Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

Cancer-selective metabolic vulnerabilities in MYC-amplified medulloblastoma.

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.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. 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.

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.

Identification of five important genes to predict glioblastoma subtypes.

BACKGROUND: Glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, has been classified into three subtypes: classical, mesenchymal, and proneural. While the original classification relied on an 840 gene-set, further clarification on true GBM subtypes uses a 150-gene signature to accurately classify GBM into the three subtypes. We hypothesized whether a machine learning approach could be used to identify a smaller gene-set to accurately predict GBM subtype. METHODS: Using a supervised machine learning approach, extreme gradient boosting (XGBoost), we developed a classifier to predict the three subtypes of glioblastoma (GBM): classical, mesenchymal, and proneural. We tested the classifier on in-house GBM tissue, cell lines, and xenograft samples to predict their subtype. RESULTS: We identified the five most important genes for characterizing the three subtypes based on genes that often exhibited high Importance Scores in our XGBoost analyses. On average, this approach achieved 80.12% accuracy in predicting these three subtypes of GBM. Furthermore, we applied our five-gene classifier to successfully predict the subtype of GBM samples at our centre. CONCLUSION: Our 5-gene set classifier is the smallest classifier to date that can predict GBM subtypes with high accuracy, which could facilitate the future development of a five-gene subtype diagnostic biomarker for routine assays in GBM samples.

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on 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.

Inhibition of relaxin autocrine signaling confers therapeutic vulnerability in ovarian cancer.

Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

RAD51-Mediated DNA Homologous Recombination Is Independent of PTEN Mutational Status.

PTEN mutation occurs in a variety of aggressive cancers and is associated with poor patient outcomes. Recent studies have linked mutational loss of PTEN to reduced RAD51 expression and function, a key factor involved in the homologous recombination (HR) pathway. However, these studies remain controversial, as they fail to establish a definitive causal link to RAD51 expression that is PTEN-dependent, while other studies have not been able to recapitulate the relationship between the PTEN expression and the RAD51/HR function. Resolution of this apparent conundrum is essential due to the clinically-significant implication that PTEN-deficient tumors may be sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) commonly used in the clinical management of BRCA-mutated and other HR-deficient (HRD) tumors. METHODS: Primary Pten-deficient (and corresponding wild-type) mouse embryonic fibroblasts (MEFs) and astrocytes and PTEN-null human tumor cell lines and primary cells were assessed for RAD51 expression (via the Western blot analysis) and DNA damage repair analyses (via alkali comet and γH2AX foci assays). RAD51 foci analysis was used to measure HR-dependent DNA repair. Xrcc2-deficient MEFs served as an HR-deficient control, while the stable knockdown of RAD51 (shRAD51) served to control for the relative RAD51/HR-mediated repair and the phospho-53BP1 foci analysis served to confirm and measure non-homologous end joining (NHEJ) activity in PTEN-deficient and shRAD51-expressing (HRD) lines. Cell proliferation studies were used to measure any potential added sensitivity of PTEN-null cells to the clinically-relevant PARPi, olaparib. RAD51 levels and DNA damage response signaling were assessed in PTEN-mutant brain tumor initiating cells (BTICs) derived from primary and recurrent glioblastoma multiforme (GBM) patients, while expression of RAD51 and its paralogs were examined as a function of the PTEN status in the RNA expression datasets isolated from primary GBM tumor specimens and BTICs. RESULTS: Pten knockout primary murine cells display unaltered RAD51 expression, endogenous and DNA strand break-induced RAD51 foci and robust DNA repair activity. Defective HR was only observed in the cells lacking Xrcc2. Likewise, human glioblastoma multiforme (GBM) cell lines with known PTEN deficiency (U87, PTEN-mutated; U251 and U373, PTEN-null) show apparent expression of RAD51 and display efficient DNA repair activity. Only GBM cells stably expressing shRNAs against RAD51 (shRAD51) display dysfunctional DNA repair activity and reduced proliferative capacity, which is exacerbated by PARPi treatment. Furthermore, GBM patient-derived BTICs displayed robust RAD51 expression and intact DNA damage response signaling in spite of PTEN-inactivating mutations. RNA expression analysis of primary GBM tissue specimens and BTICs demonstrate stable levels of RAD51 and its paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and DMC1), regardless of the PTEN mutational status. CONCLUSIONS: Our findings demonstrate definitively that PTEN loss does not alter the RAD51 expression, its paralogs, or the HR activity. Furthermore, deficiency in PTEN alone is not sufficient to impart enhanced sensitivity to PARPi associated with HRD. This study is the first to unequivocally demonstrate that PTEN deficiency is not linked to the RAD51 expression or the HR activity amongst primary neural and non-neural Pten-null cells, PTEN-deficient tumor cell lines, and primary PTEN-mutant GBM patient-derived tissue specimens and BTICs.

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

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.

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

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.

Functional genomics identifies new synergistic therapies for retinoblastoma.

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 with RB1 loss or, rarely, MYCN amplification, 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 in RB1null and RB1wt;MYCNamp orthotopic 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.

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in spheroids.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

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.

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR screens are feasible in TP53 wild-type cells.

A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.

Bmi1 regulates human glioblastoma stem cells through activation of differential gene networks in CD133+ brain tumor initiating cells.

PURPOSE: Glioblastoma (GBM) is the most aggressive adult brain cancer, with a 15 month median survivorship attributed to the existence of treatment-refractory brain tumor initiating cells (BTICs). In order to better understand the mechanisms regulating the tumorigenic properties of this population, we studied the role of the polycomb group member BMI1 in our patient-derived GBM BTICs and its relationship with CD133, a well-established marker of BTICs. METHODS: Using gain and loss-of-function studies for Bmi1 in neural stem cells (NSCs) and patient-derived GBM BTICs respectively, we assessed in vitro self-renewal and in vivo tumor formation in these two cell populations. We further explored the BMI1 transcriptional regulatory network through RNA sequencing of different GBM BTIC populations that were knocked down for Bmi1. RESULTS: There is a differential role of BMI1 in CD133-positive cells, notably involving cell metabolism. In addition, we identified pivotal targets downstream of BMI1 in CD133+ cells such as integrin alpha 2 (ITGA2), that may contribute to regulating GBM stem cell properties. CONCLUSIONS: Our work sheds light on the association of three genes with CD133-BMI1 circuitry, their importance as downstream effectors of the BMI1 signalling pathway, and their potential as future targets for tackling GBM treatment-resistant cell populations.

A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin ß6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin ß6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.

Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.

Cotargeting Ephrin Receptor Tyrosine Kinases A2 and A3 in Cancer Stem Cells Reduces Growth of Recurrent Glioblastoma.

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.