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Supplementary Figure from Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer

Resistance to targeted therapies is an important clinical problem in HER2-positive (HER2+) breast cancer. “Drug-tolerant persisters” (DTP), a subpopulation of cancer cells that survive via reversible, nongenetic mechanisms, are implicated in resistance to tyrosine kinase inhibitors (TKI) in other malignancies, but DTPs following HER2 TKI exposure have not been well characterized. We found that HER2 TKIs evoke DTPs with a luminal-like or a mesenchymal-like transcriptome. Lentiviral barcoding/single-cell RNA sequencing reveals that HER2+ breast cancer cells cycle stochastically through a “pre-DTP” state, characterized by a G0-like expression signature and enriched for diapause and/or senescence genes. Trajectory analysis/cell sorting shows that pre-DTPs preferentially yield DTPs upon HER2 TKI exposure. Cells with similar transcriptomes are present in HER2+ breast tumors and are associated with poor TKI response. Finally, biochemical experiments indicate that luminal-like DTPs survive via estrogen receptor–dependent induction of SGK3, leading to rewiring of the PI3K/AKT/mTORC1 pathway to enable AKT-independent mTORC1 activation.Significance:DTPs are implicated in resistance to anticancer therapies, but their ontogeny and vulnerabilities remain unclear. We find that HER2 TKI-DTPs emerge from stochastically arising primed cells (“pre-DTPs”) that engage either of two distinct transcriptional programs upon TKI exposure. Our results provide new insights into DTP ontogeny and potential therapeutic vulnerabilities.This article is highlighted in the In This Issue feature, p. 873

Supplementary Table from Ontogeny and Vulnerabilities of Drug-Tolerant Persisters in HER2+ Breast Cancer

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Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo. Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023–37. ©2018 AACR.

Protein expression of EphA2, EphA3, CD133, CD15, Bmi1, Sox2, FoxG1 and ITGA6 in GBM.

Patient demographics.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

List of shRNAs on the custom reductase library.

Supplementary Figure Legends from Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

Evaluation of SN30000-induced bystander cell killing in a co-culture system (S1); SN30000 clonogenic survival curves for HT-29, PANC-1 cells and HKO1 (S2); Plating efficiency in HT-29, PANC-1 and HKO2 reductase screens (S3); Density distributions and correlations of reductase screen sequencing data (S4); Log2 enrichment factors of shRNAs significantly selected in the reductase screens (S5); Depletion of POR protein by shRNAs in PANC-1 and HT-29 cells (S6); Replicate SN30000 clonogenic survival assays performed in stably transduced HT-29 and PANC-1 cells (S7); Measurement of plating efficiency in HKO1 and HKO2 whole-genome screens (S8); Coincidence of shRNAs significantly selected (Z > 1.96) in HT-29 and PANC-1 reductase screens and the HKO1 genome-wide screen (S9); Validation of POR shRNAs and clonogenic survival following SN30000 treatment of POR-knockout HCT116 cells (S10); Spatial distribution of POR and CA-IX expression in tongue SCC (S11); Quantitation of POR and CA-IX expression in HNSCC (S12); POR IHC staining in isogenic xenografts used for method development (S13); POR mRNA expression and sequence variation in the TCGA HNSCC dataset (S14).

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.

MYC-driven medulloblastoma (MB) is an aggressive pediatric brain tumor characterized by therapy resistance and disease recurrence. Here, we integrated data from unbiased genetic screening and metabolomic profiling to identify multiple cancer-selective metabolic vulnerabilities in MYC-driven MB tumor cells, which are amenable to therapeutic targeting. Among these targets, dihydroorotate dehydrogenase (DHODH), an enzyme that catalyzes de novo pyrimidine biosynthesis, emerged as a favorable candidate for therapeutic targeting. Mechanistically, DHODH inhibition acts on target, leading to uridine metabolite scarcity and hyperlipidemia, accompanied by reduced protein O-GlcNAcylation and c-Myc degradation. Pyrimidine starvation evokes a metabolic stress response that leads to cell-cycle arrest and apoptosis. We further show that an orally available small-molecule DHODH inhibitor demonstrates potent mono-therapeutic efficacy against patient-derived MB xenografts in vivo. The reprogramming of pyrimidine metabolism in MYC-driven medulloblastoma represents an unappreciated therapeutic strategy and a potential new class of treatments with stronger cancer selectivity and fewer neurotoxic sequelae.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1–3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs. Genome-wide CRISPR screens in mouse cancer cell lines are used to identify a core, conserved set of genes and pathways that govern how cancer cells evade killing by cytotoxic T lymphocytes.

