bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023–09–10
25 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. PI3K Signaling Through a Biochemical Systems Lens.
  2. Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel-Trenaunay syndrome in an Asian population : List the full names and institutional addresses for all authors.
  3. Conditional fluorescent mouse translocation reporters for ERK1/2 and AKT signaling.
  4. Single cell proteomics analysis of drug response shows its potential as a drug discovery platform.
  5. PTEN activates chaperone-mediated autophagy to regulate metabolism.
  6. eIF3d controls the persistent integrated stress response.
  7. NOTCH and AKT Signalling Interact to Drive Mammary Tumour Heterogeneity.
  8. AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.
  9. Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation.
  10. Setting standards for stem cells.
  11. A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo.
  12. Activation of the integrated stress response by inhibitors of its kinases.
  13. Aberrant PI3Kδ splice isoform as a potential biomarker and novel therapeutic target for endocrine cancers.
  14. A twin UGUA motif directs the balance between gene isoforms through CFIm and the mTORC1 signaling pathway.
  15. Whole mount of adult ear skin as a model to study vascular malformations.
  16. Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.
  17. Predicting the future.
  18. Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation.
  19. Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.
  20. An introduction to representation learning for single-cell data analysis.
  21. ComProliM: A cell growth assay robust to initial cell number in co-culture system.
  22. Parsaclisib, a PI3Kδ inhibitor, in relapsed and refractory follicular lymphoma (CITADEL-203): a phase 2 study.
  23. Studying Cellular Dynamics Using Proximity-Dependent Biotinylation: Somatic Cell Reprogramming.
  24. Coordinating culture change across the research landscape.
  25. The mutational landscape of the adult healthy parous and nulliparous human breast.
  1. J Biol Chem. 2023 Sep 04. pii: S0021-9258(23)02252-4. [Epub ahead of print] 105224
    PI3K Signaling Through a Biochemical Systems Lens.
    Ralitsa R Madsen, Alex Toker.
      
    DOI:  https://doi.org/10.1016/j.jbc.2023.105224
  2. Orphanet J Rare Dis. 2023 Sep 04. 18(1): 270
    Targeted next-generation sequencing for detection of PIK3CA mutations in archival tissues from patients with Klippel-Trenaunay syndrome in an Asian population : List the full names and institutional addresses for all authors.
    Yuki Sasaki, Kosuke Ishikawa, Kanako C Hatanaka, Yumiko Oyamada, Yusuke Sakuhara, Tadashi Shimizu, Tatsuro Saito, Naoki Murao, Tomohiro Onodera, Takahiro Miura, Taku Maeda, Emi Funayama, Yutaka Hatanaka, Yuhei Yamamoto, Satoru Sasaki.
       BACKGROUND: Klippel-Trenaunay syndrome (KTS) is a rare slow-flow combined vascular malformation with limb hypertrophy. KTS is thought to lie on the PIK3CA-related overgrowth spectrum, but reports are limited. PIK3CA encodes p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) that plays an essential role in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway. We aimed to demonstrate the clinical utility of targeted next-generation sequencing (NGS) in identifying PIK3CA mosaicism in archival formalin-fixed paraffin-embedded (FFPE) tissues from patients with KTS.
    RESULTS: Participants were 9 female and 5 male patients with KTS diagnosed as capillaro-venous malformation (CVM) or capillaro-lymphatico-venous malformation (CLVM). Median age at resection was 14 years (range, 5-57 years). Median archival period before DNA extraction from FFPE tissues was 5.4 years (range, 3-7 years). NGS-based sequencing of PIK3CA achieved an amplicon mean coverage of 119,000x. PIK3CA missense mutations were found in 12 of 14 patients (85.7%; 6/8 CVM and 6/6 CLVM), with 8 patients showing the hotspot variants E542K, E545K, H1047R, and H1047L. The non-hotspot PIK3CA variants C420R, Q546K, and Q546R were identified in 4 patients. Overall, the mean variant allele frequency for identified PIK3CA variants was 6.9% (range, 1.6-17.4%). All patients with geographic capillary malformation, histopathological lymphatic malformation or macrodactyly of the foot had PIK3CA variants. No genotype-phenotype association between hotspot and non-hotspot PIK3CA variants was found. Histologically, the vessels and adipose tissues of the lesions showed phosphorylation of the proteins in the PI3K/AKT/mTOR signaling pathway, including p-AKT, p-mTOR, and p-4EBP1.
