Publications by authors named "Shondra M Pruett-Miller"

58 Publications

PROSER1 mediates TET2 O-GlcNAcylation to regulate DNA demethylation on UTX-dependent enhancers and CpG islands.

Life Sci Alliance 2022 Jan 19;5(1). Epub 2021 Oct 19.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA

DNA methylation at enhancers and CpG islands usually leads to gene repression, which is counteracted by DNA demethylation through the TET protein family. However, how TET enzymes are recruited and regulated at these genomic loci is not fully understood. Here, we identify TET2, the glycosyltransferase OGT and a previously undescribed proline and serine rich protein, PROSER1 as interactors of UTX, a component of the enhancer-associated MLL3/4 complexes. We find that PROSER1 mediates the interaction between OGT and TET2, thus promoting TET2 O-GlcNAcylation and protein stability. In addition, PROSER1, UTX, TET1/2, and OGT colocalize on many genomic elements genome-wide. Loss of PROSER1 results in lower enrichment of UTX, TET1/2, and OGT at enhancers and CpG islands, with a concomitant increase in DNA methylation and transcriptional down-regulation of associated target genes and increased DNA hypermethylation encroachment at H3K4me1-predisposed CpG islands. Furthermore, we provide evidence that PROSER1 acts as a more general regulator of OGT activity by controlling O-GlcNAcylation of multiple other chromatin signaling pathways. Taken together, this study describes for the first time a regulator of TET2 O-GlcNAcylation and its implications in mediating DNA demethylation at UTX-dependent enhancers and CpG islands and supports an important role for PROSER1 in regulating the function of various chromatin-associated proteins via OGT-mediated O-GlcNAcylation.
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http://dx.doi.org/10.26508/lsa.202101228DOI Listing
January 2022

Cutting Edge: Caspase-8 Is a Linchpin in Caspase-3 and Gasdermin D Activation to Control Cell Death, Cytokine Release, and Host Defense during Influenza A Virus Infection.

J Immunol 2021 Oct 18. Epub 2021 Oct 18.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN; and

Programmed cell death (PCD) is essential for the innate immune response, which serves as the first line of defense against pathogens. Caspases regulate PCD, immune responses, and homeostasis. Caspase-8 specifically plays multifaceted roles in PCD pathways including pyroptosis, apoptosis, and necroptosis. However, because caspase-8-deficient mice are embryonically lethal, little is known about how caspase-8 coordinates different PCD pathways under physiological conditions. Here, we report an anti-inflammatory role of caspase-8 during influenza A virus infection. We generated viable mice carrying an uncleavable version of caspase-8 ( ). We demonstrated that caspase-8 autoprocessing was responsible for activating caspase-3, thereby suppressing gasdermin D-mediated pyroptosis and inflammatory cytokine release. We also found that apoptotic and pyroptotic pathways were activated at the same time during influenza A virus infection, which enabled the cell-intrinsic anti-inflammatory function of the caspase-8-caspase-3 axis. Our findings provide new insight into the immunological consequences of caspase-8-coordinated PCD cross-talk under physiological conditions.
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http://dx.doi.org/10.4049/jimmunol.2100757DOI Listing
October 2021

SLFN11 is Widely Expressed in Pediatric Sarcoma and Induces Variable Sensitization to Replicative Stress Caused By DNA-Damaging Agents.

Mol Cancer Ther 2021 Aug 19. Epub 2021 Aug 19.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee.

