LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Mammary epithelial cells have lineage-rooted metabolic identities.

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

MYC-induced human acute myeloid leukemia requires a continuing IL-3/GM-CSF costimulus.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Single-cell analysis of autophagy activity in normal and de novo transformed human mammary cells.

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Initiation of human mammary cell tumorigenesis by mutant KRAS requires YAP inactivation.

High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.

Resolution of Cellular Heterogeneity in Human Prostate Cancers: Implications for Diagnosis and Treatment.

Prostate cancers have a justified reputation as one of the most heterogeneous human tumours. Indeed, there are some who consider that advanced and castration-resistant prostate cancers are incurable, as a direct result of this heterogeneity. However, tumour heterogeneity can be defined in different ways. To a clinician, prostate cancer is a number of different diseases, the treatments for which remain equally heterogeneous and uncertain. To the pathologist, the histopathological appearances of the tumours are notoriously heterogeneous. Indeed, the genius of Donald Gleason in the 1960s was to devise a classification system designed to take into account the heterogeneity of the tumours both individually and in the whole prostate context. To the cell biologist, a prostate tumour consists of multiple epithelial cell types, inter-mingled with various fibroblasts, neuroendocrine cells, endothelial cells, macrophages and lymphocytes, all of which interact to influence treatment responses in a patient-specific manner. Finally, genetic analyses of prostate cancers have been compromised by the variable gene rearrangements and paucity of activating mutations observed, even in large numbers of patient tumours with consistent clinical diagnoses and/or outcomes. Research into familial susceptibility has even generated the least tractable outcome of such studies: the genetic loci are of low penetrance and are of course heterogeneous. By fractionating the tumour (and patient-matched non-malignant tissues) heterogeneity can be resolved, revealing homogeneous markers of patient outcomes.

Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer.

The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra-tumoral as well as inter-tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers.

A topological view of human CD34+ cell state trajectories from integrated single-cell output and proteomic data.

Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in ∼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.

Phenotype-independent DNA methylation changes in prostate cancer.

BACKGROUND: Human prostate cancers display numerous DNA methylation changes compared to normal tissue samples. However, definitive identification of features related to the cells' malignant status has been compromised by the predominance of cells with luminal features in prostate cancers. METHODS: We generated genome-wide DNA methylation profiles of cell subpopulations with basal or luminal features isolated from matched prostate cancer and normal tissue samples. RESULTS: Many frequent DNA methylation changes previously attributed to prostate cancers are here identified as differences between luminal and basal cells in both normal and cancer samples. We also identified changes unique to each of the two cancer subpopulations. Those specific to cancer luminal cells were associated with regulation of metabolic processes, cell proliferation and epithelial development. Within the prostate cancer TCGA dataset, these changes were able to distinguish not only cancers from normal samples, but also organ-confined cancers from those with extraprostatic extensions. Using changes present in both basal and luminal cancer cells, we derived a new 17-CpG prostate cancer signature with high predictive power in the TCGA dataset. CONCLUSIONS: This study demonstrates the importance of comparing phenotypically matched prostate cell populations from normal and cancer tissues to unmask biologically and clinically relevant DNA methylation changes.

Single-cell analysis identifies a CD33+ subset of human cord blood cells with high regenerative potential.

Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.

Mass Cytometric Analysis Reveals Viable Activated Caspase-3+ Luminal Progenitors in the Normal Adult Human Mammary Gland.

The normal adult human female mammary gland is a bilayered structure consisting of an outer basal layer and two readily distinguished subsets of cells within the inner luminal layer. We now present a validated methodology for undertaking large-scale multi-parameter mass cytometric analyses of these cell types at single-cell resolution. In addition, we show how combining this approach with in vitro clonogenic assays of the proliferative and signaling responses of normal human mammary cells to epidermal growth factor (EGF) allows additional subsets with different EGF responses to be discerned. This included the identification of a subset of cells within the phenotypically defined luminal progenitor fraction that displays an elevated content of active caspase-3, including some that generate clones in vitro in response to EGF, with immunohistochemical evidence of their presence in situ in fixed preparations of normal human breast tissue.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Dissociation of Survival, Proliferation, and State Control in Human Hematopoietic Stem Cells.

The role of growth factors (GFs) in controlling the biology of human hematopoietic stem cells (HSCs) remains limited by a lack of information concerning the individual and combined effects of GFs directly on the survival, Mitogenesis, and regenerative activity of highly purified human HSCs. We show that the initial input HSC activity of such a purified starting population of human cord blood cells can be fully maintained over a 21-day period in serum-free medium containing five GFs alone. HSC survival was partially supported by any one of these GFs, but none were essential, and different combinations of GFs variably stimulated HSC proliferation. However, serial transplantability was not detectably compromised by many conditions that reduced human HSC proliferation and/or survival. These results demonstrate the dissociated control of these three human HSC bio-responses, and set the stage for future improvements in strategies to modify and expand human HSCs ex vivo.

Distinct signaling programs control human hematopoietic stem cell survival and proliferation.

Several growth factors (GFs) that together promote quiescent human hematopoietic stem cell (HSC) expansion ex vivo have been identified; however, the molecular mechanisms by which these GFs regulate the survival, proliferation. and differentiation of human HSCs remain poorly understood. We now describe experiments in which we used mass cytometry to simultaneously measure multiple surface markers, transcription factors, active signaling intermediates, viability, and cell-cycle indicators in single CD34+ cord blood cells before and up to 2 hours after their stimulation with stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor (5 GFs) either alone or combined. Cells with a CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) phenotype (∼10% HSCs with >6-month repopulating activity in immunodeficient mice) displayed rapid increases in activated STAT1/3/5, extracellular signal-regulated kinase 1/2, AKT, CREB, and S6 by 1 or more of these GFs, and ß-catenin only when the 5 GFs were combined. Certain minority subsets within the CD49f+ compartment were poorly GF-responsive and, among the more GF-responsive subsets of CD49f+ cells, different signaling intermediates correlated with the levels of the myeloid- and lymphoid-associated transcription factors measured. Phenotypically similar, but CD90-CD49f- cells (MPPs) contained lower baseline levels of multiple signaling intermediates than the CD90+CD49f+ cells, but showed similar response amplitudes to the same GFs. Importantly, we found activation or inhibition of AKT and ß-catenin directly altered immediate CD49f+ cell survival and proliferation. These findings identify rapid signaling events that 5 GFs elicit directly in the most primitive human hematopoietic cell types to promote their survival and proliferation.

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors.

Harvesting Human Prostate Tissue Material and Culturing Primary Prostate Epithelial Cells.

In order to fully explore the biology of a complex solid tumor such as prostate cancer, it is desirable to work with patient tissue. Only by working with cells from a tissue can we take into account patient variability and tumor heterogeneity. Cell lines have long been regarded as the workhorse of cancer research and it could be argued that they are of most use when considered within a panel of cell lines, thus taking into account specified mutations and variations in phenotype between different cell lines. However, often very different results are obtained when comparing cell lines to primary cells cultured from tissue. It stands to reason that cells cultured from patient tissue represents a close-to-patient model that should and does produce clinically relevant data. This chapter aims to illustrate the methods of processing, storing and culturing cells from prostate tissue, with a description of potential uses.