Biology subjects

Nam, E.

Publications and source records attributed to Nam, E..

6 recordsLinked to original sources

bioRxiv2025

Tumors Located in the Brain Impair the Frequency and Phenotype of Dendritic Cells in Blood and Tumor

BackgroundProfessional antigen-presenting dendritic cells (DC) are critical for anti-tumor immune responses, yet patients with glioblastoma, an aggressive primary brain tumor that responds poorly to current investigational immunotherapies, appear to be deficient in DC. The extent of this deficiency, the specific DC subsets affected, and the causative mechanisms remain undefined. Furthermore, DCs in other brain tumors have not been systematically investigated. MethodsHigh-parameter flow cytometry was used to profile circulating and intra-tumoral DCs in patients with glioblastoma, low-grade gliomas, and brain metastases, and non-CNS cancers. Plasma DC growth factors were quantified using ELISA. We also evaluated single-cell RNA sequencing (scRNAseq) datasets to compare intra-tumoral DCs in brain and lung tumors, and quantified DC number and phenotype in three intracranial mouse brain tumor models. ResultsOur studies reveal a profound systemic reduction of multiple DC subsets in the blood of patients with diverse brain tumors, coupled with reduced DC activation marker expression and lower plasma levels of FLT3L and G-CSF. Furthermore, scRNAseq analyses revealed reduced intra-tumoral DCs in glioblastoma compared to lung tumors. Circulating DC numbers inversely correlated with perioperative corticosteroid dose in patients with or without a brain tumor. However, brain tumor patients not receiving corticosteroids also had reduced DCs, suggesting a direct effect of the brain tumor. This was supported by our observation of systemic DC defects in mouse brain tumor models. ConclusionsWe reveal profound DC defects in patients with brain tumors, which may contribute to current difficulties in developing effective immunotherapies for glioblastoma. SummaryWe demonstrate multiple DC defects in patients with brain tumors. This includes a profound reduction in circulating DC number, diminished activation marker expression and growth factor levels in cancer patients with brain tumors compared to those without, and reduced intra-tumoral DCs in brain compared to lung tumors. This is the first time DC subsets have been fully characterized in a range of brain tumor patients. We show that corticosteroid usage is closely associated with DC defects, highlighting the adverse effects of a standard symptomatic treatment on these critical immune cells. However, tumors located within the brain also directly contribute to DC defects. We identified several mouse brain tumor models that can be used to further the understanding of this endogenous DC deficiency and to develop approaches to restore DCs, ultimately leading to new combination immunotherapies for the treatment of brain cancers. HighlightsO_LIDCs are reduced in brain tumor patients and mice with intracranial tumors. C_LIO_LIDCs are rare within glioblastoma tumor tissue. C_LIO_LIThe presence of a brain tumor and corticosteroid use are both associated with DC defects. C_LI

immunology↗

bioRxiv2025

A Characterization of the Immune Cells in Immunocompetent and Immunodeficient Mice with Orthotopic Brain Tumors.

BackgroundGlioblastoma is characterized by poor survival with few treatment advances for over 20 years. Active research is being conducted in immunotherapies; however, this may be hampered by the lack of detailed characterization of the immune compartments of orthotopic murine brain tumor models. MethodsWe used cell lines derived from glioblastoma patients and murine glioma cell lines to produce intracranial xenograft and syngraft brain tumors in NSG or C57BL/6 mice, respectively. High-parameter flow cytometry and tissue immunofluorescence staining were used to characterize the immune cell compartment in each model. We investigated brain, spleen, lymph node, and bone marrow for the frequencies of different immune cells. Further, we investigated the effect of sub-lethal whole-body irradiation, commonly used for lymphodepletion, on immune cells in immunocompetent mice. ResultsIn our study of non-tumor-bearing mice, we observed dramatic differences between NSG and C57BL/6 mice, not only of T and B cells but also other immune cells, including decreased monocytes, dendritic cells, and eosinophils. In immunocompetent brain tumor-bearing mice, we observed significant differences in the numbers of immune cells compared to non-tumour-bearing controls, primarily in the brain; however, significant differences were also in the spleen, lymph node and bone marrow, particularly in the subsets of monocytes, T cells and dendritic cells. Sub-lethal irradiation caused lasting changes in immune composition. ConclusionsOur quantitative characterization of different immune compartments in orthotopic murine models of glioblastoma may allow for a better-informed selection of tumor models that are appropriate for the pre-clinical investigation of immunotherapies for glioblastoma.

cancer biology↗

bioRxiv2024

A circular engineered sortase for interrogating histone H3 in chromatin

Reversible modification of the histone H3 N-terminal tail is critical in regulating chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex "cut-and-paste" middle down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetics discovery and therapeutic development.

