Lentiviral shRNA Library Research Articles

LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells

Chemoresistance and plasticity of tumor-initiating stem-like cells (TICs) promote tumor recurrence and metastasis. The gut-originating endotoxin-TLR4-NANOG oncogenic axis is responsible for the genesis of TICs. This study investigated mechanisms as to how TICs arise through transcriptional, epigenetic, and post-transcriptional activation of oncogenic TLR4 pathways. Here, we expressed constitutively active TLR4 (caTLR4) in mice carrying pLAP-tTA or pAlb-tTA, under a tetracycline withdrawal-inducible system. Liver progenitor cell induction accelerated liver tumor development in caTLR4-expressing mice. Lentiviral shRNA library screening identified histone H3K4 methylase SETD7 as central to activation of TLR4. SETD7 combined with hypoxia induced TLR4 through HIF2 and NOTCH. LIN28 post-transcriptionally stabilized TLR4 mRNA via de-repression of let-7 microRNA. These results supported a LIN28-TLR4 pathway for the development of HCCs in a hypoxic microenvironment. These findings not only advance our understanding of molecular mechanisms responsible for TIC generation in HCC, but also represent new therapeutic targets for the treatment of HCC.

Suppressors of Break-Induced Replication in Human Cells.

Short tandem DNA repeats are drivers of genome instability. To identify suppressors of break-induced mutagenesis human cells, unbiased genetic screens were conducted using a lentiviral shRNA library. The recipient cells possessed fragile non-B DNA that could induce DNA double-strand breaks (DSBs), integrated at an ectopic chromosomal site adjacent to a thymidine kinase marker gene. Mutagenesis of the thymidine kinase gene rendered cells resistant to the nucleoside analog ganciclovir (GCV). The screen identified genes that have established roles in DNA replication and repair, chromatin modification, responses to ionizing radiation, and genes encoding proteins enriched at replication forks. Novel loci implicated in BIR included olfactory receptors, the G0S2 oncogene/tumor suppressor axis, the EIF3H-METTL3 translational regulator, and the SUDS3 subunit of the Sin3A corepressor. Consistent with a role in suppressing BIR, siRNA knockdown of selected candidates increased the frequency of the GCVr phenotype and increased DNA rearrangements near the ectopic non-B DNA. Inverse PCR and DNA sequence analyses showed that hits identified in the screen increased genome instability. Further analysis quantitated repeat-induced hypermutagenesis at the ectopic site and showed that knockdown of a primary hit, COPS2, induced mutagenic hotspots, remodeled the replication fork, and increased nonallelic chromosome template switches.

Synaptotagmin 11 scaffolds MKK7–JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis

BackgroundIdentifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC.MethodsIn vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient’s survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis.ResultsSYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells.ConclusionSYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

High‐content screening of Alzheimer's disease genetic risk factors based on synaptic density analysis

AbstractBackgroundSynaptic loss is one of the earliest pathological hallmarks and the strongest marker of cognitive decline in Alzheimer’s disease (AD). A strong genetic predisposition is linked to AD, and genome‐wide association studies have pointed out hundreds of genes associated with the risk of developing the disease. With this background, our objective is to develop a cell‐based screen to assess the impact of each genetic risk factor on synaptic density.MethodWe screened a lentiviral shRNA library targeting 200 AD genetic risk factors using primary rat hippocampal neurons in 384‐wells plates. Immunofluorescence was performed to reveal pre‐ and post‐synaptic compartments and the neuronal network. Synaptic connectivity was then assessed through high content analysis by assigning each post‐synaptic structure to the nearest pre‐synaptic structure using Columbus and Matlab software.ResultWe identified several AD risk genes which positively or negatively impacted synaptic density in our model.ConclusionWe have developed a high‐content screening approach which allows us to define the genetic risk factors that are involved in the pathophysiological synaptic dysregulation observed in the early stages of AD.

Selective Inhibition of Nucleocytoplasmic Transport Overcomes Ruxolitinib Resistance in Myelofibrosis

