AALL0434 that induction failure could be could be partially explained by the over-expression of ABCC1 and ABCB1. Conclusions: Taken together, these results suggest that over-expression of ABC transporters plays a contributing role in mediating treatment failure in T-ALL, and underscore the need to employ alternate treatment approaches in patients for whom induction failed or for those with relapsed disease.

PathVisio Analysis:An Application Targeting the miRNA Network Associated with the p53 Signaling Pathway in Osteosarcoma

MicroRNAs(miRNAs)are small single-stranded,non-coding RNA molecules involved in the pathogenesis and progression of cancer,including osteosarcoma.We aimed to clarify the pathways involving miRNAs using new bioinformatics tools.We applied WikiPathways and PathVisio,two open-source platforms,to analyze miRNAs in osteosarcoma using miRTar and ONCO.IO as integration tools.We found 1298 records of osteosarcoma papers associated with the word“miRNA”.In osteosarcoma patients with good response to chemotherapy,miR-92a,miR-99b,miR-193a-5p,and miR-422a expression is increased,while miR-132 is decreased.All identified miRNAs seem to be centered on the TP53 network.This is the first application of PathVisio to determine miRNA pathways in osteosarcoma.MiRNAs have the potential to become a useful diagnostic and prognostic tool in the management of osteosarcoma.PathVisio is a full pathway editor with the potentiality to illustrate the biological events,augment graphical elements,and elucidate all the physical structures and interactions with standard external database identifiers.

Molecular basis of the first cell fate determination in mouse embryogenesis

Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.

Ikaros isoforms:The saga continues

Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years-implications for clinical transplantation and stability programs

BACKGROUND Peripheral blood stem cells(PBSC)are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant.Long term cryopreservation is commonly defined as five years or longer,and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft.Clinical programs,stem cell banks,and regulatory and accrediting agencies interested in product stability would benefit from such data.Thus,we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2 Rγnull(NSG)mice.AIM To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units.METHODS PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health.These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice,and the pre-freeze and post-thaw characteristics of the units were compared.Progenitor function was assessed using standard colony-forming assays.CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function.RESULTS Ten PBSC units with mean of 17 years in cryopreservation(range 13.6-18.3 years)demonstrated a mean total cell recovery of 88%±12%(range 68%-110%)and post-thaw viability of 69%±17%(range 34%-86%).BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw.Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units.All mice demonstrated long-term engraftment at 12 wk with mean34%±24%human CD45+cells,and differentiation with presence of human CD19+,CD3+and CD33+cells.Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies.CONCLUSION We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice,signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Tissue factor/FVII regulates doxorubicin-induced apoptosis in glioblastoma via activating PI3K/Akt signaling

Objective： To investigate the role of tissue factor （TF） in chemotherapeutic reagent - induced apoptosis on human glioblastoma and explore its mechanism. Methods： The expression of TF was examined by Western blotting. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by adoxorubicin were tested by Western blotting. Results： Human glioblastoma cell line U373MG expressed high level of TF while LN-229 was with low-TF level. The chemotherapeutic reagent doxorubicin revealed stronger cytotoxic effect on high-TF U373MG cells than IowoTF LN-229 cells. Enforced strong expression of TF was achieved by transfection of TF-pcDNA3 combinant on LN-229 cells in a dose-dependent manner. Enforced TF expression in transfected LN-229 cells not only impaired the doxorubicin-induced cleavage of Caspase-3 and PARP, but also inhibited the cytotoxic effect of doxorubicin. Furthermore, activation of Akt was strong in high-TF U373MG cells but weak in IowoTF LN-229 cells. Incubation of factor VII （FVII） with enforced TF-expressing LN-229 cells increased the phosphorylaUon of Akt in a time-dependent manner. Conclusion： These results suggest that over-expression of TF on glioblastoma could inhibit doxorubicin-induced apoptosis. Interaction of FVII and TF activates the downstream PI3K/Akt pathway. Tumor-derived over-expression of TF might play a role in chemotherapy resistance in glioblastoma, at lest in part, by activating PI3K/Akt-mediated survival and anti-apoptotic mechanism through the interaction of TF/FVII signaling.

Dodging the bullet: therapeutic resistance mechanisms in pediatric cancers

While advances in the treatment of pediatric cancers have improved survival to>80%across all tumor types,drug resistance continues to limit survival for a considerable number of patients.We review the known mechanisms of resistance in pediatric cancers,including processes that impair conventional chemotherapies,newer classes of targeted small molecule antineoplastic drugs,and monoclonal antibodies.We highlight similarities and differences in treatment approach and resistance between pediatric and adult cancers.We also discuss newer areas of research into drug resistance,including extracellular and immune factors.

