bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2021‒10‒31
twenty-four papers selected by
Ralitsa Radostinova Madsen
University College London Cancer Institute

  1. JHEP Rep. 2021 Dec;3(6): 100359
      Background & Aims: Phosphatidylinositides-3 kinases (PI3Ks) are promising drug targets for cancer therapy, but blockage of PI3K-AKT signalling causes hyperglycaemia, hyperinsulinaemia, and liver damage in patients, and hepatocellular carcinoma (HCC) in mice. There are 4 PI3Ks: PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ. The role of PI3Kγ in HCC is unknown.Methods: We performed histopathological, metabolic, and molecular phenotyping of mice with genetic ablation of PI3Kγ using models where HCC was initiated by the carcinogen diethylnitrosamine (DEN) and promoted by dietary or genetic obesity (ob/ob). The role of PI3Kγ in leucocytes was investigated in mice lacking PI3Kγ in haematopoietic and endothelial cells.
    Results: Loss of PI3Kγ had no effects on the development of DEN-induced HCC in lean mice. However, in mice injected with DEN and placed on an obesogenic diet, PI3Kγ ablation reduced tumour growth, which was associated with reduced insulinaemia, steatosis, and expression of inflammatory cytokines. ob/ob mice lacking PI3Kγ, and mice with diet-induced obesity lacking PI3Kγ in leucocytes and endothelial cells did not display improved insulin sensitivity, steatosis, metabolic inflammation, or reduced tumour growth. However, these mice showed a reduced number of tumours, reduced liver infiltration by neutrophils, and reduced hepatocyte proliferation acutely induced by DEN.
    Conclusions: Loss of PI3Kγ reduces tumour development in obesity-promoted HCC through multiple cell types and mechanisms that include improved insulinaemia, steatosis, and metabolic inflammation as well as the regulation of acute neutrophil infiltration and compensatory hepatocyte proliferation. PI3Kγ-selective inhibition may represent a novel therapeutic approach to reduce HCC initiation and slow HCC progression.
    Lay summary: Class-1 phosphatidylinositides-3 kinases (PI3Ks) are critical targets in cancer therapy, but complete inhibition of all isoforms causes liver damage, hyperglycaemia, and insulinaemia. Here we show that selective ablation of the PI3Kγ isoform dampens tumour initiation and growth in a mouse model of carcinogen-initiated and obesity-promoted hepatocellular carcinoma (HCC). The effect of PI3Kγ ablation on reduced tumour growth was explained by reduced tumour cell proliferation, which was associated with reduced insulin levels, liver lipids, and reduced expression of tumour-promoting cytokines. PI3Kγ ablation in leucocytes of obese mice had no effects on tumour size. However, it reduced tumour number in association with reduced carcinogen-induced neutrophil infiltration and hepatocyte proliferation in livers of obese mice. Inhibition of PI3Kγ may thus reduce HCC initiation and growth in obese subjects by a mechanism involving reduced metabolic stress and insulinaemia and reduced carcinogen-induced neutrophil infiltration to the fatty liver.
    Keywords:  AKT; AST, aspartate aminotransferase; BMDM, bone marrow-derived macrophages; DEN, diethylnitrosamine; GTT, glucose tolerance test; HCC, hepatocellular carcinoma; HFD, high-fat diet; ITT, insulin tolerance test; Insulin; NAFLD; NASH; PI3K, phosphatidylinositides-3 kinase; PTEN, phosphatase and tensin homolog; RT, room temperature; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labelling; WT, wild-type; mTOR
  2. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01143-1. [Epub ahead of print] 101337
      The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal co-immunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD), but not with the MUC1-ED. Prior pharmacologic inhibition of NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-BA-DANA, did not diminish NEU1-MUC1-CD association. In addition, glutathione S-transferase (GST) pull-down assays utilizing deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 amino acids of the MUC1-CD. In a cell-free system, we found that purified NEU1 interacted with immobilized GST-MUC1-CD, and purified MUC1-CD associated with immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either wild-type NEU1 or a catalytically-dead NEU1 G68V mutant diminished association of the established MUC1-CD binding partner, phosphoinositide 3-kinase (PI3K), to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.
