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.

Loss of the HVEM Tumor Suppressor in Lymphoma and Restoration by Modified CAR-T Cells.

The HVEM (TNFRSF14) receptor gene is among the most frequently mutated genes in germinal center lymphomas. We report that loss of HVEM leads to cell-autonomous activation of B cell proliferation and drives the development of GC lymphomas in vivo. HVEM-deficient lymphoma B cells also induce a tumor-supportive microenvironment marked by exacerbated lymphoid stroma activation and increased recruitment of T follicular helper (TFH) cells. These changes result from the disruption of inhibitory cell-cell interactions between the HVEM and BTLA (B and T lymphocyte attenuator) receptors. Accordingly, administration of the HVEM ectodomain protein (solHVEM(P37-V202)) binds BTLA and restores tumor suppression. To deliver solHVEM to lymphomas in vivo, we engineered CD19-targeted chimeric antigen receptor (CAR) T cells that produce solHVEM locally and continuously. These modified CAR-T cells show enhanced therapeutic activity against xenografted lymphomas. Hence, the HVEM-BTLA axis opposes lymphoma development, and our study illustrates the use of CAR-T cells as "micro-pharmacies" able to deliver an anti-cancer protein.

The Eph-receptor A7 is a soluble tumor suppressor for follicular lymphoma.

Insights into cancer genetics can lead to therapeutic opportunities. By cross-referencing chromosomal changes with an unbiased genetic screen we identify the ephrin receptor A7 (EPHA7) as a tumor suppressor in follicular lymphoma (FL). EPHA7 is a target of 6q deletions and inactivated in 72% of FLs. Knockdown of EPHA7 drives lymphoma development in a murine FL model. In analogy to its physiological function in brain development, a soluble splice variant of EPHA7 (EPHA7(TR)) interferes with another Eph-receptor and blocks oncogenic signals in lymphoma cells. Consistent with this drug-like activity, administration of the purified EPHA7(TR) protein produces antitumor effects against xenografted human lymphomas. Further, by fusing EPHA7(TR) to the anti-CD20 antibody (rituximab) we can directly target this tumor suppressor to lymphomas in vivo. Our study attests to the power of combining descriptive tumor genomics with functional screens and reveals EPHA7(TR) as tumor suppressor with immediate therapeutic potential.

A Rapid Translational Immune Response Program in CD8 Memory T Lymphocytes.

The activation of memory T cells is a very rapid and concerted cellular response that requires coordination between cellular processes in different compartments and on different time scales. In this study, we use ribosome profiling and deep RNA sequencing to define the acute mRNA translation changes in CD8 memory T cells following initial activation events. We find that initial translation enables subsequent events of human and mouse T cell activation and expansion. Briefly, early events in the activation of Ag-experienced CD8 T cells are insensitive to transcriptional blockade with actinomycin D, and instead depend on the translation of pre-existing mRNAs and are blocked by cycloheximide. Ribosome profiling identifies ∼92 mRNAs that are recruited into ribosomes following CD8 T cell stimulation. These mRNAs typically have structured GC and pyrimidine-rich 5' untranslated regions and they encode key regulators of T cell activation and proliferation such as Notch1, Ifngr1, Il2rb, and serine metabolism enzymes Psat1 and Shmt2 (serine hydroxymethyltransferase 2), as well as translation factors eEF1a1 (eukaryotic elongation factor α1) and eEF2 (eukaryotic elongation factor 2). The increased production of receptors of IL-2 and IFN-γ precedes the activation of gene expression and augments cellular signals and T cell activation. Taken together, we identify an early RNA translation program that acts in a feed-forward manner to enable the rapid and dramatic process of CD8 memory T cell expansion and activation.

Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System.

A promising new approach to broad spectrum antiviral drugs is the inhibition of the eukaryotic translation initiation factor 4A (elF4A), a DEAD-box RNA helicase that effectively reduces the replication of several pathogenic virus types. Beside the antipathogenic effect, modulation of a host enzyme activity could also have an impact on the immune system. Therefore, we performed a comprehensive study on the influence of elF4A inhibition with natural and synthetic rocaglates on various immune cells. The effect of the rocaglates zotatifin, silvestrol and CR-31-B (-), as well as the nonactive enantiomer CR-31-B (+), on the expression of surface markers, release of cytokines, proliferation, inflammatory mediators and metabolic activity in primary human monocyte-derived macrophages (MdMs), monocyte-derived dendritic cells (MdDCs), T cells and B cells was assessed. The inhibition of elF4A reduced the inflammatory potential and energy metabolism of M1 MdMs, whereas in M2 MdMs, drug-specific and less target-specific effects were observed. Rocaglate treatment also reduced the inflammatory potential of activated MdDCs by altering cytokine release. In T cells, the inhibition of elF4A impaired their activation by reducing the proliferation rate, expression of CD25 and cytokine release. The inhibition of elF4A further reduced B-cell proliferation, plasma cell formation and the release of immune globulins. In conclusion, the inhibition of the elF4A RNA helicase with rocaglates suppressed the function of M1 MdMs, MdDCs, T cells and B cells. This suggests that rocaglates, while inhibiting viral replication, may also suppress bystander tissue injury by the host immune system. Thus, dosing of rocaglates would need to be adjusted to prevent excessive immune suppression without reducing their antiviral activity.

NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death.

Although diffuse large B cell lymphoma (DLBCL) cells widely express the BCL2 protein, they rarely respond to treatment with BCL2-selective inhibitors. Here we show that DLBCL cells harboring PMAIP1/NOXA gene amplification were highly sensitive to BCL2 small-molecule inhibitors. In these cells, BCL2 inhibition induced cell death by activating caspase 9, which was further amplified by caspase-dependent cleavage and depletion of MCL1. In DLBCL cells lacking NOXA amplification, BCL2 inhibition was associated with an increase in MCL1 protein abundance in a BIM-dependent manner, causing a decreased antilymphoma efficacy. In these cells, dual inhibition of MCL1 and BCL2 was required for enhanced killing. Pharmacologic induction of NOXA, using the histone deacetylase inhibitor panobinostat, decreased MCL1 protein abundance and increased lymphoma cell vulnerability to BCL2 inhibitors in vitro and in vivo. Our data provide a mechanistic rationale for combination strategies to disrupt lymphoma cell codependency on BCL2 and MCL1 proteins in DLBCL.

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer.

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Targeted PET imaging strategy to differentiate malignant from inflamed lymph nodes in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults. DLBCL exhibits highly aggressive and systemic progression into multiple tissues in patients, particularly in lymph nodes. Whole-body 18F-fluodeoxyglucose positron emission tomography ([18F]FDG-PET) imaging has an essential role in diagnosing DLBCL in the clinic; however, [18F]FDG-PET often faces difficulty in differentiating malignant tissues from certain nonmalignant tissues with high glucose uptake. We have developed a PET imaging strategy for DLBCL that targets poly[ADP ribose] polymerase 1 (PARP1), the expression of which has been found to be much higher in DLBCL than in healthy tissues. In a syngeneic DLBCL mouse model, this PARP1-targeted PET imaging approach allowed us to discriminate between malignant and inflamed lymph nodes, whereas [18F]FDG-PET failed to do so. Our PARP1-targeted PET imaging approach may be an attractive addition to the current PET imaging strategy to differentiate inflammation from malignancy in DLBCL.

RiboDiff: detecting changes of mRNA translation efficiency from ribosome footprints.

MOTIVATION: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. RESULTS: We present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy. AVAILABILITY AND IMPLEMENTATION: RiboDiff webpage http://bioweb.me/ribodiff Source code including scripts for preprocessing the FASTQ data are available at http://github.com/ratschlab/ribodiff CONTACTS: zhongy@cbio.mskcc.org or raetsch@inf.ethz.chSupplementary information: Supplementary data are available at Bioinformatics online.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation.

We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, sequenced, and subjected to a second round of validation. By using this strategy, we isolated canonical genes and microRNAs that mediate metastatic reactivation in the lung. To identify genes that oppose reactivation, we screened an expression library encoding shRNAs, and we identified target genes that encode potential enforcers of dormancy. Our screening strategy enables the identification and rapid biological validation of single genetic entities that are necessary to maintain dormancy or to induce reactivation. This technology should facilitate the elucidation of the molecular underpinnings of these processes.

Generation and characterization of a single-chain anti-EphA2 antibody.

Recombinant antibody phage library technology provides multiple advantages, including that human antibodies can be generated against proteins that are highly conserved between species. We used this technology to isolate and characterize an anti-EphA2 single-chain antibody. We show that the antibody binds the antigen with 1:1 stoichiometry and has high specificity for EphA2. The crystal structure of the complex reveals that the antibody targets the same receptor surface cavity as the ephrin ligand. Specifically, a lengthy CDR-H3 loop protrudes deep into the ligand-binding cavity, with several hydrophobic residues at its tip forming an anchor-like structure buried within the hydrophobic Eph pocket, in a way similar to the ephrin receptor-binding loop in the Eph/ephrin structures. Consequently, the antibody blocks ephrin binding to EphA2. Furthermore, it induces apoptosis and reduces cell proliferation in lymphoma cells lines. Since Ephs are important mediators of tumorigenesis, such antibodies could have applications both in research and therapy.