Myelofibrosis (MF) is a stem cell-derived hematopoietic malignancy characterized by constitutive activation of JAK/STAT signaling through mutations in JAK2, calreticulin or MPL. MF patients have reduced life expectancy due to cytopenias, progression to acute myeloid leukemia and thromboembolic events. JAK kinase inhibitors such as ruxolitinib reduce constitutive symptoms but are not curative, with persistence of malignant cells. Allogeneic stem cell transplant remains the only curative therapy for MF, but is associated with considerable morbidity and mortality, defining an urgent need for new therapies that induce more profound and durable responses.We used a barcoded lentiviral shRNA library to identify survival-critical genes for MF in HEL cells (homozygous for JAK2V617F), established from a patient with erythroleukemia. shRNAs targeting RAN and binding protein (RANBP) were significantly depleted, implicating nucleocytoplasmic transport (NCT) as one of the top survival-critical pathways. RAN is a small GTPase, whose cycling between GTP- and GDP-bound forms provides the engine for NCT. We previously presented results showing that shRNA-mediated knockdown of RAN, significantly reduced proliferation and induced apoptosis in HEL and SET-2 cells (harboring heterozygous JAK2V617F). Additionally, KPT-330 (selinexor, Karyopharm Therapeutics), an inhibitor of CRM1, the core component of NCT, also inhibited proliferation of HEL and SET-2 cells and colony formation by primary MF CD34+ cells, but not cord blood (CB). Importantly, inhibition of NCT was effective at reducing colony formation in samples from ruxolitinib-refractory MF patients, regardless of mutation status. A clinical trial to evaluate KPT-330 in ruxolitinib-refractory MF is in preparation.KPT-8602 is a second-generation inhibitor of CRM-1. Unlike KPT-330, KPT-8602 does not cross the brain-blood barrier, resulting in minimal anorexia, malaise, and weight loss [Etchin et al., Leukemia, 2017 Jan;31(1):143-150]. To determine the efficacy of KPT-8602 in MF, we tested KPT-8602 in cell lines, patient samples, and a mouse model of MF, along with ruxolitinib as comparison.In cell proliferation assays, KPT-8602 was five-fold more potent than ruxolitinib against HEL cells (IC50: 83 nM vs. 437 nM) and as potent as ruxolitinib in SET-2 cells (IC50: 95 nM vs. 85 nM). In clonogenic assays, KPT-8602 showed selectivity toward primary MF vs. CB CD34+ cells (IC50: 25 nM for MF vs. 258 nM for CB) and, in combination with ruxolitinib, resulted in a 95% reduction in colony formation. KPT-8602 inhibited colony formation by CD34+ cells from all MF patients tested, including ruxolitinib-refractory cases, irrespective of mutation status (including triple negative).To evaluate the possible use of KPT-8602 in vivo, we used a mouse transduction/transplantation model of JAK2V617F - positive myeloproliferative neoplasm (MPN). In this model, mice develop MPN resembling polycythemia vera that progresses to MF [Bumm et al., Cancer Res., 2006, 1;66(23):11156-65]. MPN was evident based on elevated blood counts and blood GFP+ cells (representing JAK2V617F allelic burden). MPN mice were randomized into four groups: vehicles, KPT-8602 (initial dose 15 mg/kg daily, orally), ruxolitinib (initial dose 50 mg/kg twice daily, orally) or combined ruxolitinib with KPT-8602 at these same concentrations, and treated for 28 days. KPT-8602 as a single agent reduced red blood cells (RBC), white blood cells (WBC), BM GFP+ cells, peripheral blood (PB) GFP+ cells and spleen size to the same extent as ruxolitinib, and the combination was no more effective than either single agent (Table 1). Spleen cytoarchitecture was fully restored by KPT-8602, but bone marrow fibrosis was not rectified in any treatment group. Mice in all groups, including the combined vehicle controls, still experienced weight loss and lethality, suggesting that toxicity may be due to vehicles, and that optimization of drug formulations and dosing is warranted.Overall, these results indicate that NCT is critical for MF survival, and that NCT inhibition may provide therapeutic benefit for MF patients. KPT-8602 is being tested in a clinical trial (NCT02649790) for refractory multiple myeloma. The results of our study provide strong rationale for a clinical trial in patients with refractory MF. [Display omitted] DisclosuresBaloglu:Karyopharm Therapeutics Inc: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deininger:Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Celgene: Research Funding; ARIAD: Consultancy; Gilead: Research Funding; BMS: Consultancy, Research Funding.

Abstract A43: Identification of novel YAP/TAZ regulators in metastatic cancer

Abstract Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the downstream effectors of the Hippo tumor inhibition pathway. Studies from us and others show that inappropriate activation of YAP and/or TAZ promotes tumor growth and metastasis. Increased YAP and/or TAZ expression and nuclear localization is observed in many human cancers and often predict poor prognosis. Mutations in Hippo-YAP/TAZ pathway exist but are not common enough to explain the frequently observed increase of YAP/TAZ expression and activity in cancer. Therefore, we hypothesize that other cancer-associated pathways cause aberrant YAP/TAZ activation in cancer cells, and identifying such pathways would provide targets for treatment of YAP/TAZ-driven cancers. Furthermore, transcriptional cofactors are often difficult to target and YAP/TAZ have important functions in adult tissues. Therefore, targeting YAP/TAZ regulators may provide a nonsystemic path of targeting YAP/TAZ that could avoid severe adverse effects. To identify novel YAP/TAZ regulators in metastatic cancer cells, we screened a lentiviral shRNA library that targets 124 cancer-associated genes in A375 MA2 (MA2) cells using a YAP/TAZ-TEAD (TEA domain family members) transcriptional reporter assay. We found that 62 genes are able to regulate YAP/TAZ activity in MA2, 24 of which are already established YAP/TAZ regulators, which supports the effectiveness of our approach. The other 38 genes have not been reported previously, which are potential novel YAP/TAZ regulators. We subsequently validated these candidate YAP/TAZ regulators by examining the expression of the YAP/TAZ target gene CTGF by qPCR and phosphorylation of YAP and TAZ by Western blot in MA2 and other metastatic cell lines. We are currently pursuing several of these novel YAP/TAZ regulators to determine the mechanism through which they regulate YAP/TAZ activity and test their role in metastasis. Our results suggest that YAP/TAZ regulators are cell type and context specific and may also functionally compensate for each other. Therefore, it is critical to identify a “core” group of YAP/TAZ regulators that are important in multiple cancer types. This could provide new strategies to more effectively inhibit YAP/TAZ activation and treat metastatic cancer. Citation Format: Yuxuan Xiao, Emily Norton, Janine Warren, John Lamar. Identification of novel YAP/TAZ regulators in metastatic 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 A43.

