Biology subjects

Brown, B. D.

Publications and source records attributed to Brown, B. D..

11 recordsLinked to original sources

bioRxiv2025

Potentiating CAR-T bystander killing by enhanced Fas/FasL signaling mitigates antigen escape in heterogeneous tumors

Antigen (Ag) escape is a frequent mechanism of relapse after CAR-T therapy, even though only [~]1% of leukemic and [~]0.1% of lymphoma cells are Ag- at baseline. In this study, we modeled extreme Ag heterogeneity (>20%) to define how Fas/FasL-dependent bystander killing contributes to tumor clearance. Across patient cohorts, Fas expression predicted survival after CD19 CAR-T therapy, particularly in CD19-low disease. In both murine and human systems, Fas-dependent bystander killing required Ag stimulation and cell contact, operated within a defined therapeutic window, and could eradicate large fractions of Ag- tumors in vivo. Pharmacologic potentiation with inhibitor of apoptosis protein antagonists or genetic stabilization of CAR-T membrane-bound FasL enhanced bystander killing but simultaneously induced CD4 T cell fratricide, which was rescued by CAR-T Fas knockout. Importantly, Fas sensitization also enabled bispecific antibody-redirected T cells to mediate bystander killing in resistant tumors. Finally, targeting tumor-associated macrophages triggered Fas-dependent clearance of neighboring tumor cells. These findings establish Fas-mediated bystander killing as a generalizable and therapeutically actionable axis to prevent Ag escape and broaden the scope of targeted T cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/677496v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@11bedorg.highwire.dtl.DTLVardef@d09d07org.highwire.dtl.DTLVardef@1f53c1dorg.highwire.dtl.DTLVardef@13d96f9_HPS_FORMAT_FIGEXP M_FIG C_FIG Fas-mediated bystander killing can be potentiated by IAP inhibition and FasL stabilization, and further improved by decoupling CD4 T cell fratricide via Fas knockout. Schematic representation of CAR-T bystander killing across three mechanistic states. Left (Baseline): Antigen-positive (CD19) tumor cells activate CAR-T cells, leading to FasL-mediated cytotoxicity of neighboring antigen-negative (CD19-) tumor cells via membrane-bound FasL. Limited FasL expression and ADAM10-mediated shedding constrain bystander killing, and CD4 T cells are largely spared. Middle (Potentiated): Enhancing Fas signaling through IAP inhibition (IAPi), increasing FasL expression, and reducing FasL shedding result in improved bystander killing but also increased CD4 T cell fratricide. Right (Potentiated & Decoupled): Combining IAPi with FasL stabilization and CAR-T Fas knockout (FasKO) augments bystander killing while eliminating CD4 T cell fratricide.

immunology↗

bioRxiv2025

Plasminogen activator inhibitors orchestrate the immunosuppressive tumor microenvironment in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix (ECM) that sustains an immunosuppressive tumor microenvironment (TME). While this protective niche has been described, the molecular determinants orchestrating its formation and dictating its immune interactions are not well defined. Using Perturb-map, we determine how dozens of different gene perturbations shape the growth and cellular environments of PDAC clones through space and time. Our study reveals dynamic, gene-specific adaptations of immune neighborhoods during clonal selection. We identified Serpinb2 (PAI2) and Serpine1 (PAI1) as key cancer-derived mediators of TME remodeling and immune evasion. These factors promote the deposition of a fibrin-rich ECM that shapes immune cell composition, locally retains and polarizes immunosuppressive macrophages and excludes cytotoxic T cells. Deletion of either Serpinb2 or Serpine1 greatly enhanced tumor response to anti-PD1 immunotherapy in an aggressive PDAC model. Transcriptomic analysis further linked their expression to distinct PDAC subtypes and poor patient survival. Our findings demonstrate that Serpinb2 and Serpine1 establish a permissive niche for tumor progression and show how PDAC cells exploit components of the fibrinolysis pathway to remodel the ECM, alter macrophage composition, and protect themselves from immune editing, ultimately reinforcing the role of extracellular factors in shaping an immune-privileged tumor niche.