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes., Mutations in RAS-MAPK pathway genes are implicated in Noonan-spectrum, yet up to 20% of cases have unknown cause. Here, the authors identify RREB1 underlying a 6p microdeletion RASopathy-like syndrome and show that RREB1, SIN3A and KDM1A form a transcriptional repressive complex to control methylation of MAPK pathway genes.

Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily withRB1loss or, rarely,MYCNamplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens inRB1nullandRB1wt;MYCNamporthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53–BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53–p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53–BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.

Single-cell RNA-sequencing (scRNA-seq) offers unprecedented insight into heterogenous biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell analytic toolbox that offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Novel methods that are introduced to facilitate scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression; (ii) cluster resolution optimization using a marker-specificity criterion; (iii) marker-based cell-type annotation with Miko scoring; and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Our unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and we provide illustrative examples of cellular and transcriptomic annotation of developmental and immunological scRNA-seq atlases. Overall, scPipeline provides a flexible computational framework for in-depth scRNA-seq analysis.

Single-cell RNA-sequencing (scRNA-seq) offers functional insight into complex biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell data analysis toolbox that builds on existing methods and offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Advances to scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression, (ii) cluster resolution optimization using a marker-specificity criterion, (iii) marker-based cell-type annotation with Miko scoring, and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Both unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and illustrative examples of cellular transcriptomic annotation of developmental and immunological scRNA-seq atlases are provided herein. Overall, scPipeline offers a flexible computational framework for in-depth scRNA-seq analysis.

Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor stem cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either an a priori, pre-existing fitness advantage, or a priori equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human GBM, and the prognostic value of interrogating the MRD state in solid cancers.

Graphical abstract, Infectious disease transmission models often stratify populations by age and geographic patches. Contact patterns between age groups and patches are key parameters in such models. Arenas et al. (2020) develop an approach to simulate contact patterns associated with recurrent mobility between patches, such as due to work, school, and other regular travel. Using their approach, mixing between patches is greater than mobility data alone would suggest, because individuals from patches A and B can form contacts if they meet in patch C. We build upon their approach to address three potential gaps that remain, outlined in the bullets below. We describe the steps required to implement our approach in detail, and present step-wise results of an example application to generate contact matrices for SARS-CoV-2 transmission modelling in Ontario, Canada. We also provide methods for deriving the mobility matrix based on GPS mobility data (appendix). • Our approach includes a distribution of contacts by age that is responsive to the underlying age distributions of the mixing populations. • Our approach maintains different age mixing patterns by contact type, such that changes to the numbers of different types of contacts are appropriately reflected in changes to overall age mixing patterns. • Our approach distinguishes between two mixing pools associated with each patch, with possible implications for the overall connectivity of the population: the home pool, in which contacts can only be formed with other individuals residing in the same patch, and the travel pool, in which contacts can be formed with some residents of, and any other visitors to the patch.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1−4 (DPH1−DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for DPH1, DPH2, or DPH3, which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that Drosophila nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.

Objectives COVID-19 has had devastating effects on long-term care homes across much of the world, and especially within Canada, with over 50% of the mortality from COVID-19 in 2020 in these homes. Understanding the way in which the virus spreads within these homes is critical to preventing further outbreaks. Design Retrospective chart review. Settings and Participants Long-term care home residents and staff in Ontario, Canada. Methods We conducted a longitudinal study of a large long-term care home COVID-19 outbreak in Ontario, Canada using electronic medical records, public health records, staff assignments and resident room locations to spatially map the outbreak through the facility. Results By analyzing the outbreak longitudinally we were able to draw three important conclusions: 1) 84.5% had typical COVID-19 symptoms and only 15.5% of residents had asymptomatic infection, 2) there was a high attack rate of 85.8%, which appeared to be explained by a high degree of interconnectedness within the home exacerbated by staffing shortages, and 3) clustering of infections within multi-bedded rooms was common. Conclusion and Implications Low rates of asymptomatic infection suggest symptom-based screening in residents remains very important for detecting outbreaks, a high degree of interconnectedness explains the high attack rate and there is a need for improved guidance for homes with multi-bedded rooms on optimizing resident room movement to mitigate spread of COVID-19 in long-term care homes., This study examines a large COVID-19 long-term care home outbreak showing low rate of asymptomatic infection, and a high rate of interconnectedness, especially between roommates, driving transmission.

