Search

Ongoing, first-in-human clinical trials illustrate the feasibility and translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson’s disease (PD). However, a major unresolved challenge in the field is the extensive cell death following transplantation with in vivo. We identified p53-mediated apoptotic cell death as major contributor to dopamine neuron loss and uncovered a causal link of TNFa-NFκB signaling in limiting cell survival. As a translationally applicable strategy to purify postmitotic dopamine neurons, we performed a cell surface marker screen that enabled purification without the need for genetic reporters. Combining cell sorting with adalimumab pretreatment, a clinically approved and widely used TNFa inhibitor, enabled efficient engraftment of postmitotic dopamine neurons leading to extensive re-innervation and functional recovery in a preclinical PD mouse model. Thus, transient TNFa inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic human PSC-derived dopamine neurons in PD.HighlightsIn vivoCRISPR-Cas9 screen identifies p53 limiting survival of grafted human dopamine neurons.TNFα-NFκB pathway mediates p53-dependent human dopamine neuron deathCell surface marker screen to enrich human dopamine neurons for translational use.FDA approved TNF-alpha inhibitor rescuesin vivodopamine neuron survival within vivofunction.

Most eukaryotes harbor two distinct pre-mRNA splicing machineries: the major spliceosome, which removes >99% of introns, and the minor spliceosome, which removes rare, evolutionarily conserved introns. Although hypothesized to serve important regulatory functions, physiologic roles of the minor spliceosome are not well understood. For example, the minor spliceosome component ZRSR2 is subject to recurrent, leukemia-associated mutations, yet functional connections among minor introns, hematopoiesis and cancers are unclear. Here, we identify that impaired minor intron excision via ZRSR2 loss enhances hematopoietic stem cell self-renewal. CRISPR screens mimicking nonsense-mediated decay of minor intron-containing mRNA species converged on LZTR1, a regulator of RAS-related GTPases. LZTR1 minor intron retention was also discovered in the RASopathy Noonan syndrome, due to intronic mutations disrupting splicing and diverse solid tumors. These data uncover minor intron recognition as a regulator of hematopoiesis, noncoding mutations within minor introns as potential cancer drivers and links among ZRSR2 mutations, LZTR1 regulation and leukemias. Loss of function of the minor spliceosome component ZRSR2 enhances hematopoietic stem cell self-renewal through minor intron retention of its target LZTR1, which is a regulator of RAS-related GTPases. Minor intron retention of LZTR1 was also identified in Noonan syndrome and diverse solid tumor types.

The maturation of human pluripotent stem cell (hPSC)-derived neurons mimics the protracted timing of human brain development, extending over months and years to reach adult-like function. Prolonged in vitro maturation presents a major challenge to stem cell-based applications in modeling and treating neurological disease. We designed a high-content imaging assay based on morphological and functional readouts in hPSC-derived cortical neurons to reveal underlying pathways and to identify chemicals capable of accelerating neuronal maturation. Probing a library of 2688 bioactive drugs, we identified multiple compounds that drive neuronal maturation including inhibitors of LSD1 and DOT1L and activators of calcium-dependent transcription. A cocktail of 4 factors GSK-2879552, EPZ-5676, NMDA and Bay K 8644, which we collectively termed GENtoniK, triggered maturation across all assays tested including measures of synaptic density, electrophysiology and transcriptomics. Remarkably, GENtoniK was similarly effective in enhancing neuronal maturation in 3D cortical organoids and in spinal motoneurons, and improved aspects of cell maturation in non-neural lineages such as melanocytes and pancreatic beta cells. These results demonstrate that the maturation of multiple hPSC-derived cell types can be enhanced by simple pharmacological intervention and suggests that some of the mechanisms controlling the timing of human maturation are shared across lineages.

