Confer Growth Advantage Research Articles

Genetic and Transcriptomic Characterization of Marrow Progenitor Cells in Patients with Chronic Lymphocytic Leukemia

Tumor formation is the result of a stepwise process through which cells acquire genetic alterations that may confer growth advantages to enable clonal expansion. One model in leukemia is that mutations accumulate in normal hematopoietic stem cells (HSC) leading to pre-leukemic cells which carry some, but not all, leukemia-specific mutations. Chronic lymphocytic leukemia (CLL) has been traditionally viewed as a mature B cell malignancy, however, recent studies have identified leukemia-specific mutations in CD34+ cells from either peripheral blood or bone marrow. These data raise the question of whether marrow progenitor cells in CLL are dysregulated even before commitment to the B cell lineage and, if so, what are the characteristics of such cells in CLL.To this end, we first quantified the absolute number of CD34+ cells from marrow collected from CLL patients (n=9) vs healthy adult volunteers (n=8). Using a florescence bead-based absolute quantification assay (BD Trucount), we detected a significant reduction of CD34+ progenitor cells in CLL compared to normal donors (Average: 55.6 vs 391/ml, p=0.002, Welch's t -test). To determine if this quantitative change was accompanied by changes in the cellular state of these cells, we performed low-input RNA sequencing (RNA-seq) from 7 marrow progenitor subpopulations derived from 5 CLL patients and 4 healthy donors, that were isolated on the basis of flow cytometric sorting. These subpopulations included HSC (Lin-CD34+CD38-CD90+CD45RA-), MPP (Lin-CD34+CD38+CD90-CD45RA-), MLP (Lin-CD34+CD38-CD90-CD45RA+), CMP (Lin-CD34+CD38+CD90-CD135+CD45RA-), MEP (Lin-CD34+CD38+CD90-CD135-CD45RA+), GMP (Lin-CD34+CD38+CD90-CD135+/-CD45RA+), and CLP (Lin-CD34+CD38+CD10+CD45RA+). This systematic analysis revealed both a CLL-specific transcriptome profile as well as unique expression signatures for each progenitor subpopulation. In total, 119 genes were identified as CLL progenitor cell-specific and were highly enriched for the regulation of mRNA catabolic processes and protein translation.To determine if leukemia-specific mutations are present in these progenitor cells and may contribute to dysregulation, we performed targeted deep-sequencing with low-input DNA derived from these subpopulations. We focused on marrow cells collected from 3 CLL patients, whose matched peripheral blood leukemia cells had previously undergone whole-exome sequencing (WES). Of the 30 somatic mutations identified by WES in CLL cells from Patient 1, we detected presence of 4 and 26 mutations in HSC and Pro-B/Pre-B cells, respectively. Likewise, we detected 4 of 34 CLL-specific mutations in HSC cells for Patient 2. In contrast, none of 10 mutations detected in Patient 3's CLL cells by WES were evident in the HSC subpopulation, but 9 of 10 mutations could be identified at the Pro-B/Pre-B stage. None of the 74 mutations from CLL cells from Patients 1-3 were detected in DNA extracted from marrow cells collected from 3 normal healthy donors.To confirm the presence of these somatic mutations in CLL progenitor cells, we utilized a microfluidics-based approach to perform multiplexed simultaneous targeted detection of up to 48 mutations and expression of up to 96 genes from cDNA of individual cells. We constructed panels of 96 genes based on the aforementioned RNA-seq data to confirm both identity of cellular differentiation stage and cellular source (normal vs CLL). We thus tested whether the single-cell marrow progenitor cells from CLL patients harbored leukemia-specific mutation while expressing progenitor cell genes. With this method, we validated the expression of 2 mutations in bona fide HSC at the single-cell level in Patient 1. Further validation studies of somatic mutation detection using low-input DNA and single-cell approach are ongoing.In summary, we present evidence of pre-leukemic cells at the HSC stage and these cells have increasing similarity to the cognate CLL at the pro/preB stage. Our DNA and single-cell analysis highlight the existence of CLL progenitor cells harboring leukemia-specific mutations while transcriptome profiling identified a CLL-specific gene signature associated with these pre-leukemic cells. We anticipate that further systematic analysis linking mutation, epigenetic changes, and dysregulated gene expression will enhance our understanding of the stepwise transformation and pathogenesis of this disease. DisclosuresNeuberg:Synta Pharmaceuticals: Other: Stock shares. Wiestner:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding. Brown:Janssen: Consultancy; Janssen Oncology: Honoraria; Redx: Consultancy; Gilead: Consultancy, Research Funding; Celgene: Consultancy; Infinity Pharmaceuticals: Consultancy; Roche/Genentech: Consultancy; Astellas Pharma: Consultancy; Pfizer: Consultancy; Sun BioPharma: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria; Pharmacyclics: Consultancy; AstraZeneca: Consultancy. Wu:Neon Therapeutics: Consultancy.

