Publications by authors named "Asen Bagashev"

20 Publications

  • Page 1 of 1

Combinatorial efficacy of entospletinib and chemotherapy in patient-derived xenograft models of infant acute lymphoblastic leukemia.

Haematologica 2020 05 15. Epub 2020 May 15.

Div of Oncology, Children Hospital and Center for Childhood Cancer Research, Philadelphia, USA;

Survival of infants with KMT2A-rearranged (R) acute lymphoblastic leukemia (ALL) remains dismal despite intensive chemotherapy. We observed constitutive phosphorylation of spleen tyrosine kinase (SYK) and associated signaling proteins in infant ALL patient-derived xenograft (PDX) model specimens and hypothesized that the SYK inhibitor entospletinib would inhibit signaling and cell growth in vitro and leukemia proliferation in vivo. We further predicted that combined entospletinib and chemotherapy could augment anti-leukemia effects. Basal kinase signaling activation and HOXA9/MEIS1 expression differed among KMT2A-R (KMT2A-AFF1 [n=4], KMT2A-MLLT3 [n=1], KMT2A-MLLT1 [n=4]) and non-KMT2A-R [n=3] ALL specimens and stratified by genetic subgroup. Incubation of KMT2A-R ALL cells in vitro with entospletinib inhibited methylcellulose colony formation and SYK pathway signaling in a dose-dependent manner. In vivo inhibition of leukemia proliferation with entospletinib monotherapy was observed in RAS-wild-type KMT2A-AFF1, KMT2A-MLLT3, and KMT2A-MLLT1 ALL PDX models with enhanced activity in combination with vincristine chemotherapy in several models. Surprisingly, entospletinib did not decrease leukemia burden in two KMT2A-AFF1 PDX models with NRAS/ or KRAS mutations, suggesting potential RAS-mediated resistance to SYK inhibition. As hypothesized, superior inhibition of ALL proliferation was observed in KMT2A-AFF1 PDX models treated with entospletinib and the MEK inhibitor selumetinib versus vehicle or inhibitor monotherapies (p<0.05). In summary, constitutive activation of SYK and associated signaling occurs in KMT2A-R ALL with in vitro and in vivo sensitivity to entospletinib. Combination therapy with vincristine or selumetinib further enhanced treatment effects of SYK inhibition. Clinical study of entospletinib and chemotherapy or other kinase inhibitors in patients with KMT2A-R leukemias may be warranted.
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http://dx.doi.org/10.3324/haematol.2019.241729DOI Listing
May 2020

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL.

J Clin Invest 2020 07;130(7):3637-3653

Division of Oncology, and.

Children and adults with Philadelphia chromosome-like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor-like factor 2-rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface μ-heavy chain (μHC). Combinatorial targeting of JAK/STAT, PI3K, and "BCR-like" signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.
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http://dx.doi.org/10.1172/JCI134424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324172PMC
July 2020

CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy.

Blood Adv 2019 11;3(22):3539-3549

Division of Oncology, Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA; and.

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19- subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)- deep remission, whereas 67 patients had a recurrence after achieving a MRD- deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19- leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19- leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19- events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD- remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.
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http://dx.doi.org/10.1182/bloodadvances.2019000692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880911PMC
November 2019

HIV-1 Tat protein promotes neuronal dysregulation by inhibiting E2F transcription factor 3 (E2F3).

J Biol Chem 2019 03 27;294(10):3618-3633. Epub 2018 Dec 27.

From the Molecular Studies of Neurodegenerative Diseases Laboratory, FELS Institute for Cancer Research and Molecular Biology,

Individuals who are infected with HIV-1 accumulate damage to cells and tissues ( neurons) that are not directly infected by the virus. These include changes known as HIV-associated neurodegenerative disorder (HAND), leading to the loss of neuronal functions, including synaptic long-term potentiation (LTP). Several mechanisms have been proposed for HAND, including direct effects of viral proteins such as the Tat protein. Searching for the mechanisms involved, we found here that HIV-1 Tat inhibits E2F transcription factor 3 (E2F3), CAMP-responsive element-binding protein (CREB), and brain-derived neurotropic factor (BDNF) by up-regulating the microRNA miR-34a. These changes rendered murine neurons dysfunctional by promoting neurite retraction, and we also demonstrate that E2F3 is a specific target of miR-34a. Interestingly, bioinformatics analysis revealed the presence of an E2F3-binding site within the promoter, which we validated with ChIP and transient transfection assays. Of note, luciferase reporter assays revealed that E2F3 up-regulates expression and that Tat interferes with this up-regulation. Further, we show that miR-34a inhibition or E2F3 overexpression neutralizes Tat's effects and restores normal distribution of the synaptic protein synaptophysin, confirming that Tat alters these factors, leading to neurite retraction inhibition. Our results suggest that E2F3 is a key player in neuronal functions and may represent a good target for preventing the development of HAND.
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http://dx.doi.org/10.1074/jbc.RA118.003744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416426PMC
March 2019