cancer biology↗

bioRxiv2025

Targetable BIRC5 dependency in therapy-resistant TP53 mutated acute myeloid leukemia

TP53 mutations across multiple cancers, including acute myeloid leukemia (AML), are associated with poor outcomes irrespective of treatment modality. However, druggable vulnerabilities beyond canonical p53 targets remain largely unexplored. We identify BIRC5 (encodes survivin), an inhibitor of the apoptosis protein (IAP) family, as a novel vulnerability in TP53 mutant AML using an unbiased, comprehensive multiomics approach -- whole-genome CRISPR knockout screen, bulk and single-cell RNA-seq, proteomics, and high-throughput drug screen. Mechanistically, BIRC5 deletion in AML restored caspase-9 and -3/7 activity and downregulated other IAPs, implicating BIRC5 as the central post-mitochondrial regulator for blocking apoptosis. p53 stabilization suppressed BIRC5 selectively in TP53 wild-type AML, explaining BIRC5 upregulation in TP53 mutant lines and AML primary tumors (n > 700). Longitudinal single-cell RNAseq (n = 22 pairs) revealed expansion of BIRC5high stem and progenitor leukemia clones in TP53 mutant AML patients post-VenAza therapy. Survivin and IAP inhibitors emerged as top combination partners with VenAza in TP53 mutant AML cells and showed potent in vivo leukemic blast inhibition in cell line and patient-derived xenograft models along with primary tumors. Beyond AML, BIRC5 was upregulated broadly across 17 of 25 TP53 mutant cancers in the TCGA cohort, and combination with survivin inhibitors overcame chemotherapy resistance in TP53 deficient triple negative breast and colorectal cancers. These findings define BIRC5 as a critical, targetable dependency and unveil survivin/IAP inhibition as a promising therapeutic axis to overcome p53-related resistance across both hematologic and solid malignancies. Key PointsO_LIBIRC5 upregulation is a novel dependency in TP53 mutant AML that mediates therapy resistance by evasion of apoptosis. C_LIO_LICombination with Survivin/IAP inhibitors overcomes venetoclax/azacitidine resistance in TP53 mutant AML. C_LI

cancer biology↗

bioRxiv2025

Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)

We report an enantioselective protein affinity selection mass spectrometry screening approach (E-ASMS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. 16 binders to 12 targets, including many proteins predicted to be "challenging to ligand", were discovered and confirmed in orthogonal biophysical assays. 7 binders to 6 targets bound in an enantioselective manner, with KD values ranging from 3 to 20 {micro}M. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselectivity was readily explained. We conclude E-ASMS can be used to identify and characterize selective and weakly-binding ligands for novel protein targets with unprecedented throughput and sensitivity.

pharmacology and toxicology↗

bioRxiv2024

ICOS limits memory-like properties and function of exhausted PD-1+ CD8 T cells

During persistent antigen stimulation, PD-1+CD8 T cells are maintained by progenitor exhausted PD-1+TCF-1+CD8 T cells (Tpex). Tpex respond to PD-1 blockade, and regulation of Tpex differentiation into more functional Tex is of major interest for cancer immunotherapies. Tpex express high levels of Inducible Costimulator (ICOS), but the role of ICOS for PD-1+CD8 T cell responses has not been addressed. In chronic infection, ICOS-deficiency increased both number and quality of virus-specific CD8 T cells, with accumulation of effector-like Tex due to enhanced survival. Mechanistically, loss of ICOS signaling potentiated FoxO1 activity and memory-like features of Tpex. In mice with established chronic infection, ICOS-Ligand blockade resulted in expansion of effector-like Tex and reduction in viral load. In a mouse model of hepatocellular carcinoma, ICOS inhibition improved cytokine production by tumor-specific PD-1+CD8 T cells and delayed tumor growth. Overall, we show that ICOS limits CD8 T cell responses during chronic antigen exposure.

