Cell Context-dependent Manner Research Articles

Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

AC0010 is a pyrrolopyrimidine-based irreversible inhibitor of epidermal growth factor receptor (EGFR), structurally distinct from previously reported pyrimidine-based irreversible EGFR inhibitors such as osimertinib and rociletinib. AC0010 selectively inhibits EGFR T790M mutation in both preclinical and clinical studies. However, AC0010 treatment eventually triggers drug resistance with unknown mechanism. To this end, we established two H1975 NSCLC-derived lines resistant to AC0010 after a series of drug exposure and selection in either nude-mice xenograft tumor (H1975-P) or cell culture (H1975-AVR) settings. Both lines obtained 100-fold resistance to AC0010 as compared to the parental lines. To elucidate underlying mechanism, we performed unbiased RNAseq-based profiling analysis and found that H1975-P cells had c-MET overexpression, whereas H1975-AVR cells had BCL-2 overexpression. AC0010 resistance was partially abrogated by targeting c-MET or BCL-2 using either pharmacological (small molecule inhibitors) and/or genetic (siRNA-based knockdown) approach, respectively. Our study shows that drug resistance to AC0010 can be developed via the different mechanism in a cell context–dependent manner and provides the proof-of-concept evidence for rational drug combinations to overcome resistance for maximal therapeutic efficacy.

Fractionated radiotherapy might induce epithelial-mesenchymal transition and radioresistance in a cellular context manner.

Despite the fact that radiotherapy is a main therapeutic modality in cancer treatment, recent evidence suggests that fractionated radiotherapy (FR) might confer radioresistance through epithelial-mesenchymal transition (EMT). Nevertheless, the effects of FR on EMT phenotype and the potential link between EMT induction and radioresistance development yet to be clarified. The aim of this study was to assess whether FR could promote EMT, and to elucidate if induction of EMT contributes to the acquisition of radioresistance. To this end, two human cancer cell lines (A549 and HT-29) were irradiated (2 Gy/day) and analyzed using wound healing, transwell migration and invasion assays, real-time polymerase chain reaction (for E-cadherin, N-cadherin, Vimentin, CD44, CD133, Snail, and Twist), clonogenic assay, Annexin V/PI, and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Irradiation of A549 (for 5 or 10 consecutive days) resulted in morphological changes including elongation of cytoplasm and nuclei and pleomorphic nuclei. Also, irradiation-enhanced migratory and invasive potential of A549. These phenotypic changes were in agreement with decreased expression of the epithelial marker (E-cadherin), enhanced expression of mesenchymal markers (N-cadherin, Vimentin, Snail, and Twist) and increased stemness factors (CD44 and CD133). Moreover, induction of EMT phenotype was accompanied with enhanced radioresistance and proliferation of irradiated A549. However, FR (for 5 consecutive days) did not increase HT-29 motility. Furthermore, molecular alterations did not resemble EMT phenotype (downregulation of E-cadherin, Vimentin, ALDH, CD44, CD133, and Snail). Eventually, FR led to enhanced radiosensitivity and decreased proliferation of HT-29. Altogether, our findings suggest that FR might induce EMT and confer radioresistance in a cell context-dependent manner.

Siva-1 emerges as a tissue-specific oncogene beyond its classic role of a proapoptotic gene

Siva-1 is a typical apoptotic protein commonly activated by the p53 tumor suppressor protein and should therefore participate in a barrier against the development of cancer. It has proapoptotic activities in various cell systems. Recent findings suggest that Siva-1 possesses several other apoptosis-independent functions and interacts with many other proteins not directly involved in apoptosis. It harbors the ARF E3 ubiquitin protein ligase activity, a property that is clearly prooncogenic and leads to p53 degradation through the upregulation of the Hdm2 protein level. Surprisingly, recent evidence shows that Siva-1 absence prevents the development of non-small cell lung carcinomas in a mouse model and reveals the oncogenic roles in the same types of human cells, indicating its unique function as an oncogene in the cell context-dependent manner. Herein, we review reported activities of Siva-1 in various experimental settings and comment on its ambiguous function in tumor biology.

