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Single-cell transcriptomics of the myeloid milieu reveals an angiogenic niche in triple-negative breast cancer.
No full textIntratumoral myeloid cells are highly heterogeneous in terms of development and function and are pivotal for forming and regulating the tumor microenvironment. However, the myeloid milieu in triple-negative breast cancer (TNBC) remains poorly understood. Here, to elucidate this myeloid milieu, we integrated in-house and public single-cell RNA sequencing data. We detected diverse neutrophil and mononuclear-phagocyte subtypes and delineated their developmental trajectories and functions. Of particular interest were the VEGF
Pluripotent cell states and fates in human embryo models.
No full textPluripotency, the capacity to generate all cells of the body, is a defining property of a transient population of epiblast cells found in pre-, peri- and post-implantation mammalian embryos. As development progresses, the epiblast cells undergo dynamic transitions in pluripotency states, concurrent with the specification of extra-embryonic and embryonic lineages. Recently, stem cell-based models of pre- and post-implantation human embryonic development have been developed using stem cells that capture key properties of the epiblast at different developmental stages. Here, we review early primate development, comparing pluripotency states of the epiblast in vivo with cultured pluripotent cells representative of these states. We consider how the pluripotency status of the starting cells influences the development of human embryo models and, in turn, what we can learn about the human pluripotent epiblast. Finally, we discuss the limitations of these models and questions arising from the pioneering studies in this emerging field
SARS-CoV-2 induced immune perturbations in infants vary with disease severity and differ from adults\u27 responses.
Get PDFDifferences in immune profiles of children and adults with COVID-19 have been previously described. However, no systematic studies have been reported from infants hospitalized with severe disease. We applied a multidimensional approach to decipher the immune responses of SARS-CoV-2 infected infants (n = 26; 10 subacute, 11 moderate and 5 severe disease; median age = 1.6 months) and matched controls (n = 14; median age = 2 months). Single cell (scRNA-seq) profiling of PBMCs revealed substantial alterations in cell composition in SARS-CoV-2 infected infants; with most cell-types switching to an interferon-stimulated gene (ISGhi) state including: (i) CD14+ monocytes co-expressing ISGs and inflammasome-related molecules, (ii) ISGhi naive CD4+ T cells, (iii) ISGhi proliferating cytotoxic CD8+ T cells, and (iv) ISGhi naive and transitional B cells. We observe increased serum concentrations of both interferons and inflammatory cytokines in infected infants. Antibody responses to SARS-CoV-2 are also consistently detected in the absence of anti-IFN autoantibodies. Compared with infected adults, infants display a similar ISG signature in monocytes but a markedly enhanced ISG signature in T and B cells. These findings provide insights into the distinct immune responses to SARS-CoV-2 in the first year of life and underscore the importance of further defining the unique features of early life immunity
Mis-splicing-derived neoantigens and cognate TCRs in splicing factor mutant leukemias.
Get PDFMutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here, we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-major histocompatibility complex (MHC) dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant-induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide proof of concept for genetically redirecting T cells to recognize these targets
Impact of antibody Fc engineering on translational pharmacology, and safety: insights from industry case studies.
Get PDFTherapeutic monoclonal antibodies (mAbs) are often designed to not only bind targets via their antigen-binding domains (Fabs) but to also engage with cell surface receptors, FcγRs and FcRn, through their Fc regions, which may result in a variety of functional outcomes, including antibody- dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC) and alteration of circulating half-lives. Engineering the Fc regions to achieve desirable pharmacology and pharmacokinetics is a widely adopted strategy in drug development. Fc regions can be modified through amino acid substitutions and glycoengineering, resulting in enhanced or reduced effector functions, preferential binding to FcR subtypes, or pH-dependent binding to FcRns. These alterations in binding and effector activities of mAbs may potentially also be accompanied by undesirable effects or safety concerns. Critical assessment of pharmacology and safety in the nonclinical setting is essential before exposing humans to the engineered mAb. For Fc-modified mAbs, the choice of in vitro and in vivo nonclinical pharmacology and safety models need to account for species differences in FcR expression and function, potentially divergent effects of Fc modifications in humans versus nonclinical species, impact of target and cognate ligand expression patterns, and potential impact of emergent anti-drug antibodies directed against the mAb. Using a variety of industry case studies, we highlight key aspects of nonclinical pharmacology and toxicology testing strategies, factors that influence choice of nonclinical models, translatability of findings, input from health authorities and suggest best practice approaches for nonclinical testing of Fc modified mAbs
Characterization of a Mouse Model of Mitophagy-Driven Mitochondrial DNA Depletion Syndrome
No full textMitochondrial DNA Depletion Syndromes (MDDS) are rare genetic disorders that reduce mitochondrial DNA (mtDNA) abundance in cells, impairing mitochondrial function. Some forms of MDDS result from excessive mitophagy, a process which destroys healthy mitochondria and results in profound metabolic dysfunction. F-Box and Leucine Rich Repeat Protein 4 (FBXL4) negatively regulates mitophagy by promoting degradation of mitophagy receptors BNIP3 and BNIP3L/NIX. While Fbxl4 knockout mice have revealed embryonic lethality, patient-derived variants that are associated with severe multi-systemic symptoms and result in early childhood mortality remain unstudied in-vivo. To address this, our lab has generated a mouse model carrying the clinically identified C584R missense mutation to investigate the mechanism by which this variant causes mitochondrial and subsequent organ dysfunction. Evidence from this characterization verifies decreased mtDNA copy number, upregulation of BNIP3 and BNIP3L/NIX, and decreased oxidative phosphorylation in Fbxl4C584R mice. Further investigation of the Fbxl4C584R mouse will provide novel insight into the pathology of FBXL4-mediated MDDS and may reveal therapeutic targets for patients with Fbxl4 mutations
Increased longevity of circulating human IgG in an NSG Fc gamma receptor-1 deficient humanized mouse model.