SUMMARYSARS-CoV-2, depends on host cell components for replication, therefore the identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection. Such host factors may be necessary for infection and replication of SARS-CoV-2 and, if druggable, presents an attractive strategy for anti-viral therapy. We performed genome wide CRISPR knockout screens in Vero E6 cells and 4 human cell lines including Calu-3, Caco-2, Hek293 and Huh7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while all other host genes identified were cell line specific including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, lipid metabolism, immune pathways and chromatin modulation. Notably, chromatin modulator genes KMT2C and KDM6A in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed. Overall, the network of host factors that have been identified will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate the development of host-directed therapies.IN BRIEFSARS-CoV-2, depends on host cell components for infection and replication. Genome-wide CRISPR screens were performed in multiple human cell lines to elucidate common host dependencies required for SARS-CoV-2 infection. Only ACE2, the cognate SARS-CoV-2 entry receptor, was common amongst cell lines, while all other host genes identified were cell line specific, several of which converged on pathways involved in cell signalling, lipid metabolism, immune pathways, and chromatin modulation. Overall, a network of host factors was identified that will be broadly applicable to understanding the impact of SARS-CoV-2 on human cells and facilitate productive targeting of host genes and pathways.HIGHLIGHTS- Genome-wide CRISPR screens for SARS-CoV-2 in multiple human cell lines- Identification of wide-ranging cell-type dependent genetic dependencies for SARS-CoV-2 infection- ACE2 is the only common host factor identified across different cell types

Background There is growing evidence supporting the efficacy of shorter courses of antibiotic therapy for common infections, however the risks of prolonged antibiotic duration are underappreciated. Objectives We sought to estimate the incremental daily risk of antibiotic-associated harms. Methods We searched three major databases to retrieve systematic reviews from 2000 to July 30, 2020 in any language. Eligibility Systematic reviews were required to evaluate shorter versus longer antibiotic therapy with fixed durations between 3 and 14 days. RCTs included for meta-analysis were identified from the systematic reviews. Participants Adult and pediatric patients from any setting. Interventions Primary outcomes were the proportion of patients experiencing adverse drug events, superinfections and antimicrobial resistance. Risk of Bias Assessment: Each RCT was evaluated for quality by extracting the assessment reported by each systematic review. Data Synthesis The daily odds ratio (OR) of antibiotic harm was estimated and pooled using random effects meta-analysis. Results Thirty-five (35) systematic reviews encompassing 71 eligible randomized controlled trials were included. Studies most commonly evaluated duration of therapy for respiratory tract (n=36, 51%) and urinary tract infections (n=29, 41%). Overall, 23,174 patients were evaluated for antibiotic-associated harms. Adverse events (n=20,345), superinfections (n=5,776), and AMR (n=2,330) were identified in 19.9% (n=4,039), 4.8% (n=280), and 10.6% (n=246) of patients, respectively. Each day of antibiotic therapy was associated with 4% increased odds of experiencing an adverse event (OR 1.04, 95% CI [1.02 to 1.07]). Daily odds of severe adverse effects also increased (OR 1.09, 95% CI [1.00 to 1.19). The daily incremental odds of superinfection and AMR were OR 0.98 (0.92 to 1.06) and OR 1.03 (0.98 to 1.07), respectively. Conclusion Each additional day of antibiotic therapy is associated with measurable antibiotic harm, particularly adverse events. These data may provide additional context for clinicians when weighing benefits versus risks of prolonged antibiotic therapy.

PurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of brain tumor initiating cells (BTICs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here we identify CD70 as a potential therapeutic target for recurrent GBM BTICs.Experimental DesignIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We utilize CD70 knockdown studies, subsequent RNAseq pathway analysis, and in vivo xenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 CAR-T therapy, which we validated in vitro and in vivo using our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosis in vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable poly-therapeutic avenue to co-target both GBM and its TIME.

Reductive power in the cytosol of pancreatic cancer cells drives a dependency on the endoplasmic reticulum protein PRDX4., There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H2O2 metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs–governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.