    Keywords:  Akt PBK; cell surface associated (MUC1); mucin 1; neuraminidase-1; phosphatidylinositide 3‐kinase (PI 3‐kinase); sialic acid; sialidase
  3. Chem Sci. 2020 Apr 07. 11(13): 3511-3515
      PI3Kα controls several cellular processes and its aberrant signalling is implicated in tumorigenesis. One of its hotspot mutations, E545K, increases PI3Kα lipid kinase activity, but its mode of action is only partially understood. Here, we perform biased and unbiased molecular dynamics simulations of PI3Kα and uncover, for the first time, the free energy landscape of the E545K PI3Kα mutant. We reveal the mechanism by which E545K leads to PI3Kα activation in atomic-level detail, which is considerably more complex than previously thought.
  4. PLoS One. 2021 ;16(10): e0259426
      ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.
  5. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01136-4. [Epub ahead of print] 101330
      CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the function of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-β receptor (TGF- βR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-βR and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-βR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-β, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.
    Keywords:  C-terminal Src kinase (CSK); SMAD3; STAT3; T cell differentiation; T cell receptor (TCR); T regulatory cell; Th17; Treg; cytokine; immunity; interleukin 6 (IL-6); mTOR; phosphoinositide 3-kinase (PI3K); phospholipid signaling; protein kinase A (PKA); proteomics; transforming growth factor β (TGF-β)
  6. BMC Cancer. 2021 Oct 28. 21(1): 1152
      BACKGROUND: Phosphatase and tensin homolog (PTEN) loss is associated with tumorigenesis, tumor progression, and therapy resistance in breast cancer. However, the clinical value of PTEN as a biomarker in these patients is controversial. We sought to determine whether the benefit of traditional biomarkers testing is improved by the analysis of PTEN status for the identification of high-risk breast cancer.METHODS: A cohort of 608 patients with breast cancer was included in this study. Based on the expression on the neoplastic cells compared to the normal internal controls by immunohistochemistry (IHC), cases were classified as PTEN-low (PTEN-L) or PTEN-retained (PTEN-WT). The former constituted the study group, while the latter the control group. Analysis of gene expression was performed on publicly available genomic data and included 4265 patients from the METABRIC and MSK cohorts retrieved from cBioPortal. The Shapiro-Wilk test was used to analyze the normal distributions of continuous variables. Relationships between PTEN status and the clinicopathologic and molecular features of the patient population were assessed using Fisher's exact test or Chi-squared/Wilcoxon rank-sum test. Survival curves were built according to the Kaplan-Meier method.
    RESULTS: Alteration in PTEN status was significantly different at protein and gene levels, where the reduced protein expression was observed in 280/608 cases (46.1%) from our group, while genetic aberrations in only 315/4265 (7.4%) cases of the METABRIC and MSK cohorts. PTEN-L tumors were significantly enriched for hormone receptors (HR) and HER2 negativity (n = 48, 17.1%) compared to PTEN-WT tumors (n = 22, 6.7%; p = 0.0008). Lack of HR with or without HER2 overexpression/amplification was significantly associated with worse overall survival (OS) in PTEN-L but not in PTEN-WT breast cancers (p < .0001). Moreover, PTEN-L protein expression but not gene alterations was related to the outcome, in terms of both OS and disease-free survival (p = 0.002).
    CONCLUSIONS: The combined analysis of PTEN, HER2, and HR status offers relevant information for a more precise risk assessment of patients with breast cancer.
    Keywords:  Biomarkers; Breast cancer; Estrogen receptor; HER2; Hormone receptors; PTEN; Progesterone receptor; Prognosis
  7. Mol Syst Biol. 2021 Oct;17(10): e10141
      Tumor relapse from treatment-resistant cells (minimal residual disease, MRD) underlies most breast cancer-related deaths. Yet, the molecular characteristics defining their malignancy have largely remained elusive. Here, we integrated multi-omics data from a tractable organoid system with a metabolic modeling approach to uncover the metabolic and regulatory idiosyncrasies of the MRD. We find that the resistant cells, despite their non-proliferative phenotype and the absence of oncogenic signaling, feature increased glycolysis and activity of certain urea cycle enzyme reminiscent of the tumor. This metabolic distinctiveness was also evident in a mouse model and in transcriptomic data from patients following neo-adjuvant therapy. We further identified a marked similarity in DNA methylation profiles between tumor and residual cells. Taken together, our data reveal a metabolic and epigenetic memory of the treatment-resistant cells. We further demonstrate that the memorized elevated glycolysis in MRD is crucial for their survival and can be targeted using a small-molecule inhibitor without impacting normal cells. The metabolic aberrances of MRD thus offer new therapeutic opportunities for post-treatment care to prevent breast tumor recurrence.