immunology↗

bioRxiv2024

A STAT3 Degrader Demonstrates Pre-clinical Efficacy in Venetoclax resistant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that continues to have poor prognosis despite recent therapeutic advances. Venetoclax (Ven), a BCL2-inhibitor has shown a high response rate in AML; however, relapse is invariable due to mitochondrial dysregulation that includes upregulation of the antiapoptotic protein MCL1, a central mechanism of Ven resistance (Ven-res). We have previously demonstrated that the transcription factor STAT3 is upregulated in AML hematopoietic stem and progenitor cells (HSPCs) and can be effectively targeted to induce apoptosis of these aberrant cells. We now show that overexpression of STAT3 alone is sufficient to initiate a strong AML phenotype in a transgenic murine model. Phospho-proteomic data from Ven treated AML patients show a strong correlation of high total STAT3 and phospho-STAT3 [both p-STAT3(Y705) and p-STAT3(S727)] expression with worse survival and reduced remission duration. Additionally, significant upregulation of STAT3 was observed in Ven-res cell lines, in vivo models and primary patient samples. A novel and specific degrader of STAT3 demonstrated targeted reduction of total STAT3 and resulting inhibition of its active p-STAT3(Y705) and p-STAT3(S727) forms. Treatment with the STAT3 degrader induced apoptosis in parental and Ven-res AML cell lines and decreased mitochondrial depolarisation, and thereby dependency on MCL1 in Ven-res AML cell line, as observed by BH3 profiling assay. STAT3 degrader treatment also enhanced differentiation of myeloid and erythroid colonies in Ven-res peripheral blood mononuclear cells (PBMNCs). Upregulation of p-STAT3(S727) was also associated with pronounced mitochondrial structural and functional dysfunction in Ven-res cell lines, that were restored by STAT3 degradation. Treatment with a clinical-stage STAT3 degrader, KT-333 resulted in a significant reduction in STAT3 and MCL1 protein levels within two weeks of treatment in a cell derived xenograft model of Ven-res AML. Additionally, this treatment significant improvement in the survival of a Ven-res patient-derived xenograft in-vivo study. Degradation of STAT3 resulting in downregulation of MCL1 and improvements in global mitochondrial dysfunction suggests a novel mechanism of overcoming Ven-res in AML. Statement of PurposeFive-year survival from AML is dismal at 30%. Our prior research demonstrated STAT3 over-expression in AML HSPCs to be associated with inferior survival. We now explore STAT3 over-expression in Ven-res AML, explain STAT3 mediated mitochondrial perturbations and describe a novel therapeutic strategy, STAT3 degradation to overcome Ven-res.

cancer biology↗

bioRxiv2024

Myeloid progenitor dysregulation fuels immunosuppressive macrophages in tumors

ABSTRACTMonocyte-derived macrophages (mo-macs) drive immunosuppression in the tumor microenvironment (TME) and tumor-enhanced myelopoiesis in the bone marrow (BM) fuels these populations. Here, we performed paired transcriptome and chromatin analysis over the continuum of BM myeloid progenitors, circulating monocytes, and tumor-infiltrating mo-macs in mice and in patients with lung cancer to identify myeloid progenitor programs that fuel pro-tumorigenic mo-macs. Analyzing chromatin accessibility and histone mark changes, we show that lung tumors prime accessibility for Nfe2l2 (NRF2) in BM myeloid progenitors as a cytoprotective response to oxidative stress. NRF2 activity is sustained and increased during monocyte differentiation into mo-macs in the lung TME to regulate oxidative stress, in turn promoting metabolic adaptation, resistance to cell death, and contributing to immunosuppressive phenotype. NRF2 genetic deletion and pharmacological inhibition significantly reduced mo-macs survival and immunosuppression in the TME, enabling NK and T cell therapeutic antitumor immunity and synergizing with checkpoint blockade strategies. Altogether, our study identifies a targetable epigenetic node of myeloid progenitor dysregulation that sustains immunoregulatory mo-macs in the TME.

