Search

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, 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 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, 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. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown 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.

SummaryGlioblastoma (GBM) treatment includes maximal safe resection of the core and MRI contrast-enhancing (CE) tumor. Complete resection of the infiltrative non-contrast-enhancing (NCE) tumor rim is rarely achieved. We established a safe, semi-automated workflow for spatially-registered sampling of MRI-defined GBM regions in 19 patients with downstream analysis and biobanking, enabling studies of NCE, wherefrom recurrence/progression typically occurs. Immunophenotyping revealed underrepresentation of myeloid cell subsets and CD8+ T cells in the NCE. While NCE T cells phenotypically and functionally resembled those in matching CE tumor, subsets of activated (CD69hi) effector memory CD8+ T cells were overrepresented. Contrarily, CD25hiTregs and other subsets were underrepresented. Overall, our study demonstrated that MRI-guided, spatially-registered, intraoperative immunosampling is feasible as part of routine GBM surgery. Further elucidation of the shared and spatially distinct microenvironmental biology of GBM will enable development of therapeutic approaches targeting the NCE infiltrative tumor to decrease GBM recurrence.

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single- cell RNA-sequencing (scRNA-seq) dataset of ∼7 million cells profiled, and a spatially resolved transcriptomic dataset of ∼4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

Variation in cortical cytoarchitecture is the basis for histology-based definition of cortical areas, such as Brodmann areas. Single cell transcriptomics enables higher-resolution characterization of cell types in human cortex, which we used to revisit the idea of the canonical cortical microcircuit and to understand functional areal specialization. Deeply sampled single nucleus RNA-sequencing of eight cortical areas spanning cortical structural variation showed highly consistent cellular makeup for 24 coarse cell subclasses. However, proportions of excitatory neuron subclasses varied strikingly, reflecting differences in intra- and extracortical connectivity across primary sensorimotor and association cortices. Astrocytes and oligodendrocytes also showed differences in laminar organization across areas. Primary visual cortex showed dramatically different organization, including major differences in the ratios of excitatory to inhibitory neurons, expansion of layer 4 excitatory neuron types and specialized inhibitory neurons. Finally, gene expression variation in conserved neuron subclasses predicts differences in synaptic function across areas. Together these results provide a refined cellular and molecular characterization of human cortical cytoarchitecture that reflects functional connectivity and predicts areal specialization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate - a key question especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. Bioinformatic analysis of CDC42 mRNA revealed a potential iron-responsive element that may contribute to the regulation of CDC42 by iron. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using GBM models, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased T cell infiltration and increased T cell exhaustion. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD1 treatment. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, which was further corroborated by in vitro exhaustion assays. Moreover, increased T cell exhaustion was observed in male GBM patients. These findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy response.Statement of significanceImmunotherapies in GBM patients have been unsuccessful due to a variety of factors including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-specific T cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve therapeutic efficacy of immunotherapy in GBM.

Hematopoietic stem cell (HSC) transplantation (HST) is a curative treatment for many hematopoietic cancers and bone marrow (BM) disorders but is currently limited by numerous complications including a lengthy recovery period, prolonged neutropenia resulting in severe infections and bleeding, and a high incidence of graft vs. host disease (GVHD). While clinical studies have demonstrated that sex mismatch, notably male recipients with female donor cells, results in increased risk of GVHD (likely due to male recipient minor histocompatibility antigens targeted by donor female T-cells 1), increased non-relapse mortality, and decreased overall survival, the mechanisms underlying sex-determinants on hematopoiesis and post-transplant recovery are not clear. In this manuscript we have identified: 1) unique expression of hematopoietic niche factors in the BM and spleens of male and female mice, 2) altered kinetics of hematopoietic reconstitution following transplantation when male vs. female BM is used as the donor cell source, 3) a sex-specific role for the recipient niche in promoting post HST recovery, and 4) a dose-dependent role for exogenous sex hormones in maintaining hematopoietic stem and progenitor cells (HSPCs). Taken together, these data demonstrate that sex-specific cellular and molecular signaling occurs during hematopoietic regeneration. Further identifying novel sex-dependent determinants of regeneration following transplantation will not only enhance understanding of steady state versus regeneration hematopoiesis but may also reveal unique (and potentially sex-specific) therapeutic targets to accelerate hematologic recovery.Key PointsMale and female mice display altered kinetics of regeneration following HST due to unique niche factors in hematopoietic compartments.Exogenous steroid sex hormones uniquely regulate the pool of hematopoietic stem and progenitor cells and may impact transplantation outcomes.