Pediatric sarcomas represent a heterogeneous group of malignancies that exhibit variable response to DNA-damaging chemotherapy. Schlafen family member 11 protein (SLFN11) increases sensitivity to replicative stress and has been implicated as a potential biomarker to predict sensitivity to DNA-damaging agents (DDA). SLFN11 expression was quantified in 220 children with solid tumors using IHC. Sensitivity to the PARP inhibitor talazoparib (TAL) and the topoisomerase I inhibitor irinotecan (IRN) was assessed in sarcoma cell lines, including SLFN11 knock-out (KO) and overexpression models, and a patient-derived orthotopic xenograft model (PDOX). SLFN11 was expressed in 69% of pediatric sarcoma sampled, including 90% and 100% of Ewing sarcoma and desmoplastic small round-cell tumors, respectively, although the magnitude of expression varied widely. In sarcoma cell lines, protein expression strongly correlated with response to TAL and IRN, with SLFN11 KO resulting in significant loss of sensitivity and Surprisingly, retrospective analysis of children with sarcoma found no association between SLFN11 levels and favorable outcome. Subsequently, high SLFN11 expression was confirmed in a PDOX model derived from a patient with recurrent Ewing sarcoma who failed to respond to treatment with TAL + IRN. Selective inhibition of BCL-xL increased sensitivity to TAL + IRN in SLFN11-positive resistant tumor cells. Although SLFN11 appears to drive sensitivity to replicative stress in pediatric sarcomas, its potential to act as a biomarker may be limited to certain tumor backgrounds or contexts. Impaired apoptotic response may be one mechanism of resistance to DDA-induced replicative stress.
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http://dx.doi.org/10.1158/1535-7163.MCT-21-0089DOI Listing
August 2021

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.

Nat Genet 2021 08 2;53(8):1177-1186. Epub 2021 Aug 2.

Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates β-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to β-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.
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http://dx.doi.org/10.1038/s41588-021-00904-0DOI Listing
August 2021

Nonsense-Mediated RNA Decay Is a Unique Vulnerability of Cancer Cells Harboring or Mutations.

Cancer Res 2021 Sep 2;81(17):4499-4513. Epub 2021 Jul 2.

Department of Cell Biology and Physiology, School of Medicine, Washington University in St. Louis, St. Louis, Missouri.

Nonsense-mediated RNA decay (NMD) is recognized as an RNA surveillance pathway that targets aberrant mRNAs with premature translation termination codons (PTC) for degradation, however, its molecular mechanisms and roles in health and disease remain incompletely understood. In this study, we developed a novel reporter system to accurately measure NMD activity in individual cells. A genome-wide CRISPR-Cas9 knockout screen using this reporter system identified novel NMD-promoting factors, including multiple components of the SF3B complex and other U2 spliceosome factors. Interestingly, cells with mutations in the spliceosome genes and , which are commonly found in myelodysplastic syndrome (MDS) and cancers, have overall attenuated NMD activity. Compared with wild-type (WT) cells, SF3B1- and U2AF1-mutant cells were more sensitive to NMD inhibition, a phenotype that is accompanied by elevated DNA replication obstruction, DNA damage, and chromosomal instability. Remarkably, the sensitivity of spliceosome mutant cells to NMD inhibition was rescued by overexpression of RNase H1, which removes R-loops in the genome. Together, these findings shed new light on the functional interplay between NMD and RNA splicing and suggest a novel synthetic lethal strategy for the treatment of MDS and cancers with spliceosome mutations. SIGNIFICANCE: This study has developed a novel NMD reporter system and identified a potential therapeutic approach of targeting the NMD pathway to treat cancer with spliceosome gene mutations.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-4016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416940PMC
September 2021

Degradation of Janus kinases in CRLF2-rearranged acute lymphoblastic leukemia.

Blood 2021 Jun 10. Epub 2021 Jun 10.

St Jude Children's Research Hospital, Memphis, Tennessee, United States.

CRLF2-rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) comprises over half of Philadelphia chromosome-like (Ph-like) ALL, is associated with poor outcome in children and adults. Overexpression of CRLF2 results in activation of JAK-STAT and parallel signaling pathways in experimental models, but existing small molecule inhibitors of Janus kinases show variable and limited efficacy. Here we evaluated the efficacy of proteolysis-targeting chimeras (PROTACs) directed against Janus kinases. Solving the structure of type I JAK inhibitors ruxolitinib and baricitinib bound to the JAK2 tyrosine kinase domain enabled the rational design and optimization of multiple series of cereblon (CRBN)-directed JAK PROTACs utilizing derivatives of JAK inhibitors, linkers and CRBN-specific molecular glues. The resulting JAK PROTACs were evaluated for target degradation, and activity tested in a panel of leukemia/lymphoma cell lines and xenograft models of kinase-driven ALL. Multiple PROTACs were developed that degraded Janus kinases and potently killed CRLF2--rearranged cell lines, the most active of which also degraded the known CRBN neosubstrate GSPT1, and suppressed proliferation of CRLF2-rearranged ALL in vivo. While dual JAK/GSPT1-degrading PROTACs were most potent, development and evaluation of multiple PROTACs in an extended panel of xenografts identified a potent JAK2-degrading GSPT1-sparing PROTAC that demonstrated efficacy in the majority of the kinase-driven xenografts which were otherwise unresponsive to type I JAK inhibitors. Together, these data show the potential of JAK-directed protein degradation as a therapeutic approach in JAK-STAT-driven ALL, and highlight the interplay of Janus kinase and GSPT1 degradation activity in this context.
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http://dx.doi.org/10.1182/blood.2020006846DOI Listing
June 2021

Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity.

