Non-genetic Mechanisms Research Articles

Modulating environmental signals to reveal mechanisms and vulnerabilities of cancer persisters.

Cancer persister cells are able to survive otherwise lethal doses of drugs through nongenetic mechanisms, which can lead to cancer regrowth and drug resistance. The broad spectrum of molecular differences observed between persisters and their treatment-naïve counterparts makes it challenging to identify causal mechanisms underlying persistence. Here, we modulate environmental signals to identify cellular mechanisms that promote the emergence of persisters and to pinpoint actionable vulnerabilities that eliminate them. We found that interferon-γ (IFNγ) can induce a pro-persistence signal that can be specifically eliminated by inhibition of type I protein arginine methyltransferase (PRMT) (PRMTi). Mechanistic investigation revealed that signal transducer and activator of transcription 1 (STAT1) is a key component connecting IFNγ’s pro-persistence and PRMTi’s antipersistence effects, suggesting a previously unknown application of PRMTi to target persisters in settings with high STAT1 expression. Modulating environmental signals can accelerate the identification of mechanisms that promote and eliminate cancer persistence.

A large scale mass spectrometry-based histone screening for assessing epigenetic developmental toxicity

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose–response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine).

L’esofagite eosinofila: quando deglutire diventa difficile

Knowledge in the genetic field of autoinflammatory diseases has progressively increased over the last decade. Starting from the genetic knowledge on a few initially known diseases (Familial Mediterranean Fever, Periodic Syndrome Associated with Tumor Necrosis Factor Receptor 1, Mevalonate Kinase Deficiency, Cryopyrinopathies), at least 40 genes associated with these conditions have been identified, and more than 100 genes are currently being tested with Next Generation Sequencing (NGS) technologies. Indeed, starting recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy, based on the Sanger method, and restricted to a few prototypic recurrent fevers. More recently, improvement of genetic technologies, in particular NGS, allowed identification of genetic variants among involved genes in an easier and more rapid way. Detection of more and more genetic variants made interpretation of results more complicated, and often a genetic diagnosis is not achieved. On the other hand, other mechanisms, namely somatic mosaicisms, epigenetic modifications, digenic inheritance, allow to explain some not genetically defined cases. Eventually, further genetic and non-genetic mechanisms will be probably identified in near future to explain underlying basis of autoinflammatory diseases, especially in a still large part of patients without a clearcut genetic basis.

Circular RNAs: novel regulators of resistance to systemic treatments in breast cancer.

Systematic treatments including chemotherapy, endocrine therapy, and HER2-targeted therapy are important therapeutic approaches to breast cancer. However, drug resistance is a major barrier to achieving a cure in breast cancer (BC) patients. Hence, it is urgent to gain insight into the drug-resistance mechanisms in order to improve the prognosis of BC patients. Genetic alternations, epigenetic alternations, and other non-genetic mechanisms such as BC stem-like cells, metabolic reprogramming, and tumor microenvironment contribute to drug resistance of BC. With the development of single-cell sequencing of circulating tumor cell and next-generation sequencing of matched pre- and post- progression tumor biopsies or ctDNA from BC patients with drug resistance, new mechanisms of resistance are being discovered. An increasing number of microRNAs and long non-coding RNAs have been found to be associated with the drug resistance of BC. However, there are few reports on the role of circular RNAs (circRNAs) as master regulators of drug resistance. Therefore, there is still much to say in the field of drug resistance-related circRNAs. In this review, we mainly focus on literature evidence for the detailed mechanisms associated with systematic treatments' resistance of BC and how circRNAs intensify or weaken drug resistance. Exogenous expression of tumor suppressive circRNAs or knockdown of oncogenic circRNAs has been verified to reverse drug resistance of BC cells, which highlights that circRNAs may function as potential biomarkers and/or therapeutic targets of BC. Treatment targeting abnormally expressed circRNAs alone or combined with other systemic treatments may be a promising approach to conquering drug resistance.

Implementation of Clinical Phosphoproteomics and Proteomics for Personalized Medicine.

