Biology subjects

Rich, J. N.

Publications and source records attributed to Rich, J. N..

8 recordsLinked to original sources

bioRxiv2025

A gel-top model for characterizing mesenchymal features of glioblastoma cells

Glioblastoma (GBM) remains one of the most aggressive adult cancers, with limited treatment options due to an incomplete understanding of its biology. GBM exhibits different transcriptional subtypes, among which the mesenchymal (mGBM) variant is associated with a particularly aggressive nature and poor outcomes. Developing models that capture mGBM phenotypes could offer new insights into its biology and therapeutic vulnerabilities. However, the lack of in vitro models for mGBM has posed challenges in investigating the aggressive GBM subtype. Here, we present a gel-top culture model in which GBM cells exhibit MES features, including distinct transcriptomic profiles, invasive phenotypes, cell morphology, and cytoskeletal organization. Furthermore, co-culturing GBM cells with microglia in this system revealed enhanced microglial recruitment and interaction, effectively recapitulating the tumor microenvironment of mGBM that traditional 2D culture fails to model. Using the bioinformatic tool, Small Molecule Suite, we identified casein kinase 2 (CK2) as a targetable molecule in GBM cells within our system, potentially driving MES features. Notably, CK2 inhibitors reduced the mesenchymal signature of mGBM cells, suggesting their potential as a targeted therapy for mGBM. Together, our findings suggest that the gel-top culture model replicates key features of mGBM, serving as a valuable platform for studying its biology and identifying novel therapeutic strategies.

bioengineering↗

bioRxiv2024

Advancing Clinical Response Against Glioblastoma: Evaluating SHP1705 CRY2 Activator Efficacy in Preclinical Models and Safety in Phase I Trials

BackgroundIt has been reported that circadian clock components, Brain and Muscle ARNT-Like 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK), are uniquely essential for glioblastoma (GBM) stem cell (GSC) biology and survival. Consequently, we developed a novel Cryptochrome (CRY) activator SHP1705, which inhibits BMAL1-CLOCK transcriptional activity. MethodsWe analyzed buffy coats isolated from Phase 1 clinical trial subjects blood to assess any changes to circadian, housekeeping, and blood transcriptome-based biomarkers following SHP1705 treatment. We utilized GlioVis to determine which circadian genes are differentially expressed in non-tumor versus GBM tissues. We employed in vitro and in vivo methods to test the efficacy of SHP1705 against patient-derived GSCs and xenografts in comparison to earlier CRY activator scaffolds. Additionally, we applied a novel-REV-ERB agonist SR29065, which inhibits BMAL1 transcription, to determine whether targeting both negative limbs of the circadian transcription-translation feedback loop (TTFL) would yield synergistic effects against various GBM cells. ResultsSHP1705 is safe and well-tolerated in Phase I clinical trials. SHP1705 has increased selectivity for the CRY2 isoform and potency against GSC viability compared to previously published CRY activators. SHP1705 prolonged survival in mice bearing GBM tumors established with GSCs. When combined with the novel REV-ERB agonist SR29065, SHP1705 displayed synergy against multiple GSC lines and differentiated GSCs (DGCs). ConclusionsThese demonstrate the efficacy of SHP1705 against GSCs, which pose for GBM patient outcomes. They highlight the potential of novel circadian clock compounds in targeting GBM as single agents or in combination with each other or current standard-of-care. KEY POINTSO_LISHP1705 is a novel CRY2 activator that has shown success in Phase 1 safety trials C_LIO_LISHP1705 has a significantly improved efficacy against GSCs and GBM PDX tumors C_LIO_LINovel REV-ERB agonist SR29065 and SHP1705 display synergistic effects against GSCs C_LI IMPORTANCE OF THE STUDYCRY2 is decreased in GBM tissues compared to CRY1 suggesting that promoting CRY2 activity will be an efficacious GBM treatment paradigm. SHP1705, a CRY2 activator that has shown success in Phase 1 safety trials, has significantly improved preclinical efficacy. Novel REV-ERB agonist SR29065 displays synergistic effects against diverse GBM cells.

cancer biology↗

bioRxiv2023

Human plasma-like medium facilitates metabolic tracing and enables upregulation of immune signaling pathways in glioblastoma explants