Abstract 348: Homeobox A4 Suppresses Vascular Smooth Muscle Cell Phenotypic Switching as a Novel Regulator of YAP/TEAD Transcriptional Activity

The Hippo signaling pathway is involved in the pathophysiology of various cardiovascular diseases. Yes-associated protein (YAP) and transcriptional enhancer activator domain (TEAD) transcriptional factors, the main transcriptional complex of the Hippo pathway, were recently identified as modulators of phenotypic switching of vascular smooth muscle cells (VSMCs). However, the intrinsic regulator of YAP/TEAD-mediated gene expressions in VSMCs remains to be elucidated, then we sought to investigate a novel regulator of YAP/TEAD transactivation involved in vascular diseases. We first investigated novel YAP/TEAD regulators using lentiviral shRNA library in HEK 293T-based TEAD-responsive reporter cell line and detected Homeobox A4 (HOXA4) as a potent repressor of YAP/TEAD transcriptional activity. HOXA4 attenuated YAP/TEAD-mediated gene expression independently of YAP phosphorylation, and co-immunoprecipitation assays revealed that HOXA4 interacts with TEADs but not YAP. Mechanistically, HOXA4 attenuated YAP/TEAD-mediated transcription by competing with YAP for TEAD binding. We also clarified that the expression of HOXA4 is relatively abundant in the vasculature, especially in VSMCs. In vitro experiments in human VSMCs showed HOXA4 maintains the differentiation state of VSMCs via inhibition of YAP/TEAD-induced phenotypic switching characterized by cell morphology, cell proliferation, and gene expression patterns. We generated Hoxa4-decifient mice and confirmed the downregulation of smooth muscle-specific contractile genes and the exacerbation of vascular remodeling after carotid artery ligation in vivo. Our results demonstrate HOXA4 is a novel repressor of VSMC phenotypic switching by inhibiting YAP/TEAD-mediated transcription. These findings give us a better understanding of the vascular pathophysiology and a novel therapeutic approach for vascular remodeling.

S111 SIRT5 IS REQUIRED FOR ACUTE MYELOID LEUKEMIA GROWTH AND SURVIVAL AND MAY REPRESENT A NOVEL THERAPEUTIC TARGET

Background:Acute myeloid leukemia (AML) is a genetically heterogeneous hematopoietic neoplasm with five‐year overall survival rates <30% on standard of care therapy. While recent progress with inhibitors of FLT‐ITD and mutated IDH1/2 validates the concept of targeting driver mutants, the efficacy of the venetoclax/5‐azacytidine combination is evidence that critical vulnerabilities can encompass multiple AML genotypes.Aims:Identify novel therapy targets in AML in a genotype‐agnostic manner.Methods:We performed an shRNA screen on patient samples (N = 12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS‐5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), a lysine desuccinylase and metabolic regulator, was amongst the top candidates.Results:We transduced 25 genotypically heterogeneous 25 AML cell lines with doxycycline (dox) ‐ inducible shSIRT5 (dox‐shSIRT5). SIRT5 KD strongly inhibited proliferation, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5‐dependent cell lines). SIRT5 dependence did not correlate with specific genotype or SIRT5 expression. Data were validated using three shSIRT5 s targeting different unique in 3 SIRT5‐dependent and 3 SIRT5–independent cell lines, with consistent results. We next transduced CD34+ cells from AML patients (n = 15) or cord blood (CB, n = 5) with dox‐shSIRT5 and plated cells in colony assays. SIRT5 KD (∼40% in AML and CB) reduced AML colonies by ∼50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3‐ITD or MLL‐AF9 retrovirus was reduced by 50–60% compared to wild type controls.Metabolic profiling showed that SIRT5 KD induced reduced oxidative phosphorylation in SIRT5‐dependent, but not SIRT5‐independent cell lines, with a steep increase in mitochondrial reactive oxygen species (ROS) that preceded apoptosis and was abrogated by ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial). Further SIRT5 KD disrupted glutamine/glutamate metabolism in SIRT5‐dependent but not SIRT5‐independent cells, while SIRT5 KD in SIRT5‐independent cells was inconsequential.We injected mice with CMK‐1 cells (SIRT5‐dependent) expressing dox‐shSIRT5 and luciferase. Mice were randomized to dox‐supplemented (dox‐water) or control water, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox‐water survived without evidence of leukemia. When mice with established leukemia were switched to dox‐water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox‐water had no effect on mice engrafted with OCI‐AML3 cells (SIRT5‐independent), all of whom died from leukemia despite downregulation of SIRT5 in leukemia cells. We next assessed SIRT5 function in a retroviral transduction/transplantation model, using MLL‐AF9 and BCR‐ABL1 in a SIRT5 null or SIRT5 wild type background. Absence of SIRT5 attenuated leukemia in both cases, with significant prolongation of survival. We also functionally characterized hematopoietic stem and progenitor subsets from young and old SIRT5 wild type and null mice, but identified no significant differences.Summary/Conclusion:AML, but not normal hematopoietic cells are dependent on SIRT5. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors.

