Integrating inflammatory biomarker analysis and artificial-intelligence-enabled image-based profiling to identify drug targets for intestinal fibrosis.

Intestinal fibrosis, often caused by inflammatory bowel disease, can lead to intestinal stenosis and obstruction, but there are no approved treatments. Drug discovery has been hindered by the lack of screenable cellular phenotypes. To address this, we used a scalable image-based morphology assay called Cell Painting, augmented with machine learning algorithms, to identify small molecules that could reverse the activated fibrotic phenotype of intestinal myofibroblasts. We then conducted a high-throughput small molecule chemogenomics screen of approximately 5,000 compounds with known targets or mechanisms, which have achieved clinical stage or approval by the FDA. By integrating morphological analyses and AI using pathologically relevant cells and disease-relevant stimuli, we identified several compounds and target classes that are potentially able to treat intestinal fibrosis. This phenotypic screening platform offers significant improvements over conventional methods for identifying a wide range of drug targets.

Genome-wide CRISPR Screening to Identify Drivers of TGF-ß-Induced Liver Fibrosis in Human Hepatic Stellate Cells.

Liver fibrosis progression in chronic liver disease leads to cirrhosis, liver failure, or hepatocellular carcinoma and often ends in liver transplantation. Even with an increased understanding of liver fibrogenesis and many attempts to generate therapeutics specifically targeting fibrosis, there is no approved treatment for liver fibrosis. To further understand and characterize the driving mechanisms of liver fibrosis, we developed a high-throughput genome-wide CRISPR/Cas9 screening platform to identify hepatic stellate cell (HSC)-derived mediators of transforming growth factor (TGF)-ß-induced liver fibrosis. The functional genomics phenotypic screening platform described here revealed the novel biology of TGF-ß-induced fibrogenesis and potential drug targets for liver fibrosis.

High-throughput CRISPR-mediated 3D enrichment platform for functional interrogation of chemotherapeutic resistance.

Cancer is a disease of somatic mutations. These cellular mutations compete to dominate their microenvironment and dictate the disease outcome. While a therapeutic approach to target-specific oncogenic driver mutations helps to manage the disease, subsequent molecular evolution of tumor cells threatens to overtake therapeutic progress. There is a need for rapid, high-throughput, unbiased in vitro discovery screening platforms that capture the native complexities of the tumor and rapidly identify mutations that confer chemotherapeutic drug resistance. Taking the example of the CDK4/6 inhibitor (CDK4/6i) class of drugs, we show that the pooled in vitro CRISPR screening platform enables rapid discovery of drug resistance mutations in a three-dimensional (3D) setting. Gene-edited cancer cell clones assembled into an organotypic multicellular tumor spheroid (MCTS), exposed to CDK4/6i caused selection and enrichment of the most drug-resistant phenotypes, detectable by next-gen sequencing after a span of 28 days. The platform was sufficiently sensitive to enrich for even a single drug-resistant cell within a large, drug-responsive complex 3D tumor spheroid. The genome-wide 3D CRISPR-mediated knockout screen (>18,000 genes) identified several genes whose disruptions conferred resistance to CDK4/6i. Furthermore, multiple novel candidate genes were identified as top hits only in the microphysiological 3D enrichment assay platform and not the conventional 2D assays. Taken together, these findings suggest that including phenotypic 3D resistance profiling in decision trees could improve discovery and reconfirmation of drug resistance mechanisms and afford a platform for exploring noncell autonomous interactions, selection pressures, and clonal competition.

A biomimetic assay platform for the interrogation of antigen-dependent anti-tumor T-cell function.

Overcoming tumor-mediated immunosuppression and enhancing cytotoxic T-cell activity within the tumor microenvironment are two central goals of immuno-oncology (IO) drug discovery initiatives. However, exploratory assays involving immune components are often plagued by low-throughput and poor clinical relevance. Here we present an innovative ultra-high-content assay platform for interrogating T-cell-mediated killing of 3D multicellular tumor spheroids. Employing this assay platform in a chemical genomics screen of 1800 annotated compounds enabled identification of small molecule perturbagens capable of enhancing cytotoxic CD8+ T-cell activity in an antigen-dependent manner. Specifically, cyclin-dependent kinase (CDK) and bromodomain (BRD) protein inhibitors were shown to significantly augment anti-tumor T-cell function by increasing cytolytic granule and type II interferon secretion in T-cells in addition to upregulating major histocompatibility complex (MHC) expression and antigen presentation in tumor cells. The described biotechnology screening platform yields multi-parametric, clinically-relevant data and can be employed kinetically for the discovery of first-in-class IO therapeutic agents.

Co-regulatory networks of human serum proteins link genetics to disease.

Proteins circulating in the blood are critical for age-related disease processes; however, the serum proteome has remained largely unexplored. To this end, 4137 proteins covering most predicted extracellular proteins were measured in the serum of 5457 Icelanders over 65 years of age. Pairwise correlation between proteins as they varied across individuals revealed 27 different network modules of serum proteins, many of which were associated with cardiovascular and metabolic disease states, as well as overall survival. The protein modules were controlled by cis- and trans-acting genetic variants, which in many cases were also associated with complex disease. This revealed co-regulated groups of circulating proteins that incorporated regulatory control between tissues and demonstrated close relationships to past, current, and future disease states.

