Persistent Cancer Cells: The Deadly Survivors.

Persistent cancer cells are the discrete and usually undetected cells that survive cancer drug treatment and constitute a major cause of treatment failure. These cells are characterized by their slow proliferation, highly flexible energy consumption, adaptation to their microenvironment, and phenotypic plasticity. Mechanisms that underlie their persistence offer highly coveted and sought-after therapeutic targets, and include diverse epigenetic, transcriptional, and translational regulatory processes, as well as complex cell-cell interactions. Although the successful clinical targeting of persistent cancer cells remains to be realized, immense progress has been made in understanding their persistence, yielding promising preclinical results.

Boosting Immunity by Targeting Post-translational Prenylation of Small GTPases.

Diseases leading to immune activation and autoinflammatory phenotypes may provide a reservoir of potentially druggable pathways for optimizing immune adjuvants or boosting antitumor immune responses. Now, Xia et al. report that lipophilic statins or biphosphonates, targeting the mevalonate pathway, act as efficient vaccine adjuvants and synergize with anti-PD1 against cancer.

Post-transcriptional polyadenylation site cleavage maintains 3'-end processing upon DNA damage.

The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

Genotoxic stress impacts pre-mRNA 3'-end processing.

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress. Here we highlight recent advances illustrating the intricate interplay between pre-mRNA processing, with a focus on 3'-end processing, and genotoxic stress response.

Reciprocal Links between Pre-messenger RNA 3'-End Processing and Genome Stability.

The 3'-end processing of most pre-messenger RNAs (pre-mRNAs) involves RNA cleavage and polyadenylation and is coupled to transcription termination. In both yeast and human cells, pre-mRNA 3'-end cleavage is globally inhibited by DNA damage. Recently, further links between pre-mRNA 3'-end processing and the control of genome stability have been uncovered, as reviewed here. Upon DNA damage, various genes related to the DNA damage response (DDR) escape 3'-end processing inhibition or are regulated through alternative polyadenylation (APA). Conversely, various pre-mRNA 3'-end processing factors prevent genome instability and are found at sites of DNA damage. Finally, the reciprocal link between pre-mRNA 3'-end processing and genome stability control seems important because it is conserved in evolution and involved in disease development.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Compartment-specific and ELAVL1-coordinated regulation of intronic polyadenylation isoforms by doxorubicin.

Intronic polyadenylation (IPA) isoforms, which contain alternative last exons, are widely regulated in various biological processes and by many factors. However, little is known about their cytoplasmic regulation and translational status. In this study, we provide the first evidence that the genome-wide patterns of IPA isoform regulation during a biological process can be very distinct between the transcriptome and translatome, and between the nucleus and cytosol. Indeed, by 3'-seq analyses on breast cancer cells, we show that the genotoxic anticancer drug, doxorubicin, preferentially down-regulates the IPA to the last-exon (IPA:LE) isoform ratio in whole cells (as previously reported) but preferentially up-regulates it in polysomes. We further show that in nuclei, doxorubicin almost exclusively down-regulates the IPA:LE ratio, whereas in the cytosol, it preferentially up-regulates the isoform ratio, as in polysomes. Then, focusing on IPA isoforms that are up-regulated by doxorubicin in the cytosol and highly translated (up-regulated and/or abundant in polysomes), we identify several IPA isoforms that promote cell survival to doxorubicin. Mechanistically, by using an original approach of condition- and compartment-specific CLIP-seq (CCS-iCLIP) to analyze ELAVL1-RNA interactions in the nucleus and cytosol in the presence and absence of doxorubicin, as well as 3'-seq analyses upon ELAVL1 depletion, we show that the RNA-binding protein ELAVL1 mediates both nuclear down-regulation and cytosolic up-regulation of the IPA:LE isoform ratio in distinct sets of genes in response to doxorubicin. Altogether, these findings reveal differential regulation of the IPA:LE isoform ratio across subcellular compartments during drug response and its coordination by an RNA-binding protein.

Non-homologous end-joining at challenged replication forks: an RNA connection?

Defective DNA replication, known as 'replication stress', is a source of DNA damage, a hallmark of numerous human diseases, including cancer, developmental defect, neurological disorders, and premature aging. Recent work indicates that non-homologous end-joining (NHEJ) is unexpectedly active during DNA replication to repair replication-born DNA lesions and to safeguard replication fork integrity. However, erroneous NHEJ events are deleterious to genome stability. RNAs are novel regulators of NHEJ activity through their ability to modulate the assembly of repair complexes in trans. At DNA damage sites, RNAs and DNA-embedded ribonucleotides modulate repair efficiency and fidelity. We discuss here how RNAs and associated proteins, including RNA binding proteins, may regulate NHEJ to sustain genome stability during DNA replication.

