In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target.

Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.

A community challenge to predict clinical outcomes after immune checkpoint blockade in non-small cell lung cancer.

BACKGROUND: Predictive biomarkers of immune checkpoint inhibitor (ICI) efficacy are currently lacking for non-small cell lung cancer (NSCLC). Here, we describe the results from the Anti-PD-1 Response Prediction DREAM Challenge, a crowdsourced initiative that enabled the assessment of predictive models by using data from two randomized controlled clinical trials (RCTs) of ICIs in first-line metastatic NSCLC. METHODS: Participants developed and trained models using public resources. These were evaluated with data from the CheckMate 026 trial (NCT02041533), according to the model-to-data paradigm to maintain patient confidentiality. The generalizability of the models with the best predictive performance was assessed using data from the CheckMate 227 trial (NCT02477826). Both trials were phase III RCTs with a chemotherapy control arm, which supported the differentiation between predictive and prognostic models. Isolated model containers were evaluated using a bespoke strategy that considered the challenges of handling transcriptome data from clinical trials. RESULTS: A total of 59 teams participated, with 417 models submitted. Multiple predictive models, as opposed to a prognostic model, were generated for predicting overall survival, progression-free survival, and progressive disease status with ICIs. Variables within the models submitted by participants included tumor mutational burden (TMB), programmed death ligand 1 (PD-L1) expression, and gene-expression-based signatures. The best-performing models showed improved predictive power over reference variables, including TMB or PD-L1. CONCLUSIONS: This DREAM Challenge is the first successful attempt to use protected phase III clinical data for a crowdsourced effort towards generating predictive models for ICI clinical outcomes and could serve as a blueprint for similar efforts in other tumor types and disease states, setting a benchmark for future studies aiming to identify biomarkers predictive of ICI efficacy. TRIAL REGISTRATION: CheckMate 026; NCT02041533, registered January 22, 2014. CheckMate 227; NCT02477826, registered June 23, 2015.

TISMO: syngeneic mouse tumor database to model tumor immunity and immunotherapy response.

Syngeneic mouse models are tumors derived from murine cancer cells engrafted on genetically identical mouse strains. They are widely used tools for studying tumor immunity and immunotherapy response in the context of a fully functional murine immune system. Large volumes of syngeneic mouse tumor expression profiles under different immunotherapy treatments have been generated, although a lack of systematic collection and analysis makes data reuse challenging. We present Tumor Immune Syngeneic MOuse (TISMO), a database with an extensive collection of syngeneic mouse model profiles with interactive visualization features. TISMO contains 605 in vitro RNA-seq samples from 49 syngeneic cancer cell lines across 23 cancer types, of which 195 underwent cytokine treatment. TISMO also includes 1518 in vivo RNA-seq samples from 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade (ICB) studies. We manually annotated the sample metadata, such as cell line, mouse strain, transplantation site, treatment, and response status, and uniformly processed and quality-controlled the RNA-seq data. Besides data download, TISMO provides interactive web interfaces to investigate whether specific gene expression, pathway enrichment, or immune infiltration level is associated with differential immunotherapy response. TISMO is available at http://tismo.cistrome.org.

Novel stop-gain RNF170 variation detected in a Chinese family with adolescent-onset hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of inherited neurodegenerative disease characterized by progressive lower limb spasticity. Recent studies revealed that biallelic variants in RNF170 gene cause autosomal recessive complicated HSP with infancy onset. Here, we report an adolescent-onset HSP patient from a consanguineous Chinese family, with lower extremity stiffness, spastic gait, and unstable straight-line walking as the main manifestations. Whole-exome sequencing identifies a novel RNF170 mutation c.190C>T (p.R64*), which co-segregates with the disease in this pedigree. Functional analysis, including quantitative real-time PCR (RT-qPCR) and Western blot, indicates that both the mRNA and protein levels of mutant RNF170 are significantly reduced, which confirms the loss-of-function mechanism. Our study expands the spectrum of RNF170-associated HSP, while the RNF170 protein-involved degradation of the inositol 1,4,5-trisphosphate receptor in neurodegenerative motor neuron disorders deserves further investigation.