No full textMonoclonal antibodies (mAbs) are powerful therapeutic tools that are used to treat multiple types of human cancer as well as a diverse set of non-malignant diseases. Humanized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice implanted with human tumors and with immune cells and tissues are widely used in studies of mAb-based therapeutics. However, due to a gain of function mutation in the Fcgr1 gene in the NOD strain background, NSG mice rapidly clear human IgG1, IgG3, and IgG4. As most mAbs are either IgG1 or IgG4 isotypes, the use of NOD-based mouse models for preclinical testing of therapeutic mAbs is limited by the reduced half-life in vivo. In order to extend the half-life of mAbs in NSG mice and create a more physiologically relevant model, we created NSG Fcγ Receptor I knock out (NSG-Fcgr1null) mice. IgG clearance was measured for three different cancer therapeutic mAbs: rituximab (IgG1), trastuzumab (IgG1), and pembrolizumab (IgG4), by comparing the levels of circulating human IgG over the course of 5 weeks post IV injection in NSG and NSG-Fcgr1null mice. Preliminary pharmacokinetic analyses found significant increases in the half-lives and exposure of each of these mAbs in the NSG-Fcgr1null mice when compared to NSG controls. Additionally, when engrafted with human hematopoietic stem cells (HSCs), NSG-Fcgr1null mice supported higher levels of serum IgG when compared to NSG controls. Overall, the NSG-Fcgr1null mouse presents a more physiologically relevant and translatable model for the in vivo testing of human therapeutic mAbs
Investigating BRCA1 Promoter Methylation using Molecular and Computational Approaches
No full textBreast Cancer is one of the most common types of cancer for women as it accounts for about 30% of all new female cancers every year. Breast cancer has many different subtypes, one of whom is Triple-Negative Breast Cancer (TNBC). Due to the lack of three main receptors, Estrogen, Progesterone, and Her2, it is one of the deadliest types of breast cancer.
BRCA1 is a critical component of the homologous recombination pathway, which is responsible for the error-free repair of DNA double stranded breaks. To put into context the importance of BRCA1, roughly half of all TNBC are linked to loss of BRCA1 activity.
Epigenetic silencing through BRCA1 promoter methylation is a common mode of BRCA1 gene inactivation as it accounts for half the BRCA1 deficient TNBC cases or around 25% of total TNBC cases. Unfortunately, the mechanism regarding how BRCA1 methylation spontaneously occurs is not very well studied due to the lack of biological models. To address this issue, the Liu lab took advantage of a rare but naturally occurring BRCA1 promoter mutation (c.-107A\u3eT), which was found in two cases of familial breast and ovarian cancer, and was associated with BRCA1 promoter methylation in cis. The Liu lab introduced the c.-107A\u3eT mutation in the induced pluripotent stem cell (iPSC) line WIBJ2 and the human mammary epithelial cell (HMEC) line AR7HT. When this mutation was introduced in WIBJ2 cells, DNA methylation at the BRCA1 promoter was observed. However, in the AR7HT cells, there was no significant gain of methylation at the BRCA1 promoter. Based on this observation, I investigated the binding activity of RNA Polymerase II to the BRCA1 promoter by performing the ChIP assay followed by a qPCR, which showed that there was RNA Polymerase II binding at the BRCA1 promoter region in both WIBJ2 and AR7HT cells. Sanger Sequencing analysis performed on the WIBJ2 ChIP-qPCR amplification products showed a relative enrichment of the wildtype BRCA1 allele in the Pol2 pulldown sample, relative to both input DNA and the igG control pulled down sample, supporting our hypothesis that RNA Polymerase II is preferentially bound to the wild-type (WT) promoter over the mutant promoter, in WIBJ2 cells.