    CONCLUSIONS: The PI3K/AKT/mTOR pathway in mesenchymal tissues was activated in patients with KTS. Amplicon-based targeted NGS could identify low-level mosaicism from low-input DNA extracted from FFPE tissues, potentially providing a diagnostic option for personalized medicine with inhibitors of the PI3K/AKT/mTOR signaling pathway.
    Keywords:  Capillary malformations; High-throughput nucleotide sequencing; Klippel–Trenaunay Syndrome; Limb hypertrophy; Lymphatic abnormalities; PIK3CA-related overgrowth spectrum; Phosphatidylinositol 3-Kinase; Segmental hypertrophy; Vascular malformations; Venous malformations
    DOI:  https://doi.org/10.1186/s13023-023-02893-1
  3. Dev Biol. 2023 Sep 01. pii: S0012-1606(23)00146-X. [Epub ahead of print]503 113-119
    Conditional fluorescent mouse translocation reporters for ERK1/2 and AKT signaling.
    Colin J Dinsmore, Philippe Soriano.
      Understanding how cells activate intracellular signaling pathways in response to external signals, such as growth factors, is a longstanding goal of cell and developmental biology. Recently, live-cell signaling reporters have greatly expanded our understanding of signaling dynamics in response to wide-ranging stimuli and chemical or genetic perturbation, both ex vivo (cell lines) and in vivo (whole embryos or animals). Among the many varieties of reporter systems, translocation reporters that change sub-cellular localization in response to pathway activation have received considerable attention for their ease of use compared to FRET systems and favorable response times compared to transcriptional reporters. We reasoned that mouse reporter lines expressed in a conditional fashion would be a useful addition to the arsenal of mouse genetic tools, as such lines remain undeveloped despite widespread use of these sensors. We therefore created and validated two novel mouse reporter lines at the ROSA26 locus. One expresses an ERK1/2 pathway reporter and a nuclear marker from a single transcript, while the second additionally expresses an AKT reporter in order to simultaneously interrogate both pathways.
    Keywords:  AKT; ERK; FOXO; Genetics; KTR; Mouse; Reporter; Signaling
    DOI:  https://doi.org/10.1016/j.ydbio.2023.08.007
  4. Mol Omics. 2023 Sep 08.
    Single cell proteomics analysis of drug response shows its potential as a drug discovery platform.
    Juerg Straubhaar, Alexandria D'Souza, Zachary Niziolek, Bogdan Budnik.
      Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us a unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments.
    DOI:  https://doi.org/10.1039/d3mo00124e
  5. Autophagy. 2023 Sep 08. 1-2
    PTEN activates chaperone-mediated autophagy to regulate metabolism.
    S Joseph Endicott, Richard A Miller.
      PTEN is a negative modulator of the INS-PI3K-AKT pathway and is an essential regulator of metabolism and cell growth. PTEN is one of the most commonly mutated tumor suppressors in cancer. However, PTEN overexpression extends the lifespan of both sexes of mice. We recently showed that PTEN is necessary and sufficient to activate chaperone-mediated autophagy (CMA) in the mouse liver and cultured cells. Selective protein degradation via CMA is required to suppress glycolysis and fatty acid synthesis when PTEN is overexpressed. Thus, activation of CMA downstream of PTEN might modulate health and metabolism through selective degradation of key metabolic enzymes.
    Keywords:  Aging; PTEN; autophagy; chaperone-mediated autophagy; metabolism
    DOI:  https://doi.org/10.1080/15548627.2023.2255966
  6. Mol Cell. 2023 Aug 30. pii: S1097-2765(23)00643-3. [Epub ahead of print]
    eIF3d controls the persistent integrated stress response.