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli.

Protein N-glycosylation is a common post-translational modification that plays significant roles on the structure, property, and function of glycoproteins. Due to N-glycan heterogeneity of naturally occurring glycoproteins, the functions of specific N-glycans on a particular glycoprotein are not always clear. Glycoprotein in vitro N-glycan engineering using purified recombinant enzymes is an attractive strategy to produce glycoproteins with homogeneous N-glycoforms to elucidate the specific functions of N-glycans and develop better glycoprotein therapeutics. Toward this goal, we have successfully expressed in E. coli glycoside hydrolases and glycosyltransferases from bacterial and human origins and developed a robust enzymatic platform for in vitro processing glycoprotein N-glycans from high-mannose-type to α2-6- or α2-3-disialylated biantennary complex type. The recombinant enzymes are highly efficient in step-wise or one-pot reactions. The platform can find broad applications in N-glycan engineering of therapeutic glycoproteins.

Enhanced tumor accumulation and therapeutic efficacy of liposomal drugs through over-threshold dosing.

BACKGROUND: Most intravenously administered drug-loaded nanoparticles are taken up by liver Kupffer cells, and only a small portion can accumulate at the tumor, resulting in an unsatisfactory therapeutic efficacy and side effects for chemotherapeutic agents. Tumor-targeted drug delivery proves to be the best way to solve this problem; however, the complex synthesis, or surface modification process, together with the astonishing high cost make its clinical translation nearly impossible. METHODS: Referring to Ouyang's work and over-threshold dosing theory in general, blank PEGylated liposomes (PEG-Lipo) were prepared and used as tumor delivery enhancers to determine whether they could significantly enhance the tumor accumulation and in vivo antitumor efficacy of co-injected liposomal ACGs (PEG-ACGs-Lipo), a naturally resourced chemotherapeutic. Here, the phospholipid dose was used as an indicator of the number of liposomes particles with similar particle sizes, and the liposomes was labelled with DiR, a near-red fluorescent probe, to trace their in vivo biodistribution. Two mouse models, 4T1-bearing and U87-bearing, were employed for in vivo examination. RESULTS: PEG-Lipo and PEG-ACGs-Lipo had similar diameters. At a low-threshold dose (12 mg/kg equivalent phospholipids), PEG-Lipo was mainly distributed in the liver rather than in the tumor, with the relative tumor targeting index (RTTI) being ~ 0.38 at 72 h after administration. When over-threshold was administered (50 mg/kg or 80 mg/kg of equivalent phospholipids), a much higher and quicker drug accumulation in tumors and a much lower drug accumulation in the liver were observed, with the RTTI increasing to ~ 0.9. The in vivo antitumor study in 4T1 tumor-bearing mice showed that, compared to PEG-ACGs-Lipo alone (2.25 mg/kg phospholipids), the co-injection of a large dose of blank PEG-Lipo (50 mg/kg of phospholipids) significantly reduced the tumor volume of the mice by 22.6% (P < 0.05) and enhanced the RTTI from 0.41 to 1.34. The intravenous injection of a low drug loading content (LDLC) of liposomal ACGs (the same dose of ACGs at 50 mg/kg of equivalent phospholipids) achieved a similar tumor inhibition rate (TIR) to that of co-injection. In the U87 MG tumor-bearing mouse model, co-injection of the enhancer also significantly promoted the TIR (83.32% vs. 66.80%, P < 0.05) and survival time of PEG-ACGs-Lipo. CONCLUSION: An over-threshold dosing strategy proved to be a simple and feasible way to enhance the tumor delivery and antitumor efficacy of nanomedicines and was benefited to benefit their clinical result, especially for liposomal drugs.

TIMER2.0 for analysis of tumor-infiltrating immune cells.

