Biology subjects

Pitson, S. M.

Publications and source records attributed to Pitson, S. M..

7 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

Novel RNA Polymerase I and Cyclin Dependent Kinase combination therapy for the treatment of aggressive Acute Myeloid Leukemia.

Despite advances in therapy, specific subtypes Acute Myeloid Leukaemia (AML) remains largely incurable. The first-in-class RNA Polymerase I (Pol I) inhibitor, CX-5461, has demonstrated promising activity in both haematological malignancies and solid tumours by selectively inhibiting ribosome biogenesis to induce nucleolar stress, a critical vulnerability in rapidly proliferating cancer cells. CX-5461 has undergone clinical trials for the treatment of both solid and haematological malignancies, and 2nd generation Pol I inhibitor PMR-116 has also entered clinical trials, underscoring the translational potential of drugs that target RNA Polymerase I. To enhance the therapeutic efficacy of Pol I inhibition and prevent the emergence of resistance, we conducted a cell line-based unbiased screen of FDA-approved drugs to identify compounds that might synergise with CX-5461. This screen revealed that the pan-CDK inhibitors Dinaciclib and Flavopiridol enhance nucleolar stress pathway (NSP) activation and are strong candidates for combinatorial therapy with CX-5461. Further analysis showed that the combination of CX-5461 and Dinaciclib acts synergistically across a genetically diverse panel of human AML cell lines. This synergy is dependent on an intact NSP, with both agents independently stabilising p53, but with distinct phenotypic outcomes: Dinaciclib induces rapid apoptosis, whereas CX-5461 primarily enforces cell cycle arrest. This functional complementarity results in efficient tumour cell clearance and likely accounts for the delayed onset of therapy resistance. Importantly, combination treatment with CX-5461 and Dinaciclib significantly improved survival in murine models of AML and reduced colony formation in primary human AML samples. Together these findings provide preclinical evidence for a novel combination treatment strategy that leverages nucleolar stress and cell cycle control to enhance treatment outcomes in AML, paving the way for clinical translation of Pol I-CDK co-targeting therapies.

cancer biology↗

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↗

bioRxiv2023

Cell Type-Specific Regulation by a Heptad of Transcription Factors in Human Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay of transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs - FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2 - bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remained unknown. We mapped genome-wide chromatin contacts and TF binding profiles in HSPC subsets (HSC, CMP, GMP, MEP) and found that heptad occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. These findings suggest that specific heptad-TF combinations play critical roles in regulating hematopoietic differentiation and provide a valuable resource for development of targeted therapies to manipulate specific HSPC subsets.

developmental biology↗

bioRxiv2023

Mitochondrial translocation of TFEB regulates complex I and inflammation

TFEB is a master regulator of autophagy, lysosome biogenesis and mitochondrial metabolism that works, and immunity, primarily through transcription controlled by cytosol-to-nuclear translocation. Emerging data indicate additional regulatory interactions at the surface of organelles such as lysosomes. Here we show that TFEB has a non-transcriptional role in mitochondria, regulating the electron transport chain complex I to down-modulate inflammation. Proteomic analysis revealed extensive TFEB co-precipitation with several mitochondrial proteins, whose interactions are disrupted upon infection with S. Typhimurium. Localization of TFEB in the mitochondrial matrix was confirmed by high resolution confocal microscopy and biochemistry with translocation dependent on a conserved N-terminal TOMM20-binding motif enhanced by mTOR inhibition. Within the mitochondria, TFEB and protease LONP1 antagonistically co-regulate complex I, reactive oxygen species and the inflammatory response. Consequently, during infection, lack of TFEB specifically in the mitochondria exacerbates the expression of pro-inflammatory cytokines, contributing to innate immune pathogenesis.

cell biology↗

bioRxiv2022

GD2-targeting CAR-T cells enhanced by transgenic IL-15 expression are an effective and clinically feasible therapy for glioblastoma

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited. MethodsWe profiled an extensive biobank of patients biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity. ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors. What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors. What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing. How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

immunology↗

bioRxiv2021

Slit-Robo Signalling Establishes a Sphingosine-1-Phosphate Gradient to Polarise Fin Mesenchyme and Establish Fin Morphology

Immigration of mesenchymal cells into the growing fin and limb buds drives distal outgrowth, with subsequent tensile forces between these cells essential for fin and limb morphogenesis. Morphogens derived from the apical domain of the fin, orientate limb mesenchyme cell polarity, migration, division and adhesion. The zebrafish mutant stomp displays defects in fin morphogenesis including blister formation and associated loss of orientation and adhesion of immigrating fin mesenchyme cells. Positional cloning of stomp identified a mutation in the gene encoding the axon guidance ligand, Slit3. We provide evidence that Slit ligands derived from immigrating mesenchyme act via Robo receptors at the Apical Ectodermal Ridge (AER) to promote release of sphingosine-1-phosphate (S1P). S1P subsequently diffuses back to the mesenchyme to promote their polarisation, orientation, positioning and adhesion to the interstitial matrix of the fin fold. We thus demonstrate coordination of the Slit-Robo and S1P signalling pathways in fin fold morphogenesis. Our work introduces a mechanism regulating the orientation, positioning and adhesion of its constituent cells.

cell biology↗