Targeting multiple signaling pathways:the new approach to acute myeloid leukemia therapy

Acute myeloid leukemia(AML)is the most common form of acute leukemia in adults and the second most common form of acute leukemia in children.Despite this,very little improvement in survival rates has been achieved over the past few decades.This is partially due to the heterogeneity of AML and the need for more targeted therapeutics than the traditional cytotoxic chemotherapies that have been a mainstay in therapy for the past 50 years.In the past 20 years,research has been diversifying the approach to treating AML by investigating molecular pathways uniquely relevant to AML cell proliferation and survival.Here we review the development of novel therapeutics in targeting apoptosis,receptor tyrosine kinase(RTK)signaling,hedgehog(HH)pathway,mitochondrial function,DNA repair,and c-Myc signaling.There has been an impressive effort into better understanding the diversity of AML cell characteristics and here we highlight important preclinical studies that have supported therapeutic development and continue to promote new ways to target AML cells.In addition,we describe clinical investigations that have led to FDA approval of new targeted AML therapies and ongoing clinical trials of novel therapies targeting AML survival pathways.We also describe the complexity of targeting leukemia stem cells(LSCs)as an approach to addressing relapse and remission in AML and targetable pathways that are unique to LSC survival.This comprehensive review details what we currently understand about the signaling pathways that support AML cell survival and the exceptional ways in which we disrupt them.

Crosstalk between the B7/CD28 and EGFR pathways:Mechanisms and therapeutic opportunities

Somatic activating mutations in the epidermal growth factor receptor(EGFR)are one of the most common oncogenic drivers in cancers such as non-small-cell lung cancer(NSCLC),metastatic colorectal cancer,glioblastoma,head and neck cancer,pancreatic cancer,and breast cancer.Molecular-targeted agents against EGFR signaling pathways have shown robust clinical efficacy,but patients inevitably experience acquired resistance.Although immune checkpoint inhibitors(ICIs)targeting PD-1/PD-L1 have exhibited durable anti-tumor responses in a subset of patients across multiple cancer types,their efficacy is limited in cancers harboring activating gene alterations of EGFR.Increasing studies have demonstrated that upregulation of new B7/CD28 family members such as B7-H3,B7x and HHLA2,is associated with EGFR signaling and may contribute to resistance to EGFR-targeted therapies by creating an immunosuppressive tumor microenvironment(TME).In this review,we discuss the regulatory effect of EGFR signaling on the PD-1/PD-L1 pathway and new B7/CD28 family member pathways.Understanding these interactions may inform combination therapeutic strategies and potentially overcome the current challenge of resistance to EGFR-targeted therapies.We also summarize clinical data of anti-PD-1/PD-L1 therapies in EGFR-mutated cancers,as well as ongoing clinical trials of combination of EGFR-targeted therapies and anti-PD-1/PD-L1 immunotherapies.

The role of the PI3K/AKT/mTOR pathway in brain tumor metastasis

The PI3K/AKT/mTOR(PAM)pathway is involved in a variety of cellular functions and often contributes to oncogenesis and cancer progression.It has been recognized that this pathway is frequently activated in the most common central nervous system cancers of adults and children,malignant gliomas and medulloblastomas(MB).In these tumors,the PAM network controls key functions necessary for cell invasion and metastasis,such as cell motility.This review summarizes the current knowledge about the role of PAM signaling in cell invasion and metastasis in gliomas and MB.Current approaches to inhibit cell invasion and metastasis by targeting the PAM pathway will also be discussed.

Simultaneous cotargeting of ATR and RNA Polymerase I transcription demonstrates synergistic antileukemic effects on acute myeloid leukemia

Dear Editor,Continued development of novel therapeutic agents is critical to improve the survival of patients with acute myeloid leukemia(AML).RNA Polymerase I(Pol I)-mediated transcription and ribosomal biogenesis become dysregulated,thereby allowing synthesis of necessary substrates to support uncontrolled cancer cell proliferation 1.The Pol I transcription rate is higher in AML cells than nonleukemic myeloid precursors 2,suggesting Pol I transcrip-tion as a therapeutic target for AML.CX-5461 is a potent Pol I transcription inhibitor and stabilizer of the DNA G-quadruplex structure,which causes G2/M-phase arrest via the ATR(ataxia telangiectasia and Rad3-related protein)-mediated DNA damage response(DDR)3–5.

Targeting multiple signaling pathways:the new approach to acute myeloid leukemia therapy

Acute myeloid leukemia(AML)is the most common form of acute leukemia in adults and the second most common form of acute leukemia in children.Despite this,very little improvement in survival rates has been achieved over the past few decades.This is partially due to the heterogeneity of AML and the need for more targeted therapeutics than the traditional cytotoxic chemotherapies that have been a mainstay in therapy for the past 50 years.In the past 20 years,research has been diversifying the approach to treating AML by investigating molecular pathways uniquely relevant to AML cell proliferation and survival.Here we review the development of novel therapeutics in targeting apoptosis,receptor tyrosine kinase(RTK)signaling,hedgehog(HH)pathway,mitochondrial function,DNA repair,and c-Myc signaling.There has been an impressive effort into better understanding the diversity of AML cell characteristics and here we highlight important preclinical studies that have supported therapeutic development and continue to promote new ways to target AML cells.In addition,we describe clinical investigations that have led to FDA approval of new targeted AML therapies and ongoing clinical trials of novel therapies targeting AML survival pathways.We also describe the complexity of targeting leukemia stem cells(LSCs)as an approach to addressing relapse and remission in AML and targetable pathways that are unique to LSC survival.This comprehensive review details what we currently understand about the signaling pathways that support AML cell survival and the exceptional ways in which we disrupt them.