    Shaoni Mukhopadhyay, Maria E Amodeo, Amy S Y Lee.
      Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.
    Keywords:  ATF4; GCN2; RNA methylation; eIF3d; integrated stress response; m(6)A; translation regulation
    DOI:  https://doi.org/10.1016/j.molcel.2023.08.008
  7. Cancers (Basel). 2023 Aug 29. pii: 4324. [Epub ahead of print]15(17):
    NOTCH and AKT Signalling Interact to Drive Mammary Tumour Heterogeneity.
    Liliana Ordonez, Giusy Tornillo, Howard Kendrick, Trevor Hay, Matthew John Smalley.
      A better understanding of the mechanisms generating tumour heterogeneity will allow better targeting of current therapies, identify potential resistance mechanisms and highlight new approaches for therapy. We have previously shown that in genetically modified mouse models carrying conditional oncogenic alleles, mammary tumour histotype varies depending on the combination of alleles, the cell type to which they are targeted and, in some cases, reproductive history. This suggests that tumour heterogeneity is not a purely stochastic process; rather, differential activation of signalling pathways leads to reproducible differences in tumour histotype. We propose the NOTCH signalling pathway as one such pathway. Here, we have crossed conditional knockout Notch1 or Notch2 alleles into an established mouse mammary tumour model. Notch1/2 deletion had no effect on tumour-specific survival; however, loss of Notch alleles resulted in a dose-dependent increase in metaplastic adenosquamous carcinomas (ASQCs). ASQCs and adenomyoepitheliomas (AMEs) also demonstrated a significant increase in AKT signalling independent of Notch status. Therefore, the NOTCH pathway is a suppressor of the ASQC phenotype, while increased PI3K/AKT signalling is associated with ASQC and AME tumours. We propose a model in which PI3K/AKT and NOTCH signalling act interact to determine mouse mammary tumour histotype.
    Keywords:  AKT; NOTCH; PI3K; PTEN; histotype; mammary tumour; mouse models
    DOI:  https://doi.org/10.3390/cancers15174324
  8. bioRxiv. 2023 Aug 24. pii: 2023.08.24.554685. [Epub ahead of print]
    AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.
    Siobhan K McRee, Abraham L Bayer, Jodie Pietruska, Philip N Tsichlis, Philip W Hinds.
      Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer, due to its highly metastatic nature. Melanomas harboring oncogenic BRAF V600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT-isoform specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo , while AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that elucidation of AKT2-specific functions in metastasis could inform therapeutic strategies to improve treatment options for melanoma patients.
    DOI:  https://doi.org/10.1101/2023.08.24.554685
  9. Cell Discov. 2023 Sep 07. 9(1): 92
    Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation.
    Terytty Yang Li, Qi Wang, Arwen W Gao, Xiaoxu Li, Yu Sun, Adrienne Mottis, Minho Shong, Johan Auwerx.
      Lysosomes are central platforms for not only the degradation of macromolecules but also the integration of multiple signaling pathways. However, whether and how lysosomes mediate the mitochondrial stress response (MSR) remain largely unknown. Here, we demonstrate that lysosomal acidification via the vacuolar H+-ATPase (v-ATPase) is essential for the transcriptional activation of the mitochondrial unfolded protein response (UPRmt). Mitochondrial stress stimulates v-ATPase-mediated lysosomal activation of the mechanistic target of rapamycin complex 1 (mTORC1), which then directly phosphorylates the MSR transcription factor, activating transcription factor 4 (ATF4). Disruption of mTORC1-dependent ATF4 phosphorylation blocks the UPRmt, but not other similar stress responses, such as the UPRER. Finally, ATF4 phosphorylation downstream of the v-ATPase/mTORC1 signaling is indispensable for sustaining mitochondrial redox homeostasis and protecting cells from ROS-associated cell death upon mitochondrial stress. Thus, v-ATPase/mTORC1-mediated ATF4 phosphorylation via lysosomes links mitochondrial stress to UPRmt activation and mitochondrial function resilience.
