Metabologenomic characterization uncovers a clinically aggressive IDH mutant glioma subtype.

Mutations in the pivotal metabolic isocitrate dehydrogenase (IDH) enzymes are recognized to drive the molecular footprint of diffuse gliomas, and patients with IDH mutant gliomas have overall favorable outcomes compared to patients with IDH wild-type tumors. However, survival still varies widely among patients with IDH mutated tumors. Here, we aimed to characterize molecular signatures that explain the range of IDH mutant gliomas. By integrating matched epigenome-wide methylome, transcriptome, and global metabolome data in 154 patients with gliomas, we identified a group of IDH mutant gliomas with globally altered metabolism that resembled IDH wild-type tumors. IDH-mutant gliomas with altered metabolism have significantly shorter overall survival from their IDH mutant counterparts that is not fully accounted for by recognized molecular prognostic markers of CDKN2A/B loss and glioma CpG Island Methylator Phenotype (GCIMP) status. IDH-mutant tumors with dysregulated metabolism harbored distinct epigenetic alterations that converged to drive proliferative and stem-like transcriptional profiles, providing a window to target novel dependencies in gliomas.

Thinking (Metastasis) outside the (Primary Tumor) Box.

The metastasis of tumor cells into vital organs is a major cause of death from diverse types of malignancies [...].

Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer.

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFß and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial.

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Increased mRNA expression of CDKN2A is a transcriptomic marker of clinically aggressive meningiomas.

Homozygous deletion of CDKN2A/B was recently incorporated into the World Health Organization classification for grade 3 meningiomas. While this marker is overall rare in meningiomas, its relationship to other CDKN2A alterations on a transcriptomic, epigenomic, and copy number level has not yet been determined. We therefore utilized multidimensional molecular data of 1577 meningioma samples from 6 independent cohorts enriched for clinically aggressive meningiomas to comprehensively interrogate the spectrum of CDKN2A alterations through DNA methylation, copy number variation, transcriptomics, and proteomics using an integrated molecular approach. Homozygous CDKN2A/B deletions were identified in only 7.1% of cases but were associated with significantly poorer outcomes compared to tumors without these deletions. Heterozygous CDKN2A/B deletions were identified in 2.6% of cases and had similarly poor outcomes as those with homozygous deletions. Among tumors with intact CDKN2A/B (without a homozygous or heterozygous deletion), we found a distinct difference in outcome based on mRNA expression of CDKN2A, with meningiomas that had elevated mRNA expression (CDKN2Ahigh) having a significantly shorter time to recurrence. The expression of CDKN2A was independently prognostic after accounting for copy number loss and consistently increased with WHO grade and more aggressive molecular and methylation groups irrespective of cohort. Despite the discordant and mutually exclusive status of the CDKN2A gene in these groups, both CDKN2Ahigh meningiomas and meningiomas with CDKN2A deletions were enriched for similar cell cycle pathways but at different checkpoints. High mRNA expression of CDKN2A was also associated with gene hypermethylation, Rb-deficiency, and lack of response to CDK inhibition. p16 immunohistochemistry could not reliably differentiate between meningiomas with and without CDKN2A deletions but appeared to correlate better with mRNA expression. These findings support the role of CDKN2A mRNA expression as a biomarker of clinically aggressive meningiomas with potential therapeutic implications.

The multiomic landscape of meningiomas: a review and update.

PURPOSE: Meningiomas are the most common primary brain tumor in adults. Traditionally they have been understudied compared to other central nervous system (CNS) tumors. However over the last decade, there has been renewed interest in uncovering the molecular topography of these tumors, with landmark studies identifying key driver alterations contributing to meningioma development and progression. Recent work from several independent research groups have integrated different genomic and epigenomic platforms to develop a molecular-based classification scheme for meningiomas that could supersede histopathological grading in terms of diagnostic accuracy, biological relevance, and outcome prediction, keeping pace with contemporary grading schemes for other CNS tumors including gliomas and medulloblastomas. METHODS: Here we summarize the studies that have uncovered key alterations in meningiomas which builds towards the discovery of consensus molecular groups in meningiomas by integrating these findings. These groups supersede WHO grade and other clinical factors in being able to accurately predict tumor biology and clinical outcomes following surgery. RESULTS: Despite differences in the nomenclature of recently uncovered molecular groups across different studies, the biological similarities between these groups enables us to likely reconciliate these groups into four consensus molecular groups: two benign groups largely dichotomized by NF2-status, and two clinically aggressive groups defined by their hypermetabolic transcriptome, and by their preponderance of proliferative, cell-cycling pathways respectively. CONCLUSION: Future work, including by our group and others are underway to validate these molecular groups and harmonize the nomenclature for routine clinical use.