Intraocular retinoblastoma treatment has changed radically over the last decade, leading to a notable improvement in ocular survival. However, eyes that relapse remain difficult to treat, as few alternative active drugs are available. More challenging is the scenario of central nervous system (CNS) metastasis, in which almost no advancements have been made. Both clinical scenarios represent an urgent need for new drugs. Using an integrated multidisciplinary approach, we developed a decision process for prioritizing drug selection for local (intravitreal [IVi], intrathecal/intraventricular [IT/IVt]), systemic, or intra-arterial chemotherapy (IAC) treatment by means of high-throughput pharmacological screening of primary cells from two patients with intraocular tumor and CNS metastasis and a thorough database search to identify clinical and biopharmaceutical data. This process identified 169 compounds to be cytotoxic; only 8 are FDA-approved, lack serious toxicities and available for IVi administration. Four of these agents could also be delivered by IT/IVt. Twelve FDA-approved drugs were identified for systemic delivery as they are able to cross the blood-brain barrier and lack serious adverse events; four drugs are of oral usage and six compounds that lack vesicant or neurotoxicity could be delivered by IAC. We also identified promising compounds in preliminary phases of drug development including inhibitors of survivin, antiapoptotic Bcl-2 family proteins, methyltransferase, and kinesin proteins. This systematic approach may be applied more broadly to prioritize drugs to be repurposed or to identify novel hits for use in retinoblastoma treatment.

To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.

TCR stimulation triggers Ca

Oxidative stress and cellular response mechanisms such as NRF2-mediated antioxidant responses play differential roles in healthy and diseased cells. Constant generation and elimination of high levels of reactive oxygen species is a hallmark of many cancer cell types; this phenomenon is not observed during steady state of healthy cells. Manipulation of NRF2 transcriptional activity and the cellular redox homeostasis therefore has potential to be therapeutically exploitable for cancer therapy by preferentially targeting cancer cells for induction of oxidative stress. We found that the NRF2 inhibitor brusatol triggered increased oxidative stress while compromising viability and proliferation of multiple myeloma cells. Using a repurposing approach we discovered that the Cdc7/CDK9 inhibitor PHA-767491 is also a potent inhibitor of NRF2 transcriptional activity. The molecule was identified by high throughput screening of a library of about 5,900 drug-like molecules. Screening assays included two cell-based assays using HepG2 hepatocellular carcinoma cells: a) A NRF2 nuclear translocation assay, and b) A NRF2 luciferase reporter assay. Validation assays were performed in multiple myeloma cells and included detection of mitochondrial superoxide levels and MTS assays. We found that PHA-767491 treatment of multiple myeloma cells was associated with inhibition of nuclear translocation of NRF2, increased mitochondrial superoxide levels and inhibition of cell growth. Our findings suggest that PHA-767491 is a promising drug candidate for cancer therapy with NRF2 inhibitory potency contributing to its anti-cancer properties.

The identity of the RNA binding proteins (RBPs) that govern cancer stem cell remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomics analysis of the MSI2 interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia and SYNCRIP was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell gene associated program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. We validated SYNCRIP as a novel RBP that controls the myeloid leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in leukemia.

We present SplashRNA, a sequential classifier to predict potent microRNA-based short hairpin RNAs (shRNAs). Trained on published and novel data sets, SplashRNA outperforms previous algorithms and reliably predicts the most efficient shRNAs for a given gene. Combined with an optimized miR-E backbone, >90% of high-scoring SplashRNA predictions trigger >85% protein knockdown when expressed from a single genomic integration. SplashRNA can significantly improve the accuracy of loss-of-function genetics studies and facilitates the generation of compact shRNA libraries.