Tumor progression and the efficacy of immunotherapy are strongly influenced by the composition and abundance of immune cells in the tumor microenvironment. Due to the limitations of direct measurement methods, computational algorithms are often used to infer immune cell composition from bulk tumor transcriptome profiles. These estimated tumor immune infiltrate populations have been associated with genomic and transcriptomic changes in the tumors, providing insight into tumor-immune interactions. However, such investigations on large-scale public data remain challenging. To lower the barriers for the analysis of complex tumor-immune interactions, we significantly improved our previous web platform TIMER. Instead of just using one algorithm, TIMER2.0 (http://timer.cistrome.org/) provides more robust estimation of immune infiltration levels for The Cancer Genome Atlas (TCGA) or user-provided tumor profiles using six state-of-the-art algorithms. TIMER2.0 provides four modules for investigating the associations between immune infiltrates and genetic or clinical features, and four modules for exploring cancer-related associations in the TCGA cohorts. Each module can generate a functional heatmap table, enabling the user to easily identify significant associations in multiple cancer types simultaneously. Overall, the TIMER2.0 web server provides comprehensive analysis and visualization functions of tumor infiltrating immune cells.

Pterostilbene nanoparticles with small particle size show excellent anti-breast cancer activityin vitroandin vivo.

Pterostilbene (PTE) is known as resveratrol of the next generation and it has attracted extensive attention in recent years. PTE can inhibit the growth of a variety of tumor cells. To overcome the problem of insolubility, PTE was loaded into nanoparticles (NPs) by anti-solvent precipitation technique using soybean lecithin (SPC) and D-α-tocopheryl polyethylene glycol succinate (TPGS) as stabilizers. The obtained PTE-NPs had an average particle size of 71.0 nm, a polydispersity index （PDI） value of 0.258, and a high zeta potential of -40.8 mV. PTE-NPs can maintain particle size stability in various physiological media. The entrapment efficiency of PTE-NPs was 98.24%. And the apparently water solubility of PTE-NPs was about 53 times higher than the solubility of PTE (54.41µg ml-1v-1s-1. 2.89 mg ml-1). M-1T-1T-1assay showed that the antitumor activity of PTE-NPs on 4T1 breast cancer cells, MCF-7 breast cancer cells and Hela cervical cancer cells was significantly increased by 4, 6 and 8 times than that of free PTE, respectively.In vivostudies have shown that PTE-NPs has a certain dose dependence. When injected intraperitoneally, PTE-NPs showed a similar therapeutic effect as paclitaxel injection (TIR was 57.53% versus 57.23%) against 4T1 tumor-bearing mice. This should be due to the improved bioavailability of the drug caused by nano-drug delivery system (nano-DDS). These results indicate that PTE-NPs may be a clinically promising anti-tumor drug for breast cancer treatment.

Soybean lecithin stabilizes disulfiram nanosuspensions with a high drug-loading content: remarkably improved antitumor efficacy.

Disulfiram (DSF) has been considered as "Repurposing drug" in cancer therapy in recent years based on its good antitumor efficacy. DSF is traditionally used as an oral drug in the treatment of alcoholism. To overcome its rapid degradation and instability, DSF nanosuspensions (DSF/SPC-NSps) were prepared using soybean lecithin (SPC) as a stabilizer of high drug-loaded content (44.36 ± 1.09%). Comprehensive characterization of the nanosuspensions was performed, and cell cytotoxicity, in vivo antitumor efficacy and biodistribution were studied. DSF/SPC-NSps, having a spherical appearance with particle size of 155 nm, could remain very stable in different physiological media, and sustained release. The in vitro MTT assay indicated that the cytotoxicity of DSF/SPC-NSps was enhanced remarkably compared to free DSF against the 4T1 cell line. The IC50 value decreased by 11-fold (1.23 vs. 13.93 µg/mL, p < 0.01). DSF/SPC-NSps groups administered via intravenous injections exhibited better antitumor efficacy compared to the commercial paclitaxel injection (PTX injection) and had a dose-dependent effect in vivo. Notably, DSF/SPC-NSps exhibited similar antitumor activity following oral administration as PTX administration via injection into a vein. These results suggest that the prepared nanosuspensions can be used as a stable delivery vehicle for disulfiram, which has potential application in breast cancer chemotherapy.