Because disease-associated microglia (DAM) and disease-associated astrocytes (DAA) are involved in the pathophysiology of Alzheimer's disease (AD), we systematically identified molecular networks between DAM and DAA to uncover novel therapeutic targets for AD. Specifically, we develop a network-based methodology that leverages single-cell/nucleus RNA sequencing data from both transgenic mouse models and AD patient brains, as well as drug-target network, metabolite-enzyme associations, the human protein–protein interactome, and large-scale longitudinal patient data. Through this approach, we find both common and unique gene network regulators between DAM (i.e., PAK1, MAPK14, and CSF1R) and DAA (i.e., NFKB1, FOS, and JUN) that are significantly enriched by neuro-inflammatory pathways and well-known genetic variants (i.e., BIN1). We identify shared immune pathways between DAM and DAA, including Th17 cell differentiation and chemokine signaling. Last, integrative metabolite-enzyme network analyses suggest that fatty acids and amino acids may trigger molecular alterations in DAM and DAA. Combining network-based prediction and retrospective case-control observations with 7.2 million individuals, we identify that usage of fluticasone (an approved glucocorticoid receptor agonist) is significantly associated with a reduced incidence of AD (hazard ratio [HR] = 0.86, 95% confidence interval [CI] 0.83–0.89, P < 1.0 × 10−8). Propensity score–stratified cohort studies reveal that usage of mometasone (a stronger glucocorticoid receptor agonist) is significantly associated with a decreased risk of AD (HR = 0.74, 95% CI 0.68–0.81, P < 1.0 × 10−8) compared to fluticasone after adjusting age, gender, and disease comorbidities. In summary, we present a network-based, multimodal methodology for single-cell/nucleus genomics-informed drug discovery and have identified fluticasone and mometasone as potential treatments in AD.

BackgroundGlioblastoma (GBM) tumor cells modulate expression of iron-associated genes to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulator (HFE) gene, encoding the iron sensing HFE protein, is upregulated in GBM and correlates with poor survival outcomes. However, the molecular mechanisms underlying these observations remain unclear. Identification of pathways for targeting iron dependence in GBM tumors is therefore a critical area of investigation.MethodsWe interrogated the impact of cell-intrinsic Hfe expression on proliferation and tumor growth through genetic loss and gain of function approaches in syngeneic mouse glioma models. We determined the expression of iron-associated genes and their relationship with survival in GBM using public datasets and identified differentially expressed pathways in Hfe knockdown cells through Nanostring transcriptional profiling.ResultsLoss of Hfe induced apoptotic cell death in vitro and inhibited tumor growth in vivo while overexpression of Hfe accelerated both proliferation and tumor growth. Analysis of iron gene signatures in Hfe knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Analyzing differentially expressed pathways further identified oxidative stress as the top pathway upregulated with Hfe loss. Enhanced 55Fe uptake and generation of reactive oxygen species (ROS) were found with Hfe knockdown, implicating toxic iron overload resulting in apoptotic cell death.ConclusionsCollectively, these findings identify a novel role for HFE in regulating iron homeostasis in GBM tumors and provide a potential avenue for future therapeutic development.Key PointsHFE is an iron sensor that is upregulated in GBM and negatively impacts survival.HFE overexpression drives proliferation and tumor growth in vivo.Loss of HFE increases production of reactive oxygen species and induces apoptosis, extending survival in vivo.Importance of StudyDysregulation of iron metabolism is an important feature of GBM contributing to tumor growth and negatively impacting survival. The identification of key iron regulators controlling this process is therefore important for therapeutic targeting. We identify HFE as an important regulator of iron homeostasis in GBM and suggest a role for sexual dimorphism in HFE-mediated tumor iron regulation that ultimately results in differential survival outcomes. Our findings demonstrate that HFE drives tumor cell proliferation and survival in GBM and may be a viable target for modulating tumor iron flux and inducing apoptosis in tumor cells.

