Search

International audience; The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

Background The treatment landscape for advanced non-small cell lung cancer (aNSCLC) has evolved rapidly since immuno-oncology (IO) therapies were introduced. This study used recent data to assess real-world treatment patterns and clinical outcomes in aNSCLC in the United Kingdom. Methods Electronic prescribing records of treatment-naive patients starting first-line (1 L) treatment for aNSCLC between June 2016 and March 2018 (follow-up until December 2018) in the United Kingdom were assessed retrospectively. Patient characteristics and treatment patterns were analyzed descriptively. Outcomes assessed included overall survival (OS), time to treatment discontinuation, time to next treatment, and real-world tumor response. Results In all, 1003 patients were evaluated (median age, 68 years [range, 28–93 years]; 53.9% male). Use of 1 L IO monotherapy (0–25.9%) and targeted therapy (11.8–15.9%) increased during the study period, but chemotherapy remained the most common 1 L treatment at all time points (88.2–58.2%). Median OS was 9.5 months (95% CI, 8.8–10.7 months) for all patients, 8.1 months (95% CI, 7.4–8.9 months) with chemotherapy, 14.0 months (95% CI, 10.7–20.6 months) with IO monotherapy, and 20.2 months (95% CI, 16.0–30.5 months) with targeted therapy. In the 28.6% of patients who received second-line treatment, IO monotherapy was the most common drug class (used in 51.6%). Conclusions Although use of 1 L IO monotherapy for aNSCLC increased in the United Kingdom during the study period, most patients received 1 L chemotherapy. An OS benefit for first-line IO monotherapy vs chemotherapy was observed but was numerically smaller than that reported in clinical trials. Targeted therapy was associated with the longest OS, highlighting the need for improved treatment options for tumors lacking targetable mutations.

SUMMARYDefects in double-strand repair mechanisms - both through germline or somatic inactivation of repair genes - is a hallmark of basal-like breast cancers. In this genetically-unstable context, a recurrent shift in cell identity occurs within the mammary epithelium. Basal-like tumors have indeed been proposed to originate from luminal progenitor (LP) cells yet tumor-initiating events remain poorly understood. Here, we map state transitions at the onset of basal-like tumorigenesis, using a Brca-1 deficient mouse model launching tumorigenesis in multiple LP cells. Combining single-cell transcriptomics to spatial multiplex imaging, we identify a population of cycling p16-expressing cells, emerging from the luminal progenitor compartment, undergoing partial epithelial-to-mesenchymal transition and losing luminal identity. Pseudo-temporal analyses position these cells as a transitory pre-tumoral state between aberrant Brca1-deficient luminal progenitors and growing tumor cells. In patients, the p16 pre-tumoral signature is found in early stage basal-like tumors, that rarely recur. Concomitant to p16 activation, we show that LP cells undergo an epigenomic crisis attested by the general re-organization of their heterochromatin. They accumulate multiple H3K27me3 micro-foci - reminiscent of the formation of senescence-associated heterochromatin foci (SAHFs) - and lose their inactive X (Xi). Both p16 activation and heterochromatin reorganization are hallmarks of human basal-like breast tumors; we propose that these events occur during initial LP transformation and are scars of an initial transitory senescent-like state.

Network meta-analysis was used to derive estimates of the relative efficacy of inclisiran, evolocumab, alirocumab, bempedoic acid, and ezetimibe in patients with hypercholesterolemia and/or at increased cardiovascular risk due to elevated low-density lipoprotein cholesterol taking maximum tolerated dose statins. Clinical trials published through February 2021 comparing percent change from baseline in low-density lipoprotein cholesterol were identified via a systematic review. Bayesian network meta-analyses were performed for patients with atherosclerotic cardiovascular disease and/or high cardiovascular risk on maximally tolerated statins in the base case, which included 23 trials. Results from the base-case analyses demonstrated that inclisiran, evolocumab, and alirocumab provide superior efficacy over placebo, bempedoic acid, and ezetimibe in terms of reduction in low-density lipoprotein cholesterol. Inclisiran was also comparable to alirocumab (mean difference: 0.78% [95% CrI: −8.35, 9.88]) and evolocumab (8.16%, [95% CrI: −1.82, 18.49]). Findings of a scenario which also included trials conducted in patients with heterozygous familial hypercholesterolemia were consistent with the base case. There was evidence of statistical heterogeneity across the included trials, roughly equivalent to variation of 5–10% change in low-density lipoprotein cholesterol, suggesting that any differences between treatments that were greater than 5–10% are generalizable. This study provides insight regarding the comparative efficacy of drugs for which no head-to-head trials exist and suggests that inclisiran, alirocumab, and evolocumab are expected to provide similar clinically meaningful improvements in low-density lipoprotein cholesterol in patients with hypercholesterolemia on maximally tolerated statins who are at increased cardiovascular risk.