3032 - The Cell Polarity and Scaffolding Protein, PAR3, Acts as A Tumour Suppressor in Acute Myeloid Leukemia Through Regulation of the Hippo Pathway

A key feature of epithelial cancers is loss of cell polarity. Polarity in epithelial cells is maintained by three complexes, the Par, Scribble, and Crumbs complexes. PAR3, a key component of the Par polarity complex, acts as a tumour suppressor or promoter in a cell and physiological context dependent manner in epithelial cancers. However, the role of PAR3 in hematopoiesis and hematopoietic malignancies is unknown. We utilised different Par3 conditional knockout mouse models in conjunction with an extensive phenotypic labelling regime and flow cytometry to investigate the role of PAR3 in leukemogenesis. We show loss of PAR3 in experimental mice conferred a strong predisposition to acute myeloid leukemia (AML) as evidenced by a significant increase in Granulocyte/Macrophage progenitors (GMP). In line with these results, interrogation of the TCGA data set revealed AML patients with low levels of PARD3 have a significantly worse prognosis. Reverse Phase Protein Array (RPPA) analyses revealed significant up-regulation of the Hippo downstream transcriptional activator, Yes-associated protein (YAP), in experimental animals compared to controls. Immunohistochemistry confirmed RPPA results and showed YAP was significantly up-regulated in the bone marrow of experimental mice compared to controls and coincided with a significant increase in proliferation as indicated by Ki67 staining. PAR3 and YAP predominantly co-localised in the nucleus further implicating PAR3 as an important component of the Hippo pathway. RNAseq in conjunction with PCR arrays are being conducted to reveal other Hippo family members deregulated in experimental mice compared to controls and we are using 7-color multiplex immunohistochemistry to identify alterations in the spatial distribution and physiology of GMP in the bone marrow upon loss of PAR3. Collectively, our studies will enhance our understanding of how PAR3 suppresses AML and may reveal potential therapeutic targets.

RIP kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome.

Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context-dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory "cytokine storm" has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase-inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.

The regulatory roles of Notch in osteocyte differentiation via the crosstalk with canonical Wnt pathways during the transition of osteoblasts to osteocytes.

Osteocytes comprise more than 90% of the cells in bone and are differentiated from osteoblasts via an unknown mechanism. Recently, it was shown that Notch signaling plays an important role in osteocyte functions. To gain insights into the mechanisms underlying the functions of Notch in regulating the transition of osteoblasts to osteocytes, we performed a luciferase assay by cloning the proximal E11 and dentin matrix acidic phosphoprotein 1 (DMP1) promotor regions into pGluc-Basic 2 vectors, which were subsequently transfected into the IDG-SW3 (osteocytes), MC3T3 (osteoblasts) and 293T (non-osteoblastic cells) cell lines. Two approaches were used to activate Notch signaling in vitro. One was a Notch1 extracellular antibody-coated cell culture plate, and the other was transfection of a Hairy/Enhancer of Split 1 (Hes1) overexpression vector. The interaction between the Notch and Wnt signaling pathways was probed by assessing the expression of a series of phosphorylated proteins involved in the cascade of both signaling pathways. Our data suggested that Notch signaling regulates E11 expression through Hes1 activity, while Hes1 solely did not initiate the expression of DMP1. The regulatory function of E11 by Hes1 was not observed in the 293T cell line, indicating a cell context-dependent manner of the Notch signaling pathway. Additionally, we found that Notch inhibited Wnt signaling at the late differentiation stage of osteocytes by both directly repressing phosphorylated Akt and preventing the nuclear aggregation of β-catenin. These findings provide profound understandings of Notch's regulatory function in osteocyte differentiation.

Crosstalk between TGF-β signaling and epigenome.

The transforming growth factor beta (TGF-β) family of ligands plays major roles in embryonic development, tissue homeostasis, adult immunity, and wound repair. Dysregulation of TGF-β signaling pathway leads to severe diseases. Its key components have been revealed over the past two decades. This family of cytokines acts by activating receptor activated SMAD (R-SMAD) transcription factors, which in turn modulate the expression of specific sets of target genes. Cells of a multicellular organism have the same genetic information, yet they show structural and functional differences owing to differential expression of their genes. Studies have demonstrated that epigenetic regulation, an integral part of the TGF-β signaling, enables cells to sense and respond to TGF-β signaling in a cell context-dependent manner. R-SMAD, as the central transcription factor of TGF-β signaling, can recruit various epigenetic regulators to shape the transcriptome. In this review, we focus on epigenetic regulatory mechanisms in the TGF-β signaling during mammalian development and diseases and discuss the central role of the interaction between R-SMAD and various epigenetic regulators in this epigenetic regulation. The crosstalk between TGF-β signaling and the epigenome could serve as a versatile fine-tuning mechanism for transcriptional regulation during embryonic development and progression of diseases, particularly cancer.