    DOI:  https://doi.org/10.1038/s41421-023-00589-1
  10. Nat Methods. 2023 Sep;20(9): 1267
    Setting standards for stem cells.
      
    DOI:  https://doi.org/10.1038/s41592-023-02016-5
  11. Nat Commun. 2023 Sep 06. 14(1): 5474
    A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo.
    Burcu Bestas, Sandra Wimberger, Dmitrii Degtev, Alexandra Madsen, Antje K Rottner, Fredrik Karlsson, Sergey Naumenko, Megan Callahan, Julia Liz Touza, Margherita Francescatto, Carl Ivar Möller, Lukas Badertscher, Songyuan Li, Silvia Cerboni, Niklas Selfjord, Elke Ericson, Euan Gordon, Mike Firth, Krzysztof Chylinski, Amir Taheri-Ghahfarokhi, Mohammad Bohlooly-Y, Mike Snowden, Menelaos Pangalos, Barrett Nuttall, Pinar Akcakaya, Grzegorz Sienski, Marcello Maresca.
      Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes.
    DOI:  https://doi.org/10.1038/s41467-023-41240-7
  12. Nat Commun. 2023 Sep 08. 14(1): 5535
    Activation of the integrated stress response by inhibitors of its kinases.
    Maria Szaruga, Dino A Janssen, Claudia de Miguel, George Hodgson, Agnieszka Fatalska, Aleksandra P Pitera, Antonina Andreeva, Anne Bertolotti.
      Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases.
    DOI:  https://doi.org/10.1038/s41467-023-40823-8
  13. Front Endocrinol (Lausanne). 2023 ;14 1190479
    Aberrant PI3Kδ splice isoform as a potential biomarker and novel therapeutic target for endocrine cancers.
    Siyoung Ha, Himali Gujrati, Bi-Dar Wang.
       Introduction: PI3K/AKT signaling pathway is upregulated in a broad spectrum of cancers. Among the class I PI3Ks (PI3Kδ/β/δ isoforms), PI3Kδ has been implicated in hematologic cancers and solid tumors. Alternative splicing is a post-transcriptional process for acquiring proteomic diversity in eukaryotic cells. Emerging evidence has highlighted the involvement of aberrant mRNA splicing in cancer development/progression.
    Methods: Our previous studies revealed that PIK3CD-S is an oncogenic splice variant that promotes tumor aggressiveness and drug resistance in prostate cancer (PCa). To further evaluate the potential of utilizing PI3Kδ-S (encoded from PIK3CD-S) as a cancer biomarker and/or drug target, comprehensive analyses were performed in a series of patient samples and cell lines derived from endocrine/solid tumors. Specifically, IHC, immunofluorescence, western blot and RT-PCR assay results have demonstrated that PI3Kδ isoforms were highly expressed in endocrine/solid tumor patient specimens and cell lines.
    Results: Differential PIK3CD-S/PIK3CD-L expression profiles were identified in a panel of endocrine/solid tumor cells. SiRNA knockdown of PIK3CD-L or PIK3CD-S differentially inhibits AKT/mTOR signaling in PCa, breast, colon and lung cancer cell lines. Moreover, siRNA knockdown of PTEN increased PI3Kδ levels and activated AKT/mTOR signaling, while overexpression of PTEN reduced PI3Kδ levels and inhibited AKT/mTOR signaling in cancer cells. Intriguingly, PI3Kδ-S levels remained unchanged upon either siRNA knockdown or overexpression of PTEN. Taken together, these results suggested that PTEN negatively regulates PI3Kδ-L and its downstream AKT/mTOR signaling, while PI3Kδ-S promotes AKT/mTOR signaling without regulation by PTEN. Lastly, PI3Kδ inhibitor Idelalisib and SRPK1/2 inhibitor SRPIN340 were employed to assess their efficacies on inhibiting the PI3Kδ-expressing endocrine/solid tumors. Our results have shown that Idelalisib effectively inhibited PI3Kδ-L (but not PI3Kδ-S) mediated AKT/mTOR signaling. In contrast, SRPIN340 reversed the aberrant mRNA splicing, thereby inhibiting AKT/mTOR signaling. In-vitro functional assays have further demonstrated that a combination of Idelalisib and SRPIN340 achieved a synergistic drug effect (with drastically reduced cell viabilities/growths of tumor spheroids) in inhibiting the advanced tumor cells.
