技术资料

Acute myeloid leukemia (AML) is fatal in the majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds,selected 4 hits targeting the IQGAP1-GRD domain,and conducted SAR of the ‘fittest hit’ to identify UR778Br,a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation,induced apoptosis,resulted in G2/M arrest,and inhibited colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows response to IQGAP1 inhibition,and UR778Br,identified through in-silico studies,selectively targeted AML cells while sparing normal marrow. Subject terms: Cancer,Cell biology,Drug discovery,Immunology,Oncology View Publication

Fibroblastic reticular cells (FRCs) are a subpopulation of stromal cells modulating the immune environments in health and disease. We have previously shown that activation of TLR9 signaling in FRC in fat-associated lymphoid clusters (FALC) regulate peritoneal immunity via suppressing immune cell recruitment and peritoneal resident macrophage (PRM) retention. However,FRCs are heterogeneous across tissues and organs. The functions of each FRC subset and the regulation of TLR9 in distinct FRC subsets are unknown. Here,we confirmed that specific deletion of TLR9 in FRC improved bacterial clearance and survival during peritoneal infection. Furthermore,using single-cell RNA sequencing,we found two subsets of FRCs (CD55 hi and CD55 lo ) in the mesenteric FALC. The CD55 hi FRCs were enriched in gene expression related to extracellular matrix formation. The CD55 lo FRCs were enriched in gene expression related to immune response. Interestingly,we found that TLR9 is dominantly expressed in the CD55 lo subset. Activation of TLR9 signaling suppressed proliferation,cytokine production,and retinoid metabolism in the CD55 lo FRC,but not CD55 hi FRC. Notably,we found that adoptive transfer of Tlr9 -/– CD55 lo FRC from mesenteric FALC more effectively improved the survival during peritonitis compared with WT-FRC or Tlr9 -/– CD55 hi FRC. Furthermore,we identified CD55 hi and CD55 lo subsets in human adipose tissue-derived FRC and confirmed the suppressive effect of TLR9 on the proliferation and cytokine production in the CD55 lo subset. Therefore,inhibition of TLR9 in the CD55 lo FRCs from adipose tissue could be a useful strategy to improve the therapeutic efficacy of FRC-based therapy for peritonitis. View Publication

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD),but the mechanism underlying this link is poorly understood. In particular,the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here,single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how,in an isoform- and lipidation-specific way,apoE modulates the aggregation,clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells,and reduces secretion of inflammatory markers and membrane damage,demonstrating a clear path to AD therapeutics. Subject terms: Protein aggregation,Nanoscale biophysics View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock,but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma,we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction,abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent,synergistic therapeutic potential agnostic of glioma subtype,leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy,opening avenues for combinatorial treatment in these lethal malignancies. Subject terms: CNS cancer,Paediatric cancer,Radiotherapy View Publication

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here,we report a novel antigen Plasmodium prohibitin “ Pf PHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach,we show that loss of Hsp70A1A in red blood cells (RBCs) or Pf PHB2 in infected red blood cells (iRBCs),respectively,inhibit Pf PHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting Pf PHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant Pf PHB2 binds to RBCs which is inhibited by anti- Pf PHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of Pf PHB2 to serve as antigen is further supported by detection of anti- Pf PHB2 antibody in patient sera. Overall,this study proposes Pf PHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment. Subject areas: biochemistry,molecular biology,immunology View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However,the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection,skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors,ligands,ion channels and metabolites to generate injury-specific neuroimmune interactomes,we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts. Subject terms: Neuroimmunology,Inflammation View Publication

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor,CDC20,preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However,the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression,we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for,gastrointestinal atrophy is detrimental. Remarkably,deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome,while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect,identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast,only overexpression of anti-apoptotic BCL2,but none of the BH3-only protein deficiencies mentioned above,can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo. View Publication

Of all gynecologic cancers,epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts,90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa,we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover,treatment with LIFR inhibitor,EC359 significantly reduced OCa cell viability and cell survival with an IC 50 ranging from 5-50 nM. Furthermore,EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro,ex vivo and in vivo models including cell-based xenografts,patient-derived explants,organoids,and xenograft tumors,we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally,EC359 therapy considerably improved tumor immunogenicity by robust CD45 + leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-,B-,and other immune cells. Collectively,our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa. Subject terms: Molecular medicine,Ovarian cancer View Publication

The development of tyrosine kinase inhibitors (TKIs) has revolutionarily increased the overall survival of patients with chronic myeloid leukemia (CML). However,drug resistance remains a major obstacle. Here,we demonstrated that a BCR-ABL1-independent long non-coding RNA,IRAIN,is constitutively expressed at low levels in CML,resulting in imatinib resistance. IRAIN knockdown decreased the sensitivity of CD34 + CML blasts and cell lines to imatinib,whereas IRAIN overexpression significantly increased sensitivity. Mechanistically,IRAIN downregulates CD44,a membrane receptor favorably affecting TKI resistance,by binding to the nuclear factor kappa B subunit p65 to reduce the expression of p65 and phosphorylated p65. Therefore,the demethylating drug decitabine,which upregulates IRAIN,combined with imatinib,formed a dual therapy strategy which can be applied to CML with resistance to TKIs. Subject areas: Molecular biology,Cell biology,Cancer View Publication

Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling in vitro . For successful differentiation,a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment,during which distinct cells in the brain and during in vitro differentiation acquire their fates in an unsynchronized manner,hinders pooled transcriptional comparisons. One approach is to “translate” chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal,due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells,but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells. Subject areas: Natural sciences,Biological sciences,Biochemistry,Molecular biology,Neuroscience,Cellular neuroscience,Cell biology View Publication