Mutations in RNA splicing factors are amongst the most common genetic alterations in myeloid malignancies. Mutations in the splicing factors SF3B1, SRSF2, and U2AF1 occur as heterozygous, missense mutations and have been shown to confer a change-of-function. In contrast, the X chromosome encoded ZRSR2 is enriched in nonsense/frameshift mutations in males, consistent with loss of function. To date however, we do not understand the basis for enrichment of ZRSR2 mutations in leukemia. Moreover, ZRSR2 is the only one of these factors that primarily functions in the minor spliceosome. While most introns are spliced by the major spliceosome, a small subset ( The rarity and conservation of minor introns offered a unique opportunity to investigate splicing factor mutations and identify potential tissue-specific roles of the minor spliceosome. Modeling loss-of-function mutations in ZRSR2 via a mouse model for induced deletion of Zrsr2 revealed strikingly enhanced self-renewal of Zrsr2-deficient male and female hematopoietic cells (Fig. A-C). This was in stark contrast to the effects of hotspot mutations in Sf3b1and Srsf2 and similar to those of Tet2 loss on increasing self-renewal and numbers of HSCs. Zrsr2 loss was also associated increased myeloid cells in the blood and long-term hematopoietic stem cells (HSCs) in the marrow (Fig. C). To understand the mechanistic basis by which ZRSR2 loss causes aberrant HSC self-renewal, we quantified transcriptome-wide splicing patterns in MDS patients. ZRSR2-mutant samples had widespread, dysfunctional recognition of minor introns- 48% of minor introns exhibiting significantly increased retention (Fig. D). We next systematically mimicked the effects of nonsense-mediated decay caused by minor intron retention in ZRSR2-mutants. Every gene containing a ZRSR2-regulated minor intron was targeted by 4 sgRNAs via a positive-enrichment CRISPR screen using pools of lentiviral sgRNAs in cytokine-dependent human and mouse hematopoietic cell lines. This identified several minor intron-containing genes whose downregulation conferred cytokine independence. Strikingly, just one gene was enriched in all lines (Fig. E): LZTR1, a cullin-3 adaptor for ubiquitin-mediated suppression of RAS-related GTPases which is subject to loss-of-function mutations in several cancers and the RASopathy Noonan Syndrome. Minor intron retention in LZTR1 correlated with reduced LZTR1 protein in MDS patients (Fig. F-G). Inducing mutations in either the protein-coding region of LZTR1 or its minor intron resulted in cytokine independence (Fig. H), reduced LZTR1, and dramatic accumulation of RIT1, a RAS GTPase substrate of LZTR1. In a Noonan Syndrome family wherein one child died of AML, the mother and all children carried an intronic mutation within LZTR1's minor intron (Fig. I-J). Fibroblasts from each family member revealed clear LZTR1 minor intron retention with impaired LZTR1 protein expression and RIT1 accumulation in subjects bearing the LZTR1 minor intron mutation (Fig. J). We next interrogated LZTR1 minor intron splicing across all cancers in the TCGA. While LZTR1's minor intron was efficiently excised in normal samples, a notable subset of tumors in almost all cancer types exhibited significantly increased retention within LZTR1's minor intron. These data indicate LZTR1 is frequently dysregulated via perturbed minor intron splicing - much more so than by protein-coding mutations alone. Here we uncover a heretofore unrecognized role of minor intron excision in regulating HSC self-renewal, a molecular link between ZRSR2 mutations and aberrant LZTR1 splicing and expression, and frequent LZTR1 minor intron retention in diverse cancers and cancer predisposition syndromes. Given frequent post-transcriptional disruption of LZTR1 in the absence of protein-coding mutations, our data additionally motivate study of other cancer-associated minor intron-containing genes which may be dysregulated via similar, and as-yet-undetected, aberrant splicing. Figure Disclosures Abdel-Wahab: Merck: Consultancy; Envisagenics Inc.: Current equity holder in private company; H3 Biomedicine Inc.: Consultancy, Research Funding; Janssen: Consultancy.

The major histocompatibility complex I (MHC-1) presents antigenic peptides to tumor-specific CD8+ T cells. The regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. Regulators of cell-surface HLA amounts were discovered using a pooled human kinome shRNA interference–based approach. Hits scoring highly were subsequently validated by additional RNAi and pharmacologic inhibitors. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of MHC-I expression and antigen presentation machinery in multiple cancer types, acting through an ERK output–dependent mechanism; the pathways responsible for increased MHC-I upon kinase inhibition were mapped. Activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I and the antigen presentation machinery. Pharmacologic inhibition of MAPK signaling also led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Cancer Immunol Res; 4(11); 936–47. ©2016 AACR.

