Transcriptional Adaptation Research Articles

Lysine methyltransferase 2 plays a key role in the encystation process in the parasite Giardia lamblia

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.

Histone H1x in mouse ventral hippocampus associates with, but does not cause behavioral adaptations to stress

Prior research has identified differential protein expression levels of linker histone H1x within the ventral hippocampus (vHipp) of stress-susceptible versus stress-resilient mice. These mice are behaviorally classified based on their divergent responses to chronic social stress. Here, we sought to determine whether elevated vHipp H1x protein levels directly contribute to these diverging behavioral adaptations to stress. First, we demonstrated that stress-susceptible mice uniquely express elevated vHipp H1x protein levels following chronic stress. Given that linker histones coordinate heterochromatin compaction, we hypothesize that elevated levels of H1x in the vHipp may impede pro-resilience transcriptional adaptations and prevent development of the resilient phenotype following social stress. To test this, 8–10-week-old male C57BL/6 J mice were randomly assigned to groups undergoing 10 days of chronic social defeat stress (CSDS) or single housing, respectively. Following CSDS, mice were classified as susceptible versus resilient based on their social interaction behaviors. We synthesized a viral overexpression (OE) vector for H1x and transduced all stressed and single housed mice with either H1x or control GFP within vHipp. Following viral delivery, we conducted social, anxiety-like, and memory-reliant behavior tests on distinct cohorts of mice. We found no behavioral adaptations following H1x OE compared to GFP controls in susceptible, resilient, or single housed mice. In sum, although we confirm elevated vHipp protein levels of H1x associate with susceptibility to social stress, we observe no significant behavioral consequence of H1x OE. Thus, we conclude elevated levels of H1x are associated with, but are not singularly sufficient to drive development of behavioral adaptations to stress.

Microglia contribute to methamphetamine reinforcement and reflect persistent transcriptional and morphological adaptations to the drug

Methamphetamine use disorder (MUD) is a chronic, relapsing disease that is characterized by repeated drug use despite negative consequences and for which there are currently no FDA-approved cessation therapeutics. Repeated methamphetamine (METH) use induces long-term gene expression changes in brain regions associated with reward processing and drug-seeking behavior, and recent evidence suggests that methamphetamine-induced neuroinflammation may also shape behavioral and molecular responses to the drug. Microglia, the resident immune cells in the brain, are principal drivers of neuroinflammatory responses and contribute to the pathophysiology of substance use disorders. Here, we investigated transcriptional and morphological changes in dorsal striatal microglia in response to methamphetamine-taking and during methamphetamine abstinence, as well as their functional contribution to drug-taking behavior. We show that methamphetamine self-administration induces transcriptional changes associated with protein folding, mRNA processing, immune signaling, and neurotransmission in dorsal striatal microglia. Importantly, many of these transcriptional changes persist through abstinence, a finding supported by morphological analyses. Functionally, we report that microglial ablation increases methamphetamine-taking, possibly involving neuroimmune and neurotransmitter regulation. In contrast, microglial depletion during abstinence does not alter methamphetamine-seeking. Taken together, these results suggest that methamphetamine induces both short and long-term changes in dorsal striatal microglia that contribute to altered drug-taking behavior and may provide valuable insights into the pathophysiology of MUD.

The hallmarks of a tradeoff in transcriptomes that balances stress and growth functions.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

Mediator kinase inhibition impedes transcriptional plasticity and prevents resistance to ERK/MAPK-targeted therapy in KRAS-mutant cancers

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Fine-Tuning Amyloid Precursor Protein Expression through Nonsense-Mediated mRNA Decay.

Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.

Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Unraveling Key Factors for Hypoxia Tolerance in Contrasting Varieties of Cotton Rose by Comparative Morpho-physiological and Transcriptome Analysis.