SummaryTriple-negative breast cancer is associated with the worst prognosis and the highest risk of recurrence among all breast cancer subtypes1. Residual disease, formed by cancer cells persistent to chemotherapy, remains one of the major clinical challenges towards full cure2,3. There is now consensus that non-genetic processes contribute to chemoresistance in various tumor types, notably through the initial emergence of a reversible chemotolerant state4–6. Understanding non-genetic tumor evolution stands now as a prerequisite for the design of relevant combinatorial approaches to delay recurrence. Here we show that the repressive histone mark H3K27me3 is a determinant of cell fate under chemotherapy exposure, monitoring epigenomes, transcriptomes and lineage with single-cell resolution. We identify a reservoir of persister basal cells with EMT markers and activated TGF-β pathway leading to multiple chemoresistance phenotypes. We demonstrate that, in unchallenged cells, H3K27 methylation is a lock to the expression program of persister cells. Promoters are primed with both H3K4me3 and H3K27me3, and removing H3K27me3 is sufficient for their transcriptional activation. Leveraging lineage barcoding, we show that depleting H3K27me3 alters tumor cell fate under chemotherapy insult – a wider variety of tumor cells tolerate chemotherapy. Our results highlight how chromatin landscapes shape the potential of unchallenged cancer cells to respond to therapeutic stress.

International audience; CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.

Aims: Model how moving from current disease-modifying drug (DMD) prescribing patterns for relapsing-remitting multiple sclerosis (RRMS) observed in the United Kingdom (UK) to prescribing patterns based on patient preferences would impact health outcomes over time. Materials and methods: A cohort-based Markov model was used to measure the effect of DMDs on long-term health outcomes for individuals with RRMS. Data from a discrete choice experiment were used to estimate the market shares of DMDs based on patient preferences (i.e. preference shares). These preference shares and real-world UK market shares were used to calculate the effect of prescribing behavior on relapses, disability progression, and quality-adjusted life-years (QALYs). The incremental benefit of patient-centered prescribing over current practices for the UK RRMS population was then estimated; scenario and sensitivity analyses were also conducted. Results: Compared to current prescribing practices, when UK patients with RRMS were treated following patient preferences, health outcomes were improved. This population was expected to experience 501,690 relapses and gain 1,003,263 discounted QALYs over 50 years under patient-centered prescribing practices compared to 538,417 relapses and 958,792 discounted QALYs under current practices (−6.8% and +4.6%, respectively). Additionally, less disability progression was observed when prescribed treatment was based on patient preferences. In a scenario analysis where only oral treatments were considered, the results were similar, although the magnitude of benefit was smaller. Number of relapses was most sensitive to how the annualized relapse rate was modeled; disability progression was most sensitive to mortality rate assumptions. Limitations: Treatment efficacy estimates applied to various models in this study were based on data derived from clinical trials, rather than real-world data; the impact of patient-centered prescribing on treatment adherence and/or switching was not modeled. Conclusions: The population of UK RRMS patients may experience overall health gains if patient preferences are better incorporated into prescribing practices.

SUMMARYCD44 is a transmembrane glycoprotein that is linked to various biological processes reliant on the epigenetic plasticity of cells, including development, inflammation, immune responses, wound healing and cancer progression. While thoroughly studied, functional regulatory roles of this so-called ‘cell surface marker’ remain elusive. Here, we report the discovery that CD44 mediates endocytosis of iron interacting with hyaluronates in tumorigenic cell lines and primary cancer cells. We found that this glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transition, unlike the canonical transferrin-dependent pathway. This transition is further characterized by molecular changes required for iron-catalyzed oxidative demethylation of the repressive histone mark H3K9me2 that governs the expression of mesenchymal genes.CD44itself is transcriptionally regulated by nuclear iron, demonstrating a positive feedback loop, which is in contrast to the negative regulation of transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This comprehensive study reveals an alternative iron uptake mechanism that prevails in the mesenchymal state of mammalian cells, illuminating a central role of iron as a rate-limiting regulator of epigenetic plasticity.