While immunotherapies have shown durable responses for multiple tumors, their efficacy remains limited in some advanced cancers, including glioblastoma. This may be due to differences in the immune landscape, as the glioblastoma microenvironment strongly favors immunosuppressive myeloid cells, which are linked to an elevation in immune-suppressive cytokines, including macrophage migration inhibitory factor (MIF). We now find that a single-nucleotide polymorphism (SNP) rs755622 in the MIF promoter associates with increased leukocyte infiltration in glioblastoma. Furthermore, we identified lactotransferrin expression as being associated with the rs755622 SNP, which could also be used as a biomarker for immune infiltrated tumors. These findings provide the first example in glioblastoma of a germline SNP that underlies differences in the immune microenvironment and identifies high lactotransferrin as a potential factor promoting immune activation.

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells results in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, causes nuclear lobulation, blebbing and micronucleation, which subsequently results in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction is dependent on VRK1 kinase activity and is rescued by ectopic VRK2 expression. Knockdown of VRK1 leads to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC therapeutic resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. The knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. Mechanistically, SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown dramatically increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of standard-of-care GBM therapies against therapeutically resistant CSCs.SummaryLauko et al. demonstrate a functional role for SerpinB3, which is elevated in glioblastoma cancer stem cells and protects against lysosomal-mediated cell death. SerpinB3 can be targeted to increase the efficacy of radiation in glioblastoma pre-clinical models.

Increased myeloid-derived suppressor cell (MDSC) frequency is associated with worse outcomes and poor therapeutic response in glioblastoma (GBM). Monocytic (m) MDSCs represent the predominant subset in the GBM microenvironment. However, the molecular basis of mMDSC enrichment in the tumor microenvironment compared to granulocytic (g) MDSCs has yet to be determined. Here, we report that mMDSCs and gMDSCs display differences in their tumoraccelerating ability, with mMDSCs driving tumor growth in GBM models. Epigenetic assessments indicate enhanced gene accessibility for cell adhesion programs in mMDSCs and higher cellsurface integrin expression in mouse and human mMDSCs. Integrin β1 blockage abrogated the tumor-promoting phenotype of mMDSCs and altered the immune profile in the tumor microenvironment. These findings suggest that integrin β1 expression underlies the enrichment of mMDSCs in tumors and represents a putative immunotherapy target to attenuate myeloid cell-driven immune suppression in GBM.SummaryMyeloid-derived suppressor cells (MDSCs) drive glioblastoma growth; however, the function of specific MDSCs subsets is unclear. Bayik et al. demonstrate that adhesion programs are enhanced in monocytic MDSCs and responsible for their GBM-promoting function.

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 CSCin vitrogrowth and self-renewal as well asin vivotumor 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

While dynamic microenvironmental interactions drive tumor growth and therapeutic resistance, the underlying direct cell-cell communication mechanisms remain poorly understood. We identified horizontal mitochondrial transfer as a mechanism that enhances tumorigenesis in glioblastoma. This transfer occurs primarily from brain-resident cells, including astrocytes, and can be appreciated in vitro and in vivo through intercellular connections between GBM cells and non-malignant host cells. The acquisition of astrocyte mitochondria drives an overall enhancement of mitochondrial membrane potential and metabolic capacity, while increasing glioblastoma cell self-renewal and tumor-initiating capacity. Collectively, our findings demonstrate that mitochondrial transfer augments the tumorigenic capacity of glioblastoma cells and reveals a previously unknown cell-cell communication mechanism that drives tumor growth.One-Sentence SummaryMitochondrial transfer from astrocytes to glioblastoma alters metabolic profile and enhances the tumor-initiation capacity.