SIRT5 As a Therapeutic Target in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm with five-year overall survival rates <30% on standard of care therapy. Relapse is common and thought to originate from AML stem cells that survive in the protective bone marrow (BM) microenvironment. Next generation sequencing (NGS) has revealed the somatic mutation spectrum of AML and the approval of midostaurin for FLT3 ITD- and enasidenib for IDH2 -mutated AML is evidence that the molecular knowledge is being translated clinically.To identify survival-critical AML genes irrespective of mutational status, we performed an shRNA screen on patient samples (N=12) using a barcoded lentiviral shRNA library. Transduced cells were cultured for 9 days on HS-5 stromal cells (to mimic the BM microenvironment) and subjected to NGS for barcode quantification. Sirtuin 5 (SIRT5), the only known enzyme with lysine desuccinylase, demalonylase, or deglutarylase activity, was amongst the top candidates. SIRT5 is implicated in regulating metabolic pathways, including energy metabolism.We first confirmed growth inhibition upon SIRT5 knockdown (KD) in cryopreserved AML cells from the original screen. Next, we transduced a panel of 25 AML cell lines with doxycycline (dox) - inducible shSIRT5 (dox-shSIRT5). SIRT5 KD strongly inhibited growth, reduced colony formation and increased apoptosis in 18/25 lines (SIRT5-dependent cell lines). Seven cell lines showed <20% colony reduction despite >80% reduction of SIRT5 protein and were considered SIRT5-independent. SIRT5 dependence was neither correlated with specific mutations nor basal SIRT5 expression. To control for off-target effects we tested three unique shSIRT5s targeting different sequences in three SIRT5-dependent and three SIRT5-independent cell lines, and confirmed the differential sensitivity for all lines and all constructs. We next transduced CD34+ cells from AML patients (n=15) or cord blood (CB, n=5) with dox-shSIRT5 and plated cells in colony assays. SIRT5 KD (~40% in AML and CB) reduced AML colonies by ~50%, with no effect on CB. Colony formation by SIRT5 null mouse bone marrow infected with FLT3-ITD or MLL-AF9 retrovirus was reduced by 50-60% compared to wild type controls.Metabolic profiling showed that SIRT5 KD induced a profound reduction of oxidative phosphorylation and glycolysis in SIRT5-dependent, but not SIRT5-independent cell lines. Mitochondrial reactive oxygen species (ROS) in SIRT5-dependent cell lines were strongly increased upon SIRT5 KD, and this increase preceded apoptosis. Ectopic expression of superoxide dismutase 2 (SOD2, mitochondrial) abrogated the increase in ROS and rescued cells from apoptosis. Metabolomics and RNAseq suggest that SIRT5-dependent cells exhibit profound metabolic disruption, including reduced TCA cycle activity, and recurrent transcriptional changes, while SIRT5 KD in SIRT5-independent cells is inconsequential.We next injected mice with CMK-1 cells (SIRT5-dependent) expressing dox-shSIRT5 and luciferase. Mice were randomized to dox-supplemented (dox-water) or control water 18 hours post injection, and monitored by luminescence imaging. Control mice showed abundant luminescence at week 3, and died before week 5, while mice receiving dox-water survived throughout the 13-week experiment without evidence of leukemia. When mice with established leukemia were switched to dox-water following week 3, luminescence rapidly decreased and the mice survived without evidence of leukemia until termination of the experiment (week 13). Dox-water had no effect on mice engrafted with OCI-AML3 cells (SIRT5-independent), all of whom died with extensive leukemic involvement despite downregulation of SIRT5 in leukemia cells. We are currently testing the requirement of SIRT5 in MLL-AF9-mediated AML using a retroviral BM transplant mouse model. Preliminary data suggest that absence of SIRT5 may prolong survival.Our data suggest that SIRT5 KD preferentially targets AML cells over normal cells. As SIRT5 null mice are viable with only minor metabolic abnormalities at steady state, these data implicate SIRT5 as a potential therapy target in AML and support the development of clinical SIRT5 inhibitors. DisclosuresDeininger:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

Abstract IA09: RiboSeq and ye shall find: Noncanonical protein translation in cancer Iimunosurveillance