Aberrant splicing in B-cell acute lymphoblastic leukemia.

Nucleic Acids Res 2018 11;46(21):11357-11369

Department of Pathology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

Aberrant splicing is a hallmark of leukemias with mutations in splicing factor (SF)-encoding genes. Here we investigated its prevalence in pediatric B-cell acute lymphoblastic leukemias (B-ALL), where SFs are not mutated. By comparing these samples to normal pro-B cells, we found thousands of aberrant local splice variations (LSVs) per sample, with 279 LSVs in 241 genes present in every comparison. These genes were enriched in RNA processing pathways and encoded ∼100 SFs, e.g. hnRNPA1. HNRNPA1 3'UTR was most pervasively mis-spliced, yielding the transcript subject to nonsense-mediated decay. To mimic this event, we knocked it down in B-lymphoblastoid cells and identified 213 hnRNPA1-regulated exon usage events comprising the hnRNPA1 splicing signature in pediatric leukemia. Some of its elements were LSVs in DICER1 and NT5C2, known cancer drivers. We searched for LSVs in other leukemia and lymphoma drivers and discovered 81 LSVs in 41 additional genes. Seventy-seven LSVs out of 81 were confirmed using two large independent B-ALL RNA-seq datasets, and the twenty most common B-ALL drivers, including NT5C2, showed higher prevalence of aberrant splicing than of somatic mutations. Thus, post-transcriptional deregulation of SF can drive widespread changes in B-ALL splicing and likely contributes to disease pathogenesis.
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http://dx.doi.org/10.1093/nar/gky946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277088PMC
November 2018

CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum.

Mol Cell Biol 2018 11 15;38(21). Epub 2018 Oct 15.

Division of Experimental Pathology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.
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http://dx.doi.org/10.1128/MCB.00383-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189457PMC
November 2018

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

Cancer Discov 2015 Dec 29;5(12):1282-95. Epub 2015 Oct 29.

Division of Cancer Pathobiology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Immunology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Cell & Molecular Biology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology & Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Unlabelled: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.

Significance: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
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http://dx.doi.org/10.1158/2159-8290.CD-15-1020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670800PMC
December 2015

Involvement of miR-196a in HIV-associated neurocognitive disorders.

Apoptosis 2014 Aug;19(8):1202-14

Molecular Studies of Neurodegenerative Diseases Lab, FELS Institute for Cancer Research & Molecular Biology, Temple University School of Medicine, PHA # 302, 3307 North Broad Street, Philadelphia, PA, 19140, USA.

Involvement of the human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) protein in neuronal deregulation and in the development of HIV-1 associated neurocognitive disorders (HAND) has been amply explored; however the mechanisms involved remain unclear. In search for the mechanisms, we demonstrated that Tat deregulates neuronal functions through a pathway that involved p73 and p53 pathway. We showed that Tat uses microRNA-196a (miR-196a) to deregulate the p73 pathway. Further, we found that the Abelson murine leukemia (c-Abl) phosphorylates p73 on tyrosine residue 99 (Tyr-99) in Tat-treated cells. Interestingly, Tat lost its ability to promote accumulation and phosphorylation of p73 in the presence of miR-196a mimic. Interestingly, accumulation of p73 did not lead to neuronal cell death by apoptosis as obtained by cell viability assay. Western blot analysis using antibodies directed against serine residues 807 and 811 of retinoblastoma (Rb) protein was also used to validate our data regarding lack of cell death. Hyperphosphorylation of RB (S807/811) is an indication of cell neuronal viability. These results highlight the key role played by p73 and microRNA in Tat-treated neurons leading to their deregulation and it deciphers mechanistically one of the pathways used by Tat to cause neuronal dysfunction that contributes to the development of HAND.
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http://dx.doi.org/10.1007/s10495-014-1003-2DOI Listing
August 2014

Noncoding RNAs and LRRFIP1 regulate TNF expression.

