Genome-Wide CRISPR-Cas9 Screen Identifies Sensitizers to LSD1 Inhibition in MLL-Translocated Human AML Cells

Abstract Lysine-specific demethylase (LSD1, also known as KDM1A) is an epigenetic regulator that has recently emerged as a potential therapeutic target in acute myeloid leukemia (AML). It is a flavin dependent monoamine oxidase which can demethylate monomethyl or dimethyl lysine 4 of histone H3. Pharmacological inhibition of LSD1 induces differentiation of blast cells in MLL-translocated AML and has shown significant promise in pre-clinical studies. With LSD1 inhibitors advancing through early-phase clinical trials, there is a strong pre-clinical rationale for the identification of genes whose protein products collaborate with LSD1 to retard differentiation in cancer and which could potentially be targeted in combination therapies for enhanced therapeutic benefit. To identify potential drug-gene synthetic lethal interactions, we performed a genome wide loss-of-function CRISPR-Cas9 screen in human THP1 AML cells treated with a potent and selective tranylcypromine-derivative LSD1 inhibitor trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine (OG86). THP1 cells exhibit a t(9;11) MLL gene rearrangement and display similar phenotypic and functional responses to those observed in primary MLL-translocated AML cells following LSD1 inhibition. The cells were transduced with lentiviral human CRISPR knockout (hGeCKOv2) library containing 122,411 sgRNAs targeting 19,050 protein coding genes and 1864 miRNA precursors in the human genome. The transduced cells were divided into two groups, treated with either DMSO or 250nM OG86 and maintained in culture for ~10 population doublings. To investigate sgRNA representation in the cell population harvested at different stages of the screen, sgRNA cassettes were PCR amplified from genomic DNA and deep sequenced; negatively selected genes were identified with the MAGeCK computational algorithm. After successful initial quality assessment of the screen, we next searched for genes selectively depleted in OG86-treated versus DMSO-treated THP1 cells in samples collected on Day 15 and Day 18. At a false discovery rate of 7.5% there were 10 expressed genes whose sgRNA representation was depleted at both time points. In particular, these included two genes coding for components of the MTOR signaling pathway: MTOR associated protein, LST8 homolog (MLST8) and Ras-related GTP-binding protein A (RRAGA). In the Day 18 comparison, an additional MTOR pathway gene LAMTOR2 scored among the ten most depleted. MLST8 is a core component of TORC1/TORC2 complex and RRAGA is involved in the activation of TORC1 by amino acids. Recruitment of both RAG proteins and TORC1 to lysosomal membranes in response to amino acids, and the consequent activation of TORC1 signalling, requires the trimeric Ragulator complex, of which LAMTOR2 is a member.Based on our screen, we hypothesized that THP1 AML cells exposed to pharmacologic inhibition of LSD1 exhibit increased sensitivity to concomitant inhibition of the amino acid sensing component of the TORC1 pathway. Using genetic knockdown and pharmacological inhibition strategies, we then validated our screen hits in combination with LSD1 inhibition in targeting human AML cells. RNAi based knockdown of RRAGA, LAMTOR2 and MLST8 in combination with LSD1 inhibition were found to promote myeloid differentiation and reduce cell proliferation in THP1 cells. Interestingly the mTORC1 pathway inhibitor everolimus (RAD001) showed at least additive effect in combination with OG86 to decrease THP1 cell proliferation and promote immunophenotypic differentiation. Comparison of transcription changes in combined versus single treatment conditions by RNA-seq analysis further confirmed a more extensive and wide-ranging upregulation of a myeloid differentiation program upon concomitant inhibition of LSD1 and mTORC1 pathway. In vitro studies performed in primary patient AML cells gave similar results. Finally, in vivo studies using AML patient-derived xenograft mouse model confirmed that combination treatment promotes a strong myeloid differentiation program. In conclusion, we report here that inhibition of mTORC1 sensitizes human MLL-translocated AML cells to LSD1 inhibitor-mediated differentiation therefore highlighting a novel combination approach for evaluation in clinical trials. Disclosures No relevant conflicts of interest to declare.