MicroRNA-128-3p is a novel oncomiR targeting PHF6 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia arises from the leukemic transformation of developing thymocytes and results from cooperative genetic lesions. Inactivation of the PHF6 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting an important tumor suppressive role for PHF6 in the pathobiology of this leukemia. Although the precise function of PHF6 is still unknown, this gene is most likely involved in chromatin regulation, a strongly emerging theme in T-cell acute lymphoblastic leukemia. In this context, our previous description of a cooperative microRNA regulatory network controlling several well-known T-cell acute lymphoblastic leukemia tumor suppressor genes, including PHF6, is of great importance. Given the high frequency of PHF6 lesions in T-cell acute lymphoblastic leukemia and the integration of PHF6 in this microRNA regulatory network, we aimed to identify novel oncogenic microRNAs in T-cell acute lymphoblastic leukemia which suppress PHF6. To this end, we performed an unbiased PHF6 3'UTR-microRNA library screen and combined the results with microRNA profiling data of samples from patients with T-cell acute lymphoblastic leukemia and normal thymocyte subsets. We selected miR-128-3p as a candidate PHF6-targeting, oncogenic microRNA and demonstrated regulation of PHF6 expression upon modulation of this microRNA in T-cell acute lymphoblastic leukemia cell lines. In vivo evidence of an oncogenic role of this microRNA in T-cell acute lymphoblastic leukemia was obtained through accelerated leukemia onset in a NOTCH1-induced T-cell acute lymphoblastic leukemia mouse model upon miR-128-3p over-expression. We conclude that miR-128-3p is a strong novel candidate oncogenic microRNA in T-cell acute lymphoblastic leukemia which targets the PHF6 tumor suppressor gene.

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.

Synthetic lethality of drug-induced polyploidy and BCL-2 inhibition in lymphoma.

Spontaneous whole genome duplication and the adaptive mutations that disrupt genome integrity checkpoints are infrequent events in B cell lymphomas. This suggests that lymphomas might be vulnerable to therapeutics that acutely trigger genomic instability and polyploidy. Here, we report a therapeutic combination of inhibitors of the Polo-like kinase 4 and BCL-2 that trigger genomic instability and cell death in aggressive lymphomas. The synthetic lethality is selective for tumor cells and spares vital organs. Mechanistically, inhibitors of Polo-like kinase 4 impair centrosome duplication and cause genomic instability. The elimination of polyploid cells largely depends on the pro-apoptotic BAX protein. Consequently, the combination of drugs that induce polyploidy with the BCL-2 inhibitor Venetoclax is highly synergistic and safe against xenograft and PDX models. We show that B cell lymphomas are ill-equipped for acute, therapy-induced polyploidy and that BCL-2 inhibition further enhances the removal of polyploid lymphoma cells.

Rapamycin-Induced Feedback Activation of eIF4E-EIF4A Dependent mRNA Translation in Pancreatic Cancer.

Pancreatic cancer cells adapt molecular mechanisms to activate the protein synthesis to support tumor growth. This study reports the mTOR inhibitor rapamycin's specific and genome-wide effect on mRNA translation. Using ribosome footprinting in pancreatic cancer cells that lack the expression of 4EBP1, we establish the effect of mTOR-S6-dependent mRNAs translation. Rapamycin inhibits the translation of a subset of mRNAs including p70-S6K and proteins involved in the cell cycle and cancer cell growth. In addition, we identify translation programs that are activated following mTOR inhibition. Interestingly, rapamycin treatment results in the translational activation of kinases that are involved in mTOR signaling such as p90-RSK1. We further show that phospho-AKT1 and phospho-eIF4E are upregulated following mTOR inhibition suggesting a feedback activation of translation by rapamycin. Next, targeting eIF4E and eIF4A-dependent translation by using specific eIF4A inhibitors in combination with rapamycin shows significant growth inhibition in pancreatic cancer cells. In short, we establish the specific effect of mTOR-S6 on translation in cells lacking 4EBP1 and show that mTOR inhibition leads to feedback activation of translation via AKT-RSK1-eIF4E signals. Therefore, targeting translation downstream of mTOR presents a more efficient therapeutic strategy in pancreatic cancer.

Frequent 4EBP1 Amplification Induces Synthetic Dependence on FGFR Signaling in Cancer.

The eIF4E translation initiation factor has oncogenic properties and concordantly, the inhibitory eIF4E-binding protein (4EBP1) is considered a tumor suppressor. The exact molecular effects of 4EBP1 activation in cancer are still unknown. Surprisingly, 4EBP1 is a target of genomic copy number gains (Chr. 8p11) in breast and lung cancer. We noticed that 4EBP1 gains are genetically linked to gains in neighboring genes, including WHSC1L1 and FGFR1. Our results show that FGFR1 gains act to attenuate the function of 4EBP1 via PI3K-mediated phosphorylation at Thr37/46, Ser65, and Thr70 sites. This implies that not 4EBP1 but instead FGFR1 is the genetic target of Chr. 8p11 gains in breast and lung cancer. Accordingly, these tumors show increased sensitivity to FGFR1 and PI3K inhibition, and this is a therapeutic vulnerability through restoring the tumor-suppressive function of 4EBP1. Ribosome profiling reveals genes involved in insulin signaling, glucose metabolism, and the inositol pathway to be the relevant translational targets of 4EBP1. These mRNAs are among the top 200 translation targets and are highly enriched for structure and sequence motifs in their 5'UTR, which depends on the 4EBP1-EIF4E activity. In summary, we identified the translational targets of 4EBP1-EIF4E that facilitate the tumor suppressor function of 4EBP1 in cancer.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5'-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5'-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.