The cotton rose (Hibiscus mutabilis) is a plant species commonly found in tropical and subtropical regions. It is remarkably resilient to waterlogging stress; however, the underlying mechanism behind this trait is yet unknown. This study used hypoxia-tolerant "Danbanhong" (DBH) and more hypoxia-sensitive "Yurui" (YR) genotypes and compared their morpho-physiological and transcriptional responses to hypoxic conditions. Notably, DBH had a higher number of adventitious roots (20.3) compared to YR (10.0), with longer adventitious roots in DBH (18.3 cm) than in YR (11.2 cm). Furthermore, the formation of aerenchyma was 3-fold greater in DBH compared to YR. Transcriptomic analysis revealed that DBH had more rapid transcriptional responses to hypoxia than YR. Identification of a greater number of differentially expressed genes (DEGs) for aerenchyma, adventitious root formation and development, and energy metabolism in DBH supported that DBH had better morphological and transcriptional adaptation than YR. DEG functional enrichment analysis indicated the involvement of variety-specific biological processes in adaption to hypoxia. Plant hormone signaling transduction, MAPK signaling pathway and carbon metabolism played more pronounced roles in DBH, whereas the ribosome genes were specifically induced in YR. These results show that effective multilevel coordination of adventitious root development and aerenchyma, in conjunction with plant hormone signaling and carbon metabolism, is required for increased hypoxia tolerance. This study provides new insights into the characterization of morpho-physiological and transcriptional responses to hypoxia in H. mutabilis, shedding light on the molecular mechanisms of its adaptation to hypoxic environments.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation

ABSTRACT Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Abstract 3303: Paralogue selective p300 degraders induce synthetic lethality in pre-clinical models of CBP-deficient and p300-dependent malignancies

Abstract EP300 (or p300) acts as histone acetyltransferase (HAT) and transcriptional adapter or co-activator regulating transcription via chromatin remodeling. Both histone and non-histone proteins are acetylated by p300. In addition to HAT function, p300 has crotonyl-transferase activities, and that p300-catalyzed histone crotonylation directly stimulates transcription to a greater degree. p300 functions by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, NFκB, c-Myc, estrogen receptor (ER) and androgen receptor (AR) and other proteins such as PD-L1 and FOXP3. Selective targeting of p300 is expected to lead to therapeutic efficacy in CBP-mutant and p300-dependent malignancies with high degree of tolerability because of sparing of the other paralog CBP in normal cells. CBP mutant cancers comprise of several solid and hematological malignancies including 10% to 15% of non-small cell and small cell lung cancers harboring loss-of-function (LOF) aberrations. p300-dependent malignancies include prostate cancer, in which p300 plays a major role for androgen-dependent and -independent transactivation of the AR, MYCN-amplified neuroblastoma and ER+ breast cancers. Since the conventional CBP/p300 inhibitors do not discriminate between CBP and p300 proteins due to high sequence homology, we adopted a degrader approach, which has the potential to lead to paralog selectivity due to differentiated ternary complex formation. Herein we report best-in-class novel, potent and paralog selective p300 degraders with excellent selectivity over CBP and and other bromodomain containing proteins such as BRD4 by implementing molecular modeling, iterative medicinal chemistry and SAR based approaches. Synthetic lethality has been demonstrated while selectively degrading p300 in the cell lines having LOF mutation of another paralogue, CBP. Identified selective p300 degraders displayed excellent selectivity towards p300 degradation across tested cell lines and are orally bioavailable. Selective antiproliferative activity was observed in a panel of cell lines with CBP mutations or p300 dependency including AR+/ER+ cell lines along with the downregulation of target genes. In summary, we have identified highly selective degraders of p300 with desirable profile. Efforts are underway to advance them further towards nominating a development candidate. Citation Format: Saravanan Thiyagarajan, Chandrasekhar Abbineni, Krishna Chaitanya T, Achala Apte, Iram Khan Iqbal, Naveen Kumar R, Aravind A B, Avinash Kumar, Dabbeeru Madhu Babu, Shwetha Shwetha, Ankita Manna, Monalisha Mandal, Rituparna Panigrahi, Sivapriya Marappan, Suraj T Gore, Mohamad Fairus, Lim Yang Jin, Subhendu Mukherjee, Girish Daginakatte, Shekar Chelur, Kavitha Nellore, Murali Ramachandra, Susanta Samajdar. Paralogue selective p300 degraders induce synthetic lethality in pre-clinical models of CBP-deficient and p300-dependent malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3303.

CF2-II Alternative Splicing Isoform Regulates the Expression of Xenobiotic Tolerance-Related Cytochrome P450 CYP6CY22 in Aphis gossypii Glover.