J Immunol 2014 Apr 24;192(7):3057-67. Epub 2014 Feb 24.

Division of Allergy Immunology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

Noncoding RNAs have been implicated in the regulation of expression of numerous genes; however, the mechanism is not fully understood. We identified bidirectional, long noncoding RNAs upstream of the TNF gene using five different methods. They arose in a region where the repressors LRRFIP1, EZH2, and SUZ12 were demonstrated to bind, suggesting a role in repression. The noncoding RNAs were polyadenylated, capped, and chromatin associated. Knockdown of the noncoding RNAs was associated with derepression of TNF mRNA and diminished binding of LRRFIP1 to both RNA targets and chromatin. Overexpression of the noncoding RNAs led to diminished expression of TNF and recruitment of repressor proteins to the locus. One repressor protein, LRRFIP1, bound directly to the noncoding RNAs. These data place the noncoding RNAs upstream of TNF gene as central to the transcriptional regulation. They appear to serve as a platform for the assembly of a repressive complex.
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http://dx.doi.org/10.4049/jimmunol.1302063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965610PMC
April 2014

Roles and functions of HIV-1 Tat protein in the CNS: an overview.

Virol J 2013 Dec 21;10:358. Epub 2013 Dec 21.

Molecular Studies of Neurodegenerative Diseases Lab, The Fels Institute for Cancer Research & Molecular Biology, Philadelphia, PA 19140, USA.

Nearly 50% of HIV-infected individuals suffer from some form of HIV-associated neurocognitive disorders (HAND). HIV-1 Tat (a key HIV transactivator of transcription) protein is one of the first HIV proteins to be expressed after infection occurs and is absolutely required for the initiation of the HIV genome transcription. In addition to its canonical functions, various studies have shown the deleterious role of HIV-1 Tat in the development and progression of HAND. Within the CNS, only specific cell types can support productive viral replication (astrocytes and microglia), however Tat protein can be released form infected cells to affects HIV non-permissive cells such as neurons. Therefore, in this review, we will summarize the functions of HIV-1 Tat proteins in neural cells and its ability to promote HAND.
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http://dx.doi.org/10.1186/1743-422X-10-358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879180PMC
December 2013

Cdk9 phosphorylates Pirh2 protein and prevents degradation of p53 protein.

Cell Cycle 2013 May 19;12(10):1569-77. Epub 2013 Apr 19.

Molecular Studies of Neurodegenerative Diseases Lab, The Fels Institute for Cancer Research & Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA.

Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long-terminal repeat (HIV-1 LTR). We recently demonstrated that through their physical interaction, cdk9 phosphorylates p53 on Ser-392, leading to p53 stability and accumulation. As a result, p53 stalled transcriptional elongation of the HIV-1 LTR and significantly reduced HIV-1 replication in primary microglia and astrocytes. Therefore, we sought to identify the mechanisms used by cdk9 to allow this p53 function. Using western blot analysis, we found that cdk9 promotes inhibition and phosphorylation of Mdm2 on Ser-395, thus preventing degradation of p53, a protein that is directly involved in promoting p53 ubiquitination. On the other hand, we showed that cdk9 phosphorylates Pirh2 on Ser-211 and Thr-217 residues through their physical interaction. Phosphorylation of Pirh2 renders it inactive and may contribute to p53-inhibition of transcriptional elongation of the HIV-1 LTR. Hence, we suggest that phosphorylation of Pirh2 may be a novel target for the inhibition of HIV-1 gene expression.
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http://dx.doi.org/10.4161/cc.24733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680536PMC
May 2013

Role of p53 in neurodegenerative diseases.

Neurodegener Dis 2012 28;9(2):68-80. Epub 2011 Oct 28.

Molecular Studies of Neurodegenerative Diseases Laboratory, Department of Neurology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

Background: p53 plays an important role in many areas of cellular physiology and biology, ranging from cellular development and differentiation to cell cycle arrest and apoptosis. Many of its functions are attributed to its role in assuring proper cellular division. However, since the establishment of its role in cell cycle arrest, damage repair, and apoptosis (thus also establishing its importance in cancer development), numerous reports have demonstrated additional functions of p53 in various cells. In particular, p53 appears to have important functions as it relates to neurodegeneration and synaptic plasticity.