    Keywords:  glycolysis; metabolic modeling; multi-omics integration; oncogenic memory; organoids
  8. Genes Dev. 2021 Oct 28.
      Understanding the genetic control of human embryonic stem cell function is foundational for developmental biology and regenerative medicine. Here we describe an integrated genome-scale loss- and gain-of-function screening approach to identify genetic networks governing embryonic stem cell proliferation and differentiation into the three germ layers. We identified a deep link between pluripotency maintenance and survival by showing that genetic alterations that cause pluripotency dissolution simultaneously increase apoptosis resistance. We discovered that the chromatin-modifying complex SAGA and in particular its subunit TADA2B are central regulators of pluripotency, survival, growth, and lineage specification. Joint analysis of all screens revealed that genetic alterations that broadly inhibit differentiation across multiple germ layers drive proliferation and survival under pluripotency-maintaining conditions and coincide with known cancer drivers. Our results show the power of integrated multilayer genetic screening for the robust mapping of complex genetic networks.
    Keywords:  genetic screening; germ layer formation; human embryonic stem cells
  9. Nat Commun. 2021 Oct 26. 12(1): 6176
      Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.
  10. Cancer Lett. 2021 Oct 22. pii: S0304-3835(21)00532-2. [Epub ahead of print]524 206-218
      Phosphatidylinositol 3-kinase (PI3K) δ-specific inhibitors have been approved for the therapy of certain types of B cell lymphoma (BCL). However, their clinical use is limited by the substantial toxicity and lack of efficacy in other types of BCL. Emerging evidence indicates that PI3Kα plays important roles in the progression of B cell lymphoma. In this study, we revealed that PI3Kα was important for the PI3K signaling and proliferation in BCL cells. A novel clinical PI3Kα-selective inhibitor CYH33 possessed superior activity against BCL compared to the marketed PI3Kα-selective inhibitor Alpelisib and PI3Kδ-selective inhibitor Idelalisib. Though CYH33 was able to inhibit PI3K/AKT signaling in tested BCL cells, differential activity against proliferation was observed. Transcriptome profiling revealed that CYH33 down-regulated "MYC-targets" gene set in sensitive but not resistant cells. CYH33 inhibited c-MYC transcription in sensitive cells, which was attributed to a decrease in acetylated H3 bound to the promoter and super-enhancer region of c-MYC. Accordingly, CYH33 treatment resulted in phosphorylation and proteasomal degradation of the histone acetyltransferase p300. An unbiased screening with drugs approved or in clinical trials for the therapy of BCL identified that the clinical BET (Bromodomain and Extra Terminal domain) inhibitor OTX015 significantly potentiated the activity of CYH33 against BCL in vitro and in vivo, which was associated with enhanced inhibition on c-MYC expression and induction of cell cycle arrest and apoptosis. Our findings provide the rationale of combined CYH33 with BET inhibitors for the therapy of B cell lymphoma.
    Keywords:  B cell lymphoma; BET; CYH33; MYC; P300; PI3K
  11. Oncogene. 2021 Oct 26.
      Intratumoral heterogeneity has been described for various tumor types and models of human cancer, and can have profound effects on tumor progression and drug resistance. This study describes an in-depth analysis of molecular and functional heterogeneity among subclonal populations (SCPs) derived from a single triple-negative breast cancer cell line, including copy number analysis, whole-exome and RNA sequencing, proteome analysis, and barcode analysis of clonal dynamics, as well as functional assays. The SCPs were found to have multiple unique genetic alterations and displayed significant variation in anchorage independent growth and tumor forming ability. Analyses of clonal dynamics in SCP mixtures using DNA barcode technology revealed selection for distinct clonal populations in different in vitro and in vivo environmental contexts, demonstrating that in vitro propagation of cancer cell lines using different culture conditions can contribute to the establishment of unique strains. These analyses also revealed strong enrichment of a single SCP during the development of xenograft tumors in immune-compromised mice. This SCP displayed attenuated interferon signaling in vivo and reduced sensitivity to the antiproliferative effects of type I interferons. Reduction in interferon signaling was found to provide a selective advantage within the xenograft microenvironment specifically. In concordance with the previously described role of interferon signaling as tumor suppressor, these findings suggest that similar selective pressures may be operative in human cancer and patient-derived xenograft models.