The expression of P450 genes is regulated by trans-regulatory factors or cis-regulatory elements and influences how endogenous or xenobiotic substances are metabolized in an organism's tissues. In this study, we showed that overexpression of the cytochrome P450 gene, CYP6CY22, led to resistance to cyantraniliprole in Aphis gossypii. The expression of CYP6CY22 increased in the midgut and remaining carcass of the CyR strain, and after repressing the expression of CYP6CY22, the mortality of cotton aphids increased 2.08-fold after exposure to cyantraniliprole. Drosophila ectopically expressing CYP6CY22 exhibited tolerance to cyantraniliprole and cross-tolerance to xanthotoxin, quercetin, 2-tridecanone, tannic acid, and nicotine. Moreover, transcription factor CF2-II (XM_027994540.2) is transcribed only as the splicing variant isoform CF2-II-AS, which was found to be 504 nucleotides shorter than CF2-II in A. gossypii. RNAi and yeast one-hybrid (Y1H) results indicated that CF2-II-AS positively regulates CYP6CY22 and binds to cis-acting element p (-851/-842) of CYP6CY22 to regulate its overexpression. The above results indicated that CYP6CY22 was regulated by the splicing isoform CF2-II-AS, which will help us further understand the mechanism of transcriptional adaption of cross-tolerance between synthetic insecticides and plant secondary metabolites mediated by P450s.

Abstract B052: The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer

Abstract Introduction: Previously we discovered that a novel metastasis-initiating cell (MIC) population is marked by LMO2, a transcriptional adaptor protein in hematopoietic stem cells, and a T-cell oncogene. Knocking down LMO2 did not affect the growth of primary tumors, but did significantly reduce the number of circulating tumor cells and lung metastases. Transcriptional analysis of LMO2 knockdown shows the HALLMARK_DNA_REPAIR pathway is upregulated, suggesting that LMO2 is involved in regulating DNA repair in breast cancer cells. Interestingly, a previous study reported that in diffuse large B-cell lymphoma high LMO2 expression results in defective repair of double-stranded DNA breaks (Parvin et al., Cancer Cell, 36, 3 [2019]). We hypothesize that in breast cancer LMO2 expression is regulating the choice between homology-directed repair (HDR), an error-free method of DNA repair, and non-homologous end-joining (NHEJ), a more error-prone method. Cells that use NHEJ to repair DNA are dependent on poly (ADP-ribose) polymerase (PARP) and can be targeted with PARP inhibitors. LMO2 could thus be used as a new biomarker for benefit from PARP inhibition. Methods: LMO2 was identified using single-cell RNAseq data from human breast cancer patients. In vitro and in vivo assays using breast cancer cell lines and patient-derived xenografts were used to characterize the effect of LMO2 on metastasis. To study the role of LMO2 in the DNA damage response, we used MDA-MB-468 and HCC1806 cells with control and 2 independent shRNAs targeting LMO2. Immunofluorescent staining for γH2AX, 53BP1, and RAD51 was performed to measure changes in DNA damage and repair markers. Control and LMO2 knockdown cells were treated with olaparib, with cellular proliferation measured with a WST-1 assay and colony formation measured with crystal violet staining. Results and Conclusion: Knocking down LMO2 expression results in decreased NHEJ and increased HDR in MDA-MB-468 and HCC1806 cells. There is no significant difference in DNA damage overall as measured by γH2AX foci/nucleus. LMO2 knockdown in MDA-MB-468 cells reduces sensitivity to PARP inhibition with olaparib, as measured by an increase in the IC50 values. Our preliminary data suggest that LMO2 expression likely regulates the choice between NHEJ and HDR. LMO2 expression introduces a bias towards NHEJ, which sensitizes cells to PARP inhibition. This suggests that LMO2+ cells could be targeted with PARP inhibition, which could thus be used to target metastasis-initiating cells. Citation Format: Isobel Fetter, Veronica Haro-Acosta, Shaheen Sikandar. The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B052.

The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum.

