Biology subjects

Green, M. R.

Publications and source records attributed to Green, M. R..

15 recordsLinked to original sources

bioRxiv2026

A novel KIR3DL2-directed CAR T-cell therapy for cutaneous and systemic T-cell lymphomas

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies and multiple myeloma, but its extension to T-cell lymphomas (TCLs) has been constrained by the paucity of antigens that can be safely targeted. While targeting pan-T antigens broadens the therapeutic reach across various TCLs, it carries a substantial risk of immunosuppression and infection. Here, we report killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 2 (KIR3DL2/CD158k) as a viable target for CAR T-cell therapy against a wide range of TCLs. Using single-cell RNA-seq data analysis and flow cytometric phenotyping, we observed that KIR3DL2 expression is restricted to a subset of mature NK cells and a minor fraction of CD8 T cells, with negligible expression in other healthy adult tissues. Among TCLs, KIR3DL2 expression was noted in various subtypes of cutaneous and systemic TCLs. Anti-KIR3DL2 CAR constructs (3DL2CARs) efficiently and selectively redirected T cells against antigen-positive cell lines but not isogenic KIR3DL2-negative counterparts, with antigen-specific signaling, cytotoxicity, proliferation, and cytokine production. Importantly, 3DL2CART cells produced sustained tumor regression and prolonged survival in two distinct cell line-derived xenograft models of cutaneous TCL and in one patient-derived xenograft model of hepatosplenic TCL. 3DL2CART cells exhibited minimal off-target activity against bystander immune cells and showed no appreciable fratricide. To mitigate the risk of tumor cell contamination in autologous CAR T-cell products, we established a two-tiered strategy that integrates immunomagnetic depletion of KIR3DL2-positive tumor cells with in vivo activation of a safety switch if inadvertently transduced malignant cells contribute to immune escape. Together, our results establish the preclinical efficacy and safety of 3DL2CART-cell therapy and support its clinical translation. This approach may offer a targeted alternative to pan-T-cell-directed CAR T-cell strategies for patients with relapsed/refractory cutaneous and systemic TCLs, addressing a critical unmet clinical need.

immunology↗

bioRxiv2026

Harnessing the PRDM1-PGC1A axis to enhance CAR T cell efficacy

Despite its promise in treating B cell malignancies, chimeric antigen receptor (CAR) T cell therapy remains limited by resistance mechanisms that impair CAR T cell stemness, persistence, and effector function. Here, we identified PRDM1, which encodes the transcriptional repressor, BLIMP-1, as a crucial regulator of CAR T cell terminal differentiation and exhaustion. PRDM1 was highly enriched in exhausted-like tumor-infiltrating CAR T cells from lymphoma patients and in pre-infusion products from clinical non-responders. These findings link increased PRDM1 expression to CAR T cell dysfunction and poor clinical outcomes. Genetic deletion of PRDM1 significantly promoted CAR T cell proliferative capacity, memory differentiation and antitumor efficacy in both hematologic and solid tumor models. Mechanistically, PRDM1 deletion increased the activity of PGC1a;, a transcriptional coactivator central to mitochondrial biogenesis and antioxidant capacity, thereby improving mitochondrial function, oxidative stress resistance, and metabolic reserve. Depletion of PGC1a; attenuated the improved efficacy of PRDM1-deficient CAR T cells, indicating an essential role for this pathway downstream of PRDM1 loss. Pharmacologic activation of the PPAR-PGC1a; program with a clinically approved agonist, bezafibrate, enhanced CAR T cell antitumor activity without direct tumoricidal effects. Notably, transient exposure to bezafibrate during CAR T cell manufacturing was sufficient to confer durable treatment benefits. Together, our study defines a PRDM1-PGC1a; axis that controls CAR T cell differentiation, mitochondrial fitness and therapeutic response, which provides a mechanistic and translational framework for advancing CAR T cell therapy across distinct tumor contexts.

immunology↗

bioRxiv2026

Proteome landscape of B-cell malignancies identifies mantle cell lymphoma protein signature