    Conclusion: In summary, our study has suggested a promising potential of utilizing PI3Kδ-S (an oncogenic isoform conferring drug resistance and exempt from PTEN regulation) as a prognostic biomarker and drug target in advanced endocrine cancers.
    Keywords:  Idelalisib resistance; PTEN; aberrant PI3Kδ splice isoform; endocrine cancers; precision prognostic biomarker; synergistic drug effect
    DOI:  https://doi.org/10.3389/fendo.2023.1190479
  14. Elife. 2023 09 04. pii: e85036. [Epub ahead of print]12
    A twin UGUA motif directs the balance between gene isoforms through CFIm and the mTORC1 signaling pathway.
    R Samuel Herron, Alexander K Kunisky, Jessica R Madden, Vivian I Anyaeche, May Z Maung, Hun-Way Hwang.
      Alternative polyadenylation (APA) generates mRNA isoforms and diversifies gene expression. Here we report the discovery that the mTORC1 signaling pathway balances the expression of two Trim9/TRIM9 isoforms through APA regulation in human and mouse. We showed that CFIm components, CPSF6 and NUDT21, promote the short Trim9/TRIM9 isoform (Trim9-S/TRIM9-S) expression. In addition, we identified an evolutionarily conserved twin UGUA motif, UGUAYUGUA, in TRIM9-S polyadenylation site (PAS) that is critical for its regulation by CPSF6. We found additional CPSF6-regulated PASs with similar twin UGUA motifs in human and experimentally validated the twin UGUA motif functionality in BMPR1B, MOB4, and BRD4-L. Importantly, we showed that inserting a twin UGUA motif into a heterologous PAS was sufficient to confer regulation by CPSF6 and mTORC1. Our study reveals an evolutionarily conserved mechanism to regulate gene isoform expression by mTORC1 and implicates possible gene isoform imbalance in cancer and neurological disorders with mTORC1 pathway dysregulation.
    Keywords:  CLIP; Tsc1; alternative polyadenylation; chromosomes; gene expression; human; isoform; mTORC1; mouse; tuberous sclerosis complex
    DOI:  https://doi.org/10.7554/eLife.85036
  15. Animal Model Exp Med. 2023 Aug;6(4): 362-368
    Whole mount of adult ear skin as a model to study vascular malformations.
    Brandee Decker-Rockefeller, Qingfen Li, Kevin Pumiglia.
       BACKGROUND: Genetic analysis in human patients has linked mutations in PIK3CA, the catalytic subunit of PI-3'Kinase, to sporadic incidences of vascular malformations.
    METHODS: We have developed a mouse model with inducible and endothelial-specific expression of PIK3CAH1047R , resulting in the development of vascular malformations. Systemic induction of this mutation in adult mice results in rapid lethality, limiting our ability to track and study these lesions; therefore, we developed a topical and local induction protocol using the active metabolite of tamoxifen, 4OH-T, on the ear skin of adults.
    RESULTS: This approach allows us to successfully model the human disease in a mature and established vascular bed and track the development of vascular malformations. To validate the utility of this model, we applied a topical rapamycin ointment, as rapamycin is therapeutically beneficial to patients in clinical trials. We found that the induced ear lesions showed significant attenuation after treatment, which was easily quantified.
    CONCLUSIONS: These data collectively provide evidence of a new model to study vascular malformations in adult tissues, which should be particularly useful in environments lacking specialized small-animal imaging facilities.