Objective: In this review, we will address p53 functions as it relates to various neurodegenerative diseases, mainly its implications in the development of HIV-associated neurocognitive disorders.

Conclusion: p53 plays a pivotal role in the development of neurodegenerative diseases through its interaction with cellular factors, viral factors, and/or small RNAs that have the ability to promote the development of these diseases. Hence, inhibition of p53 may present an ideal target to restore neuronal functions.
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http://dx.doi.org/10.1159/000329999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3304514PMC
May 2012

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs.

J Biol Chem 2011 Nov 28;286(47):41125-34. Epub 2011 Sep 28.

Department of Neurology, Molecular Studies of Neurodegenerative Diseases Laboratory, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.
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http://dx.doi.org/10.1074/jbc.M111.268466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220514PMC
November 2011

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.

J Biol Chem 2011 Oct 4;286(40):34976-85. Epub 2011 Aug 4.

Department of Neurology, Molecular Studies of Neurodegenerative Diseases Laboratory, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.
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http://dx.doi.org/10.1074/jbc.M111.241547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186354PMC
October 2011

Characterization of LRRFIP1.

Biochem Cell Biol 2010 Dec;88(6):899-906

The Children's Hospital of Philadelphia, The University of Pennsylvania School of Medicine, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.

LRRFIP1 has been identified as a regulator of toll-like receptor (TLR) pathway signaling; however, little is known about its own regulation and function. This study was undertaken to characterize the biochemical properties and its regulation. Over-expression of full length LRRFIP1 led to enhanced responses to lipopolysaccharide (LPS). We examined its expression in monocytic cell lines because they express a broad range of TLRs. We found that its level of expression was not altered by LPS or phorbol myristate acetate (PMA) but that it was up-regulated by nicotine, influenza infection, and serum starvation. Phosphorylation was examined because of the bioinformatically predicted serine phosphorylation sites. Serine phosphorylation was detected and was altered by both poly I:C and nicotine. Finally, we examined the regulation of intracellular localization in response to dsRNA and found that LRRFIP1 colocalized with labeled dsRNA in monocyte lysosomal structures but not with lysosomes lacking dsRNA. These data suggest that LRRFIP1 is phosphorylated in response to immunologic stimuli and it is directed to lysosomal structures.
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http://dx.doi.org/10.1139/O10-014DOI Listing
December 2010

Leucine-rich repeat (in Flightless I) interacting protein-1 regulates a rapid type I interferon response.

J Interferon Cytokine Res 2010 Nov 29;30(11):843-52. Epub 2010 Jun 29.

The Division of Allergy Immunology, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.
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http://dx.doi.org/10.1089/jir.2010.0017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2992405PMC
November 2010

Myeloid differentiation primary response gene 88 (MyD88) deficiency in a large kindred.

J Allergy Clin Immunol 2010 Jul 9;126(1):172-5. Epub 2010 Jun 9.

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http://dx.doi.org/10.1016/j.jaci.2010.04.014DOI Listing
July 2010

Immune dysregulation in severe influenza.

J Leukoc Biol 2009 Jun;85(6):1036-43

Divisions of Allergy and Immunology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Among previously healthy children with severe influenza, the mechanisms leading to increased pathology are not understood. We hypothesized that children with severe influenza would have high levels of circulating cytokines. To examine this, we recruited patients with severe influenza and examined plasma cytokine levels as well as the ability of peripheral blood cells to respond to stimuli. Ten patients with severe influenza were enrolled during the 2005-2007 influenza seasons. We evaluated plasma cytokine levels, circulating NK cells, and responses to TLR ligands during the illness. We compared these patients with five patients with moderate influenza, six patients with respiratory syncytial virus (RSV), and 24 noninfected controls. Patients with influenza showed depressed responses to TLR ligands when compared with RSV patients and healthy controls (P<0.05). These normalized when retested during a convalescent phase. Plasma levels of IL-6, IL-12, and IFN- were elevated in influenza patients compared with controls (P<0.05). A compromised ability to produce TNF- was reproduced by in vitro infection, and the magnitude of the effect correlated with the multiplicity of infection and induction of IFN regulatory factor 4 expression. Aberrant, systemic, innate responses to TLR ligands during influenza infection may be a consequence of specific viral attributes such as a high inoculum or rapid replication and may underlie the known susceptibility of influenza-infected patients to secondary bacterial infections.
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http://dx.doi.org/10.1189/jlb.1108710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698588PMC
June 2009