biochemistry↗

bioRxiv2024

Asymmetric Engagement of Dimeric CRL3KBTBD4 by the Molecular Glue UM171 Licenses Degradation of HDAC1/2 Complexes

UM171 is a potent small molecule agonist of ex vivo human hematopoietic stem cell (HSC) self-renewal1, a process that is tightly controlled by epigenetic regulation. By co-opting KBTBD4, a substrate receptor of the CULLIN3-RING E3 ubiquitin ligase complex, UM171 promotes the degradation of members of the CoREST transcriptional corepressor complex, thereby limiting HSC attrition2,3. However, the direct target and mechanism of action of UM171 remain unclear. Here, we reveal that UM171 acts as a molecular glue to induce high-affinity interactions between KBTBD4 and HDAC1 to promote the degradation of select HDAC1/2 corepressor complexes. Through proteomics and chemical inhibitor studies, we discover that the principal target of UM171 is HDAC1/2. Cryo-electron microscopy (cryo-EM) analysis of dimeric KBTBD4 bound to UM171 and the LSD1-HDAC1-CoREST complex unveils an unexpected asymmetric assembly, in which a single UM171 molecule enables a pair of KBTBD4 KELCH-repeat propeller domains to recruit HDAC1 by clamping on its catalytic domain. One of the KBTBD4 propellers partially masks the rim of the HDAC1 active site pocket, which is exploited by UM171 to extend the E3-neo-substrate interface. The other propeller cooperatively strengthens HDAC1 binding via a separate and distinct interface. The overall neomorphic interaction is further buttressed by an endogenous cofactor of HDAC1-CoREST, inositol hexakisphosphate, which makes direct contacts with KBTBD4 and acts as a second molecular glue. The functional relevance of the quaternary complex interaction surfaces defined by cryo-EM is demonstrated by in situ base editor scanning of KBTBD4 and HDAC1. By delineating the direct target of UM171 and its mechanism of action, our results reveal how the cooperativity offered by a large dimeric CRL E3 family can be leveraged by a small molecule degrader and establish for the first time a dual molecular glue paradigm.

molecular biology↗

bioRxiv2024

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes

Cancer mutations can create neomorphic protein-protein interactions to drive aberrant function1. As a substrate receptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex, KBTBD4 is recurrently mutated in medulloblastoma (MB)2, the most common embryonal brain tumor in children, and pineoblastoma3. These mutations impart gain-of-function to KBTBD4 to induce aberrant degradation of the transcriptional corepressor CoREST4. However, their mechanism of action remains unresolved. Here, we elucidate the mechanistic basis by which KBTBD4 mutations promote CoREST degradation through engaging HDAC1/2, the direct neomorphic target of the substrate receptor. Using deep mutational scanning, we systematically map the mutational landscape of the KBTBD4 cancer hotspot, revealing distinct preferences by which insertions and substitutions can promote gain-of-function and the critical residues involved in the hotspot interaction. Cryo-electron microscopy (cryo-EM) analysis of two distinct KBTBD4 cancer mutants bound to LSD1-HDAC1-CoREST reveals that a KBTBD4 homodimer asymmetrically engages HDAC1 with two KELCH-repeat propeller domains. The interface between HDAC1 and one of the KBTBD4 propellers is stabilized by the MB mutations, which directly insert a bulky side chain into the active site pocket of HDAC1. Our structural and mutational analyses inform how this hotspot E3-neo-substrate interface can be chemically modulated. First, our results unveil a converging shape complementarity-based mechanism between gain-of-function E3 mutations and a molecular glue degrader, UM171. Second, we demonstrate that HDAC1/2 inhibitors can block the mutant KBTBD4-HDAC1 interface, the aberrant degradation of CoREST, and the growth of KBTBD4-mutant MB models. Altogether, our work reveals the structural and mechanistic basis of cancer mutation-driven neomorphic protein-protein interactions and pharmacological strategies to modulate their action for therapeutic applications.

molecular biology↗

bioRxiv2020

Systematically optimized BCMA/CS1 bispecific CAR-T cells robustly control heterogeneous multiple myeloma

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies but also demonstrated marked vulnerability to antigen escape and tumor relapse. Here, we report the rational design and systematic optimization of bispecific CAR-T cells with robust activity against multiple myeloma (MM), including heterogeneous MM that is resistant to conventional CAR-T cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CAR-T cells exhibit significantly higher CAR expression levels and greater antigen-stimulated proliferation compared to T cells that co-express individual BCMA and CS1 CARs. Compared to single-input BCMA- or CS1-targeting CAR-T cells, BCMA/CS1 bispecific CAR-T cells significantly prolong the survival of animals bearing heterogeneous MM tumors. Combination therapy with anti-PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, but CAR-T cell treatment alone was able to achieve durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CAR-T cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

bioengineering↗