ZRANB2 and SYF2-mediated splicing programs converging on ECT2 are involved in breast cancer cell resistance to doxorubicin.

Besides analyses of specific alternative splicing (AS) variants, little is known about AS regulatory pathways and programs involved in anticancer drug resistance. Doxorubicin is widely used in breast cancer chemotherapy. Here, we identified 1723 AS events and 41 splicing factors regulated in a breast cancer cell model of acquired resistance to doxorubicin. An RNAi screen on splicing factors identified the little studied ZRANB2 and SYF2, whose depletion partially reversed doxorubicin resistance. By RNAi and RNA-seq in resistant cells, we found that the AS programs controlled by ZRANB2 and SYF2 were enriched in resistance-associated AS events, and converged on the ECT2 splice variant including exon 5 (ECT2-Ex5+). Both ZRANB2 and SYF2 were found associated with ECT2 pre-messenger RNA, and ECT2-Ex5+ isoform depletion reduced doxorubicin resistance. Following doxorubicin treatment, resistant cells accumulated in S phase, which partially depended on ZRANB2, SYF2 and the ECT2-Ex5+ isoform. Finally, doxorubicin combination with an oligonucleotide inhibiting ECT2-Ex5 inclusion reduced doxorubicin-resistant tumor growth in mouse xenografts, and high ECT2-Ex5 inclusion levels were associated with bad prognosis in breast cancer treated with chemotherapy. Altogether, our data identify AS programs controlled by ZRANB2 and SYF2 and converging on ECT2, that participate to breast cancer cell resistance to doxorubicin.

eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies.

In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K-AKT-mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E-eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E-eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma.

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR). Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-ß signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-ß (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-ß results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-ß becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-ß signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a 'drug holiday' and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

Evaluation of the efficacy of immunotherapy for non-resectable mucosal melanoma.

BACKGROUND: Immune checkpoint inhibitors are now standard-of-care treatments for metastatic cutaneous melanoma. However, for rare sub-groups, such as mucosal melanomas, few published data are available, and with no established therapeutic guidelines. Our objective was to assess the response to anti-CTLA4 and anti-PD1 immunotherapy in patients with mucosal melanomas. METHODS: We performed a single-center, prospective cohort analysis of patients with non-surgical locally advanced and/or metastatic mucosal melanoma receiving anti-CTLA4 and/or anti-PD1 immunotherapy from 2010 to 2016. RESULTS: Forty-four patients were enrolled, including 18 (40.9%) with head and neck, 12 (27.3%) with vulvo-vaginal and 14 (31.8%) with ano-rectal primary tumours. Eleven (25%) patients had stage 3 disease, and 11 (25%) had distant metastases. The first-line immunotherapy was ipilimumab in 24 patients and pembrolizumab in 20. The objective response rate (ORR) was 8.2% (one complete response) for ipilimumab and 35% (four complete responses) for pembrolizumab. No significant difference was observed for primary tumour location. The median follow-up was 24 months (range 4-73). The median progression-free survival (PFS) in the first-line ipilimumab and pembrolizumab groups was 3 months [95% confidence interval (CI) 2.5-4.6] and 5 months (95% CI 2.6-33.1), respectively (p = 0.0147). CONCLUSION: In the patients with unresectable and/or metastatic mucosal melanoma, we found ORR and PFS rates comparable to those in patients with cutaneous melanoma, with no significant differences in the types of mucosal surfaces involved. Anti-PD1 therapy has a more favorable benefit-risk ratio than ipilimumab and should be used preferentially.

Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3'-end processing and function during DNA damage.

Following DNA damage, mRNA 3'-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3'-end formation when normal mechanisms of pre-mRNA 3'-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3'-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.

DNA damage: RNA-binding proteins protect from near and far.

Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the prevention of genome instability. These studies show that RBPs prevent harmful RNA/DNA hybrids and are involved in the DNA damage response (DDR), from DNA repair to cell survival decisions. Indeed, specific RBPs allow the selective regulation of DDR genes at multiple post-transcriptional levels (from pre-mRNA splicing/polyadenylation to mRNA stability/translation) and are directly involved in DNA repair. These multiple activities are mediated by RBP binding to mRNAs, nascent transcripts, noncoding RNAs, and damaged DNA. Finally, because DNA damage modifies RBP localization and binding to different RNA/DNA molecules, we propose that upon DNA damage, RBPs coordinately regulate various aspects of both RNA and DNA metabolism.

Age at cancer onset in germline TP53 mutation carriers: association with polymorphisms in predicted G-quadruplex structures.