Simple preparation and greatly improved oral bioavailability: The supersaturated drug delivery system of quercetin based on PVP K30.

Quercetin, as a representative flavonoid, is widely present in daily diet and has been developed as a dietary supplement due to its beneficial physiological activities. However, the application of quercetin is limited due to its poor water solubility and extensive metabolism. So far, the nano-drug delivery systems designed to improve its bioavailability generally have the shortcomings of low drug loading content and difficulty in industrial production. In order to tackle these problems, quercetin supersaturated drug delivery system (QSDDS) was successfully prepared using solvent method, for which PVP K30 was employed as a crystallization and precipitation inhibitor to maintain the supersaturated state of quercetin in aqueous system. The obtained QSDDS, with a relative high drug loading content of 13%, could quickly disperse in water and form colloidal system with the mean particle size of about 200 nm, meanwhile induce the generation of supersaturated quercetin solution more than 12 h. In vivo pharmacokinetic study proved that QSDDS achieved a high absolute bioavailability of 36.05%, 10 times as that of physical quercetin suspension, which was dose-dependent with higher bioavailability at higher dose. Considering the simple preparation method, QSDDS provided a feasible strategy and a simple way to improve oral absorption of insoluble flavonoids.

Both gain- and loss-of-function variants of KCNA1 are associated with paroxysmal kinesigenic dyskinesia.

KCNA1 is the coding gene for Kv1.1 voltage-gated potassium-channel α subunit. Three variants of KCNA1 have been reported to manifest as paroxysmal kinesigenic dyskinesia (PKD), but the correlation between them remains unclear due to the phenotypic complexity of KCNA1 variants as well as the rarity of PKD cases. Using the whole exome sequencing followed by Sanger sequencing, we screen for potential pathogenic KCNA1 variants in patients clinically diagnosed with paroxysmal movement disorders and identify three previously unreported missense variants of KCNA1 in three unrelated Chinese families. The proband of one family (c.496G>A, p.A166T) manifests as episodic ataxia type 1, and the other two (c.877G>A, p.V293I and c.1112C>A, p.T371A) manifest as PKD. The pathogenicity of these variants is confirmed by functional studies, suggesting that p.A166T and p.T371A cause a loss-of-function of the channel, while p.V293I leads to a gain-of-function with the property of voltage-dependent gating and activation kinetic affected. By reviewing the locations of PKD-manifested KCNA1 variants in Kv1.1 protein, we find that these variants tend to cluster around the pore domain, which is similar to epilepsy. Thus, our study strengthens the correlation between KCNA1 variants and PKD and provides more information on genotype-phenotype correlations of KCNA1 channelopathy.

Mitochondrial-Targeted Triphenylphosphonium-Hydroxycamptothecin Conjugate and Its Nano-Formulations for Breast Cancer Therapy: In Vitro and In Vivo Investigation.