Immune checkpoint inhibitor resistance in soft tissue sarcoma

The emergence of immunotherapy as a cancer therapy has dramatically changed the treatment paradigm of systemic cancer therapy.There have been several trials evaluating immune checkpoint blockade(ICI)in soft tissue sarcoma.While there is generally a limited response in sarcoma,a subset of patients has durable responses to immunotherapy.This is attributable to a variety of factors including histologic subtype,tumor-infiltrating lymphocytes,and the tumor microenvironment among others.There is ongoing translational and clinical research evaluating ICI resistance in sarcoma and identifying therapeutic strategies to overcome this resistance.Herein,we provide a review of the current data,proposed mechanisms of resistance,and potential approaches to overcome this resistance.

In silico analysis of RNA and small RNA sequencing data from human BM-MSCs and differentiated osteocytes, chondrocytes and tenocytes

Background:Adult stem cells have a remarkable capacity of differentiating into various cell types necessary for tissue and organ regeneration.Multiple studies have focused on the differentiation potential of mesenchymal stem cells(MSCs),however little is known about the molecular characteristics of MSCs and their progenies obtained from donors of different ages.In this study,we analyzed publicly available sequencing data obtained from young(~22-year-old,n=8)and older(~65.5-year-old,n=8)donors of MSCs and their differentiated counterparts:osteocytes,chondrocytes and tenocytes.The raw mRNA and small RNA(non-coding RNA)sequencing data was downloaded from NIH BioProjects and systematically analyzed in order to identify uniquely expressed genes in MSC-derived osteocytes,chondrocytes and tenocytes of younger and older people.Results:We identified many commonly up-and downregulated genes are similar in both groups.However,the young group displayed a greater variety of differentially expressed genes in all analyzed MSC-derived cells.This discrepancy in gene expression profiles between younger and older groups may indicate a greater differentiation potential of MSCs isolated from younger donors.miRNA and mRNA integrated analysis showed key miRNAs that regulate mRNAs in both groups from all differentiated lineages.Conclusions:Our analysis provides additional information to previously reported data for identification of MSC markers of plasticity and engraftment.In addition,our data may shed light upon the molecular mechanisms of age-associated musculoskeletal diseases caused by a decreased capacity of MSCs to regenerate the locomotor system in elderly people.

输血引起的非感染性严重危害

通过对献血者进行的问卷调查和各种复杂感染性疾病血液筛选方法的改善，输血引起的感染性并发症的发生率已明显下降，与此同时，非感染性严重危害（NISHOTs）已成为输血后最常见的并发症。NISHOTs的范围很广，包括所有已被广泛认识的输血反应（溶血、发热、脓毒症以及变态反应，荨麻疹／过敏反应）以及一些还不太为人所知的并发症，如误输血、输血相关的急性肺损伤、输血引起的循环超负荷、输血后紫癜、输血相关的移植物抗宿主病、微嵌合体、输血相关的免疫调节、同种异体免疫、代谢紊乱、大量输血后凝血障碍、红细胞贮存破坏引起的并发症、过量输血或输血不足的并发症以及铁超负荷。最近，无论在新闻媒介还是在科学界，NISHOTs已受到广泛关注。随着输血潜在风险的不断增加，研究者们关注着“自由输血”和“限制输血”的并发症发生率和死亡率，以及与“新鲜血”相比“陈旧血”可能带来的潜在危害。本文就目前最新的文献和报道对NISHOTs进行回顾性分析。

Finding the perfect recipe for success

Familial hemophagocytic lymphohistiocytosis (FHLH) is a rare genetic disease for which optimal therapy includes consolidation with a hematopoietic stem cell transplant (HSCT). The HLH-1994 study demonstrated a 3-year survival of 62% in patients who received HSCT.1 The conditioning regimen was myeloablative carrying with it significant morbidity and mortality from the resulting organ toxicity. As several studies have shown that 20%–30%donor chimerism is sufficient in most cases to eliminate the clinical manifestations of FHLH, an attempt was made to reduce the toxicity of the conditioning regimen by reducing the myeloablative aspects and replacing them with more immunoablative components. Several single institution reports demonstrated the effectiveness of this approach2,3 achieving overall survival rates of 80%–100%, although this was accompanied by significant issues with mixed chimerism and nonengraftment. In the Bone Marrow Transplant Clinical Trials Network (BMT-CTN) protocol 1204 multi-institutional trial similar results were obtained with only a third of patients achieving sustained donor engraftment with the initial transplant, and the remainder requiring subsequent transplants to achieve this.4 Although the 1-year survival achieved 80%, this dropped to 67% by 18 months. Although significant progress was made in reducing transplant related mortality, clearly additional changes to the conditioning regimen are needed to achieve improved engraftment.