Abstract By displaying oligopeptides on the cell surface, class I major histocompatibility complex (MHC-I) molecules enable CD8+ T cell immunosurveillance of tumor cells. MHC-I peptide ligands derive proteins synthesized by cancer cells. A number of lines of evidence indicate that many MHC-I peptides derive from a pool of rapidly degraded defective ribosomal products (DRiPs). DRiPs include misfolded gene products resulting from errors in transcription, translation, folding, targeting, and assembly. They also encompass the translation products of “immunoribosomes,” specialized ribosomes proposed to generate antigenic peptides for the express propose of immunosurveillance. There are now several examples in which noncanonical translation efficiently generates peptides for immunosurveillance. I will discuss how the method of ribosome profiling can be used to identify novel tumor target peptides for immunotherapy arising from noncanonical translation. To determine whether ribosome heterogeneity itself can also selectively increase antigenic peptide generation, we screened a lentiviral shRNA library targeting each of the 80 human ribosomal protein genes. We identified candidate genes by alterations in steady state class I molecule expression (a measure of overall peptide supply) or in the ratio of source proteins to source protein-derived peptides. Two ribosomal proteins that either increase or decrease the efficiency of peptide generation are near neighbors on the ribosome surface, suggesting possible mechanisms for influencing peptide generation. Citation Format: Jonathan W. Yewdell. RiboSeq and ye shall find: Noncanonical protein translation in cancer Iimunosurveillance [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA09.

Abstract 84: Identification of HNRNPLL as a novel metastasis suppressor of colorectal cancer

Abstract Despite the advances in surgery and chemotherapy for colorectal cancer patients, those with distant metastasis still suffer poor prognosis. Metastasis is a complex process regulated by a number of biomolecules, which can be conceptually divided into metastasis promoters and suppressors. To identify novel targets for prevention and/or therapy of colorectal cancer metastasis, we performed an in vivo shRNA library screen for colorectal cancer metastasis suppressor genes using an orthotopic transplantation model. CMT93 cells, a murine colorectal cancer cell line with poor metastasizing activity, were transduced with lentiviral shRNA library, and then transplanted into the rectum of C57/BL6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNAs were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNAs. Among 47 candidate genes identified, we focused on Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) gene encoding a pre-mRNA binding protein involved in pre-mRNA splicing. Hnrnpll knockdown in CMT93 cells significantly increased their matrigel invasion in vitro and lung metastasis in vivo. An RNA-immunoprecipitation analysis identified Cd44 as a candidate target of Hnrnpll. The mRNA level of Cd44 variant 6 (Cd44v6), a splicing variant of Cd44 that had been shown to demarcate colon cancer stem cells driving colon cancer metastasis, was increased by knocking down Hnrnpll in colon cancer cells. Treatment with neutralizing anti-CD44v6 monoclonal antibodies suppressed matrigel invasion of colon cancer cells induced by Hnrnpll knockdown, suggesting that Hnrnpll may suppress invasion and metastasis, at least in part, by modulating the alternative splicing of Cd44. HNRNPLL expression level in colorectal cancer cells was drastically decreased upon induction of epithelial-mesenchymal transition (EMT), accompanied by increased expression of CD44v6. The link between HNRNPLL and EMT was further suggested by immunostaining analysis of clinical samples; colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression as compared with those in the tumor center. These results suggest that HNRNPLL is a novel metastasis suppressor in colorectal cancer. Possible mechanisms for regulation of HNRNPLL expression during EMT will also be discussed. Citation Format: Masahiro Aoki, Eiichi Sasaki, Kenya Kimura, Koji Komori, Yasuhiro Shimizu, Yasushi Yatabe, Keiichiro Sakuma. Identification of HNRNPLL as a novel metastasis suppressor of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 84.

Abstract 2901: Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors

Abstract Castration-resistant prostate cancer (CRPC) is frequently characterized by elevated expression of alternative nuclear receptors able to at least partially maintain the androgen receptor (AR) transcriptional program. These alternative receptors include ARv7, a ligand binding domain (LBD)-deficient, constitutively active splice variant that correlates with poor prognosis, reduced survival, and resistance to existing LBD-targeted standard of care therapy. As a nuclear hormone receptor, AR exists in a dynamic cycle with molecular chaperones (heat shock proteins, HSPs) HSP40/HSP70/HSP90 which foster proper folding and remodeling of the LBD to bind ligand. Notably, HSP90 inhibitors promote AR degradation and display efficacy in prostate cancer xenograft models. Although it has been shown that LBD-deficient ARv7 functions independently of HSP90, we have observed that loss of the LBD does not abrogate HSP40/HSP70 binding, as ARv7 interacts with HSP40/HSP70 in CRPC cells. As such, targeting either HSP40 (C86) or HSP70 (JG98/JG231) inhibits stability and function of AR/ARv7. However, this is complicated by the fact that over 40 isoforms of HSP40 (DnaJ proteins) and 13 HSP70 paralogs exist in humans. Therefore, we sought to identify the requirements for components of the HSP40/HSP70 network in mediating intracellular stability of AR/ARv7, and determine which chaperones orchestrate sensitivity to HSP40/HSP70 inhibitors. Using a doxycycline-inducible lentiviral proteostasis shRNA library, we identified genes that appear to be required for proliferation of 22Rv1 CRPC cells (expressing endogenous AR and ARv7) distinct from those found in ARv7-deficient LNCaP prostate cancer cells. These include genes from several chaperone families including DnaJA3 (HSP40), HSPA2 (HSP70-2), and HSPD1 (HSP60), which may be unique regulators of ARv7 stability. Synthetic lethal screens using C86 and JG231 were also performed to determine which genes may mediate sensitivity to these chaperone inhibitors. Knockdown of HSP40 isoforms (DnaJA1, DnaJA2), HSP70 paralog HSPA4, or stress factors (ATF6, HSF1) in combination with C86 lead to growth impairment in 22Rv1 cells. DnaJA1, DnaJA2, and DnaJA3 were all found in the cytosol and nucleus of 22Rv1 cells, and in a complex with AR/ARv7 after a biotin-C86 IP, suggesting they be required for AR/ARv7 stability, dimerization, and/or nuclear translocation. Other genes from chaperone families HSP40 (DnaJC7, DnaJC8, DnaJC16), HSP70 (HSPA5, HSPA8, HSPA9), and HSP90/HSP90 co-chaperones (HSP90AB1, Cdc37, PTGES3 [p23]) were found to be important mediators of sensitivity to JG231, revealing targets for possible combination therapies. Taken together, these data help identify HSP40/HSP70 subnetworks regulating AR/ARv7 and those mediating sensitivity to chaperone inhibitors to aid in identifying new drug targets for efficacy in CRPC. Citation Format: Michael A. Moses, Genesis Rivera-Marquez, Isabelle Taylor, Hao Shao, Jason Gestwicki, Jane Trepel, Len Neckers. Identifying synthetic lethal interactions in castration-resistant prostate cancer using HSP40 and HSP70 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2901.