Roots are fundamental for plants to adapt to variable environmental conditions. The development of a robust root system is orchestrated by numerous genetic determinants and, among them, the MADS-box gene ANR1 has garnered substantial attention. Prior research has demonstrated that, in chrysanthemum, CmANR1 positively regulates root system development. Nevertheless, the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified. In this study, we successfully identified bric-a-brac, tramtrack and broad (BTB) and transcription adapter putative zinc finger (TAZ) domain protein CmBT1 as the interacting partner of CmANR1 through a yeast-two-hybrid (Y2H) screening library. Furthermore, we validated this physical interaction through bimolecular fluorescence complementation and pull-down assays. Functional assays revealed that CmBT1 exerted a negative influence on root development in chrysanthemum. In both in vitro and in vivo assays, it was evident that CmBT1 mediated the ubiquitination of CmANR1 through the ubiquitin/26S proteasome pathway. This ubiquitination subsequently led to the degradation of the CmANR1 protein and a reduction in the transcription of CmANR1-targeted gene CmPIN2, which was crucial for root development in chrysanthemum. Genetic analysis suggested that CmBT1 modulated root development, at least in part, by regulating the level of CmANR1 protein. Collectively, these findings shed new light on the regulatory role of CmBT1 in degrading CmANR1 through ubiquitination, thereby repressing the expression of its targeted gene and inhibiting root development in chrysanthemum.

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Chemical genetic screens are a powerful tool for exploring how cancer cells' response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or nuclear factor κB (NF-κB) inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

Molecular Signaling and Metabolic Responses during the Interaction between Human Keratinocytes (HaCaT) and the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is the leading causative agent of dermatophytosis worldwide. Keratinocytes are the first line of defense that drives an immune response against fungal invasion. Host-specific pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) to trigger immunological pathways. Fungal cell wall components are the primary sources of fungal PAMPs, and some pathogens increase cell wall rearrangement to evade the immune system. Glycolysis and enhanced lactate levels are critical for improving host immune responses to fungal infections. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we evaluated the transcriptional responses of human genes involved in fungal recognition and glycolytic metabolism and fungal cell-wall-related genes in a co-culture model of human keratinocytes with T. rubrum. We observed the upregulation of several Toll-like receptors (TLRs), NOD-like receptors (NLRs), and glycolytic genes. Complementarily, we measured intra- and extracellular glucose levels and the increase in lactate production in the co-culture supernatant. We noted a distinct transcriptional regulation pattern of fungal cell-wall-related genes from fungal growth on keratin as the primary carbon source compared to co-culture with human keratinocytes. Our results showed new insights into the transcriptional adaptation of keratinocytes, particularly in regulating genes involved in sensing and metabolic processes, during the interaction with T. rubrum.

'Against all floods': plant adaptation to flooding stress and combined abiotic stresses.

Current climate change brings with it a higher frequency of environmental stresses, which occur in combination rather than individually leading to massive crop losses worldwide. In addition to, for example, drought stress (low water availability), also flooding (excessive water) can threaten the plant, causing, among others, an energy crisis due to hypoxia, which is responded to by extensive transcriptional, metabolic and growth-related adaptations. While signalling during flooding is relatively well understood, at least in model plants, the molecular mechanisms of combinatorial flooding stress responses, for example, flooding simultaneously with salinity, temperature stress and heavy metal stress or sequentially with drought stress, remain elusive. This represents a significant gap in knowledge due to the fact that dually stressed plants often show unique responses at multiple levels not observed under single stress. In this review, we (i) consider possible effects of stress combinations from a theoretical point of view, (ii) summarize the current state of knowledge on signal transduction under single flooding stress, (iii) describe plant adaptation responses to flooding stress combined with four other abiotic stresses and (iv) propose molecular components of combinatorial flooding (hypoxia) stress adaptation based on their reported dual roles in multiple stresses. This way, more future emphasis may be placed on deciphering molecular mechanisms of combinatorial flooding stress adaptation, thereby potentially stimulating development of molecular tools to improve plant resilience towards multi-stress scenarios.

Lingvokultural va tiliy tarjima aspektlari: Proper nomlarning ingliz tilidan Karakalpak tiliga tarjimasi

This thesis examines the current problems in translating different terms, especially in terms of geopolitical terms and antroponyms from English into Karakalpak language and some methods and ways of translating these terms paying attention to the peculiar feature of Karakalpak language. It also discusses useful tips and suggestions in translating proper names using transcription, transliteration and national adaptation.