The identification of biomarkers for companion diagnostics is revolutionizing the development of treatments tailored to individual patients in different disease areas including cancer. Precision medicine is most frequently based on the detection of genomic markers that correlate with the efficacy of selected targeted therapies. However, since nongenetic mechanisms also contribute to disease biology, there is a considerable interest of using proteomic techniques as additional source of biomarkers to personalize therapies. In this chapter, we describe label-free mass spectrometry methods for proteomic and phosphoproteomic analysis compatible with routine analysis of clinical samples. We also outline bioinformatic pipelines based on statistical learning that use these proteomics datasets as input to quantify kinase activities and predict drug responses in cancer cells.

Non-genetic determinants of malignant clonal fitness at single-cell resolution

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of thesecretory leukocyte peptidase inhibitorgene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues andimmune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Metabolic Plasticity in Melanoma Progression and Response to Oncogene Targeted Therapies.

Simple SummaryTargeted anti-cancer therapies have revolutionised melanoma patient care; however, cures remain uncommon due to acquired drug resistance that results in disease relapse. Recent insights from the clinic and experimental settings have identified a key role for metabolic plasticity, defined as the flexibility to utilise different nutrients and process them in different ways, in both disease progression and response to targeted therapies. Here, we discuss how this plasticity creates a moving target with important implications for identifying new combination therapies.Resistance to therapy continues to be a barrier to curative treatments in melanoma. Recent insights from the clinic and experimental settings have highlighted a range of non-genetic adaptive mechanisms that contribute to therapy resistance and disease relapse, including transcriptional, post-transcriptional and metabolic reprogramming. A growing body of evidence highlights the inherent plasticity of melanoma metabolism, evidenced by reversible metabolome alterations and flexibility in fuel usage that occur during metastasis and response to anti-cancer therapies. Here, we discuss how the inherent metabolic plasticity of melanoma cells facilitates both disease progression and acquisition of anti-cancer therapy resistance. In particular, we discuss in detail the different metabolic changes that occur during the three major phases of the targeted therapy response—the early response, drug tolerance and acquired resistance. We also discuss how non-genetic programs, including transcription and translation, control this process. The prevalence and diverse array of these non-genetic resistance mechanisms poses a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes in the quest to prevent therapy resistance.

Non-Genetic Determinants of Clonotypic T Cell Expansion Following Allogeneic Stem Cell Transplant