A loss-of-function genetic screening reveals synergistic targeting of AKT/mTOR and WTN/\u03b2-catenin pathways for treatment of AML with high PRL-3 phosphatase

BackgroundProtein tyrosine phosphatase of regenerating liver 3 (PRL-3) is overexpressed in a subset of AML patients with inferior prognosis, representing an attractive therapeutic target. However, due to relatively shallow pocket of the catalytic site of PRL-3, it is difficult to develop selective small molecule inhibitor.MethodsIn this study, we performed whole-genome lentiviral shRNA library screening to discover synthetic lethal target to PRL-3 in AML. We used specific small molecule inhibitors to validate the synthetic lethality in human PRL-3 high vs PRL-3 low human AML cell lines and primary bone marrow cells from AML patients. AML mouse xenograft model was used to examine the in vivo synergism.ResultsThe list of genes depleted in TF1-hPRL3 cells was particularly enriched for members involved in WNT/β-catenin pathway and AKT/mTOR signaling. These findings prompted us to explore the impact of AKT/mTOR signaling inhibition in PRL-3 high AML cells in combination with WNT/β-catenin inhibitor. VS-5584, a novel, highly selective dual PI3K/mTOR inhibitor, and ICG-001, a WNT inhibitor, were used as a combination therapy. A synthetic lethal interaction between mTOR/AKT pathway inhibition and WNT/β-catenin was validated by a variety of cellular assays. Notably, we found that treatment with these two drugs significantly reduced leukemic burden and prolonged survival of mice transplanted with human PRL-3 high AML cells, but not with PRL-3 low AML cells.ConclusionsIn summary, our results support the existence of cooperative signaling networks between AKT/mTOR and WNT/β-catenin pathways in PRL-3 high AML cells. Simultaneous inhibition of these two pathways could achieve robust clinical efficacy for this subtype of AML patient with high PRL-3 expression and warrant further clinical investigation.

Abstract A132: Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen

Abstract Inhibiting angiogenesis, which is a shared characteristic of all solid tumors, is a promising approach for treating cancer. However, emerging resistance after initial response is still a drawback. We sought to decipher the molecular landscape of tumor resistance to continuous treatment with VEGF-receptor tyrosine kinase inhibitor (RTKi) sunitinib (SU11248, SU). To address that, we performed serial transplantation of a syngeneic LLC tumor model in C57bl6 mice using a genome-wide lentiviral shRNA library targeting about 39,000 transcripts (150K complexity). The abundance of each single knockdown (kd) was longitudinally traced via molecular barcodes towards development of resistant tumors. After three in vivo panning rounds, distinct shRNA constructs were found to be significantly enriched in tumors passaged under SU selection pressure (p3-SU) compared to those passaged without treatment (p3-ctrl). We focused on validation of three candidate shRNA targeting Kdm5c, Csf1, and Cd44. Knockdown of each of these three genes confirmed their role in emerging resistance to sunitinib in vivo in syngeneic model. Furthermore, knockdown of Kdm5c promoted resistance in 786-0 human renal cell carcinoma xenograft model, in which sunitinib is used as the first-line therapy. Transcriptome analysis of shKdm5c, shCsf1, and shCd44 harboring vs. shCtrl cells suggested that the development of resistance is partly due to modulation of cellular pathways as well as alteration of interaction between cells and their microenvironment. In addition, shKdm5c tumors demonstrated resistance towards pazopanib, an VEGF-RTKi with different inhibitory spectrum compared to sunitinib, which suggests a general mechanism of resistance to inhibitors of VEGF signaling after Kdm5c knockdown. Furthermore, cells with shKdm5c showed an increase in survival under hypoxia induced by VEGF-RTKi. Expression profiling of these cells in hypoxia illustrated a network of genes under Kdm5c control, including three components of PRC2 epigenetic modulator, Ezh2, Eed, and Suz12. We found that knockdown of Kdm5c interrupts the imposed break on these genes under hypoxia. In conclusion, we have shown that loss of Kdm5c, CSf1, and Cd44 renders tumor cells resistant to VEGF-RTKi partly through compensatory upregulation of angiogenic factors. In addition, loss of Kdm5c leads to aberrant activation of a gene-regulatory circuitry controlling cellular response to hypoxia. Citation Format: Azadeh Fahim Golestaneh, Maoyun Sun, Christian Schwager, Zili Tang, Lili Ma, Philip Hahnfeldt, Mahmoud Moustafa, Wilko Weichert, Juergen Debus, Lynn Hlatky, Amir Abdollahi. Deciphering the molecular regulators of tumor resistance to VEGF-receptor tyrosine kinase inhibitors via in vivo genome-wide RNAi screen [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A132.