J Exp Med 2021 Aug 4;218(8). Epub 2021 Jun 4.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN.

T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage-specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cell-intrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.
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http://dx.doi.org/10.1084/jem.20202415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185987PMC
August 2021

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Nature 2021 07 2;595(7866):295-302. Epub 2021 Jun 2.

Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB. We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBB) into Makassar β-globin (HBB), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB to HBB. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB-to-HBB editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB, generates benign HBB, and minimizes the undesired consequences of double-strand DNA breaks.
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http://dx.doi.org/10.1038/s41586-021-03609-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8266759PMC
July 2021

The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.

PLoS Biol 2021 06 2;19(6):e3001281. Epub 2021 Jun 2.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.
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http://dx.doi.org/10.1371/journal.pbio.3001281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8202955PMC
June 2021

Identification of Potent, Selective, and Orally Bioavailable Small-Molecule GSPT1/2 Degraders from a Focused Library of Cereblon Modulators.

J Med Chem 2021 06 27;64(11):7296-7311. Epub 2021 May 27.

Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, United States.

Whereas the PROTAC approach to target protein degradation greatly benefits from rational design, the discovery of small-molecule degraders relies mostly on phenotypic screening and retrospective target identification efforts. Here, we describe the design, synthesis, and screening of a large diverse library of thalidomide analogues against a panel of patient-derived leukemia and medulloblastoma cell lines. These efforts led to the discovery of potent and novel GSPT1/2 degraders displaying selectivity over classical IMiD neosubstrates, such as IKZF1/3, and high oral bioavailability in mice. Taken together, this study offers compound (SJ6986) as a valuable chemical probe for studying the role of GSPT1/2 and , and it supports the utility of a diverse library of CRBN binders in the pursuit of targeting undruggable oncoproteins.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201443PMC
June 2021

Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells.

Leukemia 2021 Mar 17. Epub 2021 Mar 17.

Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Pediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.
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http://dx.doi.org/10.1038/s41375-021-01212-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8446103PMC
March 2021

Profiling chromatin accessibility in pediatric acute lymphoblastic leukemia identifies subtype-specific chromatin landscapes and gene regulatory networks.

Leukemia 2021 Mar 13. Epub 2021 Mar 13.

Hematological Malignancies Program and Center for Precision Medicine in Leukemia, St. Jude Children's Research Hospital, Memphis, TN, USA.

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy comprised of molecular subtypes largely characterized by aneuploidy or recurring chromosomal rearrangements. Despite extensive information on the ALL transcriptome and methylome, there is limited understanding of the ALL chromatin landscape. We therefore mapped accessible chromatin in 24 primary ALL cell biospecimens comprising three common molecular subtypes (DUX4/ERG, ETV6-RUNX1 and hyperdiploid) from patients treated at St. Jude Children's Research Hospital. Our findings highlight extensive chromatin reprogramming in ALL, including the identification ALL subtype-specific chromatin landscapes that are additionally modulated by genetic variation. Chromatin accessibility differences between ALL and normal B-cells implicate the activation of B-cell repressed chromatin domains and detail the disruption of normal B-cell development in ALL. Among ALL subtypes, we uncovered roles for basic helix-loop-helix, homeodomain and activator protein 1 transcription factors in promoting subtype-specific chromatin accessibility and distinct gene regulatory networks. In addition to chromatin subtype-specificity, we further identified over 3500 DNA sequence variants that alter the ALL chromatin landscape and contribute to inter-individual variability in chromatin accessibility. Collectively, our data suggest that subtype-specific chromatin landscapes and gene regulatory networks impact ALL biology and contribute to transcriptomic differences among ALL subtypes.
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http://dx.doi.org/10.1038/s41375-021-01209-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435544PMC
March 2021

A proteomics approach for the identification of cullin-9 (CUL9) related signaling pathways in induced pluripotent stem cell models.