immunology↗

bioRxiv2023

Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling

Organ homeostasis is maintained by regulated proliferation of distinct cell populations. In mouse liver, cyclin D1-positive hepatocytes in the midlobular zone repopulate the parenchyma at a constant rate to preserve liver homeostasis. The mitogenic cues that underlie this process are unknown. Hepatic stellate cells, the livers pericytes, are in close proximity to hepatocytes and have been implicated in supporting hepatocyte proliferation, but their role in liver homeostasis is unknown. Here, we employ a T cell-mediated hepatic stellate cell ablation model to remove nearly all hepatic stellate cells in the murine liver, enabling the unbiased characterization of hepatic stellate cell functions. In the normal murine liver, complete loss of hepatic stellate cells persists for up to 6 weeks and reduces liver mass. Our results show that hepatic stellate cells induce cyclin D1 in midlobular hepatocytes by release of neurotrophin-3 to promote hepatocyte proliferation via tropomyosin receptor kinase B signaling. These findings establish that hepatic stellate cells form the niche for midlobular hepatocytes and reveal a novel hepatocyte growth factor signaling pathway. One-Sentence SummaryHepatic stellate cells provide mitogenic cues for midlobular hepatocyte proliferation and metabolic zonation by secreting neurotrophin-3.

cell biology↗

bioRxiv2021

Targeting macrophages with CAR-T cells delays solid tumor progression and enhances anti-tumor immunity

Tumor-associated macrophages (TAMs) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably to PD1 blockade, and significantly extended mouse survival. Anti-tumor effects were mediated by F4.CAR-T-produced IFN-{gamma}, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigens and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof-of-principle evidence to support CAR-T targeting of TAMs as a means to enhance antitumor immunity.

immunology↗

bioRxiv2021

Perturb-map enables CRISPR genomics with spatial resolution and identifies regulators of tumor immune composition

The cellular architecture of a tumor, particularly immune composition, has a major impact on cancer outcome, and thus there is an interest in identifying genes that control the tumor microenvironment (TME). While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying gene functions operating extracellularly or within a tissue context. To address this, we developed an approach for spatial functional genomics called Perturb-map, which utilizes protein barcodes (Pro-Code) to enable spatial detection of barcoded cells within tissue. We show >120 Pro-Codes can be imaged within a tumor, facilitating spatial mapping of 100s of cancer clones. We applied Perturb-map to knockout dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Additionally, we paired Perturb-map and spatial transcriptomics for unbiased molecular analysis of Pro-Code/CRISPR lesions. Our studies found in Tgfbr2 knockout lesions, the TME was converted to a mucinous state and T-cells excluded, which was concomitant with increased TGF{beta} expression and pathway activation, suggesting Tgfbr2 loss on lung cancer cells enhanced suppressive effects of TGF{beta} on the TME. These studies establish Perturb-map for functional genomics within a tissue at single cell-resolution with spatial architecture preserved.

immunology↗

bioRxiv2021

P53 is a direct regulator of the immune co-stimulatory molecule CD80

Increasing evidence indicates oncogenes and tumor suppressors not only influence cell fitness but can also control the immunophenotype of cells. Here, we examined how 34 commonly mutated genes in colorectal cancer (CRC) may influence the expression of 8 key immunomodulatory proteins. To do this, we employed a functional genomics approach utilizing Pro-Code/CRISPR libraries for high-dimensional analysis. We introduced a library of 102 Pro-Code/gRNA combinations, targeting each of the 34 genes, in CT26 cells, a CRC cell model, and measured the expression of each of the immunomodulatory proteins by CyTOF mass cytometry. Notably, cells carrying a Pro-Code/CRISPR targeting the Trp53 lost expression of the immune co-stimulatory molecule CD80. Validation confirmed that Trp53 knockout resulted in the loss of CD80 and that activation of P53, through DNA damage or stabilization, resulted in CD80 upregulation. P53 ChIP-seq identified the CD80 promoter as a direct target of P53. CD80 regulation by P53 was identified in other cells, including normal epithelial cells and macrophages. Functionally, CD80 reduction caused by P53 loss led to a reduced capacity for CRC to prime antigen-specific T cells. These studies establish CD80, a canonical co-stimulatory molecule, as a direct target of the tumor suppressor and DNA damage response gene, P53.

immunology↗