Cell growth and proliferation require the coordinated activation of many cellular processes, including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation and set thresholds for expression of target proteins. Emerging data suggest that microRNA function is enhanced by cellular activation due in part to induction of the RNA-induced silencing complex (RISC) scaffold protein GW182. In the current study, we demonstrate that increased expression of GW182 in activated or transformed immune cells results from effects of phosphoinositol 3-kinase–Akt–mechanistic target of rapamycin (PI3K-Akt-mTOR) and Jak-Stat-Pim signaling on the translation of GW182 mRNA. Both signaling pathways enhanced polysome occupancy and eukaryotic initiation factor 4E (eIF4E) binding to the 5′ 7mG cap of GW182 mRNA. The effect of Jak-Stat-Pim signaling on polysome occupancy and expression of GW182 protein was greater than that of PI3K-Akt-mTOR signaling, likely resulting from enhanced eIF4A-dependent unwinding of G-quadruplexes in the 5′ untranslated region of GW182 mRNA. Consistent with this, GW182 expression and microRNA function were reduced by inhibition of mTOR or Pim kinases, translation initiation complex assembly, or eIF4A function. Taken together, these data provide a mechanistic link between microRNA function and cap-dependent translation that allows activated immune cells to maintain microRNA-mediated repression of targets despite enhanced rates of protein synthesis.

Integrin-mediated adhesion and signaling via focal adhesion kinase is required for mammary tumorigenesis in the PyMT mouse model. Moreover, PTK2 encoding FAK is amplified in a substantial fraction of human breast cancers. We have investigated the mechanism through which integrin signaling controls mammary tumorigenesis and found that integrin-mediated mechano-transduction controls invasive growth in stiff 3D matrices through activation of YAP/TAZ. Mechanistic studies revealed that integrin signaling induces—via activation of FAK and Src family kinases (SFKs) —tyrosine phosphorylation and inactivation of LATS. The ensuing activation of YAP/TAZ is necessary for invasive growth of HER2+ breast cancer cells. Engagement of HER2/HER3 with neuregulin impinges on SFKs to amplify activation of YAP/TAZ in HER2+ breast cancer cells, suggesting that integrin-mediated mechano-transduction functions as a rheostat to regulate HER2 oncogenic signaling. Finally, administration of dasatinib significantly increases the efficacy of HER2 inhibition in the MMTV-Neu mouse model of HER2+ breast cancer. These findings reveal a major mechanism through which the Hippo pathway is disabled in breast cancer and suggest that targeting HER2 and SFKs simultaneously may be a rational strategy in selected cases of breast cancer. This abstract is also being presented as Poster A22. Citation Format: Shimin Wang, Young-Mi Kim, Ralph Garippa, Larry Norton, Filippo G. Giancotti. Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR04.

Integrin-mediated adhesion and signaling via focal adhesion kinase is required for mammary tumorigenesis in the PyMT mouse model. Moreover, PTK2 encoding FAK is amplified in a substantial fraction of human breast cancers. We have investigated the mechanism through which integrin signaling controls mammary tumorigenesis and found that integrin-mediated mechano-transduction controls invasive growth in stiff 3D matrices through activation of YAP/TAZ. Mechanistic studies revealed that integrin signaling induces—via activation of FAK and Src family kinases (SFKs) —tyrosine phosphorylation and inactivation of LATS. The ensuing activation of YAP/TAZ is necessary for invasive growth of HER2+ breast cancer cells. Engagement of HER2/HER3 with neuregulin impinges on SFKs to amplify activation of YAP/TAZ in HER2+ breast cancer cells, suggesting that integrin-mediated mechano-transduction functions as a rheostat to regulate HER2 oncogenic signaling. Finally, administration of dasatinib significantly increases the efficacy of HER2 inhibition in the MMTV-Neu mouse model of HER2+ breast cancer. These findings reveal a major mechanism through which the Hippo pathway is disabled in breast cancer and suggest that targeting HER2 and SFKs simultaneously may be a rational strategy in selected cases of breast cancer. Citation Format: Shimin Wang, Young-Mi Kim, Ralph Garippa, Larry Norton, Filippo G. Giancotti. Integrin-mediated mechano-transduction controls HER2 oncogenic signaling and activation of YAP in breast cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA28.

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)–JUN family genes as key barriers of DE differentiation. The JNK–JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK–JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine. CRISPR screens identify JNK–JUN family genes as repressors of definitive endoderm differentiation in human pluripotent stem cells. JUN co-occupies stem cell enhancers with OCT4, NANOG, SMAD2 and SMAD3 and inhibits the exit from pluripotency.

Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRAS(G12D) driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets.

Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.