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris.

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

Deregulation of Polycomb Repressive Complex-2 in Mantle Cell Lymphoma Confers Growth Advantage by Epigenetic Suppression of cdkn2b.

The polycomb repressive complex 2 (PRC2) maintains the transcriptional repression of target genes through its catalytic component enhancer of zeste homolog 2 (EZH2). Through modulating critical gene expression, EZH2 also plays a role in cancer development and progression by promoting cancer cell survival and invasion. Mutations in EZH2 are prevalent in certain B-cell lymphoma subtypes such as diffuse large cell lymphoma and follicular lymphoma; while no EZH2 mutation has been reported in the mantle cell lymphoma (MCL). Here we demonstrate that the PRC2 components EZH2, EED and SUZ12 are upregulated in the MCL cells as compared to normal B-cells. Moreover, stably transfected cells with wild-type EZH2 or-EED showed increased cell growth and H3K27-trimehtylation. However, unlike wild-type EZH2, ectopic expression of a deletion construct of EZH2 (EZH2Δ550−738 lacking SET domain) had no growth advantage over control cells. Pharmacological inhibition of EZH2 suppressed H3K27me3 and had significant inhibitory effect on cell growth and colony forming capacity (p < 0.05) of MCL cells, and this effect was more or less comparable to the anti-proliferative effects of EZH2 inhibition in cells harboring EZH2-mutation. Mechanistically, EZH2 appears to downregulate expression of cdkn2b gene via enhanced H3K27me3, a well-known suppressive epigenetic mark, at the cdkn2b promoter region. Overall, these results highlight that deregulation of PRC2/EZH2 is associated with epigenetic suppression of cdkn2b in MCL, and in part responsible for increased cell growth, thus the EZH2 inhibitors may have therapeutic potential in the patients with MCL.

The Chromatin Regulator ZMYM2 Restricts Human Pluripotent Stem Cell Growth and Is Essential for Teratoma Formation.

SummaryChromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.

Network-based approaches elucidate differences within APOBEC and clock-like signatures in breast cancer

BackgroundStudies of cancer mutations have typically focused on identifying cancer driving mutations that confer growth advantage to cancer cells. However, cancer genomes accumulate a large number of passenger somatic mutations resulting from various endogenous and exogenous causes, including normal DNA damage and repair processes or cancer-related aberrations of DNA maintenance machinery as well as mutations triggered by carcinogenic exposures. Different mutagenic processes often produce characteristic mutational patterns called mutational signatures. Identifying mutagenic processes underlying mutational signatures shaping a cancer genome is an important step towards understanding tumorigenesis.MethodsTo investigate the genetic aberrations associated with mutational signatures, we took a network-based approach considering mutational signatures as cancer phenotypes. Specifically, our analysis aims to answer the following two complementary questions: (i) what are functional pathways whose gene expression activities correlate with the strengths of mutational signatures, and (ii) are there pathways whose genetic alterations might have led to specific mutational signatures? To identify mutated pathways, we adopted a recently developed optimization method based on integer linear programming.ResultsAnalyzing a breast cancer dataset, we identified pathways associated with mutational signatures on both expression and mutation levels. Our analysis captured important differences in the etiology of the APOBEC-related signatures and the two clock-like signatures. In particular, it revealed that clustered and dispersed APOBEC mutations may be caused by different mutagenic processes. In addition, our analysis elucidated differences between two age-related signatures—one of the signatures is correlated with the expression of cell cycle genes while the other has no such correlation but shows patterns consistent with the exposure to environmental/external processes.ConclusionsThis work investigated, for the first time, a network-level association of mutational signatures and dysregulated pathways. The identified pathways and subnetworks provide novel insights into mutagenic processes that the cancer genomes might have undergone and important clues for developing personalized drug therapies.