EphA2 signaling is impacted by carcinoembryonic antigen cell adhesion molecule 1-L expression in colorectal cancer liver metastasis in a cell context-dependent manner.

We have shown that carcinoembryonic antigen cell adhesion molecule 1 long isoform (CEACAM1-L) expression in MC38 metastatic colorectal cancer (CRC) cells results in liver metastasis inhibition via CCL2 and STAT3 signaling. But other molecular mechanisms orchestrating CEACAM1-L-mediated metastasis inhibition remain to be defined. We screened a panel of mouse and human CRC cells and evaluated their metastatic outcome after CEACAM1 overexpression or downregulation. An unbiased transcript profiling and a phospho-receptor tyrosine kinase screen comparing MC38 CEACAM1-L-expressing and non-expressing (CT) CRC cells revealed reduced ephrin type-A receptor 2 (EPHA2) expression and activity. An EPHA2-specific inhibitor reduced EPHA2 downstream signaling in CT cells similar to that in CEACAM1-L cells with decreased proliferation and migration. Human CRC patients exhibiting high CEACAM1 in combination with low EPHA2 expression benefited from longer time to first recurrence/metastasis compared to those with high EPHA2 expression. With the added interaction of CEACAM6, we denoted that CEACAM1 high- and EPHA2 low-expressing patient samples with lower CEACAM6 expression also exhibited a longer time to first recurrence/metastasis. In HT29 human CRC cells, down-regulation of CEACAM1 along with CEA and CEACAM6 up-regulation led to higher metastatic burden. Overall, CEACAM1-L expression in poorly differentiated CRC can inhibit liver metastasis through cell context-dependent EPHA2-mediated signaling. However, CEACAM1’s role should be considered in the presence of other CEACAM family members.

MTOR signaling in immune cells and its implications for cancer immunotherapy

The realization that cellular metabolism dictates immune cell development, differentiation and function affords metabolic intervention as a potential venue for cancer immunotherapy. mTOR signaling, as a metabolic master regulator, controls immune cell biology in a cell type-specific and context dependent manner. Furthermore, mTOR activity needs to be fine-tuned to maintain proper immune function. These properties may be exploited for therapeutic purpose, yet caution should be taken against radical changes of mTOR activity. Here, the intricate and complex mTOR regulation in different immune cells is reviewed, highlighting latest discoveries and opportunities for cancer immunotherapy.

Molecular genetics and targeted therapy of WNT-related human diseases (Review).

Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/β-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/β-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate β-catenin phosphorylation and β-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/β-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/β-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and β-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3β (GSK3β) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine.

The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/β-catenin pathway by inhibiting the proteasome

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Notch3 signaling-mediated melanoma–endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called ‘dynamic stemness'. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma–endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

CBFβ-SMMHC regulates ribosomal gene transcription and alters ribosome biogenesis.

The core-binding factor (CBF) complex is a heterodimeric transcription factor comprising a CBFβ subunit and a variable DNA-binding RUNX subunit, usually RUNX1 in hematopoietic cells. Aside from its critical hematopoietic functions, CBF regulates the expression of ribosomal protein genes and ribosomal RNA (rRNA) in a cell context-dependent manner.1, 2, 3 Intriguingly, this function may have implications for the pathogenesis of acute myeloid leukemia (AML), as reduced ribosome biogenesis in RUNX1-deficient hematopoietic stem cells has recently been proposed to confer a survival advantage that favors outgrowth of preleukemic RUNX1-deficient clones.3 Furthermore, AML-associated fusion proteins that arise from translocations of CBF subunit genes have been shown to occupy nucleolar organizing regions at mitotic chromosomes,4, 5 suggesting that ribosomal homeostasis might be altered in CBF-AML.

Differential roles of PKC isoforms (PKCs) and Ca2+ in GnRH and phorbol 12-myristate 13-acetate (PMA) stimulation of p38MAPK phosphorylation in immortalized gonadotrope cells

We examined the role of PKCs and Ca2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived αT3-1 and LβT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in αT3-1 and LβT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCα, PKCβII, PKCδ and PKCε in p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in αT3-1 cells is mediated by Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ mobilization, while in the differentiated LβT2 gonadotrope cells it is mediated only by Ca2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH (∼5 min). Thus, we have identified the PKCs and the Ca2+ pools involved in GnRH stimulated p38MAPK phosphorylation.

Abstract B132: Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells

Abstract Translational control is an essential regulatory mechanism in immune cells. Although it has been suggested that poly(A) tail length-mediated translational control plays important roles in regulating select cytokine production in memory T cells and macrophages, whether this is a transcriptome-wide regulatory principle in all immune cells remains unknown. We set out to perform RNA-seq, ribosome-footprint profiling and poly(A) tail length profiling across major types of immune cells to examine the relationship between poly(A) tail length and translational efficiency in these systems. We also plan to delineate the transcriptome-wide landscape of poly(A) tail length before and after immune activation, and investigate if the coupling of poly(A) tail length and translational efficiency plays a regulatory role in this process. Meanwhile, to understand the general mechanistic details of how poly(A) tail length affects translational efficiency in a cell context-dependent manner, we carried out RNA pull-down experiments and isolated proteins from known coupled (Xenopus oocytes) and uncoupled (rabbit reticulocyte lysates) systems. By quantitative mass spectrometry analysis, we identified key factors that are responsible for coupling translational efficiency to poly(A) tail length. Among these candidates, poly(A) binding protein (PABPC1) plays an essential role. Overexpression of PABPC1 in Xenopus oocytes renders the translation of reporter RNAs with either a short poly(A) tail or a long poly(A) tail equally well, turning a coupled system into an uncoupled system. We have also generated an effective degron-induced PABPC1-knockdown cell line and are performing analysis to see if the coupling can be established in a normally uncoupled system. Our results suggest that cells can modify the expression of PABPC1 to help determine if poly(A) tail length regulates translational efficiency. Citation Format: Kehui Xiang, David Bartel. Importance and mechanism of poly(A) tail length-mediated translational control in different immune cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B132.

CAP1 (Cyclase-Associated Protein 1) Exerts Distinct Functions in the Proliferation and Metastatic Potential of Breast Cancer Cells Mediated by ERK.

The actin-regulating protein CAP1 is implicated in the invasiveness of human cancers. However, the exact role remains elusive and controversial given lines of conflicting evidence. Moreover, a potential role in the proliferative transformation has largely been overlooked. Further establishing the role and dissecting underlying mechanisms are imperative before targeting CAP1 can become a possibility for cancer treatment. Here we report our findings that CAP1 exerts cell type-dependent functions in the invasiveness of breast cancer cells. Depletion of CAP1 in the metastatic MDA-MB-231 and BT-549 cancer cells stimulated the metastatic potential while it actually inhibited it in the non-metastatic MCF-7 cancer cells or in normal cells. Moreover, we demonstrate functions for CAP1 in cancer cell proliferation and anchorage-independent growth, again in a cell context-dependent manner. Importantly, we identify pivotal roles for the ERK-centered signaling in mediating both CAP1 functions. Phosphor mutants of CAP1 at the S307/S309 regulatory site had compromised rescue effects for both the invasiveness and proliferation in CAP1-knockdown cells, suggesting that CAP1 likely mediates upstream cell signals to control both functions. These novel mechanistic insights may ultimately open up avenues for strategies targeting CAP1 in the treatment of breast cancer, tailored for specific types of the highly diverse disease.

Setd1a and NURF mediate chromatin dynamics and gene regulation during erythroid lineage commitment and differentiation

The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult β-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.

SOCS1 in cancer: An oncogene and a tumor suppressor

The Suppressor Of Cytokine Signaling 1 (SOCS1) has been extensively investigated in immune cells where it works as a potent inhibitor of inflammation by negative feedback regulation of the cytokine-activated JAK–STAT signaling pathways. SOCS1 is also recognized as a tumor suppressor in numerous cancers and its critical functional relevance in non-immune cells, including epithelial cells, has just begun to emerge. Most notably, conflicting results from clinical and experimental studies suggest that SOCS1 may function as either a tumor suppressor or a tumor promoter, in a cell context-dependent manner. Here, we present an overview of the mechanisms underlying SOCS1 function as a tumor suppressor and discuss the emerging evidences of SOCS1 activity as an oncogene.