    Keywords:  PIK3CA; animal disease models; pathological angiogenesis; rapamycin; vascular malformations
    DOI:  https://doi.org/10.1002/ame2.12343
  16. Science. 2023 Sep 08. 381(6662): eabn4180
    Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.
    Xiangdong Lv, Xuan Lu, Jin Cao, Qin Luo, Yao Ding, Fanglue Peng, Apar Pataer, Dong Lu, Dong Han, Eric Malmberg, Doug W Chan, Xiaoran Wang, Sara R Savage, Sufeng Mao, Jingjing Yu, Fei Peng, Liang Yan, Huan Meng, Laure Maneix, Yumin Han, Yiwen Chen, Wantong Yao, Eric C Chang, Andre Catic, Xia Lin, George Miles, Pengxiang Huang, Zheng Sun, Bryan Burt, Huamin Wang, Jin Wang, Qizhi Cathy Yao, Bing Zhang, Jack A Roth, Bert W O'Malley, Matthew J Ellis, Mothaffar F Rimawi, Haoqiang Ying, Xi Chen.
      Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances. However, IRE1α was selectively reactivated in an ER stress-independent manner in acquired KRASi-resistant tumors, restoring proteostasis. Oncogenic KRAS promoted IRE1α protein stability through extracellular signal-regulated kinase (ERK)-dependent phosphorylation of IRE1α, leading to IRE1α disassociation from 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) E3-ligase. In KRASi-resistant tumors, both reactivated ERK and hyperactivated AKT restored IRE1α phosphorylation and stability. Suppression of IRE1α overcame resistance to KRASi. This study reveals a druggable mechanism that leads to proteostasis reprogramming and facilitates KRASi resistance.
    DOI:  https://doi.org/10.1126/science.abn4180
  17. Elife. 2023 Sep 06. pii: e91450. [Epub ahead of print]12
    Predicting the future.
    Greg M Walter, Katrina McGuigan.
      Experiments on worms suggest that a statistical measure called the G matrix can accurately predict how phenotypes will adapt to a novel environment over multiple generations.
    Keywords:  C. elegans; G-matrix; adaptation; evolutionary biology; experimental evolution; locomotion behavior; phenotypic evolution; quantitative genetics
    DOI:  https://doi.org/10.7554/eLife.91450
  18. Nat Commun. 2023 Sep 05. 14(1): 5417
    Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation.
    Han Jin, Cheng Zhang, Martin Zwahlen, Kalle von Feilitzen, Max Karlsson, Mengnan Shi, Meng Yuan, Xiya Song, Xiangyu Li, Hong Yang, Hasan Turkez, Linn Fagerberg, Mathias Uhlén, Adil Mardinoglu.
      Cell lines are valuable resources as model for human biology and translational medicine. It is thus important to explore the concordance between the expression in various cell lines vis-à-vis human native and disease tissues. In this study, we investigate the expression of all human protein-coding genes in more than 1,000 human cell lines representing 27 cancer types by a genome-wide transcriptomics analysis. The cell line gene expression is compared with the corresponding profiles in various tissues, organs, single-cell types and cancers. Here, we present the expression for each cell line and give guidance for the most appropriate cell line for a given experimental study. In addition, we explore the cancer-related pathway and cytokine activity of the cell lines to aid human biology studies and drug development projects. All data are presented in an open access cell line section of the Human Protein Atlas to facilitate the exploration of all human protein-coding genes across these cell lines.
    DOI:  https://doi.org/10.1038/s41467-023-41132-w
  19. Methods Mol Biol. 2024 ;2707 279-303
    Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.
    Sergey V Prykhozhij, Jason N Berman.
      Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines.
    Keywords:  CRISPR/Cas9; Disease model; Knock-in; Long ssDNA; Point mutation; Zebrafish; ssODN
    DOI:  https://doi.org/10.1007/978-1-0716-3401-1_19
  20. Cell Rep Methods. 2023 08 28. 3(8): 100547
    An introduction to representation learning for single-cell data analysis.
    Ihuan Gunawan, Fatemeh Vafaee, Erik Meijering, John George Lock.