Migrating cell collectives are key to embryonic development but also contribute to invasion and metastasis of a variety of cancers. Cell collectives can invade deep into tissues, leading to tumor progression and resistance to therapies. Collective cell invasion is also observed in the lethal brain tumor glioblastoma, which infiltrates the surrounding brain parenchyma leading to tumor growth and poor patient outcomes.Drosophilaborder cells, which migrate as a small cell cluster in the developing ovary, are a well-studied and genetically accessible model used to identify general mechanisms that control collective cell migration within native tissue environments. Most cell collectives remain cohesive through a variety of cell-cell adhesion proteins during their migration through tissues and organs. In this study, we first identified cell adhesion, cell junction, and associated regulatory genes that are expressed in human brain tumors. We performed RNAi knockdown of theDrosophilaorthologs in border cells to evaluate if migration and/or cohesion of the cluster was impaired. From this screen, we identified eight adhesion genes that disrupted border cell collective migration upon RNAi knockdown. Bioinformatics analyses further demonstrated that subsets of the orthologous genes were elevated in the margin and invasive edge of human glioblastoma patient tumors. These data together show that conserved cell adhesion and adhesion regulatory proteins with potential roles in tumor invasion also modulate collective cell migration. This dual screening approach for adhesion genes linked to glioblastoma and border cell migration thus may reveal conserved mechanisms that drive collective tumor cell invasion.

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.HighlightsHAUSP deubiquitinates and stabilizes SOX2 in glioma stem cells (GSCs).HAUSP is preferentially expressed by GSCs in perivascular niches in GBMs.HAUSP is required for maintaining GSC self-renewal and tumorigenic potential.Targeting HAUSP inhibited malignant growth in GSC-derived GBM xenografts.Inhibition of HAUSP synergized with radiation to suppress GBM tumor growth.

Poly-ADP Ribose Polymerase (PARP) inhibitors are clinically approved for the treatment of homologous recombination (HR) repair deficient tumors. PARP targeted therapy has limited efficacy in HR-proficient cancer. In this study, we identified the non-receptor lymphocyte-specific protein tyrosine kinase (LCK) as a novel regulator of HR repair pathways in endometrioid epithelial ovarian cancer (eEOC). Inhibition of LCK attenuates the expression of RAD51, BRCA1, and BRCA2 proteins necessary for HR-mediated DNA repair. HR repair in eEOC cells is LCK dependent. Upon DNA damage LCK expression is increased, and autophosphorylated, activated LCK is localized in the nucleus. LCK inhibition impairs RAD51 foci formation but augments {gamma}H2AX formation during DDR indicating reduced ability to repair DNA damage. DNA damage leads to direct interaction of LCK with RAD51 and BRCA1. Finally, attenuation of LCK sensitized HR-proficient eEOC cells to PARP inhibitor. Collectively, the findings identify a mechanism for expanding utility of PARP inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/433791v1_ufig1.gif" ALT="Figure 1"> View larger version (94K): org.highwire.dtl.DTLVardef@1b9069corg.highwire.dtl.DTLVardef@612304org.highwire.dtl.DTLVardef@141fca1org.highwire.dtl.DTLVardef@130c330_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefDey and colleagues identify the nonreceptor tyrosine kinase LCK as a mediator of homologous recombination directed DNA repair in ovarian cancer. The studies show that LCK inhibition (LCKi) is sufficient to augment Poly (ADP-Ribose) Polymerase inhibitor efficacy in Homologous Recombination (HR) proficient endometrioid ovarian cancer. HighlightsO_LINonreceptor tyrosine kinase LCK regulates expression of HR repair proteins RAD51, BRCA1 and BRCA2. C_LIO_LILCKi induces HR deficiency in endometrioid epithelial ovarian cancer. C_LIO_LIDNA damage leads to autophosphorylation of LCK and co-immunoprecipitation with RAD51 and BRCA1. C_LIO_LILCKi potentiates PARP targeted therapy in HR proficient ovarian cancer and expands the utility of the highly successful PARP inhibitors in the clinic. C_LI Statement of significanceThis study identifies a novel regulator and signaling pathway for maintaining HR repair during DNA damage. It further demonstrates a new opportunity to increase the utility of PARP inhibitors in HR-proficient eEOC cells.