ZNF680 was originally identified as a transcription factor critical for induction of BRCA1 methylation at the c.-107A\u3eT mutant promoter in WIBJ2 cells. Using several in silico techniques and databases, I showed that ZNF680 preferentially binds to promoter, TSS, enhancer, and heterochromatin regions of the genome. However, when compared to other transcription factors such as ZNF610 and ZNF785 who also bind at the BRCA1 promoter, ZNF680 ChIP-seq peak distribution pattern is unusual, in that the percentage of peaks mapping to TSS or promoter regions is much lower. Furthermore, when looking at DNA methylation via EPIC Array analysis and RNA Sequencing data, it appears that ZNF680 is not a master regulator of methylation on a genome-wide level, but that it may be regulating only a small number of genes. This suggests that ZNF680 effect at the BRCA1 promoter may be a very specific occurrence
Novel genomic therapeutic for desmoplastic small round cell tumor based on RNA sensor targeting of EWSR1::WT1 gene fusion
No full textThe initiating tumorigenic event for many cancers is a chromosomal translocation that produces an oncogenic gene fusion. The development of RNA sensing technology can identify cancerous cells harboring these gene fusion transcripts and initiate the delivery of a downstream therapeutic payload. Here, we aim to build a novel therapeutic with ADAR-RNA sensors to target and kill cells containing the EWSR1::WT1 gene fusion found in desmoplastic small round cell tumor (DSRCT). We designed EWSR1::WT1 gene fusion transcripts containing the presence or absence of KTS isoform (+/-KTS) in exon 9 of WT1 and eight different therapeutic ADAR-RNA sensors with combinatorial alterations of base pair spacing between engineered stop codons (27bp or 42bp), linker type (E2A or XTEN80), and payloads (EGFP, TKSR39, or NTR1.1) and transfected these into HEK293T cells to test editing efficiency and cell viability with the presence or absence of a prodrug. One sensor in particular was identified to demonstrate the most promising and consistent patterns of editing efficiency and cell death when co-transfected with either the EWSR1::WT1 (+KTS) or EWSR1::WT1 (-KTS) fusion transcripts. New sensors should be designed to increase cell death before testing on the patient-derived JN-DSRCT-1 cell line that expresses the EWSR1::WT1 fusion transcripts
The integrative genomic and functional immunological analyses of colorectal cancer initiating cells to modulate stemness properties and the susceptibility to immune responses.
Get PDFBACKGROUND: Colorectal cancer (CRC) initiating cells (CICs) possess self-renewal capabilities and are pivotal in tumor recurrence and resistance to conventional therapies, including immunotherapy. The mechanisms underlying their interaction with immune cells remain unclear.
METHODS: We conducted a multi-omics analysis-encompassing DNA methylation, total RNA sequencing, and microRNAs (miRNAs; N = 800) profiling on primary CICs and differentiated tumor cell lines, including autologous pairs. Functional immunological assays were performed to assess the impact of miRNA modulation.
RESULTS: CICs exhibited distinct methylation patterns, transcriptomic profiles, and miRNA expressions compared to differentiated tumor cells (p \u3c 0.05 or 0.01). Notably, miRNA-15a and -196a were implicated in regulating tumorigenic pathways, such as epithelial-to-mesenchymal transition (EMT), TGF-β signaling, and immune modulation. The transfection of CICs with miRNA mimics led to the downregulation of oncogenic EMT markers (CRKL, lncRNA SOX2-OT, JUNB, SMAD3) and TGF-β pathway, resulting in a significant reduction of the in vitro proliferation and the tumorigenicity and migration in a zebrafish xenograft model. Additionally, miRNA-15a enhanced the expression of antigen processing machinery and decreased the expression of immune checkpoints (PD-L1, PD-L2, CTLA-4) and immunosuppressive cytokines (IL-4). The co-culture of HLA-matched lymphocytes with CICs overexpressing the miRNA-15a, elicited robust tumor-specific immune responses, characterized by a shift toward central and effector memory T cell phenotypes and prevented their terminal differentiation and exhaustion. The combination of miRNA modulation with Indoleamine 2,3-dioxygenase blockade and immunomodulating agents further potentiated these effects.
CONCLUSIONS: Our study demonstrates that the modulation of miRNA-15a in CICs not only suppresses the tumorigenic properties but also enhances their visibility to the immune system by upregulating antigen presentation and reducing immunomodulatory molecules. These findings suggest that combining miRNA modulation with epigenetic or immunomodulatory agents holds significant promise for overcoming treatment resistance in CRC