QuaDMutNetEx: a method for detecting cancer driver genes with low mutation frequency

BackgroundCancer is caused by genetic mutations, but not all somatic mutations in human DNA drive the emergence or growth of cancers. While many frequently-mutated cancer driver genes have already been identified and are being utilized for diagnostic, prognostic, or therapeutic purposes, identifying driver genes that harbor mutations occurring with low frequency in human cancers is an ongoing endeavor. Typically, mutations that do not confer growth advantage to tumors – passenger mutations – dominate the mutation landscape of tumor cell genome, making identification of low-frequency driver mutations a challenge. The leading approach for discovering new putative driver genes involves analyzing patterns of mutations in large cohorts of patients and using statistical methods to discriminate driver from passenger mutations.ResultsWe propose a novel cancer driver gene detection method, QuaDMutNetEx. QuaDMutNetEx discovers cancer drivers with low mutation frequency by giving preference to genes encoding proteins that are connected in human protein-protein interaction networks, and that at the same time show low deviation from the mutual exclusivity pattern that characterizes driver mutations occurring in the same pathway or functional gene group across a cohort of cancer samples.ConclusionsEvaluation of QuaDMutNetEx on four different tumor sample datasets show that the proposed method finds biologically-connected sets of low-frequency driver genes, including many genes that are not found if the network connectivity information is not considered. Improved quality and interpretability of the discovered putative driver gene sets compared to existing methods shows that QuaDMutNetEx is a valuable new tool for detecting driver genes. QuaDMutNetEx is available for download from https://github.com/bokhariy/QuaDMutNetExunder the GNU GPLv3 license.

Correction: A nitric oxide synthase-like protein from Synechococcus produces NO/NO3 - from l-arginine and NADPH in a tetrahydrobiopterin- and Ca2+-dependent manner.

Nitric oxide synthases (NOSs) are heme-based monooxygenases that convert l-Arg to l-citrulline and nitric oxide (NO), a key signaling molecule and cytotoxic agent in mammals. Bacteria also contain NOS proteins, but the role of NO production within these organisms, where understood, differs considerably from that of mammals. For example, a NOS protein in the marine cyanobacterium Synechococcus sp. PCC 7335 (syNOS) has recently been proposed to function in nitrogen assimilation from l-Arg. syNOS retains the oxygenase (NOSox) and reductase (NOSred) domains present in mammalian NOS enzymes (mNOSs), but also contains an N-terminal globin domain (NOSg) homologous to bacterial flavohemoglobin proteins. Herein, we show that syNOS functions as a dimer and produces NO from l-Arg and NADPH in a tetrahydrobiopterin (H4B)-dependent manner at levels similar to those produced by other NOSs but does not require Ca2+-calmodulin, which regulates NOSred-mediated NOSox reduction in mNOSs. Unlike other bacterial NOSs, syNOS cannot function with tetrahydrofolate and requires high Ca2+ levels (>200 μm) for its activation. NOSg converts NO to NO3− in the presence of O2 and NADPH; however, NOSg did not protect Escherichia coli strains against nitrosative stress, even in a mutant devoid of NO-protective flavohemoglobin. We also found that syNOS does not have NOS activity in E. coli (which lacks H4B) and that the recombinant protein does not confer growth advantages on l-Arg as a nitrogen source. Our findings indicate that syNOS has both NOS and NO oxygenase activities, requires H4B, and may play a role in Ca2+-mediated signaling.

Tissue and Cell-Free DNA-Based Epigenomic Approaches for Cancer Detection.

Over 9 million people die of cancer each year worldwide, reflecting the unmet need for effective biomarkers for both cancer diagnosis and prognosis. Cancer diagnosis is complex because the majority of malignant tumors present with long periods of latency and lack of clinical presentation at early stages. During carcinogenesis, premalignant cells experience changes in their epigenetic landscapes, such as differential DNA methylation, histone modifications, nucleosome positioning, and higher orders of chromatin changes that confer growth advantage and contribute to determining the biologic phenotype of human cancers. Recent progress in microarray platforms and next-generation sequencing approaches has allowed the characterization of abnormal epigenetic patterns genome wide in a large number of cancer cases. The sizable amount of processed data also comes with challenges regarding data management and assessment for effective biomarker exploration to be further applied in prospective clinical trials. Epigenetics-based single or panel tests of genes are being explored for clinical management to fulfill unmet needs in oncology. The advance of these tests to the clinical routine will depend on rigorous, extensive, and independent validation in well-annotated cohort of patients and commercial development of clinical routine-friendly and adequate procedures. In this review we discuss the analytic validation of tissue and cell-free DNA-based epigenomic approaches for early cancer detection, diagnosis, and treatment monitoring and the clinical utility of candidate epigenetic alterations applied to colorectal, glioblastoma, breast, prostate, bladder, and lung cancer management.