Diffuse large B cell lymphoma (DLBCL) frequently harbors genetic alterations that activate the B cell receptor (BCR) and TLR pathways, which converge to activate NF-κB. While selective inhibition of BTK with ibrutinib causes clinical responses in relapsed DLBCL patients, most responses are partial and of a short duration. Here, we demonstrated that MyD88 silencing enhanced ibrutinib efficacy in DLBCL cells harboring MyD88 L265P mutations. Chemical downregulation of MyD88 expression with HDAC inhibitors also synergized with ibrutinib. We demonstrate that HDAC inhibitor regulation of MyD88 expression is mediated by STAT3. In turn, STAT3 silencing caused a decrease in MyD88 mRNA and protein levels, and enhanced the ibrutinib antilymphoma effect in MyD88 mutant DLBCL cells. Induced mutations in the STAT3 binding site in the MyD88 promotor region was associated with a decrease in MyD88 transcriptional activity. We also demonstrate that treatment with the HDAC inhibitor panobinostat decreased phosphorylated STAT3 binding to the MyD88 promotor. Accordingly, combined treatment with panobinostat and ibrutinib resulted in enhanced inhibition of NF-κB activity and caused regression of DLBCL xenografts. Our data provide a mechanistic rationale for combining HDAC inhibitors and ibrutinib for the treatment of DLBCL.

For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.

SummaryThe use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.

The recent discovery of the CRISPR/Cas system and repurposing of this technology to edit a variety of different genomes have revolutionized an array of scientific fields, from genetics and translational research, to agriculture and bioproduction. In particular, the prospect of rapid and precise genome editing in laboratory animals by CRISPR/Cas has generated an immense interest in the scientific community. Here we review current in vivo applications of CRISPR/Cas and how this technology can improve our knowledge of gene function and our understanding of biological processes in animal models.

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity., Phenotype switching is a form of plasticity that allows melanoma cancer cells that leave the primary tumour to invade secondary sites, to switch from an invasive to a proliferative state. Here the authors identify EDN3, and its synthetic enzyme ECE2, as a regulator of melanoma plasticity in the microenvironment.

The recently described clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has proven to be an exquisitely powerful and invaluable method of genetic manipulation and/or modification. As such, many researchers have realized the potential of using the CRISPR/Cas9 system as a novel screening method for the identification of important proteins in biological processes and have designed short guide RNA libraries for an in vitro screening. The seminal papers describing these libraries offer valuable information regarding methods for generating the short guide RNA libraries, creating cell lines containing these libraries, and specific details regarding the screening workflow. However, certain considerations are often overlooked that may be important when planning and performing a screen, including which CRISPR library to use and how to best analyze the resulting screen data. In this review, we offer suggestions to answer some of these questions that are not covered as deeply in the papers describing the available CRISPR libraries for an in vitro screening.