      Single-cell-resolved systems biology methods, including omics- and imaging-based measurement modalities, generate a wealth of high-dimensional data characterizing the heterogeneity of cell populations. Representation learning methods are routinely used to analyze these complex, high-dimensional data by projecting them into lower-dimensional embeddings. This facilitates the interpretation and interrogation of the structures, dynamics, and regulation of cell heterogeneity. Reflecting their central role in analyzing diverse single-cell data types, a myriad of representation learning methods exist, with new approaches continually emerging. Here, we contrast general features of representation learning methods spanning statistical, manifold learning, and neural network approaches. We consider key steps involved in representation learning with single-cell data, including data pre-processing, hyperparameter optimization, downstream analysis, and biological validation. Interdependencies and contingencies linking these steps are also highlighted. This overview is intended to guide researchers in the selection, application, and optimization of representation learning strategies for current and future single-cell research applications.
    Keywords:  deep learning; dimension reduction; hyperparameter; manifold learning; omics; systems microscopy
    DOI:  https://doi.org/10.1016/j.crmeth.2023.100547
  21. Heliyon. 2023 Sep;9(9): e19433
    ComProliM: A cell growth assay robust to initial cell number in co-culture system.
    Yunda Song, Subo Zhang.
      Cell growth is conventionally quantified using CCK-8 or MTT assays, but these methods display considerable sensitivity to initial cell quantities. Inherent sampling errors during cell counting and seeding make it impossible to achieve an absolute equivalence of initial cell numbers, potentially confounding the results of CCK-8 or MTT assays. In the present study, we introduce a novel cell proliferation assay, ComProliM, predicated on cell competition theory. Both numeral simulations and empirical testing demonstrate that ComProliM index (CPMI) reliably represents cell growth rate and is not influenced by variations in initial cell number. Intriguingly, two adherent cells of differing fluorescence states are co-cultured, suggesting that ComProliM can be successfully employed in co-culture system cell proliferation assays, including, for instance, the exploration of subclone interactions. We anticipate ComProliM will provide a viable alternative for quantifying adherent cell growth rates, particularly in cases where conventional methodologies deliver inconsistent or ambiguous results.
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e19433
  22. EClinicalMedicine. 2023 Sep;63 102130
    Parsaclisib, a PI3Kδ inhibitor, in relapsed and refractory follicular lymphoma (CITADEL-203): a phase 2 study.
    Marek Trněný, Abraham Avigdor, Matthew S McKinney, Shankara Paneesha, Björn E Wahlin, John S Hrom, David Cunningham, Nicholas Morley, Miguel Canales, Mariana Bastos-Oreiro, David Belada, Liliana Devizzi, Fred Zheng, Douglas J DeMarini, Wei Jiang, Ping Jiang, Ryan C Lynch.
       Background: Parsaclisib, a potent and highly selective PI3Kδ inhibitor, has shown clinical benefit in patients with relapsed or refractory (R/R) B-cell malignancies. This phase 2 study (CITADEL-203; NCT03126019, EudraCT 2017-001624-22) assessed efficacy and safety of parsaclisib monotherapy in patients with R/R follicular lymphoma (FL).
    Methods: Patients ≥18 years of age with histologically confirmed R/R FL (grade 1-3a) and prior treatment with ≥2 systemic therapies received parsaclisib 20 mg once daily (QD) for 8 weeks then parsaclisib 20 mg once weekly (weekly dosing group [WG]) or parsaclisib 20 mg QD for 8 weeks then parsaclisib 2.5 mg QD (daily dosing group [DG]); DG was selected for further assessment. Primary endpoint was objective response rate (ORR).