Functional profiling of microtumors to identify cancer associated fibroblast-derived drug targets.

Recent advances in chemotherapeutics highlight the importance of molecularly-targeted perturbagens. Although these therapies typically address dysregulated cancer cell proteins, there are increasing therapeutic modalities that take into consideration cancer cell-extrinsic factors. Targeting components of tumor stroma such as vascular or immune cells has been shown to represent an efficacious approach in cancer treatment. Cancer-associated fibroblasts (CAFs) exemplify an important stromal component that can be exploited in targeted therapeutics, though their employment in drug discovery campaigns has been relatively minimal due to technical logistics in assaying for CAF-tumor interactions. Here we report a 3-dimensional multi-culture tumor:CAF spheroid phenotypic screening platform that can be applied to high-content drug discovery initiatives. Using a functional genomics approach we systematically profiled 1,024 candidate genes for CAF-intrinsic anti-spheroid activity; identifying several CAF genes important for development and maintenance of tumor:CAF co-culture spheroids. Along with previously reported genes such as WNT, we identify CAF-derived targets such as ARAF and COL3A1 upon which the tumor compartment depends for spheroid development. Specifically, we highlight the G-protein-coupled receptor OGR1 as a unique CAF-specific protein that may represent an attractive drug target for treating colorectal cancer. In vivo, murine colon tumor implants in OGR1 knockout mice displayed delayed tumor growth compared to tumors implanted in wild type littermate controls. These findings demonstrate a robust microphysiological screening approach for identifying new CAF targets that may be applied to drug discovery efforts.

Challenges and opportunities toward enabling phenotypic screening of complex and 3D cell models.

Increasingly, organotypic cellular platforms are being recognized as useful tools in drug discovery. This review offers an industry-centric perspective on the benefits of emerging complex cell models over conventional 2D systems, as well as the challenges and opportunities for incorporating these multidimensional platforms into high-density formats. We particularly highlight the need for novel chemical sensors to noninvasively quantitate 3D structures in real time, and we contend that the use of more focused chemical and genomics libraries will enable screening of complex cell models derived from primary and induced pluripotent stem cells. Finally, we offer outlooks on several emerging technologies that show great potential for future integration of complex cell systems into contemporary drug screening.

Therapeutic antagonists of microRNAs deplete leukemia-initiating cell activity.

Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.

An HTS-compatible 3D colony formation assay to identify tumor-specific chemotherapeutics.

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laser-scanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.

Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets.

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was used to discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

Growth factor independence 1 antagonizes a p53-induced DNA damage response pathway in lymphoblastic leukemia.

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

Gfi1 integrates progenitor versus granulocytic transcriptional programming.

In patients with severe congenital neutropenia (SCN) and mice with growth factor independent-1 (Gfi1) loss of function, arrested myeloid progenitors accumulate, whereas terminal granulopoiesis is blocked. One might assume that Gfi-null progenitors accumulate because they lack the ability to differentiate. Instead, our data indicate that Gfi1 loss of function deregulates 2 separable transcriptional programs, one of which controls the accumulation and lineage specification of myeloid progenitors, but not terminal granulopoiesis. We demonstrate that Gfi1 directly represses HoxA9, Pbx1, and Meis1 during normal myelopoiesis. Gfi1-/- progenitors exhibit elevated levels of HoxA9, Pbx1 and Meis1, exaggerated HoxA9-Pbx1-Meis1 activity, and progenitor transformation in collaboration with oncogenic K-Ras. Limiting HoxA9 alleles corrects, in a dose-dependent manner, in vivo and in vitro phenotypes observed with loss of Gfi1 in myeloid progenitor cells but did not rescue Gfi1-/- blocked granulopoiesis. Thus, Gfi1 integrates 2 events during normal myeloid differentiation; the suppression of a HoxA9-Pbx1-Meis1 progenitor program and the induction of a granulopoietic transcription program.

Mutations in growth factor independent-1 associated with human neutropenia block murine granulopoiesis through colony stimulating factor-1.

Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.

The potential regulation of L1 mobility by RNA interference.

The hypothesis that RNA interference constrains L1 mobility seems inherently reasonable: L1 mobility can be dangerous and L1 RNA, the presumed target of RNAi, serves as a critical retrotransposition intermediate. Despite its plausibility, proof for this hypothesis has been difficult to obtain. Studies attempting to link the L1 retrotransposition frequency to alterations in RNAi activity have been hampered by the long times required to measure retrotransposition frequency, the pleiotropic and toxic effects of altering RNAi over similar time periods, and the possibility that other cellular machinery may contribute to the regulation of L1s. Another problem is that the commonly used L1 reporter cassette may serve as a substrate for RNAi. Here we review the L1-RNAi hypothesis and describe a genetic assay with a modified reporter cassette that detects approximately 4 times more L1 insertions than the conventional retrotransposition assay.

Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay.

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy.