eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer.

The majority of human breast cancers are dependent on hormone-stimulated estrogen receptor alpha (ER) and are sensitive to its inhibition. Treatment resistance arises in most advanced cancers due to genetic alterations that promote ligand independent activation of ER itself or ER target genes. Whereas re-targeting of the ER ligand binding domain (LBD) with newer ER antagonists can work in some cases, these drugs are largely ineffective in many genetic backgrounds including ER fusions that lose the LBD or in cancers that hyperactivate ER targets. By identifying the mechanism of ER translation, we herein present an alternative strategy to target ER and difficult to treat ER variants. We find that ER translation is cap-independent and mTOR inhibitor insensitive, but dependent on 5' UTR elements and sensitive to pharmacologic inhibition of the translation initiation factor eIF4A, an mRNA helicase. EIF4A inhibition rapidly reduces expression of ER and short-lived targets of ER such as cyclin D1 and other components of the cyclin D-CDK complex in breast cancer cells. These effects translate into suppression of growth of a variety of ligand-independent breast cancer models including those driven by ER fusion proteins that lack the ligand binding site. The efficacy of eIF4A inhibition is enhanced when it is combined with fulvestrant-an ER degrader. Concomitant inhibition of ER synthesis and induction of its degradation causes synergistic and durable inhibition of ER expression and tumor growth. The clinical importance of these findings is confirmed by results of an early clinical trial (NCT04092673) of the selective eIF4A inhibitor zotatifin in patients with estrogen receptor positive metastatic breast cancer. Multiple clinical responses have been observed on combination therapy including durable regressions. These data suggest that eIF4A inhibition could be a useful new strategy for treating advanced ER+ breast cancer.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

FUS reads histone H3K36me3 to regulate alternative polyadenylation.

Complex organisms generate differential gene expression through the same set of DNA sequences in distinct cells. The communication between chromatin and RNA regulates cellular behavior in tissues. However, little is known about how chromatin, especially histone modifications, regulates RNA polyadenylation. In this study, we found that FUS was recruited to chromatin by H3K36me3 at gene bodies. The H3K36me3 recognition of FUS was mediated by the proline residues in the ZNF domain. After these proline residues were mutated or H3K36me3 was abolished, FUS dissociated from chromatin and bound more to RNA, resulting in an increase in polyadenylation sites far from stop codons genome-wide. A proline mutation corresponding to a mutation in amyotrophic lateral sclerosis contributed to the hyperactivation of mitochondria and hyperdifferentiation in mouse embryonic stem cells. These findings reveal that FUS is an H3K36me3 reader protein that links chromatin-mediated alternative polyadenylation to human disease.

Proteogenomic characterization of primary colorectal cancer and metastatic progression identifies proteome-based subtypes and signatures.

Metastatic progression of colorectal adenocarcinoma (CRC) remains poorly understood and poses significant challenges for treatment. To overcome these challenges, we performed multiomics analyses of primary CRC and liver metastases. Genomic alterations, such as structural variants or copy number alterations, were enriched in oncogenes and tumor suppressor genes and increased in metastases. Unsupervised mass spectrometry-based proteomics of 135 primary and 123 metastatic CRCs uncovered distinct proteomic subtypes, three each for primary and metastatic CRCs, respectively. Integrated analyses revealed that hypoxia, stemness, and immune signatures characterize these 6 subtypes. Hypoxic CRC harbors high epithelial-to-mesenchymal transition features and metabolic adaptation. CRC with a stemness signature shows high oncogenic pathway activation and alternative telomere lengthening (ALT) phenotype, especially in metastatic lesions. Tumor microenvironment analysis shows immune evasion via modulation of major histocompatibility complex (MHC) class I/II and antigen processing pathways. This study characterizes both primary and metastatic CRCs and provides a large proteogenomics dataset of metastatic progression.

Protocol for the quantitative identification of endogenously ISGylated proteins from mammalian cell lines.

Ubiquitin-like protein ISG15 plays an important role in an array of cellular functions via its covalent attachment to target proteins (ISGylation). Here, we present a protocol for the identification of ISGylated proteins that avoids the caveats associated with ISG15 overexpression and minimizes the likelihood of false positives. We describe steps for the tagging of endogenous ISG15, followed by genotyping and clone selection. We then detail steps for ISGylation induction, the isolation of ISGylated proteins, and their identification via quantitative mass spectrometry. For complete details on the use and execution of this protocol, please refer to Wardlaw and Petrini.1.

Integrated Metabolomics Approach Reveals the Dynamic Variations of Metabolites and Bioactivities in Paeonia ostii 'Feng Dan' Leaves during Development.

Paeonia ostii 'Feng Dan' is widely cultivated in China for its ornamental, medicinal, and edible properties. The whole plant of tree peony is rich in bioactive substances, while the comprehensive understanding of metabolites in the leaves is limited. In this study, an untargeted metabolomics strategy based on UPLC-ESI-TOF-MS was conducted to analyze the dynamic variations of bioactive metabolites in P. ostii 'Feng Dan' leaves during development. A total of 321 metabolites were rapidly annotated based on the GNPS platform, in-house database, and publications. To accurately quantify the selected metabolites, a targeted method of HPLC-ESI-QQQ-MS was used. Albiflorin, paeoniflorin, pentagalloylglucose, luteolin 7-glucoside, and benzoylpaeoniflorin were recognized as the dominant bioactive compounds with significant content variations during leaf development. Metabolite variations during the development of P. ostii 'Feng Dan' leaves are greatly attributed to the variations in antioxidant activities. Among all tested bacteria, the leaf extract exhibited exceptional inhibitory effects against Streptococcus hemolytis-ß. This research firstly provides new insights into tree peony leaves during development. The stages of S1-S2 may be the most promising harvesting time for potential use in food or pharmaceutical purposes.

Systematic evaluation of AML-associated antigens identifies anti-U5 SNRNP200 therapeutic antibodies for the treatment of acute myeloid leukemia.

Despite recent advances in the treatment of acute myeloid leukemia (AML), there has been limited success in targeting surface antigens in AML, in part due to shared expression across malignant and normal cells. Here, high-density immunophenotyping of AML coupled with proteogenomics identified unique expression of a variety of antigens, including the RNA helicase U5 snRNP200, on the surface of AML cells but not on normal hematopoietic precursors and skewed Fc receptor distribution in the AML immune microenvironment. Cell membrane localization of U5 snRNP200 was linked to surface expression of the Fcγ receptor IIIA (FcγIIIA, also known as CD32A) and correlated with expression of interferon-regulated immune response genes. Anti-U5 snRNP200 antibodies engaging activating Fcγ receptors were efficacious across immunocompetent AML models and were augmented by combination with azacitidine. These data provide a roadmap of AML-associated antigens with Fc receptor distribution in AML and highlight the potential for targeting the AML cell surface using Fc-optimized therapeutics.

Harnessing transcriptionally driven chromosomal instability adaptation to target therapy-refractory lethal prostate cancer.

Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.

Radiomics signatures for predicting the Ki-67 level and HER-2 status based on bone metastasis from primary breast cancer.

This study explores the potential of radiomics to predict the proliferation marker protein Ki-67 levels and human epidermal growth factor receptor 2 (HER-2) status based on MRI images of patients with spinal metastasis from primary breast cancer. A total of 110 patients with pathologically confirmed spinal metastases from primary breast cancer were enrolled between Dec. 2017 and Dec. 2021. All patients underwent T1-weighted contrast-enhanced MRI scans. The PyRadiomics package was used to extract features from the MRI images based on the intraclass correlation coefficient and least absolute shrinkage and selection operator. The most predictive features were used to develop the radiomics signature. The Chi-Square test, Fisher's exact test, Student's t-test, and Mann-Whitney U test were used to evaluate the clinical and pathological characteristics between the high- and low-level Ki-67 groups and the HER-2 positive/negative groups. The radiomics models were compared using receiver operating characteristic curve analysis. The area under the receiver operating characteristic curve (AUC), sensitivity (SEN), and specificity (SPE) were generated as comparison metrics. From the spinal MRI scans, five and two features were identified as the most predictive for the Ki-67 level and HER-2 status, respectively. The developed radiomics signatures generated good prediction performance for the Ki-67 level in the training (AUC = 0.812, 95% CI: 0.710-0.914, SEN = 0.667, SPE = 0.846) and validation (AUC = 0.799, 95% CI: 0.652-0.947, SEN = 0.722, SPE = 0.833) cohorts. Good prediction performance for the HER-2 status was also achieved in the training (AUC = 0.796, 95% CI: 0.686-0.906, SEN = 0.720, SPE = 0.776) and validation (AUC = 0.705, 95% CI: 0.506-0.904, SEN = 0.733, SPE = 0.762) cohorts. The results of this study provide a better understanding of the potential clinical implications of spinal MRI-based radiomics on the prediction of Ki-67 levels and HER-2 status in breast cancer.

Are Older People Really More Susceptible to SARS-CoV-2?

Since the outbreak, COVID-19 has spread rapidly across the globe due to its high infectivity and lethality. Age appears to be one of the key factors influencing the status and progression of SARS-CoV-2 infection, as multiple reports indicated that the majority of COVID-19 infections and severe cases are elderly. Most people simply assume that the elderly are more susceptible to SARS-CoV-2 than the young, but the mechanism behind it is still open to question. The older and younger people are at similar risk of infection because their infection process is the same and they must be exposed to the virus first. However, whether they will get sick after exposure to the virus and how their disease progresses depend on their immune mechanisms. In older populations, inflammation and immune aging reduce their ability to resist SARS-CoV-2 infection. Meanwhile, under the influence of comorbidities, ACE2 receptor and various cytokines undergo corresponding changes, thus accelerating the entry, replication, and transmission of SARS-CoV-2 in the body, promoting disease progression, and leading to severe illness and even death. In addition, the relatively fragile mental state of the elderly can also affect their timely recovery from COVID-19. Therefore, once older people are infected with SARS-CoV-2, they are more prone to severe illness and death with a poor prognosis, and they should strengthen protection to avoid exposure to the virus.

Positive end-expiratory pressure and risk of postoperative pulmonary complications in patients living at high altitudes and undergoing surgery at low altitudes: a single-centre, retrospective observational study in China.

OBJECTIVES: To examine whether a high positive end-expiratory pressure (PEEP ≥5 cmH2O) has a protective effect on the risk of postoperative pulmonary complications (PPCs) in a cohort of patients living at high altitudes and undergoing general anaesthesia. DESIGN: Retrospective, observational study. SETTING: A tertiary hospital in China. PARTICIPANTS: Adult Tibetan patients living at high altitudes (≥3000 m) and who went to the low-altitude plain to undergo non-cardiothoracic surgery under general anaesthesia, from January 2018 to April 2020. MEASUREMENTS: This study included 1905 patients who were divided according to the application of an intraoperative PEEP: low PEEP (<5 cmH2O, including 0 cmH2O) or high PEEP (≥5 cmH2O). The primary outcome was a composite of PPCs within the first 7 postoperative days. The secondary outcomes included reintubation and unplanned intensive care unit (ICU) admission within the first 7 postoperative days and total hospital stays (day). RESULTS: The study included 1032 patients in the low PEEP group and 873 in the high PEEP group. There were no differences in the incidence of PPCs between the high and low PEEP groups (relative risk (RR) 0.913; 95% CI 0.716 to 1.165; p=0.465). After propensity score matching, 643 patients remained in each group, and the incidence of PPCs in the low PEEP group (18.0%) was higher than in the high PEEP group (13.7%; RR 0.720; 95% CI 0.533 to 0.974; p=0.033). There were no differences in the incidence of reintubation, unplanned ICU admission or hospital stays. The risk factors of PPCs derived from multiple regression showed that the application of >5 cmH2O PEEP during intraoperative mechanical ventilation was associated with a significantly lower risk of PPCs in patients from a high altitude (OR=0.725, 95% CI 0.530 to 0.992; p=0.044). CONCLUSIONS: The application of PEEP ≥5 cmH2O during intraoperative mechanical ventilation in patients living at high altitudes and undergoing surgery at low altitudes may be associated with a lower risk of PPCs. Prospective longitudinal studies are needed to further investigate perioperative lung protection ventilation strategies for patients from high altitudes. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR2100044260).

HLA-independent T cell receptors for targeting tumors with low antigen density.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

Efficacy and Safety of "Three Chinese Patent Medicines and Three TCM Prescriptions" for COVID-19: A Systematic Review and Network Meta-Analysis.

OBJECTIVE: To systematically evaluate the efficacy, safety, and precision of TMTP for COVID-19. METHODS: Randomized controlled trials and retrospective studies were searched in 11 electronic databases. This network meta-analysis included trials using TMTP to treat patients with COVID-19. The traditional pairwise meta-analysis was done by using Stata 15, and Bayesian network meta-analysis was done with WinBUGS. RESULTS: 18 trials were included with 2036 participants and 7 drugs. The results showed that LHQW had the most significant effects on improving expectoration, shortness of breath, sore throat, nausea, emesis, inappetence, muscle soreness, and headache, and it could produce the least adverse reactions. XBJ was the best drug for fever, fatigue, and diarrhea, which showed great advantages in lowering WBC levels. XFBD was the most effective drug for cough and chest distress, which had the least exacerbation rate. JHQG was the most effective for rhinobyon and rhinorrhea, while QFPD was the best drug in decreasing CRP levels. CONCLUSION: This study was the first most large-scale and comprehensive research of TMTP for COVID-19. The results showed that LHQW had good efficacy without obvious adverse reactions. Therefore, we believe that it should be firstly recommended for COVID-19 treatment. In addition, XBJ is recommended for patients with a severe fever, fatigue, and diarrhea, and JHQG is recommended for patients with obvious rhinobyon and rhinorrhea; then, XFBD is recommended for patients with cough and chest tightness as the main manifestation. Our findings will help experts develop new COVID-19 treatment guidelines to better guide clinical medication for protecting the health of COVID-19 patients.

Copper depletion modulates mitochondrial oxidative phosphorylation to impair triple negative breast cancer metastasis.

Copper serves as a co-factor for a host of metalloenzymes that contribute to malignant progression. The orally bioavailable copper chelating agent tetrathiomolybdate (TM) has been associated with a significant survival benefit in high-risk triple negative breast cancer (TNBC) patients. Despite these promising data, the mechanisms by which copper depletion impacts metastasis are poorly understood and this remains a major barrier to advancing TM to a randomized phase II trial. Here, using two independent TNBC models, we report a discrete subpopulation of highly metastatic SOX2/OCT4+ cells within primary tumors that exhibit elevated intracellular copper levels and a marked sensitivity to TM. Global proteomic and metabolomic profiling identifies TM-mediated inactivation of Complex IV as the primary metabolic defect in the SOX2/OCT4+ cell population. We also identify AMPK/mTORC1 energy sensor as an important downstream pathway and show that AMPK inhibition rescues TM-mediated loss of invasion. Furthermore, loss of the mitochondria-specific copper chaperone, COX17, restricts copper deficiency to mitochondria and phenocopies TM-mediated alterations. These findings identify a copper-metabolism-metastasis axis with potential to enrich patient populations in next-generation therapeutic trials.

The Oncogenic Action of NRF2 Depends on De-glycation by Fructosamine-3-Kinase.

The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape.

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.

Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response.

lncRNAs make up a majority of the human transcriptome and have key regulatory functions. Here we perform unbiased de novo annotation of transcripts expressed during the human humoral immune response to find 30% of the human genome transcribed during this process, yet 58% of these transcripts manifest striking differential expression, indicating an lncRNA phylogenetic relationship among cell types that is more robust than that of coding genes. We provide an atlas of lncRNAs in naive and GC B-cells that indicates their partition into ten functionally categories based on chromatin features, DNase hypersensitivity and transcription factor localization, defining lncRNAs classes such as enhancer-RNAs (eRNA), bivalent-lncRNAs, and CTCF-associated, among others. Specifically, eRNAs are transcribed in 8.6% of regular enhancers and 36.5% of super enhancers, and are associated with coding genes that participate in critical immune regulatory pathways, while plasma cells have uniquely high levels of circular-RNAs accounted for by and reflecting the combinatorial clonal state of the Immunoglobulin loci.

Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo.

The efficacy of chimeric antigen receptor (CAR) T cell therapy against poorly responding tumors can be enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to coexpress factors that boost CAR-T cell function in the tumor microenvironment. We modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells acted in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. The efficacy was similar to or better than that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach may improve safety, as the secreted scFvs remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

BCL6 Antagonizes NOTCH2 to Maintain Survival of Human Follicular Lymphoma Cells.

Although the BCL6 transcriptional repressor is frequently expressed in human follicular lymphomas (FL), its biological role in this disease remains unknown. Herein, we comprehensively identify the set of gene promoters directly targeted by BCL6 in primary human FLs. We noted that BCL6 binds and represses NOTCH2 and NOTCH pathway genes. Moreover, BCL6 and NOTCH2 pathway gene expression is inversely correlated in FL. Notably, BCL6 upregulation is associated with repression of NOTCH2 and its target genes in primary human and murine germinal center (GC) cells. Repression of NOTCH2 is an essential function of BCL6 in FL and GC B cells because inducible expression of Notch2 abrogated GC formation in mice and killed FL cells. Indeed, BCL6-targeting compounds or gene silencing leads to the induction of NOTCH2 activity and compromises survival of FL cells, whereas NOTCH2 depletion or pathway antagonists rescue FL cells from such effects. Moreover, BCL6 inhibitors induced NOTCH2 expression and suppressed growth of human FL xenografts in vivo and primary human FL specimens ex vivo These studies suggest that established FLs are thus dependent on BCL6 through its suppression of NOTCH2Significance: We show that human FLs are dependent on BCL6, and primary human FLs can be killed using specific BCL6 inhibitors. Integrative genomics and functional studies of BCL6 in primary FL cells point toward a novel mechanism whereby BCL6 repression of NOTCH2 drives the survival and growth of FL cells as well as GC B cells, which are the FL cell of origin. Cancer Discov; 7(5); 506-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 443.