PLoS One 2021 11;16(3):e0248000. Epub 2021 Mar 11.

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

CUL9 is a non-canonical and poorly characterized member of the largest family of E3 ubiquitin ligases known as the Cullin RING ligases (CRLs). Most CRLs play a critical role in developmental processes, however, the role of CUL9 in neuronal development remains elusive. We determined that deletion or depletion of CUL9 protein causes aberrant formation of neural rosettes, an in vitro model of early neuralization. In this study, we applied mass spectrometric approaches in human pluripotent stem cells (hPSCs) and neural progenitor cells (hNPCs) to identify CUL9 related signaling pathways that may contribute to this phenotype. Through LC-MS/MS analysis of immunoprecipitated endogenous CUL9, we identified several subunits of the APC/C, a major cell cycle regulator, as potential CUL9 interacting proteins. Knockdown of the APC/C adapter protein FZR1 resulted in a significant increase in CUL9 protein levels, however, CUL9 does not appear to affect protein abundance of APC/C subunits and adapters or alter cell cycle progression. Quantitative proteomic analysis of CUL9 KO hPSCs and hNPCs identified protein networks related to metabolic, ubiquitin degradation, and transcriptional regulation pathways that are disrupted by CUL9 deletion in both hPSCs. No significant changes in oxygen consumption rates or ATP production were detected in either cell type. The results of our study build on current evidence that CUL9 may have unique functions in different cell types and that compensatory mechanisms may contribute to the difficulty of identifying CUL9 substrates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951927PMC
October 2021

Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL).

Sci Rep 2021 Mar 4;11(1):5154. Epub 2021 Mar 4.

Department of Computational Biology, St Jude Children's Research Hospital, 262 Danny Thomas Place, MS321, Memphis, TN, 38105, USA.

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.
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http://dx.doi.org/10.1038/s41598-021-84647-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933146PMC
March 2021

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

Elife 2021 Feb 11;10. Epub 2021 Feb 11.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, United States.

Morphogens function in concentration-dependent manners to instruct cell fate during tissue patterning. The cytoneme morphogen transport model posits that specialized filopodia extend between morphogen-sending and responding cells to ensure that appropriate signaling thresholds are achieved. How morphogens are transported along and deployed from cytonemes, how quickly a cytoneme-delivered, receptor-dependent signal is initiated, and whether these processes are conserved across phyla are not known. Herein, we reveal that the actin motor Myosin 10 promotes vesicular transport of Sonic Hedgehog (SHH) morphogen in mouse cell cytonemes, and that SHH morphogen gradient organization is altered in neural tubes of mice. We demonstrate that cytoneme-mediated deposition of SHH onto receiving cells induces a rapid, receptor-dependent signal response that occurs within seconds of ligand delivery. This activity is dependent upon a novel Dispatched (DISP)-BOC/CDON co-receptor complex that functions in ligand-producing cells to promote cytoneme occurrence and facilitate ligand delivery for signal activation.
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http://dx.doi.org/10.7554/eLife.61432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968926PMC
February 2021

Antitumor Effects of CAR T Cells Redirected to the EDB Splice Variant of Fibronectin.

Cancer Immunol Res 2021 03 22;9(3):279-290. Epub 2020 Dec 22.

Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, Tennessee.

Chimeric antigen receptor (CAR) T-cell therapy has had limited success in early-phase clinical studies for solid tumors. Lack of efficacy is most likely multifactorial, including a limited array of targetable antigens. We reasoned that targeting the cancer-specific extra domain B (EDB) splice variant of fibronectin might overcome this limitation because it is abundantly secreted by cancer cells and adheres to their cell surface. , EDB-CAR T cells recognized and killed EDB-positive tumor cells. , 1 × 10 EDB-CAR T cells had potent antitumor activity in both subcutaneous and systemic tumor xenograft models, resulting in a significant survival advantage in comparison with control mice. EDB-CAR T cells also targeted the tumor vasculature, as judged by IHC and imaging, and their antivascular activity was dependent on the secretion of EDB by tumor cells. Thus, targeting tumor-specific splice variants such as EDB with CAR T cells is feasible and has the potential to improve the efficacy of CAR T-cell therapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925432PMC
March 2021

Therapeutic gene editing strategies using CRISPR-Cas9 for the β-hemoglobinopathies.