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DNA Methylation at ATP11A cg11702988 Is a Biomarker of Lung Disease Severity in Cystic Fibrosis: A Longitudinal Study

Genes ◽

10.3390/genes12030441 ◽

2021 ◽

Vol 12 (3) ◽

pp. 441

Author(s):

Fanny Pineau ◽

Davide Caimmi ◽

Sylvie Taviaux ◽

Maurane Reveil ◽

Laura Brosseau ◽

...

Keyword(s):

Cystic Fibrosis ◽

Dna Methylation ◽

Clinical Trials ◽

Lung Disease ◽

Disease Severity ◽

Genome Wide ◽

A Genome ◽

Lung Disease Severity ◽

Epigenetic Biomarker

Cystic fibrosis (CF) is a chronic genetic disease that mainly affects the respiratory and gastrointestinal systems. No curative treatments are available, but the follow-up in specialized centers has greatly improved the patient life expectancy. Robust biomarkers are required to monitor the disease, guide treatments, stratify patients, and provide outcome measures in clinical trials. In the present study, we outline a strategy to select putative DNA methylation biomarkers of lung disease severity in cystic fibrosis patients. In the discovery step, we selected seven potential biomarkers using a genome-wide DNA methylation dataset that we generated in nasal epithelial samples from the MethylCF cohort. In the replication step, we assessed the same biomarkers using sputum cell samples from the MethylBiomark cohort. Of interest, DNA methylation at the cg11702988 site (ATP11A gene) positively correlated with lung function and BMI, and negatively correlated with lung disease severity, P. aeruginosa chronic infection, and the number of exacerbations. These results were replicated in prospective sputum samples collected at four time points within an 18-month period and longitudinally. To conclude, (i) we identified a DNA methylation biomarker that correlates with CF severity, (ii) we provided a method to easily assess this biomarker, and (iii) we carried out the first longitudinal analysis of DNA methylation in CF patients. This new epigenetic biomarker could be used to stratify CF patients in clinical trials.

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A Genome-Wide Analysis of Variants Influencing Methotrexate Clearance Replicates SLCO1B1.

Blood ◽

10.1182/blood.v120.21.2466.2466 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2466-2466 ◽

Cited By ~ 1

Author(s):

Laura B. Ramsey ◽

John C Panetta ◽

Colton Smith ◽

Wenjian Yang ◽

Yiping Fan ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Organic Anion ◽

Organic Anion Transporter ◽

High Dose ◽

Genome Wide Analysis ◽

Anion Transporter ◽

Genome Wide ◽

A Genome

Abstract Abstract 2466 High-dose methotrexate (HDMTX) is an important element of chemotherapy for acute lymphoblastic leukemia (ALL) and other malignancies. Methotrexate clearance influences cure and toxicity in children with acute lymphoblastic leukemia (ALL). HDMTX schedules and doses vary widely among treatment protocols. The Children's Oncology Group (COG) tested the efficacy of 6 courses of 2 g/m2 over 4 hours versus 1 g/m2 over 24 hours (P9904 and P9905 protocols). Patients were assigned to one of four arms for consolidation: A, 24-hour methotrexate infusion (1 g/m2) and no delayed intensification (DI); B, 4-hour methotrexate infusion (2 g/m2) with no DI; C, 24-hour methotrexate infusion with DI; D, 4-hour methotrexate infusion with DI. We estimated methotrexate clearance for 1279 patients treated on these protocols, with two plasma MTX concentrations per course, using a Bayesian pharmacokinetic modeling approach. Germline genetic variation was assessed using the Affymetrix 6.0 array, and other single nucleotide polymorphisms (SNPs) were imputed based on 1000 Genomes reference data, yielding 5.2 million SNP genotypes evaluable per patient. Average MTX clearance was highly variable, with a median (range) of 164 (65–355) and 109 (49–290) ml/min/m2 for the 24-hour and 4-hour infusions, respectively. Methotrexate clearance was lower in older children (p = 7 × 10−7), girls (p = 2.7 × 10−4), and patients who received a delayed intensification phase during consolidation (p = 0.0022). Adjusting for age, gender, race, and treatment arm, a genome-wide analysis showed that methotrexate clearance was associated with polymorphisms in SLCO1B1(p = 2.1 × 10−11), a gene that encodes for an organic anion transporter that is known to transport methotrexate. This replicates our previous findings (Trevino et al, J Clin Oncol. 2009;27(35):5972-8) that polymorphisms in SLCO1B1 influence methotrexate clearance in ALL patients treated on St. Jude protocols with three different HDMTX schedules. In a combined meta-analysis including the 1279 COG patients and 699 St. Jude patients, and adjusting for age, gender, race, and treatment arm, the association of methotrexate clearance with SLCO1B1 SNP rs4149056 yields a p-value of 3.1 × 10−19 (Figure). Even after adjustment for the rs4149056 SNP, other polymorphisms in SLCO1B1 remained significantly related to methotrexate clearance, indicating that there are multiple variants in SLCO1B1 that can influence methotrexate clearance. Validation of the association of this gene with five different treatment regimens of methotrexate solidifies the robustness of this pharmacogenomic determinant of methotrexate clearance. Disclosures: No relevant conflicts of interest to declare.

