Automatic Annotation of Spatial Expression Patterns via Sparse Bayesian Factor Models

Advances in reporters for gene expression have made it possible to document and quantify expression patterns in 2D–4D. In contrast to microarrays, which provide data for many genes but averaged and/or at low resolution, images reveal the high spatial dynamics of gene expression. Developing computational methods to compare, annotate, and model gene expression based on images is imperative, considering that available data are rapidly increasing. We have developed a sparse Bayesian factor analysis model in which the observed expression diversity of among a large set of high-dimensional images is modeled by a small number of hidden common factors. We apply this approach on embryonic expression patterns from a Drosophila RNA in situ image database, and show that the automatically inferred factors provide for a meaningful decomposition and represent common co-regulation or biological functions. The low-dimensional set of factor mixing weights is further used as features by a classifier to annotate expression patterns with functional categories. On human-curated annotations, our sparse approach reaches similar or better classification of expression patterns at different developmental stages, when compared to other automatic image annotation methods using thousands of hard-to-interpret features. Our study therefore outlines a general framework for large microscopy data sets, in which both the generative model itself, as well as its application for analysis tasks such as automated annotation, can provide insight into biological questions

High-dimensional immune monitoring for chimeric antigen receptor T cell therapies

Purpose of Review: high-dimensional flow cytometry experiments have become a method of choice for high-throughput integration and characterization of cell populations. Here, we present a summary of state-of-the-art R-based pipelines used for differential analyses of cytometry data, largely based on Chimeric Antigen Receptor (CAR) T cell therapies. Recent Findings: in recent years, existing tools tailored to analyze complex high-dimensional data such as single cell RNA sequencing (scRNAseq) have been successfully ported to cytometry studies due to the similar nature of flow cytometry and scRNAseq platforms. Existing environments like Cytobank24, FlowJo27 and FCS Express 28 already offer a variety of these ported tools, but they either come at a premium or are fairly complicated to manage by an inexperienced user. To mitigate these limitations, experienced cytometrists and bioinformaticians usually incorporate these functions into an R-Shiny 17 application that ultimately offers a user-friendly, intuitive environment that can be used to analyze flow cytometry data. Summary: computational tools and Shiny based tools are the perfect answer to the ever-growing dimensionality and complexity of flow cytometry data, by offering a dynamic, yet user friendly exploratory space, tailored to bridge the space between the lab experimental world and the computational, machine learning space

Detection of Dysregulated Protein Association Networks by High-Throughput Proteomics Predicts Cancer Vulnerabilities

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this ‘interactome mapping by high-throughput quantitative proteome analysis’ (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network impacts to cellular fitness. Furthermore, these aberrant interactions serve as biomarkers predicting drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems

Small molecules facilitate rapid and synchronous iPSC generation

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2, and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is protracted and inefficient, resulting in heterogeneous cell populations. Here we show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-beta inhibitor (termed “AGi”) facilitates more synchronous and rapid iPSC formation from a variety of mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 hours of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations

Decade-long Remissions of Leukemia Sustained by the Persistence of Activated CD4 CAR T Cells

The adoptive transfer of T cells reprogrammed to target tumor cells has demonstrated significant potential in various malignancies. However, little is known about the long-term memory potential and the clonal stability of the infused cells. Here, we studied the fate of CD19 redirected chimeric antigen receptor (CAR19) T-cells in two leukemia patients who achieved and sustained a complete remission almost a decade ago. CAR T cells were still detectable 9+ years post-infusion. Surprisingly, a prominent, highly activated CD4+ population developed in both subjects in the years post-infusion, dominating the CAR T cell population at the late time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by few clones. Single-cell profiling of CAR T-cells obtained 9 years post-infusion demonstrated that these long-persisting CD4+ CAR T cells exhibited cytotoxic characteristics along with strong evidence of ongoing functional activation and proliferation. Given data that CD19 directed CAR T with a CD28 signaling domain do not persist long term, our data provide important insight into the development of long-term anti-tumor responses necessary for sustained remission in leukemia following CAR T-cell therapy

Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment

<div><p>A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1) transporter inhibition. We identified drugs sensitizing cell lines that are BRAF^V600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR) and platelet derived growth factor receptor (PDGFR) family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis <i>in vitro</i> and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs), demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK) kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.</p></div

Tisagenlecleucel cellular kinetics, dose, and immunogenicity in relation to clinical factors in relapsed/refractory DLBCL

The anti-CD19 chimeric antigen receptor (CAR)-T cell therapy tisagenlecleucel was evaluated in the global, phase 2 JULIET study in adult patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). We correlated tisagenlecleucel cellular kinetics with clinical/product parameters in 111 patients treated in JULIET. Tisagenlecleucel persistence in responders and nonresponders, respectively, was demonstrated for 554 and 400 days maximum by flow cytometry and for 693 and 374 days maximum by quantitative polymerase chain reaction (qPCR). No relationships were identified between cellular kinetics (qPCR) and product characteristics, intrinsic/extrinsic factors, dose, or immunogenicity. Most patients with 3-month response had detectable transgene at time of response and continued persistence for >= 6 months. Expansion (maximal expansion of transgene/CAR-positive T-cell levels in vivo postinfusion [C-max]) was potentially associated with response duration but this did not reach statistical significance (hazard ratio for a twofold increase in C-max, 0.79; 95% confidence interval, 0.61-1.01). Tisagenlecleucel expansion was associated with cytokine-release syndrome (CRS) severity and tocilizumab use; no relationships were observed with neurologic events. Transgene levels were associated with B-cell levels. Dose was associated with CRS severity, but this was not statistically significant after adjusting for baseline tumor burden. In contrast to the results from B-cell precursor acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia, similar exposure was observed in DLBCL in this study regardless of response and expansion was lower in DLBCL than B-ALL, likely from differences in cancer location and/or T-cell intrinsic factors. Relationships between expansion and CRS severity, and lack of relationships between dose and exposure, were similar between DLBCL and B-ALL. Tisagenlecleucel cellular kinetics in adult relapsed/refractory DLBCL improve current understanding of in vivo expansion and its relationships with safety/efficacy endpoints

Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia

Tolerance to self-antigens prevents the elimination of cancer by the immune system 1,2 . We used synthetic chimeric antigen receptors (CARs) to overcome immunological tolerance and mediate tumor rejection in patients with chronic lymphocytic leukemia (CLL). Remission was induced in a subset of subjects, but most did not respond. Comprehensive assessment of patient-derived CAR T cells to identify mechanisms of therapeutic success and failure has not been explored. We performed genomic, phenotypic and functional evaluations to identify determinants of response. Transcriptomic profiling revealed that CAR T cells from complete-responding patients with CLL were enriched in memory-related genes, including IL-6/STAT3 signatures, whereas T cells from nonresponders upregulated programs involved in effector differentiation, glycolysis, exhaustion and apoptosis. Sustained remission was associated with an elevated frequency of CD27+CD45RO-CD8+ T cells before CAR T cell generation, and these lymphocytes possessed memory-like characteristics. Highly functional CAR T cells from patients produced STAT3-related cytokines, and serum IL-6 correlated with CAR T cell expansion. IL-6/STAT3 blockade diminished CAR T cell proliferation. Furthermore, a mechanistically relevant population of CD27+PD-1-CD8+ CAR T cells expressing high levels of the IL-6 receptor predicts therapeutic response and is responsible for tumor control. These findings uncover new features of CAR T cell biology and underscore the potential of using pretreatment biomarkers of response to advance immunotherapies

Cediranib synergizes with PLX4720 <i>in vivo</i>.

<p>(A) ISTMel1 xenografts were generated in immunodeficient mice (<i>n</i> = 8 per group), which were treated with PLX4720 or control chow and given cediranib or water by oral gavage. ISTMel1 xenografts showed an initial response to PLX4720 treatment alone after approximately two weeks, with some additional but non-significant response to cediranib treatment. Values are shown as mean +/- S.E.M. ranges, with significant differences in mean tumor size of both PLX4720 and PLX4720 and cediranib-treated mice compared to control treatment by ANOVA. However, the combination significantly delayed progression (defined as > 500 mm³ size) beyond this initial response (right, <i>p</i> < 0.002 between PLX4720 and PLX4720 + cediranib arms by log-rank test). (B) In contrast to ISTMel1 xenografts, RPMI7951 xenografts showed a significant difference by ANOVA test in initial response to PLX4720 versus PLX4720 and cediranib treatment after three weeks, and, right, showed a prolonged delay of progression (defined as tumor > 250 mm³) of completely PLX4720-resistant tumors (<i>p</i> < 0.0001 between PLX4720 and PLX4720 and cediranib arms by log-rank test). (C) KDR staining of tumor biopsies from patients entering clinical trials for BRAF with or without MEK inhibitors demonstrated some had strong membrane KDR staining (top) throughout the tumor (inset showing membrane staining, at arrowhead), while others were negative for KDR staining (bottom) except for expected endothelial staining. (D) Comparison of progression-free survival of patients with (<i>n</i> = 6) and without (<i>n</i> = 10) membrane KDR staining showed a significant reduction in PFS (9.3 vs. 3.8 months, <i>p</i> < 0.01 by Student’s t-test) if the patient’s biopsy expressed KDR.</p

Ultra high-throughput screen to identify synergistic combinations in melanoma cells.

<p>(A) Summary of clinical development stage of 108 drugs included in the combination drug panel. (B) Example of raw UHTS data generated, demonstrating cell count information collected from DAPI channel and apoptosis data from cleaved PARP immunofluorescence; positive control of HSP90 inhibitor 17-AAG treatment is shown. (C) Summary matrix of combinatorial drug data, with each point representing the effect of one of 5,778 combinations at the standard drug concentration, as the median effect of the drug combination across all 36 melanoma cell lines on the relative cell count (left) and the calculated Bliss synergy score for that combination (right). (D) Histogram of number of cell lines a given drug combination showed synergy. Peak number of synergies were seen in one cell line, indicating many synergies are private. (E) As in (C), showing median effect of the drug combination (at standard concentration) on the relative cPARP positive proportion (left) and the calculated Bliss synergy for that cPARP level (right). (F) Graphical representation of drug combinations (drug pairs connected by an edge) that showed a significant unexpectedly high cPARP over a predicted level at the given cell count. Node size indicates the number of drug pairs that the given drug appears with other drugs on the “unexpectedly apoptotic” list. Edge color indicates the drug pair concentration (standard or low) where the elevated cPARP was found; edge pattern indicates whether the elevated cPARP was found in the setting of low cell count or normal cell count (> 80% control), with elevated cPARP in the setting of normal viability potentially representing “slow” death kinetics for that combination.</p