Background:The immunologic basis of acute GVHD fundamentally involves alloreactive donor T cells that recognize foreign major histocompatibility complex (MHC)-peptide structures derived from both major and minor antigen mismatches with the host. Within this paradigm, the relationship between the donor and recipient genetics represents a closed system that dictates the potential ability of any given T cell receptor (TCR) to expand, raising the question of whether there are predictable aspects of TCR reconstitution at a clonal level. Take a hypothetical example - if genetically identical twins were to receive allogeneic grafts from the same donor and both recipients develop GVHD, would one expect similar TCRs to be clonally expanded? It has been challenging to rigorously explore this phenomenon, however, because of the vast combinatorial diversity of αβ TCRs, the high prevalence of low copy number TCRs, and sampling constraints - all of which render tracking and comparing TCR expansion between the donor and host difficult.Methods:We address these challenges in order to better understand the predictability TCR clonal dynamics through an analysis platform utilizing 1) a series of matched and mismatched murine transplant experiments where genetically identical littermates receive T cells from the same polyclonal donor pool, thus creating multiple transplant replicates simulating the twin transplant system describe above (Fig 1), and 2) probabilistic modeling of individual TCR frequencies to account for partitioning stochasticity (variation in how low copy number TCRs are distributed from donor to recipient). We conduct high-throughput DNA-based TCR amplicon sequencing for both donor and post-transplant recipient samples to generate over 20 million TCRs and model the expansion rates of all identifiable TCRs in each transplant system using a Bayesian approach.Results:While overall V and J gene usage were similar amongst identical recipients (Fig 2), we find that a small fraction of TCR clonotypes appears to have widely disparate clone counts amongst identical recipients receiving the same donor T cell pool. For example, we saw 9,739, 129 and 0 copies of a particular TCR in 3 different recipients in our B6->B6D2F1 system (Fig 3). In order to distinguish whether TCR count discrepancies seen across identical recipients is simply a reflection of donor partitioning stochasticity or true differential expansion (Fig 4), we apply a Bayesian algorithm to identify differential expanders, which represent TCRs that are asymmetrically expanded between recipients of a genetically identical pair (Fig 5). These TCRs can be generated from both memory and naïve T cell compartments.The presence of these differentially expanded clones amongst identical recipients suggests that non-genetic dependent mechanisms may influence which TCRs expand post-transplant. We next show that broad gut decontamination of microbiota with peri-transplant vancomycin, gentamicin, cefoxitin and metronidazole dramatically reduced the fraction of differential expanders (p<0.0001). However, the change in inflammation from microbiome depletion did not appear to drive this difference, as 1) MyD88/TRIF double knockout recipients (deficient TLR signaling) did not show a reduction in differential expanders, and 2) altering conditioning intensity (900cGy to 1300 cGy TBI) also did not change the fraction of differential expanders. Rather, the difference is likely antigenically driven, as differential expanders are enriched in antigen specificity compared to other TCR sequences (p<0.0001) based on published algorithm that identify TCRs with similar amino acid sequence overlap.Conclusions:These results refine our current understanding of clonal T cell selection and expansion after allogeneic BMT and suggests that for a given transplant system, individual TCR selection is not solely dictated by genetic donor and recipient major and/or minor histocompatibility disparities. Rather, microbiota-derived molecules appear to behave as minor antigens to direct systemic clonal TCR selection. These data suggest a novel mechanism by which the microbiome may modulate transplant outcome, challenging current paradigms suggesting the microbiota primarily drive inflammation via their PAMP activities. [Display omitted] DisclosuresHill: Applied Molecular Transport: Research Funding; Syndax Pharmaceuticals: Research Funding; Compass Therapeutics: Research Funding; NapaJun Pharma: Consultancy; Generon corporation: Consultancy; iTeos Therapeutics: Consultancy, Research Funding; Neoleukin Therapeutics: Consultancy; Roche: Research Funding.

The tumor suppressor activity of DLC1 requires the interaction of its START domain with Phosphatidylserine, PLCD1, and Caveolin-1

BackgroundDLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown.MethodsLipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START.ResultsWe identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties.ConclusionThese observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.

NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation.

The nongenetic mechanisms required to sustain malignant tumor state are poorly understood. During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation and acquire invasive features. Using transcriptional profiling of cancer stem cells from benign tumors and malignant skin squamous cell carcinoma (SCC), we identified the nuclear receptor NR2F2 as uniquely expressed in malignant SCC. Using genetic gain of function and loss of function in vivo, we show that NR2F2 is essential for promoting the malignant tumor state by controlling tumor stemness and maintenance in mouse and human SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, epithelial-mesenchymal transition and invasive features, while repressing tumor differentiation and immune cell infiltration by regulating a common transcriptional program in mouse and human SCCs. Altogether, we identify NR2F2 as a key regulator of malignant cancer stem cell functions that promotes tumor renewal and restricts differentiation to sustain a malignant tumor state.

Role of environmentally induced epigenetic transgenerational inheritance in evolutionary biology: Unified Evolution Theory.

The current evolutionary biology theory primarily involves genetic alterations and random DNA sequence mutations to generate the phenotypic variation required for Darwinian natural selection to act. This neo-Darwinian evolution is termed the Modern Evolution Synthesis and has been the primary paradigm for nearly 100 years. Although environmental factors have a role in neo-Darwinian natural selection, Modern Evolution Synthesis does not consider environment to impact the basic molecular processes involved in evolution. An Extended Evolutionary Synthesis has recently developed that extends the modern synthesis to consider non-genetic processes. Over the past few decades, environmental epigenetics research has been demonstrated to regulate genetic processes and directly generate phenotypic variation independent of genetic sequence alterations. Therefore, the environment can on a molecular level through non-genetic (i.e. epigenetic) mechanisms directly influence phenotypic variation, genetic variation, inheritance and adaptation. This direct action of the environment to alter phenotype that is heritable is a neo-Lamarckian concept that can facilitate neo-Darwinian (i.e. Modern Synthesis) evolution. The integration of genetics, epigenetics, Darwinian theory, Lamarckian concepts, environment, and epigenetic inheritance provides a paradigm shift in evolution theory. The role of environmental-induced epigenetic transgenerational inheritance in evolution is presented to describe a more unified theory of evolutionary biology.