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SHQ1 Regulates MYC mRNA Splicing and Promotes T Cell Leukemogenesis

Blood ◽

10.1182/blood.v128.22.440.440 ◽

2016 ◽

Vol 128 (22) ◽

pp. 440-440

Author(s):

Hexiu Su ◽

Yufeng Hu ◽

Jue Jiang ◽

Zhichao Chen ◽

Hudan Liu

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Functional Role ◽

Conflicts Of Interest ◽

Mrna Splicing ◽

Transcriptional Level ◽

Normal Peripheral Blood ◽

Genome Wide ◽

T Cell Leukemogenesis

Abstract T-acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors resulting from the malignant transformation of T cell progenitors. In this context, constitutive activation of NOTCH1 signaling is the most prominent oncogenic pathway in T cell transformation. Yet functional role(s) of NOTCH1 in T-ALL pathogenesis and precise mechanism(s) of action remain to be fully determined. SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins which are required for spliceosomal small nuclear RNA (snRNA) maturation. We here identify SHQ1 as a NOTCH1 downstream target that plays a pivotal role in maintaining MYC splicing fidelity and promoting T-ALL cell proliferation in vitro and in vivo. We identified SHQ1 as a NOTCH1-regulated gene from multiple human T-ALL genome-wide expression studies. To validate this finding, we analyzed SHQ1 expression in a spectrum of human T-ALL cells upon NOTCH1 pathway inhibition. NOTCH1 inactivation caused a marked downregulation of SHQ1 in all T-ALL cell lines tested. We further demonstrated NOTCH1 bound to the canonical CSL binding sites in the SHQ1 promoter and directly activated the transcription. We next systematically analyzed the SHQ1 expression in 174 T-ALL primary samples and found that SHQ1 expression was significantly elevated in T-ALL compared with normal peripheral blood. To explore the functional role of SHQ1, we knocked it down in T-ALL using specific shRNAs. SHQ1 depletion profoundly inhibited T-ALL cell proliferation and induced massive apoptotic cell death. Consistent with these in vitro findings, SHQ1 depletion in a human T-ALL xenograft significantly delayed leukemia onset and prolonged survival. To decipher the molecular mechanism whereby SHQ1 contributes to T cell leukemogenesis, we performed genome-wide RNA-Seq and analyzed alternative splicing across the genome. Approximately 70% of genes exhibited abnormal intron retention upon SHQ1 depletion. Among those whose expression levels and mRNA splicing were prominently altered by SHQ1, MYC gained our attention because of its vital role in T-ALL. Depletion of SHQ1 resulted in marked downregulation of mature MYC mRNA and protein. Endogenous mRNA analysis and mini-gene experiments showed aberrant accumulation of MYC pre-mRNA in SHQ1-depleted cells. Expectedly, SHQ1 knockdown had minimal effects on T-ALL cells constitutively expressing a human MYC protein. In addition to the well-documented transcriptional control of MYC by NOTCH1, we herein report a previously unsuspected mechanism in which NOTCH1 modulates MYC splicing by activation of SHQ1. Our findings not only shed new insights in the molecular pathology of NOTCH1-induced T-ALL but also provide a new layer of regulation of oncogene MYC at the post-transcriptional level, mechanism of which may apply to other MYC involved tumors. Disclosures No relevant conflicts of interest to declare.