BackgroundIn vivo stable isotope tracing is useful for natively surveying glioma metabolism but can be difficult to implement. Stable isotope tracing is tractable using in vitro glioma models, but most models lack nutrient conditions and cell populations relevant to human gliomas. This limits our ability to study glioma metabolism in the presence of an intact tumor microenvironment (TME) and immune-metabolic crosstalk. MethodsWe optimized an in vitro stable isotope tracing approach for human glioma explants and glioma stem-like cell (GSC) lines that integrates human plasma-like medium (HPLM). We performed 15N2-glutamine tracing in GSC monocultures and human IDH-wildtype glioblastoma explants and developed an analytical framework to evaluate microenvironment-dependent metabolic features that distinguish them. We also conducted spatial transcriptomics to assess transcriptional correlates to metabolic activities. ResultsHPLM culture preserved glioma explant viability and stemness while unmasking metabolic and immune programs suppressed by conventional culture conditions. Stable isotope tracing in HPLM revealed TME-dependent and TME-independent features of tumor metabolism. Tissue explants recapitulated tumor cell-intrinsic metabolic activities, such as synthesis of immunomodulatory purines. Unlike GSC monocultures, tissue explants captured tumor cell- extrinsic activities associated with stromal cell metabolism, as exemplified by astrocytic GDP- mannose production in heterocellular explants. Finally, glioma explants displayed tumor subtype-specific metabolic reprogramming, including robust pyrimidine degradation in mesenchymal cells. ConclusionsWe present a tractable approach to assess glioma metabolism in vitro under physiological nutrient levels and in the presence of an intact TME. This platform opens new avenues to interrogate glioma metabolism and its interplay with the immune microenvironment. Importance of the StudyMetabolic reprogramming is a hallmark of tumor biology, but in vitro studies of glioma metabolism often fail to replicate the nutrient complexity and cellular heterogeneity of the TME. We developed a method to perform stable isotope tracing in glioma explants grown in HPLM to analyze metabolism in a nutrient context that reflects in vivo conditions. By comparing metabolic activities between glioma cell monocultures and explanted tumor tissues, our approach captures features of tumor metabolism that are driven by the microenvironment. We show that HPLM not only sustains cell fitness and identity in tumor explants but also evokes distinct metabolic patterns and immune activation signatures repressed by standard culture conditions. Our approach offers a tractable and scalable way to study tumor cell intrinsic and microenvironmental metabolism in faithful tissue culture glioma models, complementing powerful yet low-throughput in vivo stable isotope tracing approaches. Key PointsO_LIHPLM supports culture of glioma explants and stimulates metabolic and immune transcriptional responses. C_LIO_LIStable isotope tracing in glioma explants reveals contributions of tumor cells, stromal cells, and gene expression programs to tumor metabolism. C_LI

cancer biology↗

bioRxiv2021

The WRAD complex represents a therapeutically exploitable target for cancer stem cells in glioblastoma

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/461125v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@bc5c5eorg.highwire.dtl.DTLVardef@545a2borg.highwire.dtl.DTLVardef@1ebd3c9org.highwire.dtl.DTLVardef@19d5d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Leveraging Allele-Specific Expression for Therapeutic Response Gene Discovery in Glioblastoma.

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. Here, we interrogated allele-specific expression (ASE) in 43 patient-derived glioblastoma stem cells (GSCs) to identify recurrently dysregulated genes in glioblastoma. We identified 118 genes with recurrent ASE preferentially found in GSCs compared to normal tissues. These genes were enriched for apoptotic regulators, including Schlafen Family Member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and poly ADP ribose polymerase inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika, which suggests a potential alternative treatment strategy for chemotherapy resistant GBMs.