Mutations in genes encoding RNA splicing factors constitute the most common class of genetic alterations in patients with myelodysplastic syndromes (MDS). These occur as heterozygous point mutations at specific amino acid residues in SF3B1, SRSF2, and U2AF1, and are almost always mutually exclusive with one another. Recent studies have identified that mutations in each of these genes results in activation of the innate immune signaling through altered splicing of mRNAs encoding key enzymes in this pathway. Now, through an unbiased genetic screen as well as focused genetic studies, we have identified that SF3B1-mutant MDS depends on aberrant immune signaling for cell survival. Recent work has identified that aberrant splicing of MAP3K7 (also known as TAK1; TGF-b Activating Kinase 1) is pervasive across SF3B1-mutant human and mouse cells and results in reduced MAP3K7 protein expression and increased NF-κB signaling. Consistent with this, Map3k7 haploinsufficiency in myeloid cells is known to cause myeloproliferation, while at the same time, complete loss of Map3k7 is intolerable for hematopoietic cells. We therefore hypothesized that partial inhibition of MAP3K7 might preferentially impact SF3B1-mutant cells. To test this hypothesis, we generated mice with inducible deletion of 1 or 2 copies of Map3k7 (Mx1-cre Map3k7fl/+,Mx1-cre Map3k7fl/fl) alone or in the presence of mutant Sf3b1K700E (Mx1-cre Map3k7fl/+Sf3b1K700E/+,Mx1-cre Map3k7fl/flSf3b1K700E/+), along with all controls (Mx1-cre Sf3b1+/+ Map3k7+/+ (Wildtype; WT) and Mx1-cre Sf3b1K700E/+ mice). We then performed bone marrow transplantation (BMT) to assess the effect of Map3k7 deletion on aberrant hematopoiesis driven by mutant SF3B1. Consistent with prior reports, heterozygous deletion of Map3k7did not affect repopulating potential in BMT assays compared to controls while homozygous deletion of Map3k7 resulted in complete failure of hematopoiesis (Figure A). Interestingly, however, in the presence of Sf3b1K700E mutation, deletion of a single copy of Map3k7 completely rescued the hematopoietic defects characteristic of mutant SF3B1 in both mature and immature cells (Figure B-C). These data suggest that inhibition of residual MAP3K7 function may preferentially target SF3B1-mutant MDS cells. In parallel to the above studies, we also performed a negative selection RNAi screen to uncover novel genetic dependencies in SF3B1-mutant myeloid neoplasms. We performed pooled lentiviral infection of shRNAs targeting ~2,200 genes encoding proteins which are drug targets ("The Druggable Genome") under the control of a doxycycline-inducible vector in isogenic K562 cells expressing the two most commonly occurring SF3B1 mutations, SF3B1K666N and SF3B1K700E, from the endogenous SF3B1 locus. Two individual clones per SF3B1-mutant line were used to improve the robustness of the screen. On Day 21 following shRNA activation, genes with ≥3 shRNAs depleted in SF3B1-mutant cells while remaining unchanged in parental K562 cells were selected. This identified 101 candidates that are potentially synthetic-lethal with SF3B1 mutation (Figure D). Interestingly, pathway analysis of these potential candidates revealed of genes involved in immune and inflammatory signaling as well as in metabolic processes (Figure E). Further target validation was performed using in vitro competitive growth assay in K562 cells, and another set of SF3B1 isogenic lymphoid leukemia cell lines (NALM-6) expressing the same mutations. This revealed consistent dependency of SF3B1-mutant cells on STAT1, an essential component of the interferon (IFN) signaling pathway (Figure F). Upon exposure to Type-I IFNs, SF3B1-mutant K562 cells showed increased transcriptional response in IFN-responsive genes containing interferon stimulated response elements (ISREs) compared with SF3B1 WT cells (Figure G). These data highlight that SF3B1-mutant cells are hyper-responsive to IFN signaling and require intact IFN-signaling responses for cell survival. Taken together, the above studies indicate that sustained IFN signaling as well as activated innate immune signaling downstream of TAK1 are required for the survival of SF3B1-mutant myeloid cells. These results therefore have important therapeutic implications as they suggest that pharmacologic inhibition of STAT1/Type I IFN activation and/or TAK1 may serve as important therapeutic agents for SF3B1-mutant MDS. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc-substituted-phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac-[Lys12,Nle17,X22,Val26,Thr28]-VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up to 300-fold. A few analogs, all possessing electron-donating substituents, retained nearly full potency. Two compounds, 3-F,4-OH-Phe, 42 and 3-OCH3,4-OH-Phe, 43, were found to be 1.5- and 3.4-fold more potent than the parent compound, which equates to being 8.9- and 20-fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2-fold enhanced potency and a significantly longer duration of action (> 20 min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.

A novel series of orally active pyrimido[5,4-3][1,2,4]triazine-5,7-diamine-based hypoglycemic agents have been identified. These compounds show non-selective inhibitory properties against a panel of protein tyrosine phosphatases including PTP1B. Compounds 12 and 13 display oral glucose lowering effects in ob/ob mice.