Sex differences in the risk of SARS-CoV-2 infection have been controversial and the underlying mechanisms of COVID-19 sexual dimorphism remain understudied. Here we inspected sex differences in SARS-CoV-2 positivity, hospitalization, admission to the intensive care unit (ICU), sera immune profiling, and two single-cell RNA-sequencing (snRNA-seq) profiles from nasal tissues and peripheral blood mononuclear cells (PBMCs) of COVID-19 patients with varying degrees of disease severity. Our propensity score-matching observations revealed that male individuals have a 29% increased likelihood of SARS-CoV-2 positivity, with a hazard ration (HR) 1.32 (95% confidence interval [CI] 1.18-1.48) for hospitalization and HR 1.51 (95% CI 1.24-1.84) for admission to ICU. Sera from male patients at hospital admission had decreased lymphocyte count and elevated inflammatory markers (C-reactive protein, procalcitonin, and neutrophils). We found that SARS-CoV-2 entry factors, including ACE2, TMPRSS2, FURIN and NRP1, have elevated expression in nasal squamous cells from males with moderate and severe COVID-19. Cell-cell network proximity analysis suggests possible epithelium-immune cell interactions and immune vulnerability underlying a higher mortality in males with COVID-19. Monocyte-elevated expression of Toll like receptor 7 (TLR7) and Bruton tyrosine kinase (BTK) is associated with severe outcomes in males with COVID-19. These findings provide basis for understanding immune responses underlying sex differences, and designing sex-specific targeted treatments and patient care for COVID-19.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. However, the regulatory mechanisms of self-renewal and interactions with the vascular niche within tumor microenvironment are currently under investigation. Here, we demonstrate that BCSCs are enriched within arteriolar niche within the tumor microenvironment of estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the LPA/PKD-1 signaling pathway, which promotes arteriolar differentiation and self-renewal. Furthermore, this pathway directly promotes stemness features. These findings suggest that targeting LPA/PKD-1 signaling may disrupt the arteriolar niche within the tumor microenvironment and concomitantly eradicate BCSCs, thereby attenuating BC progression.

SummaryHyperthermic intraperitoneal chemotherapy (HIPEC) has emerged as a clinical regimen that prolongs overall survival for patients with advanced Epithelial Ovarian Cancer (EOC). However, the mechanism of action of HIPEC remains poorly understood. To provide insights into the rapid changes that accompany HIPEC, tumors from five patients with high grade serous ovarian cancer were harvested from the omentum at time of debulking and after 90 minutes of HIPEC treatment. Specimens were rapidly dissociated into single cells and processed for single cell RNA-seq. Unbiased clustering identified 19 cell clusters that were annotated based on cellular transcriptome signatures to identify the epithelial, stromal, T and B immune cells, macrophages, and natural killer cell populations. Hallmark pathway analysis revealed heat shock, metabolic reprogramming, inflammatory, and EMT pathway enrichment in distinct cell populations upon HIPEC treatment. Collectively, our findings provide the foundation for mechanistic studies focused on how HIPEC orchestrates the ovarian cancer tissue response.

Systematic identification of molecular networks in disease relevant immune cells of the nervous system is critical for elucidating the underlying pathophysiology of Alzheimer’s disease (AD). Two key immune cell types, disease-associated microglia (DAM) and disease-associated astrocytes (DAA), are biologically involved in AD pathobiology. Therefore, uncovering molecular determinants of DAM and DAA will enhance our understanding of AD biology, potentially identifying novel therapeutic targets for AD treatment. Here, we present an integrative, network-based methodology to uncover conserved molecular networks between DAM and DAA. Specifically, we leverage single-cell and single-nucleus RNA sequencing data from both AD transgenic mouse models and AD patient brains, drug-target networks, metabolite-enzyme associations, and the human protein-protein interactome, along with large-scale patient data validation from the MarketScan Medicare Supplemental Database. We find that common and unique molecular network regulators between DAM (i.e, PAK1, MAPK14, and SYK) and DAA (i.e., NFKB1, FOS, and JUN) are significantly enriched by multiple neuro-inflammatory pathways and well-known genetic variants (i.e., BIN1) from genome-wide association studies. Further network analysis reveal shared immune pathways between DAM and DAA, including Fc gamma R-mediated phagocytosis, Th17 cell differentiation, and chemokine signaling. Furthermore, integrative metabolite-enzyme network analyses imply that fatty acids (i.e., elaidic acid) and amino acids (i.e., glutamate, serine, and phenylalanine) may trigger molecular alterations between DAM and DAA. Finally, we prioritize repurposed drug candidates for potential treatment of AD by agents that specifically reverse dysregulated gene expression of DAM or DAA, including an antithrombotic anticoagulant triflusal, a beta2-adrenergic receptor agonist salbutamol, and the steroid medications (fluticasone and mometasone). Individuals taking fluticasone (an approved anti-inflammatory and inhaled corticosteroid) displayed a significantly decreased incidence of AD (hazard ratio (HR) = 0.858, 95% confidence interval [CI] 0.829-0.888, P < 0.0001) in retrospective case-control validation. Furthermore, propensity score matching cohort studies also confirmed an association of mometasone with reduced incidence of AD in comparison to fluticasone (HR =0.921, 95% CI 0.862-0.984, P < 0.0001).