J Biomed Res 2020 Nov;35(2):115-134

Department of Cellular and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

With advancements in gene editing technologies, our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate, paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases. CRISPR-Cas9, short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells. This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic. The β-hemoglobinopathies are a group of monogenic diseases, which despite their high prevalence and chronic debilitating nature, continue to have few therapeutic options available. In this review, we will discuss our existing comprehension of the genetics and current state of treatment for β-hemoglobinopathies, consider potential genome editing therapeutic strategies, and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.
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http://dx.doi.org/10.7555/JBR.34.20200096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8038529PMC
November 2020

Assessing Off-Target Editing of CRISPR-Cas9 Systems.

CRISPR J 2020 12;3(6):430-432

Department of Cell and Molecular Biology, Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

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http://dx.doi.org/10.1089/crispr.2020.29116.smiDOI Listing
December 2020

In Vivo CRISPR/Cas9-Based Targeted Disruption and Knockin of a Long Noncoding RNA.

Methods Mol Biol 2021 ;2254:305-321

Department of Physiology and Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA.

The CRISPR/Cas9 system has been widely used as an efficient genome-editing tool for studying physiological functions of long noncoding RNAs (lncRNAs). In this chapter, we describe the experimental procedures for using the CRISPR/Cas9 system to genetically modify a long noncoding RNA in vivo through the targeted disruption and knockin approaches.
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http://dx.doi.org/10.1007/978-1-0716-1158-6_19DOI Listing
March 2021

The Heme-Regulated Inhibitor Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics.

Mol Cancer Res 2021 04 7;19(4):636-650. Epub 2020 Dec 7.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee.

Antiapoptotic is one of the most frequently amplified genes in human cancers and elevated expression confers resistance to many therapeutics including the BH3-mimetic agents ABT-199 and ABT-263. The antimalarial, dihydroartemisinin (DHA) translationally represses MCL-1 and synergizes with BH3-mimetics. To explore how DHA represses MCL-1, a genome-wide CRISPR screen identified that loss of genes in the heme synthesis pathway renders mouse BCR-ABL B-ALL cells resistant to DHA-induced death. Mechanistically, DHA disrupts the interaction between heme and the eIF2α kinase heme-regulated inhibitor (HRI) triggering the integrated stress response. Genetic ablation of , which encodes HRI, blocks MCL-1 repression in response to DHA treatment and represses the synergistic killing of DHA and BH3-mimetics compared with wild-type leukemia. Furthermore, BTdCPU, a small-molecule activator of HRI, similarly triggers MCL-1 repression and synergizes with BH3-mimetics in mouse and human leukemia including both Ph and Ph-like B-ALL. Finally, combinatorial treatment of leukemia bearing mice with both BTdCPU and a BH3-mimetic extended survival and repressed MCL-1 . These findings reveal for the first time that the HRI-dependent cellular heme-sensing pathway can modulate apoptosis in leukemic cells by repressing MCL-1 and increasing their responsiveness to BH3-mimetics. This signaling pathway could represent a generalizable mechanism for repressing MCL-1 expression in malignant cells and sensitizing them to available therapeutics. IMPLICATIONS: The HRI-dependent cellular heme-sensing pathway can modulate apoptotic sensitivity in leukemic cells by repressing antiapoptotic MCL-1 and increasing their responsiveness to BH3-mimetics.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026502PMC
April 2021

FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis.

Blood 2021 01;137(2):155-167

Department of Hematology.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors. Erythroblasts lacking FBXO11 are developmentally delayed, with reduced expression of maturation-associated genes, most of which harbor bivalent histone marks at their promoters. In FBXO11-/- erythroblasts, these gene promoters bind BAHD1 and fail to recruit the erythroid transcription factor GATA1. The BAHD1 complex interacts physically with PRC2, and depletion of either component restores FBXO11-deficient erythroid gene expression. Our studies identify BAHD1 as a novel effector of PRC2-mediated repression and reveal how a single E3 ubiquitin ligase eliminates PRC2 repression at many developmentally poised bivalent genes during erythropoiesis.
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http://dx.doi.org/10.1182/blood.2020007809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820877PMC
January 2021

MEKK3-MEK5-ERK5 signaling promotes mitochondrial degradation.