Germline TP53 mutations predispose to multiple cancers defining Li-Fraumeni/Li-Fraumeni-like syndrome (LFS/LFL), a disease with large individual disparities in cancer profiles and age of onset. G-quadruplexes (G4s) are secondary structural motifs occurring in guanine tracks, with regulatory effects on DNA and RNA. We analyzed 85 polymorphisms within or near five predicted G4s in TP53 in search of modifiers of penetrance of LFS/LFL in Brazilian cancer families with (n = 35) or without (n = 110) TP53 mutations. Statistical analyses stratified on family structure showed that cancer tended to occur ~15 years later in mutation carriers who also carried the variant alleles of two polymorphisms within predicted G4-forming regions, rs17878362 (TP53 PIN3, 16 bp duplication in intron 3; P = 0.082) and rs17880560 (6 bp duplication in 3' flanking region; P = 0.067). Haplotype analysis showed that this inverse association was driven by the polymorphic status of the remaining wild-type (WT) haplotype in mutation carriers: in carriers with a WT haplotype containing at least one variant allele of rs17878362 or rs17880560, cancer occurred ~15 years later than in carriers with other WT haplotypes (P = 0.019). No effect on age of cancer onset was observed in subjects without a TP53 mutation. The G4 in intron 3 has been shown to regulate alternative p53 messenger RNA splicing, whereas the biological roles of predicted G4s in the 3' flanking region remain to be elucidated. In conclusion, this study demonstrates that G4 polymorphisms in haplotypes of the WT TP53 allele have an impact on LFS/LFL penetrance in germline TP53 mutation carriers.

Genome-wide analysis of host mRNA translation during hepatitis C virus infection.

In the model of Huh-7.5.1 hepatocyte cells infected by the JFH1 hepatitis C virus (HCV) strain, transcriptomic and proteomic studies have revealed modulations of pathways governing mainly apoptosis and cell cycling. Differences between transcriptomic and proteomic studies pointed to regulations occurring at the posttranscriptional level, including the control of mRNA translation. In this study, we investigated at the genome-wide level the translational regulation occurring during HCV infection. Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Translationally regulated mRNAs were found to correspond to genes enriched in specific pathways, including vesicular transport and posttranscriptional regulation. Interestingly, the strongest translational regulation was found for mRNAs encoding proteins involved in pre-mRNA splicing, mRNA translation, and protein folding. Strikingly, these pathways were not previously identified, through transcriptomic studies, as being modulated following HCV infection. Importantly, the observed changes in host mRNA translation were directly due to HCV replication rather than to HCV entry, since they were not observed in JFH1-infected Huh-7.5.1 cells treated with a potent HCV NS3 protease inhibitor. Overall, this study highlights the need to consider, beyond transcriptomic or proteomic studies, the modulation of host mRNA translation as an important aspect of HCV infection.

Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair.

Amino acid bioavailability impacts mRNA translation in a codon-dependent manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This coincides with the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNA replication and repair. Consequently, cells that survive MAPKi degrade aspartate and glutamate likely to generate energy, which simultaneously decreases their requirement for amino acids due to the downregulation of aspartate- and glutamate-rich proteins involved in cell proliferation. Concomitantly, the downregulation of aspartate- and glutamate-rich proteins involved in DNA repair increases DNA damage loads. Thus, DNA repair defects, and therefore mutations, are at least in part a secondary effect of the metabolic adaptation of cells exposed to MAPKi.

Post-transcriptional gene regulation: From mechanisms to RNA chemistry and therapeutics.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

Post-transcriptional checkpoints in autoimmunity.

Post-transcriptional regulation is a fundamental process in gene expression that has a role in diverse cellular processes, including immune responses. A core concept underlying post-transcriptional regulation is that protein abundance is not solely determined by transcript abundance. Indeed, transcription and translation are not directly coupled, and intervening steps occur between these processes, including the regulation of mRNA stability, localization and alternative splicing, which can impact protein abundance. These steps are controlled by various post-transcription factors such as RNA-binding proteins and non-coding RNAs, including microRNAs, and aberrant post-transcriptional regulation has been implicated in various pathological conditions. Indeed, studies on the pathogenesis of autoimmune and inflammatory diseases have identified various post-transcription factors as important regulators of immune cell-mediated and target effector cell-mediated pathological conditions. This Review summarizes current knowledge regarding the roles of post-transcriptional checkpoints in autoimmunity, as evidenced by studies in both haematopoietic and non-haematopoietic cells, and discusses the relevance of these findings for developing new anti-inflammatory therapies.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments. Conversely, depletion of FEN1, resulting in the accumulation of uncleaved RNA primers, increases 53BP1 levels at replication forks, suggesting that RNA primers contribute to the recruitment of 53BP1 at the lagging DNA strand. 53BP1 depletion induces an accumulation of S-phase poly(ADP-ribose), which constitutes a sensor of unligated Okazaki fragments. Collectively, our data indicate that 53BP1 is anchored at nascent DNA through its RNA-binding activity, highlighting the role of an RNA-protein interaction at replication forks.