RNAi Screens for Maintenance of Hematopoietic Stem Cell Phenotype During Culture

Successful ex vivo expansion of hematopoietic stem cells (HSCs) could greatly enhance cell- and gene therapy applications in treatment of malignant and inherited hematological disorders. It has therefore been a long-standing goal in the field to gain a better understanding of the genes and pathways that regulate the self-renewal ability of HSCs. We have developed RNAi screening strategies to identify modifiers of self-renewal/proliferation in human hematopoietic stem- and progenitor cells (HSCPs). Employing pooled lentiviral shRNA libraries targeted to human cord blood derived CD34+ cells, we use the limited persistence of HSPCs under ex vivo culture conditions as a basis for functional selection of shRNAs conferring prolonged maintenance or expansion of undifferentiated cells. From the screens we have been able to identify directly targetable genes such as p38 MAPK, which can be pharmacologically inhibited to enhance the stem cell output of cultured cord blood cells. Additionally, we have found several epigenetic regulators among the top-scoring genes, including genes of the cohesin family, which modulate chromatin architecture, and JARID2, an important modifier of polycomb repressive complex 2 (PRC2). Depletion of either the cohesin genes or JARID2 by shRNA impairs differentiation and enhances both in vitro expansion and the in vivo reconstitution capacity of human HSPCs. However, whether these genes can be targeted pharmacologically to support HSC expansion remains to be explored. The screens have further identified a number of shRNAs that display a remarkable ability to expand HSPCs in culture, but whose gene targets have not been validated, indicating that they are affecting one or more genes in a non-specific manner. We performed gene expression profiling of cells transduced with these off-target shRNAs to gain insight about the molecular context under which HSPCs are propagated ex vivo. A common expression signature was the down-modulation of cellular stress response genes, such as p38 MAPK (previously identified in the screens) as well as genes involved in NF-□B signaling. Indeed, we found that pharmacological inhibition of NF-□B signaling leads to a significant improvement of stem cell function, from ex vivo cultured cord blood derived CD34+ cells, as assessed by transplantation to NSG mice. The effect of NF-□B inhibition was most critical early during the culture where it reduced the levels of several inflammatory and stress-related cytokines that are induced as an immediate response to culture initiation. Taken together, findings from our RNAi screens indicate that ex vivo HSC expansion is facilitated by targeting two distinct gene categories: cellular stress response genes activated by the culture conditions, as well as epigenetic regulators controlling the balance between renewal and differentiation. In order to define the most optimal expansion conditions we are currently assessing the combined targeting of several of these factors in conjunction with other compounds such as SR1 and UM171 using cellular barcoding. Here, cells are genetically marked by lentivirally delivered barcode sequences prior to expansion culture, which enables a precise detection and comparison of the stem cell output from multiple different expansion conditions in a directly competitive manner within a single xenografted animal. Disclosures No relevant conflicts of interest to declare.

Synthetic lethal short hairpin RNA screening reveals that ring finger protein 183 confers resistance to trametinib in colorectal cancer cells

BackgroundThe mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor trametinib has shown promising therapeutic effects on melanoma, but its efficacy on colorectal cancer (CRC) is limited. Synthetic lethality arises with a combination of two or more separate gene mutations that causes cell death, whereas individual mutations keep cells alive. This study aimed to identify the genes responsible for resistance to trametinib in CRC cells, using a synthetic lethal short hairpin RNA (shRNA) screening approach.MethodsWe infected HT29 cells with a pooled lentiviral shRNA library and applied next-generation sequencing to identify shRNAs with reduced abundance after 8-day treatment of 20 nmol/L trametinib. HCT116 and HT29 cells were used in validation studies. Stable ring finger protein 183 (RNF183)-overexpressing cell lines were generated by pcDNA4-myc/his-RNF183 transfection. Stable RNF183-knockdown cell lines were generated by infection of lentiviruses that express RNF183 shRNA, and small interference RNA (siRNA) was used to knock down RNF183 transiently. Quantitative real-time PCR was used to determine the mRNA expression. Western blotting, immunohistochemical analysis, and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the protein abundance. MTT assay, colony formation assay, and subcutaneous xenograft tumor growth model were used to evaluate cell proliferation.ResultsIn the primary screening, we found that the abundance of RNF183 shRNA was markedly reduced after treatment with trametinib. Trametinib induced the expression of RNF183, which conferred resistance to drug-induced cell growth repression and apoptotic and non-apoptotic cell deaths. Moreover, interleukin-8 (IL-8) was a downstream gene of RNF183 and was required for the function of RNF183 in facilitating cell growth. Additionally, elevated RNF183 expression partly reduced the inhibitory effect of trametinib on IL-8 expression. Finally, xenograft tumor model showed the synergism of RNF183 knockdown and trametinib in repressing the growth of CRC cells in vivo.ConclusionThe RNF183-IL-8 axis is responsible for the resistance of CRC cells to the MEK1/2 inhibitor trametinib and may serve as a candidate target for combined therapy for CRC.

