Epigenetic loss of the transfer RNA-modifying enzyme TYW2 induces ribosome frameshifts in colon cancer.

Transfer RNA (tRNA) activity is tightly regulated to provide a physiological protein translation, and tRNA chemical modifications control its function in a complex with ribosomes and messenger RNAs (mRNAs). In this regard, the correct hypermodification of position G37 of phenylalanine-tRNA, adjacent to the anticodon, is critical to prevent ribosome frameshifting events. Here we report that the tRNA-yW Synthesizing Protein 2 (TYW2) undergoes promoter hypermethylation-associated transcriptional silencing in human cancer, particularly in colorectal tumors. The epigenetic loss of TYW2 induces guanosine hypomodification in phenylalanine-tRNA, an increase in -1 ribosome frameshift events, and down-regulation of transcripts by mRNA decay, such as of the key cancer gene ROBO1. Importantly, TYW2 epigenetic inactivation is linked to poor overall survival in patients with early-stage colorectal cancer, a finding that could be related to the observed acquisition of enhanced migration properties and epithelial-to-mesenchymal features in the colon cancer cells that harbor TYW2 DNA methylation-associated loss. These findings provide an illustrative example of how epigenetic changes can modify the epitranscriptome and further support a role for tRNA modifications in cancer biology.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

In vivo CRISPR-Cas9 inhibition of hepatic LDH as treatment of primary hyperoxaluria.

Genome-editing strategies, especially CRISPR-Cas9 systems, have substantially increased the efficiency of innovative therapeutic approaches for monogenic diseases such as primary hyperoxalurias (PHs). We have previously demonstrated that inhibition of glycolate oxidase using CRISPR-Cas9 systems represents a promising therapeutic option for PH type I (PH1). Here, we extended our work evaluating the efficacy of liver-specific inhibition of lactate dehydrogenase (LDH), a key enzyme responsible for converting glyoxylate to oxalate; this strategy would not be limited to PH1, being applicable to other PH subtypes. In this work, we demonstrate a liver-specific inhibition of LDH that resulted in a drastic reduction of LDH levels in the liver of PH1 and PH3 mice after a single-dose delivery of AAV8 vectors expressing the CRISPR-Cas9 system, resulting in reduced urine oxalate levels and kidney damage without signs of toxicity. Deep sequencing analysis revealed that this approach was safe and specific, with no off-targets detected in the liver of treated animals and no on-target/off-tissue events. Altogether, our data provide evidence that in vivo genome editing using CRISPR-Cas9 systems would represent a valuable tool for improved therapeutic approaches for PH.

SimiC enables the inference of complex gene regulatory dynamics across cell phenotypes.

Single-cell RNA-Sequencing has the potential to provide deep biological insights by revealing complex regulatory interactions across diverse cell phenotypes at single-cell resolution. However, current single-cell gene regulatory network inference methods produce a single regulatory network per input dataset, limiting their capability to uncover complex regulatory relationships across related cell phenotypes. We present SimiC, a single-cell gene regulatory inference framework that overcomes this limitation by jointly inferring distinct, but related, gene regulatory dynamics per phenotype. We show that SimiC uncovers key regulatory dynamics missed by previously proposed methods across a range of systems, both model and non-model alike. In particular, SimiC was able to uncover CAR T cell dynamics after tumor recognition and key regulatory patterns on a regenerating liver, and was able to implicate glial cells in the generation of distinct behavioral states in honeybees. SimiC hence establishes a new approach to quantitating regulatory architectures between distinct cellular phenotypes, with far-reaching implications for systems biology.

CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Epigenetic loss of the endoplasmic reticulum-associated degradation inhibitor SVIP induces cancer cell metabolic reprogramming.

The endoplasmic reticulum (ER) of cancer cells needs to adapt to the enhanced proteotoxic stress associated with the accumulation of unfolded, misfolded and transformation-associated proteins. One way by which tumors thrive in the context of ER stress is by promoting ER-Associated Degradation (ERAD), although the mechanisms are poorly understood. Here, we show that the Small p97/VCP Interacting Protein (SVIP), an endogenous inhibitor of ERAD, undergoes DNA hypermethylation-associated silencing in tumorigenesis to achieve this goal. SVIP exhibits tumor suppressor features and its recovery is associated with increased ER stress and growth inhibition. Proteomic and metabolomic analyses show that cancer cells with epigenetic loss of SVIP are depleted in mitochondrial enzymes and oxidative respiration activity. This phenotype is reverted upon SVIP restoration. The dependence of SVIP hypermethylated cancer cells on aerobic glycolysis and glucose was also associated with sensitivity to an inhibitor of the glucose transporter GLUT1. This could be relevant to the management of tumors carrying SVIP epigenetic loss, because these occur in high-risk patients who manifest poor clinical outcomes. Overall, our study provides insights into how epigenetics helps deal with ER stress and how SVIP epigenetic loss in cancer may be amenable to therapies that target glucose transporters.