Mitochondria are involved in various stages of cancer cell diffusion and metastasis. Therefore, targeting tumor mitochondria with antineoplastic medicines to cause mitochondria to initiate apoptosis may be an effective strategy for cancer therapy. Here, in order to enhance the anti-tumor efficacy of the antineoplastic agent hydroxycamptothecin (HCPT), the mitochondrial targeting ligand 4-(carboxybutyl) triphenylphosphine bromide (TPP) was attached to HCPT by an ester linkage. The resultant TPP-HCPT (TH) conjugate could self-assemble into nano-aggregates, with a mean particle size of 203.2 nm and a polydispersity index (PDI) value of 0.312. The TH conjugate could also co-assembly with mPEG3000-PLGA5000 into nanoparticles (TH-NPs), with a mean diameter of 86.41 nm, a PDI value of 0.256 and a zeta potential of -0.125 mV. In contrast to HCPT injections, TH aggregates displayed enhanced cellular uptake, mitochondria-targetability and cytotoxicity against 4T1 cells, while TH-NPs showed even better improvement than TH aggregates. In the in vivo study, TH aggregates displayed higher anti-tumor efficacy in 4T1 tumor-bearing mice than HCPT injections (tumor inhibition rate, 55.71% vs. 69.17%), and TH-NPs displayed more superior anti-tumor effects (tumor inhibition rate, 80.02%). In conclusion, our research demonstrated that the TPP-HCPT conjugate and its nano-formulations, including TH aggregates and TH-NPs, may be a promising mitochondria-targeting anticancer medicine for cancer therapy. As far as we know, this is the first report in which TPP and HCPT have been conjugated directly for this aim.

A γ-Glutamyl Transpeptidase (GGT)-Triggered Charge Reversal Drug-Delivery System for Cervical Cancer Treatment: In Vitro and In Vivo Investigation.

Neutral/negatively charged nanoparticles are beneficial to reduce plasma protein adsorption and prolong their blood circulation time, while positively charged nanoparticles easily transverse the blood vessel endothelium into a tumor and easily penetrate the depth of the tumor via transcytosis. Γ-Glutamyl transpeptidase (GGT) is overexpressed on the external surface of endothelial cells of tumor blood vessels and metabolically active tumor cells. Nanocarriers modified by molecules containing γ-glutamyl moieties (such as glutathione, G-SH) can maintain a neutral/negative charge in the blood, as well as can be easily hydrolyzed by the GGT enzymes to expose the cationic surface at the tumor site, thus achieving good tumor accumulation via charge reversal. In this study, DSPE-PEG2000-GSH (DPG) was synthesized and used as a stabilizer to generate paclitaxel (PTX) nanosuspensions for the treatment of Hela cervical cancer (GGT-positive). The obtained drug-delivery system (PTX-DPG nanoparticles) was 164.6 ± 3.1 nm in diameter with a zeta potential of -9.85 ± 1.03 mV and a high drug-loaded content of 41.45 ± 0.7%. PTX-DPG NPs maintained their negative surface charge in a low concentration of GGT enzyme (0.05 U/mL), whereas they showed a significant charge-reversal property in the high-concentration solution of GGT enzyme (10 U/mL). After intravenous administration, PTX-DPG NPs mainly accumulated more in the tumor than in the liver, achieved good tumor-targetability, and significantly improved anti-tumor efficacy (68.48% vs. 24.07%, tumor inhibition rate, p < 0.05 in contrast to free PTX). This kind of GGT-triggered charge-reversal nanoparticle is promising to be a novel anti-tumor agent for the effective treatment of such GGT-positive cancers as cervical cancer.

Ginsenoside Rb1 stabilized and paclitaxel / protopanaxadiol co-loaded nanoparticles for synergistic treatment of breast tumor.

Ginsenosides are the major and key components for ginseng to exert its wide and beneficial therapeutic efficacy in clinic. Meanwhile, many ginsenosides and their metabolites showed in vitro an in vivo anti-tumor activity, among which ginsenoside Rb1 has attracted much attention due to its good solubility and amphipathy. In this study, the self-assembly behavior of Rb1 was investigated and the Rb1 nano-assembly could further stabilize or encapsulated hydrophobic drugs such as protopanaxadiol (PPD) and paclitaxel (PTX) to form nanoparticles, based on which, a natural nanoscale drug delivery system, ginsenoside Rb1 stabilized and PTX/PPD co-loaded nanoparticles (GPP NPs) were prepared. The resultant GPP NPs exhibited a small particle size of 126.2 nm, a narrow size distribution (PDI=0.145), and a zeta potential of -27.3 mV. PTX loading content was 11.06% with an encapsulation efficiency of 93.86%. GPP NPs were spherical and stable in normal saline, 5% glucose, PBS, plasma, or on-shelf storage for 7 days. Both PTX and PPD existed in an amorphous state in GPP NPs and were released in a sustained pattern. GPP NPs showed 10-fold higher in vitro anti-tumor activity of than PTX injections. In the in vivo experiment, GPP NPs achieved a much higher tumor inhibition rate than PTX injections (64.95% vs 43.17%, P < 0.01) and certain tumor target ability. In conclusion, GPP NPs had significantly enhanced anti-tumor efficacy and improved tumor microenvironment, thus were promising to be developed into a novel anti-tumor agent for the treatment of breast tumor.