Cell Death Discov 2020 20;6:107. Epub 2020 Oct 20.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 USA.

Mitochondria are vital organelles that coordinate cellular energy homeostasis and have important roles in cell death. Therefore, the removal of damaged or excessive mitochondria is critical for maintaining proper cellular function. The PINK1-Parkin pathway removes acutely damaged mitochondria through a well-characterized mitophagy pathway, but basal mitochondrial turnover occurs via distinct and less well-understood mechanisms. Here we report that the MEKK3-MEK5-ERK5 kinase cascade is required for mitochondrial degradation in the absence of exogenous damage. We demonstrate that genetic or pharmacological inhibition of the MEKK3-MEK5-ERK5 pathway increases mitochondrial content by reducing lysosome-mediated degradation of mitochondria under basal conditions. We show that the MEKK3-MEK5-ERK5 pathway plays a selective role in basal mitochondrial degradation but is not required for non-selective bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis. This illuminates the MEKK3-MEK5-ERK5 pathway as a positive regulator of mitochondrial degradation that acts independently of exogenous mitochondrial stressors.
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http://dx.doi.org/10.1038/s41420-020-00342-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576125PMC
October 2020

14-3-3ζ-TRAF5 axis governs interleukin-17A signaling.

Proc Natl Acad Sci U S A 2020 10 23;117(40):25008-25017. Epub 2020 Sep 23.

Department of Physiology & Pharmacology, College of Medicine & Life Sciences, University of Toledo, Toledo, OH 43614;

IL-17A is a therapeutic target in many autoimmune diseases. Most nonhematopoietic cells express IL-17A receptors and respond to extracellular IL-17A by inducing proinflammatory cytokines. The IL-17A signal transduction triggers two broad, TRAF6- and TRAF5-dependent, intracellular signaling pathways to produce representative cytokines (IL-6) and chemokines (CXCL-1), respectively. Our limited understanding of the cross-talk between these two branches has generated a crucial gap of knowledge, leading to therapeutics indiscriminately blocking IL-17A and global inhibition of its target genes. In previous work, we discovered an elevated expression of 14-3-3 proteins in inflammatory aortic disease, a rare human autoimmune disorder with increased levels of IL-17A. Here we report that 14-3-3ζ is essential for IL-17 signaling by differentially regulating the signal-induced IL-6 and CXCL-1. Using genetically manipulated human and mouse cells, and ex vivo and in vivo rat models, we uncovered a function of 14-3-3ζ. As a part of the molecular mechanism, we show that 14-3-3ζ interacts with several TRAF proteins; in particular, its interaction with TRAF5 and TRAF6 is increased in the presence of IL-17A. In contrast to TRAF6, we found TRAF5 to be an endogenous suppressor of IL-17A-induced IL-6 production, an effect countered by 14-3-3ζ. Furthermore, we observed that 14-3-3ζ interaction with TRAF proteins is required for the IL-17A-induced IL-6 levels. Together, our results show that 14-3-3ζ is an essential component of IL-17A signaling and IL-6 production, an effect that is suppressed by TRAF5. To the best of our knowledge, this report of the 14-3-3ζ-TRAF5 axis, which differentially regulates IL-17A-induced IL-6 and CXCL-1 production, is unique.
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http://dx.doi.org/10.1073/pnas.2008214117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547158PMC
October 2020

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

J Clin Invest 2020 12;130(12):6677-6687

Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, Maryland, USA.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.
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http://dx.doi.org/10.1172/JCI140189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685754PMC
December 2020

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Mol Cell 2020 08 20;79(4):645-659.e9. Epub 2020 Jul 20.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA. Electronic address:

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.
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http://dx.doi.org/10.1016/j.molcel.2020.06.037DOI Listing
August 2020

The Zα2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.