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SLC19A1 is an importer of the immunotransmitter cGAMP

10.1101/539247 ◽

2019 ◽

Cited By ~ 2

Author(s):

Anthony F. Cordova ◽

Christopher Ritchie ◽

Gaelen T. Hess ◽

Michael C. Bassik ◽

Lingyin Li

Keyword(s):

Immune Response ◽

Clinical Trials ◽

Cell Membrane ◽

Cancer Therapeutics ◽

Host Cells ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Sting Pathway ◽

Insight Into

Abstract2’3’-cyclic-GMP-AMP (cGAMP) is a second messenger that activates the antiviral Stimulator of Interferon Genes (STING) pathway. We recently identified a novel role for cGAMP as a soluble, extracellular immunotransmitter that is produced and secreted by cancer cells. Secreted cGAMP is then sensed by host cells, eliciting an antitumoral immune response. Due to the antitumoral effects of cGAMP, other CDN-based STING agonists are currently under investigation in clinical trials for metastatic solid tumors. However, it is unknown how cGAMP and other CDNs cross the cell membrane to activate intracellular STING. Using a genome-wide CRISPR screen we identified SLC19A1 as the first known importer of cGAMP and other CDNs, including the investigational new drug 2′3′-bisphosphosphothioate-cyclic-di-AMP (2′3′-CDAS). These discoveries will provide insight into cGAMP’s role as an immunotransmitter and aid in the development of more targeted CDN-based cancer therapeutics.

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How to make a rodent giant: Genomic basis and tradeoffs of gigantism in the capybara, the world’s largest rodent

10.1101/424606 ◽

2018 ◽

Cited By ~ 6

Author(s):

Santiago Herrera-Álvarez ◽

Elinor Karlsson ◽

Oliver A. Ryder ◽

Kerstin Lindblad-Toh ◽

Andrew J. Crawford

Keyword(s):

Cell Proliferation ◽

Body Size ◽

T Cell ◽

Growth Regulation ◽

Large Body ◽

Mutation Load ◽

Large Body Size ◽

Genome Wide ◽

A Genome ◽

Cancer Suppression

AbstractGigantism is the result of one lineage within a clade evolving extremely large body size relative to its small-bodied ancestors, a phenomenon observed numerous times in animals. Theory predicts that the evolution of giants should be constrained by two tradeoffs. First, because body size is negatively correlated with population size, purifying selection is expected to be less efficient in species of large body size, leading to a genome-wide elevation of the ratio of non-synonymous to synonymous substitution rates (dN/dS) or mutation load. Second, gigantism is achieved through higher number of cells and higher rates of cell proliferation, thus increasing the likelihood of cancer. However, the incidence of cancer in gigantic animals is lower than the theoretical expectation, a phenomenon referred to as Peto’s Paradox. To explore the genetic basis of gigantism in rodents and uncover genomic signatures of gigantism-related tradeoffs, we sequenced the genome of the capybara, the world’s largest living rodent. We found that dN/dS is elevated genome wide in the capybara, relative to other rodents, implying a higher mutation load. Conversely, a genome-wide scan for adaptive protein evolution in the capybara highlighted several genes involved in growth regulation by the insulin/insulin-like growth factor signaling (IIS) pathway. Capybara-specific gene-family expansions included a putative novel anticancer adaptation that involves T cell-mediated tumor suppression, offering a potential resolution to Peto’s Paradox in this lineage. Gene interaction network analyses also revealed that size regulators function simultaneously as growth factors and oncogenes, creating an evolutionary conflict. Based on our findings, we hypothesize that gigantism in the capybara likely involved three evolutionary steps: 1) Increase in body size by cell proliferation through the ISS pathway, 2) coupled evolution of growth-regulatory and cancer-suppression mechanisms, possibly driven by intragenomic conflict, and 3) establishment of the T cell-mediated tumor suppression pathway as an anticancer adaptation. Interestingly, increased mutation load appears to be an inevitable outcome of an increase in body size.Author SummaryThe existence of gigantic animals presents an evolutionary puzzle. Larger animals have more cells and undergo exponentially more cell divisions, thus, they should have enormous rates of cancer. Moreover, large animals also have smaller populations making them vulnerable to extinction. So, how do gigantic animals such as elephants and blue whales protect themselves from cancer, and what are the consequences of evolving a large size on the ‘genetic health’ of a species? To address these questions we sequenced the genome of the capybara, the world’s largest rodent, and performed comparative genomic analyses to identify the genes and pathways involved in growth regulation and cancer suppression. We found that the insulin-signaling pathway was involved in the evolution of gigantism in the capybara. We also found a putative novel anticancer mechanism mediated by the detection of tumors by T-cells, offering a potential solution to how capybaras mitigated the tradeoff imposed by cancer. Furthermore, we show that capybara genome harbors a higher proportion of slightly deleterious mutations relative to all other rodent genomes. Overall, this study provides insights at the genomic level into the evolution of a complex and extreme phenotype, and offers a detailed picture of how the evolution of a giant body size in the capybara has shaped its genome.