Skeletal phenotypes in secreted frizzled-related protein 4 gene knockout mice mimic skeletal architectural abnormalities in subjects with Pyle's disease from SFRP4 mutations.

Mutations in SFRP4 cause Pyle's bone disease with wide metaphyses and increased skeletal fragility. The WNT signaling pathway plays important roles in determining skeletal architecture and SFRP4 is a secreted Frizzled decoy receptor that inhibits WNT signaling. Seven cohorts of male and female Sfrp4 gene knockout mice, examined through 2 years of age, had a normal lifespan but showed cortical and trabecular bone phenotypes. Mimicking human Erlenmeyer flask deformities, bone cross-sectional areas were elevated 2-fold in the distal femur and proximal tibia but only 30% in femur and tibia shafts. Reduced cortical bone thickness was observed in the vertebral body, midshaft femur and distal tibia. Elevated trabecular bone mass and numbers were observed in the vertebral body, distal femur metaphysis and proximal tibia metaphysis. Midshaft femurs retained extensive trabecular bone through 2 years of age. Vertebral bodies had increased compressive strength, but femur shafts had reduced bending strength. Trabecular, but not cortical, bone parameters in heterozygous Sfrp4 mice were modestly affected. Ovariectomy resulted in similar declines in both cortical and trabecular bone mass in wild-type and Sfrp4 KO mice. SFRP4 is critical for metaphyseal bone modeling involved in determining bone width. Sfrp4 KO mice show similar skeletal architecture and bone fragility deficits observed in patients with Pyle's disease with SFRP4 mutations.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity.

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single-cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation-related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at a single-cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

Temporal profiling of therapy resistance in human medulloblastoma identifies novel targetable drivers of recurrence.

Medulloblastoma (MB) remains a leading cause of cancer-related mortality among children. The paucity of MB samples collected at relapse has hindered the functional understanding of molecular mechanisms driving therapy failure. New models capable of accurately recapitulating tumor progression in response to conventional therapeutic interventions are urgently needed. In this study, we developed a therapy-adapted PDX MB model that has a distinct advantage of generating human MB recurrence. The comparative gene expression analysis of MB cells collected throughout therapy led to identification of genes specifically up-regulated after therapy, including one previously undescribed in the setting of brain tumors, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4). Subsequent functional validation resulted in a markedly diminished in vitro proliferation, self-renewal, and longevity of MB cells, translating into extended survival and reduced tumor burden in vivo. Targeting endothelial nitric oxide synthase, a downstream substrate of BPIFB4, impeded growth of several patient-derived MB lines at low nanomolar concentrations.

A clinically applicable integrative molecular classification of meningiomas.

Meningiomas are the most common primary intracranial tumour in adults1. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas2. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.

MicroCT analyses of mouse femoral neck architecture.

Hip fractures at the femoral neck are a major cause of morbidity and mortality, but aside from biomechanical strength testing, little is known about femoral neck architecture in mice. Procedures were optimized to analyze high-resolution (6 µm voxel size) microCT scans of the mouse femoral neck to provide bone mass and architectural information. Similar to histomorphometric observations in rats, the boundary between cortical and trabecular bone is difficult to identify in the mouse femoral mid-neck and these compartments were not analyzed separately. Analyses included total area, mineralized bone area, and bone volume fraction (BV/TV). Femoral neck architecture varies in C57BL/6J, 129/SvEv and BALB/c mouse strains. Bone cross sectional area and BV/TV were low in Lrp5 but elevated in Sost gene knockout mice. Sfrp4 gene knockout resulted in high total area, normal bone area, low BV/TV and, as indicated by BS/BV values, greater trabecularization. Femoral neck BV/TV declined with age and ovariectomy, but increased with teriparatide treatment. These findings demonstrate that the architecture of the mouse femoral neck mimics phenotypes and treatment effects observed at other skeletal sites and is a relevant bone site for translational studies examining osteoporosis therapies.