Cancer Cell Resistance to IFNγ Can Occur via Enhanced Double-Strand Break Repair Pathway Activity.

The pleiotropic cytokine interferon-gamma (IFNγ) is associated with cytostatic, antiproliferation, and proapoptotic functions in cancer cells. However, resistance to IFNγ occurs in many cancer cells, and the underlying mechanism is not fully understood. To investigate potential IFNγ-resistance mechanisms, we performed IFNγ-sensitivity screens in more than 40 cancer cell lines and characterized the sensitive and resistant cell lines. By applying CRISPR screening and transcriptomic profiling in both IFNγ-sensitive and IFNγ-resistant cells, we discovered that activation of double-strand break (DSB) repair genes could result in IFNγ resistance in cancer cells. Suppression of single-strand break (SSB) repair genes increased the dependency on DSB repair genes after IFNγ treatment. Furthermore, inhibition of the DSB repair pathway exhibited a synergistic effect with IFNγ treatment both in vitro and in vivo. The relationship between the activation of DSB repair genes and IFNγ resistance was further confirmed in clinical tumor profiles from The Cancer Genome Atlas (TCGA) and immune checkpoint blockade (ICB) cohorts. Our study provides comprehensive resources and evidence to elucidate a mechanism of IFNγ resistance in cancer and has the potential to inform combination therapies to overcome immunotherapy resistance.

Tutorial: integrative computational analysis of bulk RNA-sequencing data to characterize tumor immunity using RIMA.

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification. We describe the RNA-seq IMmune Analysis (RIMA) pipeline integrating many of these tools to streamline RNA-seq analysis. We also developed a comprehensive and user-friendly guide in the form of a GitBook with text and video demos to assist users in analyzing bulk RNA-seq data for immune characterization at both individual sample and cohort levels by using RIMA.

Discovery of Targets for Immune-Metabolic Antitumor Drugs Identifies Estrogen-Related Receptor Alpha.

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune-metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti-PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms. SIGNIFICANCE: BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms. This article is highlighted in the In This Issue feature, p. 517.

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

A simple but efficient tumor-targeted nanoparticle delivery system constructed by oleic acid.

Oleic acid (OA) is a kind of monounsaturated omega-3 fatty acid that abounds in plants and animals which can induce apoptosis and has broad-spectrum inhibitory activity against a variety of tumor cell lines. However, OA is quite insoluble and thus inconvenient to be efficiently delivered in vivo. In this work, OA was fabricated into nanoparticles to generate OA elastic nanoparticles (OA-ENPs) with a particle size of 185.6 nm and good stability in various physiological media. OA-ENPs alone achieved a high tumor inhibition rate of 60.3% without significant side effect. More surprisingly, the resultant OA-ENPs displayed dose-dependent tumor targetability. Low dose of OA-ENPs (10 mg/kg) mainly distributed in the liver after intravenous injection, while high dose of OA-ENPs mainly distributed in tumor. At the high dose of 90 mg/kg, OA-ENPs accumulation in tumor reached nearly twice as that in the liver. Here we provide a simple but effective way to achieve excellent tumor targetability without the need of any surface modification of nanoparticles.