Technological and computational advances in genomics and interactomics have made it possible to identify rapidly how disease mutations perturb interaction networks within human cells. In this study, we investigate at large-scale the effects of network perturbations caused by disease mutations within the human three-dimensional (3D), structurally-resolved macromolecular interactome. We show that disease-associated germline mutations are significantly enriched in sequences encoding protein-protein interfaces compared to mutations identified in healthy subjects from the 1000 Genomes and ExAC projects; these interface mutations correspond to protein-protein interaction (PPI)-perturbing alleles including p.Ser127Arg in PCSK9 at the PCSK9-LDLR interface. In addition, somatic missense mutations are significantly enriched in PPI interfaces compared to non-interfaces in 10,861 human exomes across 33 cancer subtypes/types from The Cancer Genome Atlas. Using a binomial statistical model, we computationally identified 470 PPIs harboring a statistically significant excess number of missense mutations at protein-protein interfaces (termed putative oncoPPIs) in pan-cancer analysis. We demonstrate that the oncoPPIs, including histone H4 complex in individual cancer types, are highly correlated with patient survival and drug resistance/sensitivity in human cancer cell lines and patient-derived xenografts. We experimentally validate the network effects of 13 oncoPPIs using a systematic binary interaction assay. We further showed that ALOX5 p.Met146Lys at the ALOX5-MAD1L1 interface and RXRA p.Ser427Phe at the RXRA-PPARG interface promote significant tumor cell growth using cell line-based functional assays, providing a functional proof-of-concept. In summary, if broadly applied, this human 3D interactome network analysis offers a powerful tool for prioritizing alleles with mutations altering PPIs that may contribute to the pathobiology of human diseases, and may offer disease-specific targets for genotype-informed therapeutic discovery.

Recent advances in cancer immunotherapy have created a greater appreciation of potential anti-tumoral impacts by the immune system; however, individual patient responses have been variable. While immunotherapy is often given after standard-of-care treatment, the effects of initial interventions on the ability of the immune system to mount a response are not well understood and this may contribute to the variable response. For glioblastoma (GBM), initial disease management includes surgical resection, perioperative high-dose steroid therapy, chemotherapy, and radiation treatment. While new discoveries regarding the impact of chemotherapy and radiation on immune response have been made and translated to clinical trial design, the impact of surgical resection and steroids on the anti-tumor immune response has yet to be determined. Further, it is now accepted that steroid usage needs to be closely evaluated in the context of GBM and immunotherapy trials. To better model the clinical scenario in GBM, we developed a mouse model that integrates tumor resection and steroid treatment to understand how these therapies affect local and systemic immune responses. Using this model, we observed a systemic reduction in lymphocytes associated with surgical resection and identified a correlation between increased tumor volume and decreased circulating lymphocytes, a relationship that was obviated by dexamethasone treatment. Furthermore, we investigated the possibility of there being similar relationships in a cohort of patients with GBM and found that prior to steroid treatment, circulating lymphocytes inversely correlated with tumor volume. Lastly, correlating GBM patient data and outcomes demonstrated that peripherally circulating lymphocyte content varies with progression-free and overall survival, independent of tumor volume, steroid use, or tumor molecular profiles. These results highlight the systemic immunosuppressive effects that initial therapies can have on patients. Such effects should be considered when designing current and future immunotherapy clinical trials and underscore the importance of circulating lymphocytes as a possible correlate of GBM disease progression.