Harnessing PROTAC technology to combat stress hormone receptor activation

Counteracting the overactivation of glucocorticoid receptors (GR) is an important therapeutic goal in stress-related psychiatry and beyond. The only clinically approved GR antagonist lacks selectivity and induces unwanted side effects. To complement existing tools of small-molecule-based inhibitors, we present a highly potent, catalytically-driven GR degrader, KH-103, based on proteolysis-targeting chimera technology. This selective degrader enables immediate and reversible GR depletion that is independent of genetic manipulation and circumvents transcriptional adaptations to inhibition. KH-103 achieves passive inhibition, preventing agonistic induction of gene expression, and significantly averts the GR’s genomic effects compared to two currently available inhibitors. Application in primary-neuron cultures revealed the dependency of a glucocorticoid-induced increase in spontaneous calcium activity on GR. Finally, we present a proof of concept for application in vivo. KH-103 opens opportunities for a more lucid interpretation of GR functions with translational potential.

A Rare Cell State Underpins Diverse Mechanisms of Adaptive Resistance to CAR T-Cell Therapy in B-Cell Acute Lymphoblastic Leukemia

The most common pattern of treatment failure of B-cell acute lymphoblastic leukemia (B-ALL) following CD19-targeting chimeric antigen receptor T cell therapy (CAR T), is a measurable residual disease (MRD) negative response followed by relapse. Whilst in a fraction of patients relapse is associated with loss of function mutations &/or deletions of the CD19 locus, a significant proportion of patients relapse with CD19+ disease. We hypothesised that this arises from a rare CAR T tolerant persister population that is able to survive the strong therapeutic pressure and seed relapse. To address this, we investigated the clonal origin and transcriptional dynamics of “CAR T tolerant persister cells” via time resolved clonal tracing of the progeny of individual B-ALL clones, using expressed molecular barcodes in an immunocompetent mouse model of CD19 targeting CAR T. We demonstrate that relapse is a pre-determined fate of rare B-ALL clones, which each adopt diverse, yet clone-specific, transcriptional phenotypes at relapse. Moreover, each diverse relapse phenotype appears to be underpinned by a shared, rare “origin of relapse” cell state that is common to all relapse-fated clones. Our immunocompetent mouse model of murine CAR T cells targeting CD19 (ID3-h41bb-3z) on a syngeneic, BCR::ABL1(p190) driven B-ALL recapitulates the clinical scenario of MRD-negative remission followed by CD19+ relapse. We labelled B-ALL cells with SPLINTR (Fennell & Vassiliadis et al. Nature 2022), a lentiviral lineage tracing reagent comprising expressed molecular barcodes that are read out by single cell RNA sequencing (scRNAseq), to allow dynamic profiling of the clonal origin and transcriptome of individual B-ALL clones over time, across multiple therapeutic timepoints including pre-treatment, remission and relapse. Tumour initiating frequency in this model of B-ALL is high with 23% of transplanted clones able to establish fulminant B-ALL. Only 0.7% of clones, however, were able to persist following CAR-T pressure and seed relapse. In fact, >99% of post-CAR T relapsed disease was comprised by just 3 (0.3%) individual clones and in one mouse disease relapse was driven entirely a single clone. Remarkably, we found that the same clones were able to drive relapse in multiple mice, consistent with a the presence of pre-determined, heritable cell-intrinsic properties capable of causing relapse following CAR T. To further study this phenomenon, we performed scRNAseq on these murine B-ALL samples to examine the transcriptional profiles of these rare, relapse fated clones prior to and following CAR T. Whilst prior to therapy, relapse fated clones were transcriptionally identical to the CAR T sensitive clones, following CAR T we observed that relapse fated clones adopted distinct transcriptional profiles compared to their transcriptome in the absence of therapeutic pressure, suggesting transcriptional adaptation underpins their ability to survive therapy. We noted that each resistant clone had a unique adaptive response and consequent transcriptional signature. Remarkably however, the adaptive response of each clone in different mice was essentially identical, indicative of strong, conserved clone-intrinsic mechanisms of adaptation. Although each CAR T resistant clone adopted disparate transcriptional states in the bulk ALL, we identified a single rare cell state (~1% of the bulk B-ALL) that was shared between all clones, highly suggestive of a common point of origin from which transcriptional adaptation arises. This rare cell state demonstrates metabolic hallmarks of therapy resistance and a distinct developmental transcriptional profile most analogous to the recently characterised preBCR-dependant stage of normal B cell development, raising the prospect that these cells represent an “origin of relapse” cell state. Preliminary data suggests that this rare cell state may also be conserved in human Ph+ B-ALL. In summary, by modelling CD19+ B-ALL relapse following MRD negative remission after CAR T, we have identified a rare cell state that our analysis suggests serves as a common and necessary point from which a transcriptionally diverse range of B-ALL phenotypes can arise and be maintained. We have demonstrated that relapse following CAR T emerges due to clone-specific transcriptional differences from this rare cell state, a phenomenon which may extend to a diverse range of therapies in B-ALL.