  12. Methods Mol Biol. 2022 ;2377 1-27
      Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete. Before tackling the complexities of lineage differentiation with genetic screens, it is critical to catalogue the general genetic requirements for cell fitness and proliferation in the pluripotent state and assess their plasticity under commonly used culture conditions.We describe a method to map essential genetic determinants of hPSC fitness and pluripotency, herein defined as cell reproduction, by genome-scale loss-of-function CRISPR screens in an inducible S. pyogenes Cas9 H1 hPSC line. To address questions of context-dependent gene essentiality, we include protocols for screening hPSCs cultured on feeder cells and laminin, two commonly used growth substrates. This method establishes parameters for genome-wide screens in hPSCs, making human stem cells amenable for functional genomics approaches to facilitate investigation of hPSC biology.
    Keywords:  Essential genes; Functional genomics; Genome-wide CRISPR screen; Growth substrate; Human pluripotent stem cells
  13. Cell Syst. 2021 Oct 21. pii: S2405-4712(21)00382-3. [Epub ahead of print]
      Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.
    Keywords:  Cathepsin L; GCN2; PDAC; lysosomes; macropinocytosis; protein scavenging; protein synthesis; translation
  14. Nat Methods. 2021 Oct 28.
      Charting an organs' biological atlas requires us to spatially resolve the entire single-cell transcriptome, and to relate such cellular features to the anatomical scale. Single-cell and single-nucleus RNA-seq (sc/snRNA-seq) can profile cells comprehensively, but lose spatial information. Spatial transcriptomics allows for spatial measurements, but at lower resolution and with limited sensitivity. Targeted in situ technologies solve both issues, but are limited in gene throughput. To overcome these limitations we present Tangram, a method that aligns sc/snRNA-seq data to various forms of spatial data collected from the same region, including MERFISH, STARmap, smFISH, Spatial Transcriptomics (Visium) and histological images. Tangram can map any type of sc/snRNA-seq data, including multimodal data such as those from SHARE-seq, which we used to reveal spatial patterns of chromatin accessibility. We demonstrate Tangram on healthy mouse brain tissue, by reconstructing a genome-wide anatomically integrated spatial map at single-cell resolution of the visual and somatomotor areas.
  15. Nat Commun. 2021 Oct 29. 12(1): 6246
      Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.
  16. Nat Commun. 2021 Oct 28. 12(1): 6216
      The risks associated with re-identification of human genetic data are severely limiting open data sharing in life sciences, even in studies where donor-related genetic variant information is not of primary interest. Here, we developed BAMboozle, a versatile tool to eliminate critical types of sensitive genetic information in human sequence data by reverting aligned reads to the genome reference sequence. Applying BAMboozle to functional genomics data, such as single-cell RNA-seq (scRNA-seq) and scATAC-seq datasets, confirmed the removal of donor-related single nucleotide polymorphisms (SNPs) and indels in a manner that did not disclose the altered positions. Importantly, BAMboozle only removes the genetic sequence variants of the sample (i.e., donor) while preserving other important aspects of the raw sequence data. For example, BAMboozled scRNA-seq data contained accurate cell-type associated gene expression signatures, splice kinetic information, and can be used for methods benchmarking. Altogether, BAMboozle efficiently removes genetic variation in aligned sequence data, which represents a step forward towards open data sharing in many areas of genomics where the genetic variant information is not of primary interest.
  17. Nat Genet. 2021 Oct 28.
      Genome-wide association studies (GWASs) have identified many variants associated with complex traits, but identifying the causal gene(s) is a major challenge. In the present study, we present an open resource that provides systematic fine mapping and gene prioritization across 133,441 published human GWAS loci. We integrate genetics (GWAS Catalog and UK Biobank) with transcriptomic, proteomic and epigenomic data, including systematic disease-disease and disease-molecular trait colocalization results across 92 cell types and tissues. We identify 729 loci fine mapped to a single-coding causal variant and colocalized with a single gene. We trained a machine-learning model using the fine-mapped genetics and functional genomics data and 445 gold-standard curated GWAS loci to distinguish causal genes from neighboring genes, outperforming a naive distance-based model. Our prioritized genes were enriched for known approved drug targets (odds ratio = 8.1, 95% confidence interval = 5.7, 11.5). These results are publicly available through a web portal ( ), enabling users to easily prioritize genes at disease-associated loci and assess their potential as drug targets.