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Genome Wide Profiling of USF1 and USF2 Occupancy in Human Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1456.1456 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1456-1456

Author(s):

Laurie A Steiner ◽

Vincent Schulz ◽

Yelena Maksimova ◽

David Tuck ◽

Patrick G. Gallagher

Keyword(s):

Mrna Expression ◽

Binding Sites ◽

Conflicts Of Interest ◽

Beta Globin ◽

Erythroid Cells ◽

Genome Wide ◽

A Genome ◽

E Box ◽

Globin Promoter

Abstract Abstract 1456 Poster Board I-479 USF1 and USF2 are ubiquitously expressed basic helix-loop-helix leucine zipper proteins that participate in a large number of biologic processes. USF1 and USF2 bind DNA as homodimers or heterodimers, typically binding E box consensus motifs. One role of USF proteins is functioning as transcription factors. Although ubiquitously expressed, they regulate expression of many cell-type and developmental-stage specific genes, such as hepcidin in hepatocytes and surfactant protein A in fetal lung cells. Another role of USF proteins is in the maintenance of chromatin architecture in barrier insulator elements, such as the well characterized 5'HS4 insulator element in the chicken beta-globin locus. In mammalian erythroid cells, USF1 and USF2 participate in the regulation of beta-globin transcription, interacting both at hypersensitive site 2 (HS2) of the beta-globin locus control region (LCR) and at the beta-globin promoter. Depletion of USF proteins leads to decreased beta-globin production. We hypothesize that in addition to beta-globin, USF proteins are important for regulation of many erythroid expressed genes. To address this hypothesis, chromatin immunoprecipitation with antibodies against USF1 and USF2 was coupled with ultra high throughput, massively parallel sequencing (Illumina Solexa sequencing, ChIP-seq) to generate a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells. To generate cells for ChIP and mRNA expression profiling, human CD34+ cells isolated from peripheral blood were cultured in serum free media with erythropoietin to induce erythroid differentiation. After 14 days in culture, FACS analysis was used to confirm cells were positive for both CD 71 and glycophorin A (the R3/R4 stage of erythroid development). mRNA transcript analyses were performed using Illumina human V6-2 expression arrays and quantitative real time RT-PCR. ChIP-seq experiments for USF1 and USF2 were done in duplicate and only binding sites present in both ChIP-seq replicates were included in data analyses. A total of 20450 USF1 and 21128 USF2 sites of occupancy were identified. Co-localization of USF1 and USF2 was common, with 16739 sites binding both USF1 and USF2 (81.9% of USF1 sites and 79.2% of USF2 sites). In an analysis of a subset of erythroid expressed focus genes, USF binding was associated with active transcription. In agreement with previous studies, there was binding of USF proteins in the beta-globin LCR, and beta-globin promoter. USF binding most commonly occurred close to annotated genes, with 48.5% of USF1 sites, 44.6% of USF 2 sites and 53.0% of sites of USF1-USF2 co-localization located within 1 kb of a transcription start site (TSS), supporting the role of USF proteins as a transcription factor in these locations. A small, but significant, number of USF binding sites were located in intergenic regions > 100 kb from any annotated TSS. (1206 USF1, 1408 USF2, and 776 USF1-USF2). Interestingly, at sites of intergenic binding, USF1 and USF2 were much less likely to co-localize, (64% of USF1 and 55% of USF2 sites), implying that the USF proteins serve a different function at these remote binding sites than at sites of binding in close proximity to a TSS. USF proteins can bind DNA in an E-box dependent or independent manner. The Weeder Algorithm (Pavesi, Bioinformatics, 2001) was used to determine the most common binding motifs for USF1 and USF2. Over-represented motifs at sites of USF1 and USF2 binding were similar, with the most common sequences being a canonical E-box, CACGTG, as well as the related sequences ACGTGA and TCACGT. This genome-wide map of USF binding correlated with mRNA expression data indicates that USF proteins serve several different, important functions throughout the human genome and support the hypothesis that USF proteins participate in the regulation of many erythroid-expressed genes. Disclosures No relevant conflicts of interest to declare.