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway.

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.

Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation

Recurrent mutations in the components of the cohesin complex (RAD21, SMC1A, SMC3, and STAG2) have been identified in human AML and other myeloid malignancies, and have been shown to occur as pre-leukemic mutations in HSC. Cohesin functions to hold chromatin strands within a ring-like structure composed of the four core components, and although its best-established role is to maintain the polarity of sister chromatids during mitosis, cohesin is also involved in double-stranded DNA damage repair and regulation of transcription. As little is known about their contributions to leukemogenesis, we sought to investigate the effects of cohesin mutants on human hematopoiesis, particularly hematopoietic stem and progenitor cells (HSPC).Introduction of mutant cohesin into AML cell lines and primary human HSPC resulted in a differentiation block with an increased frequency of CD34+ progenitor cells. A similar phenotype was observed with knockdown of core component RAD21 both in vitro and in vivo, indicating that mutant cohesin can act either through haploinsufficiency or dominant-negative mechanisms. Mutant cohesin increased the serial replating ability of HSPC in vitro and showed enrichment for HSC and leukemia stem cell gene expression programs, indicating an effect to enforce stem cell functions. Furthermore, we observed a skewing toward the myeloid lineage in cohesin mutant colonies cultured in methylcellulose, which was recapitulated by a strong myeloid skewing of human engrafted cells in vivo. Thus, mutant cohesin enforces stem cell programs and impairs human hematopoietic progenitor differentiation.Since cohesin complex mutations were identified in pre-leukemic HSC in many of the cases we investigated, we hypothesized that they may impart their phenotype in a cell context-dependent manner. To investigate this hypothesis, six human HSPC subpopulations (HSC, MPP, LMPP, CMP, GMP, and MEP) were isolated from cord blood, and these cells were transduced with cohesin WT, cohesin mutant, RAD21 shRNA, or control lentivirus. Transduced cells were then cultured in either myeloid differentiation or erythroid differentiation-promoting conditions. Strikingly, a strong myeloid differentiation block was only observed with cohesin mutant-transduced HSC and MPP, but not GMP. Similarly, a strong erythroid differentiation block was also observed in HSC and MPP, but not MEP. These results indicate that the effect of mutant cohesin is context dependent and restricted to the most immature HSPC.We next sought to elucidate the mechanism by which cohesin mutants exert their effects on human HSPC. Since the cohesin complex functions to establish and maintain DNA accessibility, and knockdown of cohesin can led to a decrease in chromatin accessibility at transcription factor (TF) clustered regions (Yan et al., 2013), we hypothesized that cohesin mutants impart their phenotypic effects through modulation of chromatin accessibility. To investigate this hypothesis, we used a newly developed method known as ATAC-Seq (Buenrostro et al., 2013) to assess genome-wide accessibility in cohesin WT and mutant HSPC. As expected, we found that cohesin mutants exhibited globally reduced chromatin accessibility at transcriptional regulatory elements. However, we detected increased chromatin accessibility at motifs for transcription factors known to be highly expressed in and critical regulators of HSPC including ERG, GATA2 and RUNX1. Further footprinting analysis, a proxy for ChIP-Seq experiments, showed a strong enrichment of binding of these factors in the mutant cells compared to WT cells.Based on these results, we developed a model in which the functional effects of mutant cohesin on human HSPC are mediated by transcription factors exhibiting increased chromatin accessibility such as ERG, GATA2 and RUNX1. From this model, we hypothesized that knockdown of these transcription factors would prevent the enforcement of stem cell programs and increase in CD34-expressing cells observed with cohesin mutants. As predicted, knockdown of ERG, GATA2, or RUNX1, but not GATA1 or PU.1, in the presence of cohesin mutants completely prevented the increase in CD34-expressing cells. These results strongly support our proposed model that mutant cohesin impairs hematopoietic differentiation and enforces stem cell programs through the modulation of ERG, GATA2, and RUNX1 chromatin accessibility, expression, and activity. DisclosuresMajeti:Forty Seven, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.