  18. Dev Biol. 2021 Oct 21. pii: S0012-1606(21)00224-4. [Epub ahead of print]481 129-138
      Development is often driven by signaling and lineage-specific cues, yielding highly uniform and reproducible outcomes. Development also involves mechanisms that generate noise in gene expression and random patterns across tissues. Cells sometimes randomly choose between two or more cell fates in a mechanism called stochastic cell fate specification. This process diversifies cell types in otherwise homogenous tissues. Stochastic mechanisms have been extensively studied in prokaryotes where noisy gene activation plays a pivotal role in controlling cell fates. In eukaryotes, transcriptional repression stochastically limits gene expression to generate random patterns and specify cell fates. Here, we review our current understanding of repressive mechanisms that produce random patterns of gene expression and cell fates in flies, plants, mice, and humans.
    Keywords:  Adcy3; Cell fate; ComK; E(var); Ebf; Greek islands; Klumpfuss; LSD1; Lhx2; Olfactory sensory neuron; Opsin; Persister; Position effect variegation; R7 photoreceptor; Repression; Spineless; Stochasticity; Su(var); X-inactivation; Xist
  19. Annu Rev Physiol. 2021 Oct 27.
      Complex multicellular organisms have evolved specific mechanisms to replenish cells in homeostasis and during repair. Here, we discuss how emerging technologies (e.g., single-cell RNA sequencing) challenge the concept that tissue renewal is fueled by unidirectional differentiation from a resident stem cell. We now understand that cell plasticity, i.e., cells adaptively changing differentiation state or identity, is a central tissue renewal mechanism. For example, mature cells can access an evolutionarily conserved program (paligenosis) to reenter the cell cycle and regenerate damaged tissue. Most tissues lack dedicated stem cells and rely on plasticity to regenerate lost cells. Plasticity benefits multicellular organisms, yet it also carries risks. For one, when long-lived cells undergo paligenotic, cyclical proliferation and redifferentiation, they can accumulate and propagate acquired mutations that activate oncogenes and increase the potential for developing cancer. Lastly, we propose a new framework for classifying patterns of cell proliferation in homeostasis and regeneration, with stem cells representing just one of the diverse methods that adult tissues employ. Expected final online publication date for the Annual Review of Physiology, Volume 84 is February 2022. Please see for revised estimates.
  20. Med Res Rev. 2021 Oct 25.
      Precision oncology benefits from effective early phase drug discovery decisions. Recently, drugging inactive protein conformations has shown impressive successes, raising the cardinal questions of which targets can profit and what are the principles of the active/inactive protein pharmacology. Cancer driver mutations have been established to mimic the protein activation mechanism. We suggest that the decision whether to target an inactive (or active) conformation should largely rest on the protein mechanism of activation. We next discuss the recent identification of double (multiple) same-allele driver mutations and their impact on cell proliferation and suggest that like single driver mutations, double drivers also mimic the mechanism of activation. We further suggest that the structural perturbations of double (multiple) in cis mutations may reveal new surfaces/pockets for drug design. Finally, we underscore the preeminent role of the cellular network which is deregulated in cancer. Our structure-based review and outlook updates the traditional Mechanism of Action, informs decisions, and calls attention to the intrinsic activation mechanism of the target protein and the rewired tumor-specific network, ushering innovative considerations in precision medicine.
    Keywords:  K-Ras4B; KRAS; cancer network; driver mutations; drug discovery; inhibitor; kinases
  21. Nature. 2021 Oct 28.
      Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.
  22. Nat Cancer. 2021 Jun;2(6): 658-671
      Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+) breast cancer patients but its value in earlier stage patients is unclear. We examined evolutionary trajectories of early-stage breast cancer tumors, using single cell RNA sequencing (scRNAseq) of serial biopsies from the FELINE clinical trial (#NCT02712723) of endocrine therapy (letrozole) alone or combined with the CDK inhibitor ribociclib. Despite differences in subclonal diversity evolution across patients and treatments, common resistance phenotypes emerged. Resistant tumors treated with combination therapy showed accelerated loss of estrogen signaling with convergent up-regulation of JNK signaling through growth factor receptors. In contrast, cancer cells maintaining estrogen signaling during mono- or combination therapy showed potentiation of CDK4/6 activation and ERK upregulation through ERBB4 signaling. These results indicate that combination therapy in early-stage ER+ breast cancer leads to emergence of resistance through a shift from estrogen to alternative growth signal-mediated proliferation.
  23. Nat Protoc. 2021 Oct 29.
      Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.
  24. iScience. 2021 Nov 19. 24(11): 103177
      The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation-deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells.
    Keywords:  Biochemistry; Molecular biology; Protein