Mantle cell lymphoma (MCL) is one of the deadliest forms of Non-Hodgkins B-cell lymphoma. Typically, patients present with both overexpression of CyclinD1 and secondary mutations identified by genomic sequencing. Although MCL patients may initially respond to treatment, they eventually relapse and succumb to disease, highlighting the essential need to identify new targets for treatment. Here we performed proteomic profiling of healthy B cells and three different forms of B-cell malignancies, including MCL, to define the proteomic signature of MCL. We compared the proteome of each to MCL and identified 10 proteins that are specifically upregulated in MCL. Of these 10 proteins, seven of them show no transcriptional changes and have been overlooked by conventional RNA expression analysis. Further analysis of the proteomic signature reveals potential avenues for dual targeting in CAR T-cell therapy and provides guidance for personalized therapeutics based on protein expression. STATEMENT OF SIGNIFICANCEWe present a resource defining the protein landscape of MCL, CLL, and FL as compared to healthy b cells identified utilizing quantitative proteomics from primary patient samples. Applied to MCL, our results identify 10 proteins specifically upregulated in MCL that may prove to be therapeutic targets to treat the disease.

cancer biology↗

bioRxiv2026

A patient derived xenograft repository capturing clinical and molecular heterogeneity of large B-cell lymphoma

Large B-cell lymphomas (LBCLs) are a clinically and molecularly diverse group of malignancies with a rapidly evolving therapeutic landscape that has introduced new areas of clinical need such as post-CD19 chimeric antigen receptor T (CART19) progression. Patient derived xenograft (PDX) models are an important tool for mechanistic studies and preclinical evaluation of new therapies and can be generated from a variety of clinical contexts that capture tumor-intrinsic resistance mechanisms. We therefore undertook a comprehensive effort to generate PDX models that encompass the molecular landscape of LBCLs and include important clinical scenarios for new drug development. Here we describe the first 48 models within this publicly available repository, capturing the transcriptional and genetic subsets of LBCL. These models also include 23 generated from post-CART progression biopsies which reproduce patterns of progression driven by CD19 mutation or expression loss, as well as tumor cell-intrinsic CART19 resistance that we validate in vivo. STATEMENT OF SIGNIFICANCEYang et al. describe X-LYMPH (Xenografts of Lymphoma), a publicly available and molecularly annotated PDX repository that captures the heterogeneity of large B-cell lymphoma. X-LYMPH includes models of chimeric antigen receptor T cell resistance, providing a shared foundation for mechanistic research and therapeutic development for lymphomas.

cancer biology↗

bioRxiv2025

Signaling induced biophysical disruption of repressed chromatin domains drives immune cell fate

Cell fate transitions require signal-induced chromatin derepression, yet mechanisms governing transitions from repressed to active chromatin states are poorly understood. We discover, at fate-defining genes across immune cell types, a signal-induced histone code, and describe domains of H3 serine 28 phosphorylation (H3S28ph) spanning architectural features, often coincident with repressive H3 lysine 27 trimethylation (H3K27me3). Employing biophysical, single cell, and functional approaches to study signal-induced cell differentiation in the immune system, we uncover epigenomic transitions and cell fate choices precipitated by histone phosphorylation (H3ph). Mechanistically, H3ph overrides Polycomb Repressive Complex 2 (PRC2) chromatin repression, biophysically disrupts polynucleosome compaction, and promotes loss of H3K27me3, while increasing activating H3K27 acetylation and H3K36 dimethylation to drive domain interactivity and stabilize transcription. We demonstrate the activity of H3ph in several cell fate transitions and illuminate biophysical mechanisms enabling rapid signal-activated chromatin derepression, processes with general relevance for cellular differentiation and activation.

immunology↗

bioRxiv2025

A Bivalent Molecular Glue Linking Lysine Acetyltransferases to Oncogene-induced Cell Death

Developing cancer therapies that induce robust death of the malignant cell is critical to prevent relapse. Highly effective strategies, such as immunotherapy, exemplify this observation. Here we provide the structural and molecular underpinnings for an approach that leverages chemical induced proximity to produce specific cell killing of diffuse large B cell lymphoma, the most common non-Hodgkins lymphoma. We develop KAT-TCIPs (lysine acetyltransferase transcriptional/epigenetic chemical inducers of proximity) that redirect p300 and CBP to activate programmed cell death genes normally repressed by the oncogenic driver, BCL6. Acute treatment rapidly reprograms the epigenome to initiate apoptosis and repress c-MYC. The crystal structure of the chemically induced p300-BCL6 complex reveals how chance interactions between the two proteins can be systematically exploited to produce the exquisite potency and selectivity of KAT-TCIPs. Thus, the malignant function of an oncogenic driver can be co-opted to activate robust cell death, with implications for precision epigenetic therapies.