    Findings: At data cut-off (January 15, 2021), 126 patients had been treated (WG: n = 23; DG: n = 103). ORR (95% confidence interval [CI]) was 77.7% (68.4-85.3) with a complete response rate (95% CI) of 19.4% (12.3-28.4) in DG; median (95% CI) duration of response was 14.7 months (10.4-not estimable [NE]), median progression-free survival was 15.8 months (11.0-NE), and median overall survival was not reached. The most common any-grade treatment-emergent adverse events (TEAEs) among all treated patients included diarrhoea (n = 48, 38.1%), nausea (n = 31, 24.6%), and cough (n = 28, 22.2%); the most common grade ≥3 TEAEs were diarrhoea (n = 15, 11.9%), neutropenia (n = 13, 10.3%), and colitis (n = 7, 5.6%). Dose interruption, reduction, and discontinuation from TEAEs occurred in 46.8% (n = 59), 17.5% (n = 22), and 23.8% (n = 30) of patients, respectively.
    Interpretation: Treatment with parsaclisib demonstrated rapid and durable responses, and a manageable safety profile in patients with R/R FL.
    Funding: Incyte Corporation.
    Keywords:  Follicular lymphoma; Non-Hodgkin lymphoma; PI3K inhibitor; Parsaclisib
    DOI:  https://doi.org/10.1016/j.eclinm.2023.102130
  23. Methods Mol Biol. 2023 ;2718 23-52
    Studying Cellular Dynamics Using Proximity-Dependent Biotinylation: Somatic Cell Reprogramming.
    Reuben Samson, Francesco Zangari, Anne-Claude Gingras.
      Assessing the reorganization of proteins and organelles following the induction of reprogramming and differentiation programs is crucial to understand the mechanistic underpinning of morphological and fate changes associated with these processes. The advent of proximity-dependent biotinylation (PDB) methods has overcome some of the limitations of biochemical purification methods, enabling proteomic characterization of most subcellular compartments. The first-generation PDB enzyme, the biotin ligase BirA* used in BioID, has now been used in multiple studies determining the cellular context in which proteins reside, typically under standard growth conditions and using long labeling (usually 8-24 h) times. Capitalizing on the generation of more active PDB enzymes such as miniTurbo that can generate strong biotinylation signals in minutes rather than hours, as well as the development of an inducible lentiviral toolkit for BioID, we define here protocols for time-resolved PDB in primary cells. Here, we report the optimization and application of lentivirally delivered miniTurbo constructs to a mouse fibroblast model of somatic cell reprogramming, allowing the study of this dynamic process. This detailed protocol also provides a baseline reference for researchers who wish to adapt these techniques to other dynamic cellular processes.
    Keywords:  BioID; Biotin; Fibroblast; Lentivirus; Mass spectrometry; Protein-protein interaction; Proteomics; Proximity labeling; Proximity-dependent biotinylation; Reprogramming; Streptavidin
    DOI:  https://doi.org/10.1007/978-1-0716-3457-8_3
  24. Front Res Metr Anal. 2023 ;8 1134082
    Coordinating culture change across the research landscape.
    Leslie D McIntosh, Cynthia Hudson Vitale.
      Scientific integrity necessitates applying scientific methods properly, collecting and analyzing data appropriately, protecting human subjects rightly, performing studies rigorously, and communicating findings transparently. But who is responsible for upholding research integrity, mitigating misinformation, and increasing trust in science beyond individual researchers? We posit that supporting the scientific reputation requires a coordinated approach across all stakeholders: funding agencies, publishers, scholarly societies, research institutions, and journalists and media, and policy-makers.
    Keywords:  cross stakeholder coordination; culture change; disinformation; misinformation; research integrity
    DOI:  https://doi.org/10.3389/frma.2023.1134082
  25. Nat Commun. 2023 Sep 06. 14(1): 5136
    The mutational landscape of the adult healthy parous and nulliparous human breast.
    Biancastella Cereser, Angela Yiu, Neha Tabassum, Lisa Del Bel Belluz, Sladjana Zagorac, Kenneth Russell Zapanta Ancheta, Rongrong Zhong, Cristian Miere, Alicia Rose Jeffries-Jones, Nina Moderau, Benjamin Werner, Justin Stebbing.
      The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.
    DOI:  https://doi.org/10.1038/s41467-023-40608-z
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