The Genomic Processes of Biological Invasions: From Invasive Species to Cancer Metastases and Back Again

The concept of invasion is useful across a broad range of contexts, spanning from the fine scale landscape of cancer tumors up to the broader landscape of ecosystems. Invasion biology provides extraordinary opportunities for studying the mechanistic basis of contemporary evolution at the molecular level. Although the field of invasion genetics was established in ecology and evolution more than 50 years ago, there is still a limited understanding of how genomic level processes translate into invasive phenotypes across different taxa in response to complex environmental conditions. This is largely because the study of most invasive species is limited by information about complex genome level processes. We lack good reference genomes for most species. Rigorous studies to examine genomic processes are generally too costly. On the contrary, cancer studies are fortified with extensive resources for studying genome level dynamics and the interactions among genetic and non-genetic mechanisms. Extensive analysis of primary tumors and metastatic samples have revealed the importance of several genomic mechanisms including higher mutation rates, specific types of mutations, aneuploidy or whole genome doubling and non-genetic effects. Metastatic sites can be directly compared to primary tumor cell counterparts. At the same time, clonal dynamics shape the genomics and evolution of metastatic cancers. Clonal diversity varies by cancer type, and the tumors’ donor and recipient tissues. Still, the cancer research community has been unable to identify any common events that provide a universal predictor of “metastatic potential” which parallels findings in evolutionary ecology. Instead, invasion in cancer studies depends strongly on context, including order of events and clonal composition. The detailed studies of the behavior of a variety of human cancers promises to inform our understanding of genome level dynamics in the diversity of invasive species and provide novel insights for management.

Cohesin Core Complex Gene Dosage Contributes to Germinal Center Derived Lymphoma Phenotypes and Outcomes.

The cohesin complex plays critical roles in genomic stability and gene expression through effects on 3D architecture. Cohesin core subunit genes are mutated across a wide cross-section of cancers, but not in germinal center (GC) derived lymphomas. In spite of this, haploinsufficiency of cohesin ATPase subunit Smc3 was shown to contribute to malignant transformation of GC B-cells in mice. Herein we explored potential mechanisms and clinical relevance of Smc3 deficiency in GC lymphomagenesis. Transcriptional profiling of Smc3 haploinsufficient murine lymphomas revealed downregulation of genes repressed by loss of epigenetic tumor suppressors Tet2 and Kmt2d. Profiling 3D chromosomal interactions in lymphomas revealed impaired enhancer-promoter interactions affecting genes like Tet2, which was aberrantly downregulated in Smc3 deficient lymphomas. Tet2 plays important roles in B-cell exit from the GC reaction, and single cell RNA-seq profiles and phenotypic trajectory analysis in Smc3 mutant mice revealed a specific defect in commitment to the final steps of plasma cell differentiation. Although Smc3 deficiency resulted in structural abnormalities in GC B-cells, there was no increase of somatic mutations or structural variants in Smc3 haploinsufficient lymphomas, suggesting that cohesin deficiency largely induces lymphomas through disruption of enhancer-promoter interactions of terminal differentiation and tumor suppressor genes. Strikingly, the presence of the Smc3 haploinsufficient GC B-cell transcriptional signature in human patients with GC-derived diffuse large B-cell lymphoma (DLBCL) was linked to inferior clinical outcome and low expression of cohesin core subunits. Reciprocally, reduced expression of cohesin subunits was an independent risk factor for worse survival int DLBCL patient cohorts. Collectively, the data suggest that Smc3 functions as a bona fide tumor suppressor for lymphomas through non-genetic mechanisms, and drives disease by disrupting the commitment of GC B-cells to the plasma cell fate.