Modulation of chromatin structure via histone modification is a major epigenetic mechanism and regulator of gene expression. However, the contribution of chromatin features to tumor heterogeneity and evolution remains unknown. Here we describe a high-throughput droplet microfluidics platform to profile chromatin landscapes of thousands of cells at single-cell resolution. Using patient-derived xenograft models of acquired resistance to chemotherapy and targeted therapy in breast cancer, we found that a subset of cells within untreated drug-sensitive tumors share a common chromatin signature with resistant cells, undetectable using bulk approaches. These cells, and cells from the resistant tumors, have lost chromatin marks-H3K27me3, which is associated with stable transcriptional repression-for genes known to promote resistance to treatment. This single-cell chromatin immunoprecipitation followed by sequencing approach paves the way to study the role of chromatin heterogeneity, not just in cancer but in other diseases and healthy systems, notably during cellular differentiation and development.

Specimens produced from technically pure iron and two different heats of ferritic/martensitic steel T91 are investigated after exposure to oxygen-containing flowing lead–bismuth eutectic (LBE) at 400 °C, 10−7 mass% dissolved oxygen, and flow velocity of 2 m/s, for exposure times between around 1000 and 13,000 h. The occurring phenomena are analyzed and quantified using metallographic cross sections prepared after exposure. While pure iron mostly shows solution underneath or in the absence of a detached and buckled oxide scale, solution in T91 occurs only in a few spots on the sample surface. However, in the case of one of the investigated heats, a singular event of exceptionally severe solution-based corrosion is observed. The results are compared especially with findings at 450 and 550 °C and otherwise similar conditions as well as austenitic steels tested in the identical experimental run.

Highly selective base-pair recognition makes DNA a suitable building block for orderly self-assembled structures. For some applications in nanotechnology DNA complexes need to be fixed onto surfaces. To fulfil this requirement on lipid membranes we have synthesised a thymidine monomer modified with a cholesterol moiety. Solution studies show that the melting temperature (Tm) of the duplex, with adjacent cholesterols on each strand, is much higher than that of the unmodified duplex.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipids bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper describes the anchoring of novel cholesterol-modified DNA-18mers in supported lipid bilayers (SLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: we compared the behavior of two anchoring functionalities differing in the number of cholesterol units and in the length of a spacer group. Quartz Crystal Microbalance with impedance monitoring (QCM-Z) measurements showed that both oligonucleotides insert into the bilayer membrane through cholesterol anchoring; however, dramatic differences, in terms of surface organization and thickness, are found as the number of anchoring units increases. In the case of multiple cholesterol units, a peculiar three-regimes concentration dependence was revealed and correlated to the effective size of the adsorbing units. Interestingly, for high oligonucleotide concentration, the adsorption process was rationalized in terms of a compaction model of amphiphilic DNA molecules. QCM-Z temperature cycles of the SLB-anchored double strands provided clear evidence for reversible hybridization at the bilayer interface.

Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo., Author Summary Replication polymerases are responsible for genome duplication; they are ubiquitous and show high levels of functional and structural conservation across all species. The HolC-HolD (χψ) complex is a component of the replicative polymerase in the model bacteria Escherichia coli, and is crucial for normal growth. We isolated suppressor mutations that restore the viability of the holD mutant and we found that inactivating the Trk system, responsible for the main pathway of potassium import, renders the entire χψ complex dispensable for growth. Activation of alternative pathways of potassium import abolishes the suppression. The viability of the holD trk mutant is due in large part to a better capacity of the χψ-less polymerase to compete with other polymerases. Potassium glutamate is the major intracellular ionic osmolyte in E. coli, and we propose that mutations that affect potassium concentration in vivo stabilize the χψ-less polymerase by increasing electrostatic interactions between the different polymerase subunits and between polymerase and DNA. Stabilized by the lower intracellular potassium concentration, the χψ-less polymerase becomes functional in trk mutants, to a level that permits cell growth with this defective polymerase. Our results imply that K+ import can play an important role in the stability of protein complexes on DNA.

In the present series of papers, we describe the results of a systematic study on the anchoring of cholesterol-tagged oligonucleotides to phospholipid bilayers followed by membrane-assisted hybridization of the complementary strand in solution. This paper compares the behavior of two cholesteryl modified oligonucleotides, differing in the architecture and hydrophobicity of the lipophilic moiety, in the self-aggregation, hybridization, and insertion in phospholipid vesicle membranes. We have focused our attention on a singly substituted derivative (SC-ON(1)) and a multicholesterol (MC-ON(1)) derivative, where the cholesteryl units are inserted at the desired positions along a noncoupling T-sequence. The self-aggregation properties in solution are also explicitly taken into account and evaluated as competitive with respect to the adsorption at fluid or solid interfaces and to hybridization with the complementary ON(2) sequence. By exploring a wide range of ON derivative concentrations, different peculiar scenarios emerge for different hydrophobicity of the amphiphilic DNA guest molecules on the vesicles, in terms of distribution and conformation of the single strand and consequent coupling properties with the complementary strand in solution.