Differentiation State Plasticity As a Mechanism of BCL2 Inhibitor Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is composed of a heterogeneous blast population that exhibits characteristics of normal myeloid cells, including immature, HSC-like cells and differentiated, monocyte-like cells. The balance of immature and differentiated blasts within a patient is important clinically, as immature AML blasts are acutely sensitive to treatment with venetoclax, a BH3 mimetic that blocks the anti-apoptotic activity of BCL2. BCL2-inhibitor-based therapeutic strategies are the standard of care among elderly patients with untreated or relapsed/refractory AML. While these strategies result in a remission rate of nearly 70%, the majority of patients who do achieve remission ultimately relapse. Frequently, the relapsed disease is composed of differentiated, monocyte-like blasts. Given the importance of BCL2 inhibitors to the clinical management of AML, it is critical to understand the mechanisms by which BCL2 inhibitors result in monocytic relapse. Disease relapse has been traditionally thought to emerge from intrinsically resistant cells that confer a survival advantage under therapeutic pressure. Several studies have suggested that differentiated AML blasts have a survival advantage in the presence of BCL2 inhibition due to a reliance on anti-apoptotic proteins other than BCL2. Immature AML blasts express high levels of BCL2, whereas blasts that are primed for lympho-myeloid or granulocytic-monocytic progenitor cell fates express high levels of MCL-1 and BCL-XL, respectively. However, these studies do not explain why patients with dominant, differentiated subclones frequently achieve long-lasting remission with BCL2-inhibitor-based strategies. Therefore, simple selection of differentiated blasts appears to be an insufficient model to describe BCL2 inhibitor resistance. Alternative models of therapeutic resistance have been identified where cancer cells adapt to a resistant phenotype and acquire a survival advantage under therapeutic pressure. In the context of BCL2 inhibitor resistance, it is unclear the extent to which the emergence of differentiated blasts is the result of selective pressure or blast differentiation state plasticity in response to BCL2 inhibition. In our work, we have investigated the adaptive mechanism of BCL2 inhibition resistance in AML. We have explored this mechanism using an AML cell line model in which immature AML cells continuously generate differentiated AML cells (OCI-AML8227). We used fluorescence-activated cell sorting to isolate immature CD34+/CD38- OCI-AML8227 cells. After 72 hours in liquid culture, we observed that these cells recapitulated a pool of differentiated CD34-/CD38+/CD14+ cells. Furthermore, we used single cell ATAC sequencing to confirm that the unfractionated pool of OCI-AML8227 cells display chromatin accessibility profiles resembling cells across the continuum of myeloid differentiation. To understand whether this model is appropriate to investigate the differentiation-state-specific differences in sensitivity to BLC2 inhibition, we measured the viability of immature CD34+ and differentiated CD34- OCI-AML8227 cells following BCL2 inhibition. Consistent with previous studies, immature CD34+ cells were significantly more sensitive to BCL2 inhibition than differentiated CD34- cells. In addition, BLC2 inhibition of unfractionated OCI-AML8227 cells resulted in an 82% increase in the number of differentiated CD34-/CD38+ cells. While the number of immature CD34+/CD38- cells was reduced following BCL2 inhibition, it was only reduced by 27%. Furthermore, there was no change in the apoptosis marker Annexin V among immature CD34+/CD38- cells. These results support an adaptive resistance model where AML cells acquire resistance through differentiation state plasticity. We will explore this mechanism by following the temporal dynamics of immature and differentiated blasts following BCL2 inhibition. Using single cell barcoding, we will not only be able to trace the trajectories of these populations but also investigate transcriptional adaptation of the immature and differentiated populations. With an understanding of the adaptive mechanism of resistance to BCL2 inhibition, we can nominate alternative targets to prevent the outgrowth of resistance populations and design more effective, rational BLC2-inhibitor-based therapeutic strategies.