Urban bat pups take after their mothers and are bolder and faster learners than rural pups

BackgroundUrbanization is rapidly changing our planet and animals that live in urban environments must quickly adjust their behavior. One of the most prevalent behavioral characteristics of urban dwelling animals is an increased level of risk-taking. Here, we aimed to reveal how urban fruitbats become risk-takers, and how they differ behaviorally from rural bats, studying both genetic and non-genetic factors that might play a role in the process. We assessed the personality of newborn pups from both rural and urban colonies before they acquired experience outdoors, examining risk-taking, exploration, and learning rates.ResultsUrban pups exhibited significantly higher risk-taking levels, they were faster learners, but less exploratory than their rural counterparts. A cross-fostering experiment revealed that pups were more similar to their adoptive mothers, thus suggesting a non-genetic mechanism and pointing towards a maternal effect. We moreover found that lactating urban mothers have higher cortisol levels in their milk, which could potentially explain the transmission of some personality traits from mother to pup.ConclusionsYoung bats seem to acquire environment suitable traits via post-birth non-genetic maternal effects. We offer a potential mechanism for how urban pups can acquire urban-suitable behavioral traits through hormonal transfer from their mothers.

The interplay between sperm-mediated and care-mediated paternal effects in threespine sticklebacks

The environment experienced by one generation can influence the phenotypes of future generations. Because parental cues can be conveyed to offspring at multiple points in time, ranging from fertilization to posthatching/parturition, offspring can potentially receive multiple cues from their parents via different mechanisms. We have relatively little information regarding how different mechanisms operate in isolation and in tandem, but it is possible, for example, that offspring phenotypes induced by nongenetic changes to gametes may be amplified by, mitigated by, or depend upon parental care. Here, we manipulated paternal experience with predation risk prior to fertilization in threespine stickleback, Gasterosteus aculeatus, and then examined the potential of paternal care to mitigate and/or amplify sperm-mediated paternal effects. Specifically, we compared (1) offspring of predator-exposed fathers who were reared without paternal care, (2) offspring of predator-exposed fathers who were reared with paternal care, (3) offspring of control (unexposed) fathers who were reared without paternal care and (4) offspring of control fathers who were reared with paternal care. We found that offspring of predator-exposed fathers were less active and had higher cortisol following a simulated predator attack. Although predator-exposed males shifted their paternal care behaviours - reduced fanning early in egg development and increased fanning right before egg hatching compared to control males - this shift in paternal behavior did not appear to affect offspring traits. This suggests that paternal care neither amplifies nor compensates for these phenotypic effects induced by sperm and that nongenetic changes induced by sperm may occur independently of nongenetic changes induced by paternal care. Overall, these results underscore the importance of considering how parents may have multiple nongenetic mechanisms by which they can influence offspring.

Cycling cancer persister cells arise from lineages with distinct programs.

Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure1, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment2. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.

Generational effects of a chronic exposure to a low environmentally relevant concentration of glyphosate on rainbow trout, Oncorhynchus mykiss

In the past few decades, glyphosate became the most used herbicide substance worldwide. As a result, the substance is ubiquitous in surface waters. Concerns have been raised about its ecotoxicological impact, but little is known about its generational toxicity. In this study, we investigate the impact of an environmentally relevant concentration of glyphosate and its co-formulants on an F2 generation issued from exposed generations F0 and F1. Trans, inter and multigenerational toxicity of 1 μgL−1 of the active substance was evaluated on early stages of development and juvenile rainbow trout (Oncorhynchus mykiss) using different molecular, biochemical, immuno-hematologic, and biometric parameters, behavior analysis, and a viral challenge. Reproductive parameters of generation F1 were not affected. However, developmental toxicity in generation F2 due to glyphosate alone or co-formulated was observed with head size changes (e.g. head surface up to +10%), and metabolic disruptions (e.g. 35% reduction in cytochrome-c-oxidase). Moreover, larvae exposed transgenerationally to Viaglif and intergenerationally to glyphosate and Roundup presented a reduced response to light, potentially indicating altered escape behavior. Overall methylation was, however, not altered and further experiments using gene-specific DNA metylation analyses are required. After several months, biochemical parameters measured in juvenile fish were no longer impacted, only intergenerational exposure to glyphosate drastically increased the susceptibility of rainbow trout to hematopoietic necrosis virus. This result might be due to a lower antibody response in exposed fish. In conclusion, our results show that generational exposure to glyphosate induces developmental toxicity and increases viral susceptibility. Co-formulants present in glyphosate-based herbicides can modulate the toxicity of the active substance. Further investigations are required to study the specific mechanisms of transmission but our results suggest that both non-genetic mechanisms and exposure during germinal stage could be involved.