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A Genome Wide shRNA Screen In Primary Human AML Cells Identifies ROCK1 As a Novel Therapeutic Target

Blood ◽

10.1182/blood.v122.21.170.170 ◽

2013 ◽

Vol 122 (21) ◽

pp. 170-170

Author(s):

Martin Wermke ◽

Aylin Camgoz ◽

Maciej Paszkowski-Rogacz ◽

Sebastian Thieme ◽

Malte von Bonin ◽

...

Keyword(s):

Conflicts Of Interest ◽

Therapeutic Targets ◽

Host Cells ◽

Leukemic Cells ◽

Therapeutic Implications ◽

Genome Wide ◽

A Genome ◽

Shrna Library ◽

Shrna Screen ◽

Novel Therapeutic

Abstract In spite of recent advances, the prognosis especially of elderly AML patients remains unsatisfactory with survival rates of less than 10 % at 10 years. Genome-wide RNA-interference screens systematically interrogating the specific vulnerabilities of leukemic cells could be a valuable tool to identify novel therapeutic targets in this patient population. So far, such screens have only been done in immortalized cell lines and / or at sub-genome scale, which limits their transferability to individual patients. Therefore, we set out to establish an unbiased genome-wide pooled shRNA screen in primary human AML cells to prove the feasibility and test the possible clinical implications of such an approach. Lentiviral transduction of primary leukemic blasts from a 67-year old patient with AML FAB M1 with a pooled shRNA library (Mission TRC shRNA library SP1, Sigma) according to a specifically optimized protocol resulted in a transduction rate of 25 %, thus rendering multiple integrants unlikely. An aliquot of the cells was separated for DNA extraction directly after removal of viral supernatant (day 0) and after 9 days of suspension culture (day 9). ShRNA barcodes integrated into the genome of the host cells were read out using PCR-coupled next-generation sequencing (HiSeq 2000, Illumina). Of 7709 shRNA contained in the library, 6626 were recovered with at least 10 reads in the day 0 sample. After 9 days of culture, 25 shRNA targeting a total of 12 genes were identified as potentially lethal to the patient's AML-cells (Table 1). All of these shRNA were subjected to single-shRNA transduction experiments using leukemic cells from the same donor. In fact, 18 of 25 shRNA were validated with respect to viability. Knockdown specificity was documented for all validated shRNA by qPCR. For further analyses we focused only on those 7 genes in which more than 50% of the shRNA identified in the pooled screen could be validated (Table 1). These genes were assessed for druggability using publicly available databases. For exploration of the potential therapeutic implications of our screen we chose ROCK1 as a potential target, because Fasudil, a specific ROCK1 inhibitor, has already been licensed for the treatment of pulmonary hypertension in humans.Table.Table1No. shRNAs >100 reads day 0No. scoring shRNA in pooled screenNo. valdiated shRNA in single shRNA experimentsOverall gene validation statusBNIPL322ValidatedC7orf16322ValidatedCCRL1321Not validatedDGAT2321Not validatedDUSP14320Not validatedMAP3K6422ValidatedROCK1532ValidatedRPS13322ValidatedSF3A1321Not validatedSNX27422ValidatedSTK3422ValidatedWDHD1220Not validated Knockdown of ROCK1 in primary leukemic blasts led to rapid cell-cycle arrest and cell-death. Treatment with Fasudil proved to be equally effective in killing leukemic cells. Compared to primary leukemic cells from the original as well as from other AML patients, Fasudil seemed to be less toxic to hematopoietic cells derived from healthy volunteer donors. RNA-sequencing revealed that in comparison to the healthy controls none of the studied AML patients demonstrated a significant overexpression of ROCK1. Moreover there was no indication for a functional ROCK1 mutation in the analyzed AML samples. Feeder based long-term culture initiating cell (LTC-IC) assays further suggested that Fasudil had a significant negative effect on the self-renewal capacity of primary human leukemic stem/progenitor cells (Figure 1). Studies in xenograft-models to assess the stem cell toxicity of ROCK1 inhibition in more detail are currently ongoing.Figure.Figure. Taken together our results show that pooled shRNA screens in primary patient-derived leukemic cells are feasible and able to pinpoint novel therapeutic targets, which might be missed in mutation- or overexpression-based approaches. Further optimization of transduction and screening protocols might enable such screens to assist physicians in the selection of optimal therapeutic strategies especially in poor risk AML. Disclosures: No relevant conflicts of interest to declare.

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Cognitive Decline in Alzheimer’s Disease: Limited Clinical Utility for GWAS or Polygenic Risk Scores in a Clinical Trial Setting

Genes ◽

10.3390/genes11050501 ◽

2020 ◽

Vol 11 (5) ◽

pp. 501

Author(s):

Jack Euesden ◽

Sivakumar Gowrisankar ◽

Angela Xiaoyan Qu ◽

Pamela St. Jean ◽

Arlene R. Hughes ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Clinical Trials ◽

Cognitive Decline ◽

Clinical Utility ◽

Phase Iii ◽

Polygenic Risk Score ◽

Polygenic Risk ◽

Genome Wide ◽

A Genome

Introduction: Alzheimer’s disease (AD) is a progressive and irreversible neurological disease. The genetics and molecular mechanisms underpinning differential cognitive decline in AD are not well understood; the genetics of AD risk have been studied far more assiduously. Materials and Methods: Two phase III clinical trials measuring cognitive decline over 48 weeks using Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog, n = 2060) and Clinical Dementia Rating-Sum of Boxes (CDR-SB, n = 1996) were retrospectively genotyped. A Genome-Wide Association Study (GWAS) was performed to identify and replicate genetic variants associated with cognitive decline. The relationship between polygenic risk score (PRS) and cognitive decline was tested to investigate the predictive power of aggregating many variants of individually small effect. Results: No loci met candidate gene or genome-wide significance. PRS explained a very small percentage of variance in rates of cognitive decline (ADAS-cog: 0.54%). Conclusions: These results suggest that incorporating genetic information in the prediction of cognitive decline in AD currently appears to have limited utility in clinical trials, consistent with small effect sizes estimated elsewhere. If AD progression is more heritable soon after disease onset, genetics may have more clinical utility.

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Genome-wide association studies detects candidate genes for wool traits by re-sequencing in Chinese fine-wool sheep

BMC Genomics ◽

10.1186/s12864-021-07399-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Hongchang Zhao ◽

Tingting Guo ◽

Zengkui Lu ◽

Jianbin Liu ◽

Shaohua Zhu ◽

...

Keyword(s):

Cell Proliferation ◽

Candidate Genes ◽

Fiber Diameter ◽

Significant Snps ◽

Genome Wide Association ◽

Genome Wide Association Studies ◽

Merino Sheep ◽

Genome Wide ◽

A Genome ◽

Staple Length

Abstract Background The quality and yield of wool determine the economic value of the fine-wool sheep. Therefore, discovering markers or genes relevant to wool traits is the cornerstone for the breeding of fine-wool sheep. In this study, we used the Illumina HiSeq X Ten platform to re-sequence 460 sheep belonging to four different fine-wool sheep breeds, namely, Alpine Merino sheep (AMS), Chinese Merino sheep (CMS), Aohan fine-wool sheep (AHS) and Qinghai fine-wool sheep (QHS). Eight wool traits, including fiber diameter (FD), fiber diameter coefficient of variance (FDCV), fiber diameter standard deviation (FDSD), staple length (SL), greasy fleece weight (GFW), clean wool rate (CWR), staple strength (SS) and staple elongation (SE) were examined. A genome-wide association study (GWAS) was performed to detect the candidate genes for the eight wool traits. Results A total of 8.222 Tb of raw data was generated, with an average of approximately 8.59X sequencing depth. After quality control, 12,561,225 SNPs were available for analysis. And a total of 57 genome-wide significant SNPs and 30 candidate genes were detected for the desired wool traits. Among them, 7 SNPs and 6 genes are related to wool fineness indicators (FD, FDCV and FDSD), 10 SNPs and 7 genes are related to staple length, 13 SNPs and 7 genes are related to wool production indicators (GFW and CWR), 27 SNPs and 10 genes associated with staple elongation. Among these candidate genes, UBE2E3 and RHPN2 associated with fiber diameter, were found to play an important role in keratinocyte differentiation and cell proliferation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results, revealed that multitude significant pathways are related to keratin and cell proliferation and differentiation, such as positive regulation of canonical Wnt signaling pathway (GO:0090263). Conclusion This is the first GWAS on the wool traits by using re-sequencing data in Chinese fine-wool sheep. The newly detected significant SNPs in this study can be used in genome-selective breeding for the fine-wool sheep. And the new candidate genes would provide a good theoretical basis for the fine-wool sheep breeding.

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Identification of Genetic Vulnerabilities and Synthetic-Lethal Targets in NSD2-High Multiple Myeloma

Blood ◽

10.1182/blood-2019-125951 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3757-3757

Author(s):

Amin Sobh ◽

Charlotte Kaestner ◽

Alberto Riva ◽

Jonathan D. Licht

Keyword(s):

Multiple Myeloma ◽

Rna Binding ◽

Conflicts Of Interest ◽

Chemotherapeutic Agents ◽

Nucleotide Metabolism ◽

Loss Of Function ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Dexamethasone Resistance

The histone methyltransferase NSD2 is overexpressed in 15-20% of multiple myeloma (MM) patients due to the t(4;14) chromosomal translocation. NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. Despite strong efforts by academia and industry, there remains no tool compound or drug that inhibits NSD2 enzymatic activity. Therefore we explored the molecular and biochemical consequences of NSD2 overexpression in MM cells and investigated genetic vulnerabilities associated with high NSD2 levels as well as synthetic-lethal drug-gene interactions in t(4;14)-positive MM cells. We utilized the well-studied isogenic pair of human MM cells derived from the t(4;14)-positive KMS-11 cell line, where NSD2 is disrupted by knocking out either the translocated overexpressed allele (TKO; NSD2-low) or the wild-type non-translocated allele (NTKO; NSD2-high). Untargeted metabolomic profiling of TKO and NTKO cells revealed that NSD2 overexpression substantially alters nucleotide metabolism. NSD2-high cells exhibited an increase in purine synthesis and a decrease in pyrimidine synthesis. In addition, a genome-wide loss-of-function CRISPR gene editing screen using the 76,000 guide Brunello library uncovered genes differentially essential between NSD2-high and low MM cells. Intriguingly, one of the genes whose disruption is selectively lethal in NSD2-high cells encodes Adenylate Kinase 2 (AK2), an enzyme involved in purine metabolism, indicating that defective nucleotide metabolism associated with increased NSD2 expression introduces genetic vulnerabilities that can be therapeutically exploited. We further explored liabilities that can improve therapeutic outcomes in t(4;14) MM. A genome-wide CRISPR screen was performed in KMS-11 MM cells to identify mechanisms of sensitivity and resistance to dexamethasone, a steroid commonly used in MM treatment. As expected, inactivating the gene encoding the glucocorticoid receptor (GR) resulted in remarkable dexamethasone tolerance. We then identified and validated multiple genes/pathways that can alter response of MM cells to dexamethasone when disrupted. For example, inactivation of interleukin 10 (IL10) signaling by disrupting either subunit of the IL10 receptor or components of the downstream JAK/STAT pathway considerably enhances dexamethasone sensitivity. In addition, disruption of many components of heparan sulfate or glycosaminoglycans synthesis pathways, whose targeting has been previously shown to increase sensitivity to conventional MM chemotherapeutic agents, increases susceptibility to dexamethasone. By contrast, genetic perturbations leading to dexamethasone resistance correspond to GR co-chaperones including FKBP4 and PTGES3 and transcriptional coactivators like the nuclear receptor transactivator 1 (NCOA1). Interestingly, disruption of genes encoding proteins implicated in RNA stability and translation such as the N6-methyladenosine (m6A)-containing RNA binding protein YTHDF2 and the PAN2-PAN3 deadenylase complex results in dexamethasone resistance. How these proteins affect the expression of pro or anti-apoptotic genes in response to dexamethasone is under investigation. Our work reveals insight into novel molecular-based treatment options for t(4;14) MM that are independent on direct NSD2 inhibition which remains unsuccessful. Disclosures No relevant conflicts of interest to declare.

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