The transcribed pseudogene RPSAP52 enhances the oncofetal HMGA2-IGF2BP2-RAS axis through LIN28B-dependent and independent let-7 inhibition.

One largely unknown question in cell biology is the discrimination between inconsequential and functional transcriptional events with relevant regulatory functions. Here, we find that the oncofetal HMGA2 gene is aberrantly reexpressed in many tumor types together with its antisense transcribed pseudogene RPSAP52. RPSAP52 is abundantly present in the cytoplasm, where it interacts with the RNA binding protein IGF2BP2/IMP2, facilitating its binding to mRNA targets, promoting their translation by mediating their recruitment on polysomes and enhancing proliferative and self-renewal pathways. Notably, downregulation of RPSAP52 impairs the balance between the oncogene LIN28B and the tumor suppressor let-7 family of miRNAs, inhibits cellular proliferation and migration in vitro and slows down tumor growth in vivo. In addition, high levels of RPSAP52 in patient samples associate with a worse prognosis in sarcomas. Overall, we reveal the roles of a transcribed pseudogene that may display properties of an oncofetal master regulator in human cancers.

Epigenetic prediction of response to anti-PD-1 treatment in non-small-cell lung cancer: a multicentre, retrospective analysis.

BACKGROUND: Anti-programmed death-1 (PD-1) treatment for advanced non-small-cell lung cancer (NSCLC) has improved the survival of patients. However, a substantial percentage of patients do not respond to this treatment. We examined the use of DNA methylation profiles to determine the efficacy of anti-PD-1 treatment in patients recruited with current stage IV NSCLC. METHODS: In this multicentre study, we recruited adult patients from 15 hospitals in France, Spain, and Italy who had histologically proven stage IV NSCLC and had been exposed to PD-1 blockade during the course of the disease. The study structure comprised a discovery cohort to assess the correlation between epigenetic features and clinical benefit with PD-1 blockade and two validation cohorts to assess the validity of our assumptions. We first established an epigenomic profile based on a microarray DNA methylation signature (EPIMMUNE) in a discovery set of tumour samples from patients treated with nivolumab or pembrolizumab. The EPIMMUNE signature was validated in an independent set of patients. A derived DNA methylation marker was validated by a single-methylation assay in a validation cohort of patients. The main study outcomes were progression-free survival and overall survival. We used the Kaplan-Meier method to estimate progression-free and overall survival, and calculated the differences between the groups with the log-rank test. We constructed a multivariate Cox model to identify the variables independently associated with progression-free and overall survival. FINDINGS: Between June 23, 2014, and May 18, 2017, we obtained samples from 142 patients: 34 in the discovery cohort, 47 in the EPIMMUNE validation cohort, and 61 in the derived methylation marker cohort (the T-cell differentiation factor forkhead box P1 [FOXP1]). The EPIMMUNE signature in patients with stage IV NSCLC treated with anti-PD-1 agents was associated with improved progression-free survival (hazard ratio [HR] 0·010, 95% CI 3·29 × 10-4-0·0282; p=0·0067) and overall survival (0·080, 0·017-0·373; p=0·0012). The EPIMMUNE-positive signature was not associated with PD-L1 expression, the presence of CD8+ cells, or mutational load. EPIMMUNE-negative tumours were enriched in tumour-associated macrophages and neutrophils, cancer-associated fibroblasts, and senescent endothelial cells. The EPIMMUNE-positive signature was associated with improved progression-free survival in the EPIMMUNE validation cohort (0·330, 0·149-0·727; p=0·0064). The unmethylated status of FOXP1 was associated with improved progression-free survival (0·415, 0·209-0·802; p=0·0063) and overall survival (0·409, 0·220-0·780; p=0·0094) in the FOXP1 validation cohort. The EPIMMUNE signature and unmethylated FOXP1 were not associated with clinical benefit in lung tumours that did not receive immunotherapy. INTERPRETATION: Our study shows that the epigenetic milieu of NSCLC tumours indicates which patients are most likely to benefit from nivolumab or pembrolizumab treatments. The methylation status of FOXP1 could be associated with validated predictive biomarkers such as PD-L1 staining and mutational load to better select patients who will experience clinical benefit with PD-1 blockade, and its predictive value should be evaluated in prospective studies. FUNDING: "Obra Social" La Caixa, Cellex Foundation, and the Health and Science Departments of the Generalitat de Catalunya.