Programmed death ligand-1 (PD-L1) expression in meningioma; prognostic significance and its association with hypoxia and NFKB2 expression.

Management of clinically aggressive meningiomas is a considerable challenge. PD-L1 induced immune suppression has increasingly gained attention in clinical management of cancer; however, to date, the clinical significance and regulatory mechanisms of PD-L1 in meningioma is not yet fully characterized. We sought to characterize PD-L1 expression in meningioma and elucidate its regulatory mechanisms. Immunohistochemical staining of PD-L1 expression in meningiomas showed 43% positivity in both tumor and immune cells and we observed intra and inter tumoral heterogeneity. Univariate and multivariate analyses confirmed that PD-L1 protein expression is an independent prognostic marker for worse recurrence free survival in meningioma. Furthermore, our transcriptomic analysis revealed a strong association between PD-L1 expression and that of NFKB2 and carbonic anhydrase 9 (CA9). We also demonstrated that both of these markers, when co-expressed with PD-L1, predict tumor progression. Our studies on several meningioma cell lines cultured in hypoxic conditions validated the association of CA9 and PD-L1 expression. Here we show the clinical significance of PD-L1 in meningioma as a marker that can predict tumor recurrence. We also show an association PD-L1 expression with NFKB2 expression and its induction under hypoxic conditions. These findings may open new avenues of molecular investigation in pathogenesis of meningioma.

Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor ß (TGF-ß) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.

Modeling germline mutations in pineoblastoma uncovers lysosome disruption-based therapy.

Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.

Nanoparticle Uptake in a Spontaneous and Immunocompetent Woodchuck Liver Cancer Model.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based in vivo experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14+ cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.

NOTUM inhibition increases endocortical bone formation and bone strength.

The disability, mortality and costs caused by non-vertebral osteoporotic fractures are enormous. Existing osteoporosis therapies are highly effective at reducing vertebral but not non-vertebral fractures. Cortical bone is a major determinant of non-vertebral bone strength. To identify novel osteoporosis drug targets, we phenotyped cortical bone of 3 366 viable mouse strains with global knockouts of druggable genes. Cortical bone thickness was substantially elevated in Notum -/- mice. NOTUM is a secreted WNT lipase and we observed high NOTUM expression in cortical bone and osteoblasts but not osteoclasts. Three orally active small molecules and a neutralizing antibody inhibiting NOTUM lipase activity were developed. They increased cortical bone thickness and strength at multiple skeletal sites in both gonadal intact and ovariectomized rodents by stimulating endocortical bone formation. Thus, inhibition of NOTUM activity is a potential novel anabolic therapy for strengthening cortical bone and preventing non-vertebral fractures.

Ketoconazole and Posaconazole Selectively Target HK2-expressing Glioblastoma Cells.

PURPOSE: Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM.Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro. RESULTS: This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells. CONCLUSIONS: Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Induction of rod versus cone photoreceptor-specific progenitors from retinal precursor cells.

During development, multipotent progenitors undergo temporally-restricted differentiation into post-mitotic retinal cells; however, the mechanisms of progenitor division that occurs during retinogenesis remain controversial. Using clonal analyses (lineage tracing and single cell cultures), we identify rod versus cone lineage-specific progenitors derived from both adult retinal stem cells and embryonic neural retinal precursors. Taurine and retinoic acid are shown to act in an instructive and lineage-restricted manner early in the progenitor lineage hierarchy to produce rod-restricted progenitors from stem cell progeny. We also identify an instructive, but lineage-independent, mechanism for the specification of cone-restricted progenitors through the suppression of multiple differentiation signaling pathways. These data indicate that exogenous signals play critical roles in directing lineage decisions and resulting in fate-restricted rod or cone photoreceptor progenitors in culture. Additional factors may be involved in governing photoreceptor fates in vivo.