RNA binding proteins (RBPs) tightly control mRNA abundance, stability and translation while mutations or altered expression of specific factors can drive malignancy. Yet, the identity of the RBPs that govern myeloid stem cells remains poorly characterized. We and others have recently demonstrated that MUSASHI-2 (MSI2) is a central regulator of the cancer stem cell program in myeloid leukemia. Therefore, we curated a list of 127 MSI2 direct protein interactors and associated genes to perform an in vivo shRNA screen using MLL-AF9 leukemia cells. We identified shRNAs corresponding to 24 genes that were significantly depleted in vivo after sequencing and comparing their representation from day 16 to day 0. We confirmed knockdown and demonstrated marked reduction in myeloid colony formation in vitro after depleting 7 hits identified in our screen. Additionally, we tested these genes in normal bone marrow c-Kit positive cells and found that the most differentially required gene in leukemia cells compared to normal cells was SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein). SYNCRIP is an RNA binding protein that has been implicated in various RNA regulatory processes but its role in the hematopoietic system is virtually unknown. Depletion of SYNCRIP with shRNAs in murine MLL-AF9 leukemia cells resulted in an increase in myeloid differentiation, apoptosis and delayed leukemogenesis in vivo (median survival of 35 days; control versus 61 days shRNA#1 knockdown was selected against, and "not reached" shRNA#2). To further assess SYNCRIP function in vivo, we developed a germline Syncrip knockout (KO) by injecting Cas9-DNA and Syncrip - guides RNAs into embryos and harvested E13 fetal liver cells. After Syncrip deletion was verified by immunoblotting, we observed normal numbers of HSCs and equivalent engraftment in lethally irradiated animals in both primary and secondary transplants. In contrast, we observed a delay in leukemeogenesis (median survival of 87.5 days; WT versus 118 days KO) in recipient mice after transplantation of MLL-AF9 transformed LSKs. Notably, non-deleted leukemia cells outcompeted the SYNCRIP deleted cells based on a reemergence of SYNCRIP expression. These data suggest that SYNCRIP is differentially required in myeloid leukemia cells compared to normal cells. Furthermore, we found that SYNCRIP was highly expressed in wide variety of human AML cell lines and in primary AML patients (n=4/5). SYNCRIP depletion with shRNAs resulted in reduced cell proliferation and the induction of apoptosis in human AML cell lines (MOLM13, NOMO-1, KASUMI-1 and NB4) and a marked decrease in engraftment of primary AML patient cells. To gain insights into SYNCRIP function, we performed RNA-sequencing of leukemia cells depleted for SYNCRIP. Gene set enrichment analysis (GSEA) negatively enriched for the MLL-AF9, HOXA9 and stem cell programs in SYNCRIP-KD cells and positively enriched for MSI2's direct mRNA binding targets and a MSI2 deficient LSC signature. Reciprocal immunoblotting in the presence or absence of RNAse demonstrated that SYNCRIP and MSI2 interaction is RNA dependent. We validated their shared targets by performing SYNCRIP RNA-immunoprecipitation (RIP) for previously identified MSI2's direct mRNAs targets (HOXA9 and c-MYC). SYNCRIP depletion resulted in reduced protein abundance of HOXA9 and c-MYC. Forced MSI2 expression partially rescued the colony formation and HOXA9 expression in SYNCRIP-KD cells. To assess the functional downstream targets of SYNCRIP in leukemia, we overexpressed HOXA9 and c-MYC in SYNCRIP-KD cells and observed that HOXA9 expression but not c-MYC partially rescues the effect of SYNCRIP depletion on myeloid colony formation. Mechanistically, we showed that SYNCRIP regulates translation of HOXA9 without affecting HoxA9 mRNA stability. Overall, we provide a strategy for interrogating the functional RNA binding network in leukemia using shRNA screening. Additionally, we validated SYNCRIP as a novel RBP that controls the leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in myeloid leukemia. Disclosures Melnick: Janssen: Research Funding. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Järås:Cantargia AB: Equity Ownership.

The major histocompatability complex (MHC) is a central receptor in the adaptive immune response and is the underlying target of several effective therapies for cancer. Druggable kinases may provide the opportunity to modulate the immune response toward MHC. However, the regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. The entire human kinome was screened using a pooled shRNA interference-based approach in a human mesothelioma cell line to uncover kinase regulators of MHC-I. Negative and positive regulators of cell surface HLA levels were discovered. A subset of highly scoring positive and negative kinase hits were subsequently validated by additional RNAi, and pharmacologic inhibitors when available. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of HLA expression in multiple cancer types. We mapped the pathways responsible for increased HLA upon kinase inhibition. Interestingly, inhibition of the MAP Kinase pathway broadly influenced expression of other components of the antigen presentation machinery. Moreover, DDR2 and MINK1 were shown to positively regulate HLA-A*02:01. This had therapeutic relevance, as shown with a therapeutic TCR mimic antibody to a MHC/peptide complex. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Citation Format: Elliott J. Brea, Claire Oh, Eusebio Manchado, Ron Gejman, George Mo, Patrizia Mondello, Ralph Garippa, Neal Rosen, David A. Scheinberg. The regulation by kinases of the expression of human major histocompatibility class I molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4899.