cancer biology↗

bioRxiv2023

Borrowing Transcriptional Kinases to Activate Apoptosis

Protein kinases are disease drivers whose therapeutic targeting traditionally centers on inhibition of enzymatic activity. Here chemically induced proximity is leveraged to convert kinase inhibitors into context-specific activators of therapeutic genes. Bivalent molecules that link ligands of the transcription factor B-cell lymphoma 6 (BCL6) to ATP-competitive inhibitors of cyclin-dependent kinases (CDKs) were developed to re-localize CDK to BCL6-bound loci on chromatin and direct phosphorylation of RNA Pol II. The resulting BCL6-target proapoptotic gene expression translated into killing of diffuse large B-cell lymphoma (DLBCL) cells at 72 h with EC50s of 0.9 - 10 nM and highly specific ablation of the BCL6-regulated germinal center response in mice. The molecules exhibited 10,000-fold lower cytotoxicity in normal lymphocytes and are well tolerated in mice. Genomic and proteomic evidence corroborated a gain-of-function mechanism where, instead of global enzyme inhibition, a fraction of total kinase activity is borrowed and re-localized to BCL6-bound loci. The strategy demonstrates how kinase inhibitors can be used to context-specifically activate transcription, accessing new therapeutic space.

cancer biology↗

bioRxiv2023

Inhibiting the GPI Transamidase Subunit GPAA1 Abolishes CD24 Surface Localization and Enhances Macrophage-Mediated Phagocytosis of Ovarian Cancer Cells

The CD24-Siglec10 signaling axis is an immune checkpoint pathway that shields ovarian cancer cells from phagocytosis by tumor-associated macrophages (TAMs), making it an appealing immunotherapeutic target. Here, we investigate factors influencing CD24 cell surface expression and assess their suitability as drug targets. Using a CRISPR-based knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment-1) as a positive regulator of CD24 cell surface expression. GPAA1 is a crucial component of the multi-subunit GPI transamidase complex, which facilitates the attachment of GPI lipid anchor to the C-terminus of CD24, enabling its surface localization. Reducing the activity of GPAA1 in ovarian cancer cells, either by genetic ablation or targeting with an aminopeptidase inhibitor bestatin, disrupts GPI attachment to CD24. This disruption impairs CD24 cell surface localization, enhances phagocytosis by TAMs, and suppresses tumor growth in mice. Our study highlights the potential of GPAA1 targeting as a therapeutic approach for CD24-positive ovarian cancers.

cancer biology↗

bioRxiv2023

CREBBP lysine acetyltransferase domain mutations create zombie enzymes that alter chromatin loading dynamics and prevent EP300 redundancy

Epigenetic regulation of gene expression is fundamental to cell state transitions. A prominent example of this is the epigenetically-regulated generation of transitory cells states within the germinal center (GC) reaction that are required for humoral immunity. The deregulation of these processes through somatic mutations can give rise to B-cell lymphoma, thus studying these mutations can provide insight into fundamental mechanisms underlying epigenetic regulation of gene expression as well as GC B-cell and lymphoma biology. Here we show that classes of mutations in the CREBBP lysine acetyltransferase gene result in different structure, catalytic activity and function. We discover that CREBBP and its paralog EP300 are dynamically and reciprocally loaded onto chromatin during a signal-responsive cell state transitions within the GC. Mutation of CREBBP in GC-derived lymphoma interrupt signal-responsive CREBBP loading onto chromatin, while also inhibiting EP300 redundancy. These observations lead to a model in which CREBBP or EP300 loss of function through acetyltransferase domain mutations, which occur frequently in both hematopoietic and solid tumors, create a zombie enzyme that retards the function of the active paralog by binding limiting concentrations of transcription factor substrate and preventing signal-responsive enhancer activation.

cancer biology↗

bioRxiv2023

CD8 T-cell dysfunction is linked with CAR T-cell failure and can be mitigated by a non-alpha IL-2 agonist, pegenzileukin

Chimeric antigen receptor (CAR) T-cell therapy has been a breakthrough for relapsed or refractory large B-cell lymphoma (rrLBCL). However, suboptimal CAR T-cell activity can lead to therapeutic failure and dismal outcome. Using single cell RNA-sequencing of rrLBCL tumors, we identify a prominent population of clonally expanded dysfunctional CAR+ CD8 T-cells indicative of ongoing tumor cell engagement, proliferation, and dysfunction at the time of progression from CAR T-cell therapy. Furthermore, we show that rrLBCL patient-derived CAR T-cells are more prone to dysfunction and loss of cytotoxicity compared to healthy donor-derived CAR T-cells. Using both antigen-driven and CAR-driven models of T-cell dysfunction, we show that pegenzileukin, a non-alpha IL2 agonist, can prevent T-cell dysfunction. In both in vitro and in vivo CAR T-cell models, pegenzileukin improved T-cell expansion and tumor control. This provides pre-clinical rational for use of pegenzileukin in combatting T-cell dysfunction, a central mechanism of CAR T-cell failure. HIGHLIGHTSO_LITumor-infiltrating CD8 CAR T-cells show clonal expansion and dysfunction at the time of progression. C_LIO_LIrrLBCL patient-derived CAR T-cells are more prone to dysfunction compared to healthy-donor-derived CAR T-cells. C_LIO_LIPegenzileukin, a non-alpha IL2 agonist, rescues antigen- and CAR-driven CD8 T-cell dysfunction and improves CAR T-cell responses in vivo. C_LI

immunology↗

bioRxiv2021

Clonal hematopoiesis is associated with increased toxicity in large B-cell lymphoma patients treated with chimeric antigen receptor T cell therapy

To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [≥]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [≥]3 ICANS [58.9% vs. 25%, p=0.02] and grade [≥]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028). Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.

genetics↗

bioRxiv2021

Immune microenvironment subtypes and association with tumor cell mutations and antigen expression in follicular lymphoma.

Follicular lymphoma (FL) is a B-cell lymphoma with a complex tumor microenvironment that is rich in non-malignant immune cells. We applied single-cell RNA-sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T-cells including a novel cytotoxic CD4 T-cell population. Their relative proportions of T-cells defined four major FL subtypes, characterized by differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced antigen presentation on FL cells. In turn, expression of MHC class II genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics. This provides a classification framework of the FL microenvironment, their association with FL genotypes and antigen presentation, and informs different potential immunotherapeutic strategies based upon tumor cell MHC class II expression. Statement of significanceWe have characterized the FL-infiltrating T-cells, identified cytotoxic CD4 T-cells as an important component, showed that the abundance of these T-cell populations is associated with tumor-cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T-cells that differed between FLs with normal versus low antigen presentation.

cancer biology↗

bioRxiv2020

Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naive and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities. SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

cancer biology↗

bioRxiv2020

dagLogo: an R/Bioconductor package for identifying and visualizing differential amino acid group usage in proteomics data

Sequence logos have been widely used as graphical representations of conserved nucleic acid and protein motifs. Due to the complexity of the amino acid (AA) alphabet, rich post-translational modification, and diverse subcellular localization of proteins, few versatile tools are available for effective identification and visualization of protein motifs. In addition, various reduced AA alphabets based on physicochemical, structural, or functional properties have been valuable in the study of protein alignment, folding, structure prediction, and evolution. However, there is lack of tools for applying reduced AA alphabets to the identification and visualization of statistically significant motifs. To fill this gap, we developed an R/Bioconductor package dagLogo, which has several advantages over existing tools. First, dagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.

bioinformatics↗

bioRxiv2019

E3 Ligase UBR5 HECT domain mutations in lymphoma control maturation of B cells via alternative splicing

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5 with the majority of mutations within the HECT domain of the protein which can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B cell maturation and activation we generated a conditional mutant disrupting UBR5s C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and up-regulation of proteins associated with mRNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B cell maturation by regulating alternative splicing of key transcripts during B cell development and suggests UBR5 mutations may promote mantle cell lymphoma initiation.\n\nKEY POINTSO_LIUtilizing a novel mouse model mimicking MCL patient mutations, the loss of UBR5s HECT domain causes alterations in B cell development.\nC_LIO_LIUBR5 mutations lead to stabilization of UBR5 and aberrant splicing.\nC_LI

immunology↗