Rarely Recognized Antibody Diversification in Covid-19 Evolution to Counteract Advanced SARS-CoV-2 Evasion Strategies, and Implications for Prophylactic Treatment.

The ongoing Covid-19 pandemic underscores the importance of finding effective and safe ways to combat the virus, and to optimally understand the immune response elicited upon natural infection. This likely involves all components of the immune system, both innate and adaptive. The impetus for the rapid development of prophylactic treatment options has led to an intense focus on neutralizing antibodies (Abs), and many novel and specialized platforms have been designed to achieve that goal. B-cell immunity relies on the generation of a diverse repertoire of Abs. Their structural variation is defined in terms of amino acid composition that is encoded in the genome or acquired through somatic mutations. Yet, key examples of frequently neglected antibody diversification mechanisms involving post-translational modifications such as N- or O-linked glycosylation are present in significant portions of the population. During the last few years, these and other beyond gene sequence determined humoral immune response mechanisms have in some specific cases revealed their potent immunomodulatory effects. Nonetheless, such more unusual mechanisms have not received much attention in the context of SARS-CoV-2. Thus, with specific focus on the latter, this paper presents, (1) the rationale for considering beyond sequence determined strategies, (2) evidence for their possible involvement in Covid-19 disease evolution, (3) consequences for vaccine design exemplified by one of the vaccine candidates that is currently undergoing trial, and (4) more general implications. Based on a critical interpretation of published literature, the hypotheses developed in this study point to a crucial role of non-genetic antibody diversification mechanisms in disease evolution to counteract unique immunogenicity determinants of SARS-CoV-2 infection. The involvement of post translational mechanisms may also help explain the widely varied immune response observed, not only among different patient groups, but also in terms of their observed incompatibility with SARS-CoV-2 infection in several human cell types. The article highlights potentials and challenges of these refined humoral immune response mechanisms to most optimally target non-genetic viral evasion strategies.

Evolutionary predictability of genetic versus nongenetic resistance to anticancer drugs in melanoma.

Therapy resistance arises from heterogeneous drug-tolerant persister cells or minimal residual disease (MRD) through genetic and nongenetic mechanisms. A key question is whether specific molecular features of the MRD ecosystem determine which of these two distinct trajectories will eventually prevail. We show that, in melanoma exposed to mitogen-activated protein kinase therapeutics, emergence of a transient neural crest stem cell (NCSC) population in MRD concurs with the development of nongenetic resistance. This increase relies on a glial cell line-derived neurotrophic factor-dependent signaling cascade, which activates the AKT survival pathway in a focal adhesion kinase (FAK)-dependent manner. Ablation of the NCSC population through FAK inhibition delays relapse in patient-derived tumor xenografts. Strikingly, all tumors that ultimately escape this treatment exhibit resistance-conferring genetic alterations and increased sensitivity to extracellular signal-regulated kinase inhibition. These findings identify an approach that abrogates the nongenetic resistance trajectory in melanoma and demonstrate that the cellular composition of MRD deterministically imposes distinct drug resistance evolutionary paths.

Is resistance to targeted therapy in cancer inevitable?

Resistance to targeted therapies is a major challenge in cancer care and occurs via genetic and non-genetic mechanisms. In this issue of Cancer Cell, Marin-Bejar etal. demonstrate that melanomas recurrently select genetic or non-genetic resistance trajectories and that targeting neural crest stem cell-like cells prevents non-genetic, but not genetic, resistance.