Background: Ovarian cancer is the most fatal gynecologic malignancy in the United States. While chemotherapy is effective in the vast majority of ovarian cancer patients, recurrence and resistance to standard systemic therapy is nearly inevitable. We discovered that CD55 via activation of the non-receptor tyrosine kinase Lymphocyte Cell-Specific Protein-Tyrosine Kinase (LCK) promoted cisplatin resistance. Here, we hypothesized that treating high grade, platinum resistant endometrioid cancer cells with an LCK inhibitor (LCKi) followed by co-treatment with cisplatin would lead to increased cisplatin efficacy. Our objective was to assess clinical outcomes associated with increased LCK expression, test our hypothesis of utilizing LCKi as pre-treatment followed by co-treatment with cisplatin in platinum resistant ovarian cancer in vitro, and evaluate our findings in vivo to assess LCKi applicability as a therapeutic agent. Results: Kaplan-Meier (KM) plotter data indicated LCK expression is associated with significantly worse median progression-free survival (HR 3.19, p=0.02), and a trend toward decreased overall survival in endometrioid ovarian tumors with elevated LCK expression (HR 2.45, p=0.41). In vitro, cisplatin resistant ovarian endometrioid cells treated first with LCKi followed by combination LCKi-cisplatin treatment showed decreased cell viability and increased apoptosis. Immunoblot studies revealed LCKi led to increased expression of phosphorylated H2A histone family X (-H2AX), a marker for DNA damage. In vivo results demonstrate treatment with LCKi followed by LCKi-cisplatin leads to significantly slowed tumor growth.Conclusions: We identified a strategy to therapeutically target cisplatin resistant endometrioid ovarian cancer leading to chemosensitization to platinum chemotherapy via treatment with LCKi followed by co-treatment with LCKi-cisplatin.

Background: Epithelial Ovarian Cancer (EOC) is the second most common gynecologic malignancy in the United States, but the leading cause of gynecologic cancer death. Despite many achieving remission with first-line therapy, up to 80% of patients will recur and require additional treatment. Antibiotic therapy is frequently used during cancer treatments for both prophylaxis and treatment of infections, although this profoundly impacts the gut microbiome. Multiple studies suggest that an unperturbed gut microbiome may provide a protective microenvironment, and disruption may be permissive to tumor growth and chemotherapy resistance, including platinum agents. Experimental Design: We assessed whether antibiotic therapy would impact growth of EOC and sensitivity to cisplatin in murine models. Immune competent or compromised mice were given control or antibiotic (ABX) containing water (metronidazole, ampicillin, vancomycin, and neomycin) for two weeks before being intraperitoneally injected with murine ID8 or ID8-VEGF EOC cells. Tumors were monitored and cisplatin therapy was administered weekly until endpoint. Stool was collected throughout the study to assess for microbial population effects over time. Cecal microbiota transplants (CMTs) of contents derived from ABX or control treated donor mice were performed on ABX treated recipient mice. Results: Both immune competent and immune compromised ID8 and ID8 VEGF tumor-bearing mice demonstrated a decreased response to cisplatin therapy in ABX treated groups with an increase in overall tumor burden. RNAseq analysis showed enrichment of multiple cell proliferation and stem cell pathways, and stem cell genes SOX2, WNT and PAX2. The self-renewal of cancer cells derived from tumors of ABX treated mice was increased compared to tumors from control mice, indicative of a microbiota derived tumor suppressor. Conclusion: Collectively, these studies indicate an intact microbiome provides a tumor suppressor and maintains chemosensitivity that is disrupted by ABX treatment.