The CRISPR/Cas9 system allows for unprecedented ease and control when editing the genome. Its potential impact on drug discovery is vast, including enabling gene and cell replacement therapies, identifying novel drug targets through functional genomic screens, and simplifying the production of disease models using permanent knockouts for validating therapy targets and testing drug efficacy. But in practical terms, how is CRISPR/Cas9 currently being applied, and where might the future challenges and pitfalls be? Furthermore, how do assays based on the new CRISPR/Cas9 technology compare with current screening methodologies, particularly those using small interfering RNA (siRNA)? View the Webinar

This report explores the potential side-chain functional groups required for interaction of the bronchodilator neuropeptide, vasoactive intestinal peptide (VIP), with its receptor. The binding affinity and biological activity of native VIP have been found to be sensitive to the removal of amino- and carboxyl-terminal residues. This data suggests that elements within the entire primary sequence of the VIP molecule appear to be necessary for recognition by VIP receptors. The introduction of alanine residues substituted into the VIP molecule is utilized to probe for side-chain functional groups that are crucial for eliciting high receptor binding affinity in vitro and high biological potency in vivo. The VIP pharmacophore appears to be identical in guinea pig lung and human lung and consists of multiple binding sites most likely involving positions Asp3, Phe6, Thr7, Tyr10, Tyr22, and Leu23. These findings could be exploited to enhance the biological potency of VIP by increasing the binding energy at these positions.

It is early to fully reflect on the state of the art in high content screening (HCS), because it is still a relatively new approach in drug discovery. Although the development of the first microscopes are a century old and the first confocal microscope is only 20 yr old, the fluorescent probes used within HCS along with the combination of robotic automation and integrated software technologies are quite new. HCS will require a few more years to fully demonstrate its potential power in drug discovery. Within the last year, however, one has seen this ever-expanding field lure participants in from all areas of science, introducing newer versions of instruments and reagents such that the combined efforts result in platforms and tools that meet many organizational goals in multiple ways. The potential of HCS today lies in its versatility. HCS can be used for primary screening, basic research, target identification, biomarkers, cytotoxicity, and helping to predict clinical outcomes. HCS is being applied to stem cells, patient cells, primary hepatocytes, and immortalized cultured cells. We have noted for individual specialized assays, there are multiple solutions just as there are for those standardized universally accepted assays. Whether we have needed to query cellular processes under live conditions or wanted to follow kinetically the course of a compound's effects on particular cellular reactions, we have been hampered by only a few limitations. This chapter offers a glimpse inside the use of HCS in our drug discovery environment.

Ligand‐activated G protein‐coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a ∼800 member superfamily of seven transmembrane‐spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems‐based biological approach, one that combines in silico bioinformatics, genomics, high‐throughput screening, and high‐content cell‐based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor β ‐arrestin–green fluorescent protein translocation assay in which full‐length human orphan GPCRs were stably expressed in a U‐2 OS cell background. These cells lend themselves to high‐speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation‐assisted plate and liquid‐handling techniques, an optimized image analysis algorithm, and a high‐speed confocal microscope with sophisticated data analysis tools.

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.

Methacholine (Mch), when injected near the ostia of the coronary arteries, induces an intense coronary vasospasm which can be measured by the degree of S wave elevation monitored from an electrocardiogram (EGG). The observed EGG changes resemble those occurring in patients with variant angina. The effects of Mch were blocked by atropine, but not by d-tubocurarine, hexamethonium, adrenergic receptor blockade, or prior reserpinization, indicating that Mch is acting directly on muscarinic receptors to produce a vasoconstriction of the coronary arteries. This model of Mch-induced coronary vasospasm appears to be useful for testing spasmolytic agents which might be of benefit in variant angina.