Abstract 4391: Identification of novel cancer driver genes in hepatocellular carcinoma cells by RNAi screening method

Abstract Recent studies revealed that EpCAM is one of the molecular markers for hepatic cancer initiating cells. However, the mechanism how the EpCAM expression in the liver cancer cell is regulated still remains to be delineated. In this study, we identified candidate genes critical for the maintenance of EpCAM positive hepatocellular carcinoma (HCC) cells using a global RNAi screening method. HCC cells were infected with pools of ~200,000 lentiviral shRNA library, then they were sorted to EpCAM positive or negative group. The abundance of shRNA in each group was analyzed using a microarray hybridization method. As a result, 76 candidate shRNAs with significantly different expression between EpCAM positive and negative populations were identified by class comparison analysis. Unsupervised clustering analysis of these genes in 247 HCC samples clearly separated EpCAM positive HCC cases from negative cases. Furthermore, we found that the EpCAM-dependent gene signature can predict survival in epithelial-like HCC cases. We focused on shRNA for PMPCB gene, which has an important role for processing mitochondrial protein, since it ranks as the top differentially altered shRNA-targeted gene between EpCAM positive and negative cells. PMPCB shRNA efficiently reduced EpCAM positive population and effectively inhibited cell proliferation both in vitro and in vivo. Interestingly, PMPCB shRNA specifically induced cell death in EpCAM positive HCC cells, but not in EpCAM negative cells. These data suggest that EpCAM positive cells functionally depend on PMPCB expression. On the other hand, we confirmed that PMPCB shRNA induced physiological dysregulation of mitochondrial function, leading to the increase of reactive oxygen species (ROS) production. We found that PMPCB shRNA significantly reduced TCF4 activity as well as the expression level of c-myc and cyclin D1, which are the downstream molecules of Wnt/β-catenin pathway. Although total β-catenin levels in the cytosol and nuclei fractions were not affected by PMPCB knockdown, overexpression of constitutively activated mutant β-catenin partially rescued the cells from PMPCB-induced apoptosis. Forkhead box O (FOXO) is known to be induced by the ROS production, bind β-catenin and decrease TCF activity, leading to the induction of apoptosis. We found that the nuclear translocation of FOXO family molecules, such as FOXO1 and FOXO3, were facilitated by PMPCB shRNA in EpCAM positive cells. These results suggest that the activation of FOXOs induced by PMPCB knockdown-mediated ROS production suppressed the activity of Wnt/β-catenin pathway, leading to the death of EpCAM positive HCC cells. In conclusion, we discovered a novel candidate target molecule of EpCAM positive HCC cells using a genome-wide RNAi screening method. Our study shed light on the mechanisms how EpCAM positive HCC cells are maintained and identified a new target molecule that has therapeutic vulnerability in EpCAM positive HCC cells. Citation Format: Atsushi Takai, Hien Dang, Naoki Oishi, Xin Wei Wang. Identification of novel cancer driver genes in hepatocellular carcinoma cells by RNAi screening method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2017-4391

InVivo RNAi Screen Unveils PPARγ as a Regulator of Hematopoietic Stem Cell Homeostasis.

SummaryHematopoietic stem cell (HSC) defects can cause repopulating impairment leading to hematologic diseases. To target HSC deficiency in a disease setting, we exploited the repopulating defect of Fanconi anemia (FA) HSCs to conduct an in vivo short hairpin RNA (shRNA) screen. We exposed Fancd2−/− HSCs to a lentiviral shRNA library targeting 947 genes. We found enrichment of shRNAs targeting genes involved in the PPARγ pathway that has not been linked to HSC homeostasis. PPARγ inhibition by shRNA or chemical compounds significantly improves the repopulating ability of Fancd2−/− HSCs. Conversely, activation of PPARγ in wild-type HSCs impaired hematopoietic repopulation. In mouse HSCs and patient-derived lymphoblasts, PPARγ activation is manifested in upregulating the p53 target p21. PPARγ and co-activators are upregulated in total bone marrow and stem/progenitor cells from FA patients. Collectively, this work illustrates the utility of RNAi technology coupled with HSC transplantation for the discovery of novel genes and pathways involved in stress hematopoiesis.

HNRNPLL, a newly identified colorectal cancer metastasis suppressor, modulates alternative splicing of CD44 during epithelial-mesenchymal transition

ObjectiveDespite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or...