J Biol Chem 2020 06 29;295(24):8325-8330. Epub 2020 Apr 29.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Z-DNA-binding protein 1 (ZBP1) is an innate immune sensor of nucleic acids that regulates host defense responses and development. ZBP1 activation triggers inflammation and pyroptosis, necroptosis, and apoptosis (PANoptosis) by activating receptor-interacting Ser/Thr kinase 3 (RIPK3), caspase-8, and the NLRP3 inflammasome. ZBP1 is unique among innate immune sensors because of its N-terminal Zα1 and Zα2 domains, which bind to nucleic acids in the Z-conformation. However, the specific role of these Zα domains in orchestrating ZBP1 activation and subsequent inflammation and cell death is not clear. Here we generated mice that express ZBP1 lacking the Zα2 domain and demonstrate that this domain is critical for influenza A virus-induced PANoptosis and underlies perinatal lethality in mice in which the RIP homotypic interaction motif domain of RIPK1 has been mutated ( ). Deletion of the Zα2 domain in ZBP1 abolished influenza A virus-induced PANoptosis and NLRP3 inflammasome activation. Furthermore, deletion of the Zα2 domain of ZBP1 was sufficient to rescue mice from perinatal lethality caused by ZBP1-driven cell death and inflammation. Our findings identify the essential role of the Zα2 domain of ZBP1 in several physiological functions and establish a link between Z-RNA sensing via the Zα2 domain and promotion of influenza-induced PANoptosis and perinatal lethality.
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http://dx.doi.org/10.1074/jbc.RA120.013752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294087PMC
June 2020

The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth.

Elife 2020 04 16;9. Epub 2020 Apr 16.

Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, United States.

The mitotic deacetylase complex (MiDAC) is a recently identified histone deacetylase (HDAC) complex. While other HDAC complexes have been implicated in neurogenesis, the physiological role of MiDAC remains unknown. Here, we show that MiDAC constitutes an important regulator of neural differentiation. We demonstrate that MiDAC functions as a modulator of a neurodevelopmental gene expression program and binds to important regulators of neurite outgrowth. MiDAC upregulates gene expression of pro-neural genes such as those encoding the secreted ligands SLIT3 and NETRIN1 (NTN1) by a mechanism suggestive of H4K20ac removal on promoters and enhancers. Conversely, MiDAC inhibits gene expression by reducing H3K27ac on promoter-proximal and -distal elements of negative regulators of neurogenesis. Furthermore, loss of MiDAC results in neurite outgrowth defects that can be rescued by supplementation with SLIT3 and/or NTN1. These findings indicate a crucial role for MiDAC in regulating the ligands of the SLIT3 and NTN1 signaling axes to ensure the proper integrity of neurite development.
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http://dx.doi.org/10.7554/eLife.57519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192582PMC
April 2020

Cancer Screens: Better Together.

CRISPR J 2020 02;3(1):12-14

Department of Cell and Molecular Biology, Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, Tennessee.

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http://dx.doi.org/10.1089/crispr.2020.29084.spmDOI Listing
February 2020

MYCN amplification and ATRX mutations are incompatible in neuroblastoma.

Nat Commun 2020 02 14;11(1):913. Epub 2020 Feb 14.

Basic Science Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41467-020-14682-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021759PMC
February 2020

Schizophrenia-related microdeletion causes defective ciliary motility and brain ventricle enlargement via microRNA-dependent mechanisms in mice.

Nat Commun 2020 02 14;11(1):912. Epub 2020 Feb 14.

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.

Progressive ventricular enlargement, a key feature of several neurologic and psychiatric diseases, is mediated by unknown mechanisms. Here, using murine models of 22q11-deletion syndrome (22q11DS), which is associated with schizophrenia in humans, we found progressive enlargement of lateral and third ventricles and deceleration of ciliary beating on ependymal cells lining the ventricular walls. The cilia-beating deficit observed in brain slices and in vivo is caused by elevated levels of dopamine receptors (Drd1), which are expressed in motile cilia. Haploinsufficiency of the microRNA-processing gene Dgcr8 results in Drd1 elevation, which is brought about by a reduction in Drd1-targeting microRNAs miR-382-3p and miR-674-3p. Replenishing either microRNA in 22q11DS mice normalizes ciliary beating and ventricular size. Knocking down the microRNAs or deleting their seed sites on Drd1 mimicked the cilia-beating and ventricular deficits. These results suggest that the Dgcr8-miR-382-3p/miR-674-3p-Drd1 mechanism contributes to deceleration of ciliary motility and age-dependent ventricular enlargement in 22q11DS.
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http://dx.doi.org/10.1038/s41467-020-14628-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021727PMC
February 2020
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