cancer biology↗

bioRxiv2021

HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

Glioblastoma (GBM) is the most lethal brain tumor containing glioma stem cells (GSCs) that promote malignant growth and therapeutic resistance. The self-renewal and tumorigenic potential of GSCs are maintained by core stem cell transcription factors including SOX2. Defining the posttranslational regulation of SOX2 may offer new insights into GSC biology and potential therapeutic opportunity. Here, we discover that HAUSP stabilizes SOX2 through deubiquitination to maintain GSC self-renewal and tumorigenic potential. HAUSP is preferentially expressed in GSCs in perivascular niches in GBMs. Disrupting HAUSP by shRNA or its inhibitor P22077 promoted SOX2 degradation, induced GSC differentiation, impaired GSC tumorigenic potential, and suppressed GBM tumor growth. Importantly, pharmacological inhibition of HAUSP synergized with radiation to inhibit GBM growth and extended animal survival, indicating that targeting HAUSP may overcome GSC-mediated radioresistance. Our findings reveal an unappreciated crucial role of HAUSP in the GSC maintenance and provide a promising target for developing effective anti-GSC therapeutics to improve GBM treatment. HighlightsO_LIHAUSP deubiquitinates and stabilizes SOX2 in glioma stem cells (GSCs). C_LIO_LIHAUSP is preferentially expressed by GSCs in perivascular niches in GBMs. C_LIO_LIHAUSP is required for maintaining GSC self-renewal and tumorigenic potential. C_LIO_LITargeting HAUSP inhibited malignant growth in GSC-derived GBM xenografts. C_LIO_LIInhibition of HAUSP synergized with radiation to suppress GBM tumor growth. C_LI

cancer biology↗

bioRxiv2021

Targeting Glioblastoma Signaling and Metabolism with A Re-Purposed Brain-Penetrant Drug

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify SMPD1, an enzyme that regulates the conversion of sphingomyelin to ceramide and a critical regulator of plasma membrane structure and organization, as an actionable drug target in glioblastoma. We show that the safe and highly brain-penetrant antidepressant fluoxetine, potently inhibits SMPD1 activity, killing GBMs, in vitro and in patient-derived xenografts, through inhibition of EGFR signaling and via activation of lysosomal stress. Combining fluoxetine with the chemotherapeutic agent temozolomide, a standard of care for GBM patients, causes massive increases in GBM cell death, and complete and long-lived tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases, reveals significantly increased survival in glioblastoma patients treated with fluoxetine, which was not seen in patients treated with other SSRI anti-depressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for GBM patients and suggest prospective randomized clinical trials.

cancer biology↗

bioRxiv2020

PI3Kγ inhibition suppresses microglia/TAM accumulation in glioblastoma microenvironment to promote exceptional temozolomide response

Precision medicine in oncology leverages clinical observations of exceptional response. Towards an understanding of the molecular features that define this response, we applied an integrated, multi-platform analysis of RNA profiles derived from clinically annotated glioblastoma samples. This analysis suggested that specimens from exceptional responders are characterized by decreased accumulation of microglia/macrophages in the glioblastoma microenvironment. Glioblastoma-associated microglia/macrophages secreted interleukin 11 (IL11) to activate STAT3-MYC signaling in glioblastoma cells. This signaling induced stem cell states that confer enhanced tumorigenicity and resistance to the standard-of-care chemotherapy, temozolomide (TMZ). Targeting a myeloid cell restricted isoform of PI3K, PI3K{gamma}, by pharmacologic inhibition or genetic inactivation, disrupted this signaling axis by suppressing microglia/macrophage accumulation and associated IL11 secretion in the tumor microenvironment. Mirroring the clinical outcomes of exceptional responders, PI3K{gamma} inhibition synergistically enhanced the anti-neoplastic effects of TMZ in orthotopic murine glioblastoma models. Moreover, inhibition or genetic inactivation of PI3K{gamma} in murine glioblastoma models recapitulated expression profiles observed in clinical specimens isolated from exceptional responders. Our results suggest key contributions from tumor-associated microglia/macrophages in exceptional responses and highlight the translational potential for PI3K{gamma} inhibition as a glioblastoma therapy. Significance StatementUnderstanding the basis for exceptional responders represents a key pillar in the framework of precision medicine. In this study, we utilized distinct informatics platforms to analyze the expression profiles of clinically annotated tumor specimens derived from patients afflicted with glioblastoma, the most common form of primary brain cancer. These analyses converged on prognostic contributions from glioblastoma-associated microglia/macrophages. Glioblastoma-associated microglia secreted interleukin 11 (IL11) to activate a STAT3-MYC signaling axis in glioblastoma cells, facilitating resistance to the standard-of-care chemotherapy, temozolomide. Microglia recruitment and IL11 secretion were dependent on the myeloid specific phosphoinositide-3-kinase gamma isoform (PI3K{gamma}). Inhibition or genetic inactivation of PI3K{gamma} in murine glioblastoma models recapitulated expression profiles observed in specimens derived from exceptional responders, suggesting potential for clinical translation.

cancer biology↗