Progenitor division and cell autonomous neurosecretion are required for rod photoreceptor sublaminar positioning.

Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.

Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses.

The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.

Loss of Epigenetic Regulation Disrupts Lineage Integrity, Induces Aberrant Alveogenesis, and Promotes Breast Cancer.

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.

Translational feasibility and efficacy of nasal photodynamic disinfection of SARS-CoV-2.

The lack of therapeutic options to fight Covid-19 has contributed to the current global pandemic. Despite the emergence of effective vaccines, development of broad-spectrum antiviral treatment remains a significant challenge, in which antimicrobial photodynamic therapy (aPDT) may play a role, especially at early stages of infection. aPDT of the nares with methylene blue (MB) and non-thermal light has been successfully utilized to inactivate both bacterial and viral pathogens in the perioperative setting. Here, we investigated the effect of MB-aPDT to inactivate human betacoronavirus OC43 and SARS-CoV-2 in vitro and in a proof-of-principle COVID-19 clinical trial to test, in a variety of settings, the practicality, technical feasibility, and short-term efficacy of the method. aPDT yielded inactivation of up to 6-Logs in vitro, as measured by RT-qPCR and infectivity assay. From a photo-physics perspective, the in vitro results suggest that the response is not dependent on the virus itself, motivating potential use of aPDT for local destruction of SARS-CoV-2 and its variants. In the clinical trial we observed variable effects on viral RNA in nasal-swab samples as assessed by RT-qPCR attributed to aPDT-induced RNA fragmentation causing falsely-elevated counts. However, the viral infectivity in clinical nares swabs was reduced in 90% of samples and undetectable in 70% of samples. This is the first demonstration based on quantitative clinical viral infectivity measurements that MB-aPDT is a safe, easily delivered and effective front-line technique that can reduce local SARS-CoV-2 viral load.

Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application.

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the coronavirus disease 2019 (COVID-19) pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human ß-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values ≤ 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct ≤ 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Mapping transgene insertion sites reveals the α-Cre transgene expression in both developing retina and olfactory neurons.

The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in Gα0+ olfactory neurons. Most α-Cre+ olfactory neurons do not express Pax6, implicating the influence of neighboring regulatory elements. RT-PCR and buried food pellet test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities.

Progression through G1/S phase of the cell cycle is coordinated by cyclin-dependent kinase (CDK) activities. Here, we find that the requirement for different CDK activities and cyclins in driving cancer cell cycles is highly heterogeneous. The differential gene requirements associate with tumor origin and genetic alterations. We define multiple mechanisms for G1/S progression in RB-proficient models, which are CDK4/6 independent and elicit resistance to FDA-approved inhibitors. Conversely, RB-deficient models are intrinsically CDK4/6 independent, but exhibit differential requirements for cyclin E. These dependencies for CDK and cyclins associate with gene expression programs that denote intrinsically different cell-cycle states. Mining therapeutic sensitivities shows that there are reciprocal vulnerabilities associated with RB1 or CCND1 expression versus CCNE1 or CDKN2A. Together, these findings illustrate the complex nature of cancer cell cycles and the relevance for precision therapeutic intervention.

Targeting the Ubiquitin-Proteasome System Using the UBA1 Inhibitor TAK-243 is a Potential Therapeutic Strategy for Small-Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with an overall 5-year survival rate of less than 10%. Treatment for SCLC with cisplatin/etoposide chemotherapy (C/E) ± radiotherapy has changed modestly over several decades. The ubiquitin-proteasome system is an underexplored therapeutic target for SCLC. We preclinically evaluated TAK-243, a first-in-class small molecule E1 inhibitor against UBA1. EXPERIMENTAL DESIGN: We assessed TAK-243 in 26 SCLC cell-lines as monotherapy and combined with C/E, the PARP-inhibitor, olaparib, and with radiation using cell viability assays. We interrogated TAK-243 response with gene expression to identify candidate biomarkers. We evaluated TAK-243 alone and in combination with olaparib or radiotherapy with SCLC patient-derived xenografts (PDX). RESULTS: Most SCLC cell lines were sensitive to TAK-243 monotherapy (EC50 median 15.8 nmol/L; range 10.2 nmol/L-367.3 nmol/L). TAK-243 sensitivity was associated with gene-sets involving the cell cycle, DNA and chromatin organization, and DNA damage repair, while resistance associated with cellular respiration, translation, and neurodevelopment. These associations were also observed in SCLC PDXs. TAK-243 synergized with C/E and olaparib in vitro across sensitive and resistant SCLC cell lines. Considerable TAK-243-olaparib synergy was observed in an SCLC PDX resistant to both drugs individually. TAK-243 radiosensitization was also observed in an SCLC PDX. CONCLUSIONS: TAK-243 displays efficacy in SCLC preclinical models. Enrichment of gene sets is associated with TAK-243 sensitivity and resistance. TAK-243 exhibits synergy when combined with genotoxic therapies in cell lines and PDXs. TAK-243 is a potential therapeutic strategy to improve SCLC patient outcomes, both as a single agent and in combination with existing therapies.

Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes.

Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.

Simplifying cancer: binary pan-cancer superclasses stratified by opposite YAP/TEAD effects.

The inherent complexity of cancer complicates treatment. Identifying higher-order principles that govern cancer biology can circumvent this problem and pinpoint broadly applicable treatment options. We recently found that opposite expression and pro- versus anti-cancer activity of a single transcriptional complex functionally stratifies cancer into binary superclasses.

Lentiviral-mediated ectopic expression of YAP and TAZ in YAPoff cancer cell lines.

Ectopic/overexpression systems are important for studying protein function, but care must be taken to avoid artifacts due to excessively high levels of overexpression. To study the function of YAP/TAZ in YAP/TAZ-deficient (YAPoff) cancers, we developed a lentiviral system using weak, constitutive promoters to ectopically express YAP/TAZ to physiologically relevant levels. We detail this system along with protocols to assess YAP/TAZ expression by flow cytometry and quantitative western blotting. This system can also be easily adapted for the study of other proteins. For complete details on the use and execution of this protocol, please refer to Pearson et al. (2021).

Photoreceptor nanotubes mediate the in vivo exchange of intracellular material.

Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.

InVision: An optimized tissue clearing approach for three-dimensional imaging and analysis of intact rodent eyes.

The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.

Binary pan-cancer classes with distinct vulnerabilities defined by pro- or anti-cancer YAP/TEAD activity.

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.

Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods.

BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.

A multiplexed, next generation sequencing platform for high-throughput detection of SARS-CoV-2.

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

Pou2f1 and Pou2f2 cooperate to control the timing of cone photoreceptor production in the developing mouse retina.

Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated 'The people behind the papers' interview.

The NEMP family supports metazoan fertility and nuclear envelope stiffness.

Human genome-wide association studies have linked single-nucleotide polymorphisms (SNPs) in NEMP1 (nuclear envelope membrane protein 1) with early menopause; however, it is unclear whether NEMP1 has any role in fertility. We show that whole-animal loss of NEMP1 homologs in Drosophila, Caenorhabditis elegans, zebrafish, and mice leads to sterility or early loss of fertility. Loss of Nemp leads to nuclear shaping defects, most prominently in the germ line. Biochemical, biophysical, and genetic studies reveal that NEMP proteins support the mechanical stiffness of the germline nuclear envelope via formation of a NEMP-EMERIN complex. These data indicate that the germline nuclear envelope has specialized mechanical properties and that NEMP proteins play essential and conserved roles in fertility.