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

SUMMARYThe isocortex and hippocampal formation are two major structures in the mammalian brain that play critical roles in perception, cognition, emotion and learning. Both structures contain multiple regions, for many of which the cellular composition is still poorly understood. In this study, we used two complementary single-cell RNA-sequencing approaches, SMART-Seq and 10x, to profile ∼1.2 million cells covering all regions in the adult mouse isocortex and hippocampal formation, and derived a cell type taxonomy comprising 379 transcriptomic types. The completeness of coverage enabled us to define gene expression variations across the entire spatial landscape without significant gaps. We found that cell types are organized in a hierarchical manner and exhibit varying degrees of discrete or continuous relatedness with each other. Such molecular relationships correlate strongly with the spatial distribution patterns of the cell types, which can be region-specific, or shared across multiple regions, or part of one or more gradients along with other cell types. Glutamatergic neuron types have much greater diversity than GABAergic neuron types, both molecularly and spatially, and they define regional identities as well as inter-region relationships. For example, we found that glutamatergic cell types between the isocortex and hippocampal formation are highly distinct from each other yet possess shared molecular signatures and corresponding layer specificities, indicating their homologous relationships. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation for the first time, and begins to shed light on its underlying relationship with the development, evolution, connectivity and function of these two brain structures.

Glioblastoma (GBM) remains among the deadliest of human malignancies. The emergence of the cancer stem cell (CSC) phenotype represents a major challenge to disease management and durable treatment response. The extrinsic, environmental, and lifestyle factors that result in CSC enrichment are not well understood. The CSC state endows cells with a fluid metabolic profile, enabling the utilization of multiple nutrient sources. Therefore, to test the impact of diet on CSC enrichment, we evaluated disease progression in tumor-bearing mice fed an obesity-inducing high-fat diet (HFD) versus an energy-balanced, low-fat control diet. HFD consumption resulted in hyper-aggressive disease that was accompanied by CSC enrichment and shortened survival. HFD consumption also drove intracerebral accumulation of saturated fats, which in turn inhibited the production and signaling of the gasotransmitter hydrogen sulfide (H2S). H2S is an endogenously produced bio-active metabolite derived from sulfur amino acid catabolism. It functions principally through protein S-sulfhydration and regulates a variety of programs including mitochondrial bioenergetics and cellular metabolism. Inhibition of H2S synthesis resulted in increased proliferation and chemotherapy resistance, whereas treatment with H2S donors led to cytotoxicity and death of cultured GBM cells. Compared to non-cancerous controls, patient GBM specimens were reduced in overall protein S-sulfhydration, which was primarily lost from proteins regulating cellular metabolism. These findings support the hypothesis that diet-regulated H2S signaling serves to suppress GBM by restricting metabolic adaptability, while its loss triggers CSC enrichment and disease acceleration. Interventions augmenting H2S bioavailability concurrent with GBM standard of care may improve outcomes for GBM patients.One Sentence SummaryConsumption of a high-fat diet (HFD) accelerates glioblastoma (GBM) by inhibiting the production and signaling of the tumor-suppressive metabolite hydrogen sulfide (H2S).

Single cell transcriptomics has transformed the characterization of brain cell identity by providing quantitative molecular signatures for large, unbiased samples of brain cell populations. With the proliferation of taxonomies based on individual datasets, a major challenge is to integrate and validate results toward defining biologically meaningful cell types. We used a battery of single-cell transcriptome and epigenome measurements generated by the BRAIN Initiative Cell Census Network (BICCN) to comprehensively assess the molecular signatures of cell types in the mouse primary motor cortex (MOp). We further developed computational and statistical methods to integrate these multimodal data and quantitatively validate the reproducibility of the cell types. The reference atlas, based on more than 600,000 high quality single-cell or -nucleus samples assayed by six molecular modalities, is a comprehensive molecular account of the diverse neuronal and non-neuronal cell types in MOp. Collectively, our study indicates that the mouse primary motor cortex contains over 55 neuronal cell types that are highly replicable across analysis methods, sequencing technologies, and modalities. We find many concordant multimodal markers for each cell type, as well as thousands of genes and gene regulatory elements with discrepant transcriptomic and epigenomic signatures. These data highlight the complex molecular regulation of brain cell types and will directly enable design of reagents to target specific MOp cell types for functional analysis.