The actinic dysplasia syndrome - diagnostic approaches defining a new concept in field carcinogenesis with multiple cSCC.

Usually, SCC lesions are surrounded by a number of clinically visible and non-visible (subclinical) areas of actinically damaged skin containing cells with dysplasia, and thus may be designated actinic dysplasia syndrome. The epithelial damage is caused mainly by UV radiation, inducing mutations in keratinocytes that may confer growth advantages resulting in preneoplastic fields. The development of visible dysplastic lesions (actinic keratosis - AK) and subsequent progression to invasive SCC requires further mutations in cancer-associated genes, like tumour suppressor genes and cell cycle regulators. Reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) represent a considerable advantage for the investigation of field cancerization. In addition, imaging allows the non-invasive monitoring of topical treatments for AKs. RCM provides invivo horizontal skin sections with a high, 1-μm lateral resolution (similar to histopathology) but with a limited penetration (about 200μm), which can hamper the visualization of important areas such as the dermal-epidermal junction. Conventional OCT has better penetration (1-2mm) at the expense of a more limited resolution (much lower than histopathology). Line-field confocal OCT (LC-OCT) combines the high precision of RCM and the good penetration of OCT in a single device and therefore appears to be very useful in diagnosing/managing AKs.

Lgr5 Functions As a Critical Negative Regulator of Wnt/β-Catenin Signaling and Is Essential for B-Lymphopoiesis and Malignant B-Cell Transformation

Introduction: When early B-cell precursors have successfully undergone V(D)J rearrangement of immunoglobulin genes, they receive a strong positive selection signal, which initiates clonal expansion and the first wave of B-lymphopoiesis. Interestingly, this expansion following successful V(D)J recombination is marked by massive up-regulation of Leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5). Results: To test the function of Lgr5 at this particular stage and later in B-cell development, we crossed Lgr5fl/fl mice with Mb1cre and CD21cre mice respectively. While Lgr5 deletion at the time of V(D)J recombination resulted in near complete ablation of the B cell lineage in the bone marrow and periphery, deletion at later stages of B-cell development had no significant impact on B-cell survival and proliferation. Here, we identified Lgr5 as a B-cell specific negative regulator of Wnt/β-catenin signaling, which is essential for normal B-lymphopoiesis but also for initiation of B-cell lineage acute lymphoblastic leukemia (B-ALL). Indeed, in children with B-ALL, higher than median mRNA levels of LGR5 at the time of diagnosis was identified as a predictor of poor clinical outcome (COG P9906, n=207, p=0.008). LGR5 over-expression conferred growth advantage to B-ALL cells, enhanced serial plating capacity and the ability to initiate leukemia in NSG mice. Limiting dilution transplant experiments showed that LGR5 overexpression increased LIC-frequencies by 4-fold. Inducible deletion of Lgr5 in BCR-ABL1- or NRASG12D-transformed B-ALL cell lines led to growth arrest, abolished colony forming capacity and compromised the ability of leukemia cells to initiate fatal disease in NSG recipients. Lgr5 is thought to function as a potentiator of Wnt/β-catenin signaling in epithelial cells. Upon deletion of Lgr5 in B-ALL cells, however, we observed massive accumulation of nuclear β-catenin and increased expression of β-catenin target genes, which suggests that Lgr5 acts as an essential negative regulator of β-catenin in the B-cell context. Phosphoproteomic approaches identified β-catenin to be differentially phosphorylated on S675 upon Lgr5-deletion, which enhances nuclear translocation and transcriptional activity of β-catenin. Deletion of LGR5 in human colon cancer cell lines did not result in β-catenin accumulation, which indicates a lineage specific role of Lgr5 in B-lymphocytes. Studying activating CTNNB1 mutations in 89,325 samples from 23 types of cancer, we found 7,777 activating mutations (8.7%). However, among 2,375 B-cell tumor samples, we did not observe a single activating mutation. Indeed, compared to solid tumors, B-ALL and B-cell lymphomas have very low amounts of nuclear β-catenin. B-ALL cells are extremely sensitive to β-catenin activation, resulting in cell cycle arrest and loss of colony forming ability, which parallels the phenotype observed in the absence of Lgr5. CRISPR-Cas9 mediated deletion of Ctnnb1 in Lgr5 knock-out B-ALL cells strikingly rescued proliferation and colony formation capacity, further corroborating the role of Lgr5 in promoting B-cell leukemogenesis by opposing aberrant activation of β-catenin. Therapeutic implication: Given that Lgr5 is required for leukemia-initiation and marks LICs, we tested an Lgr5-MMAE antibody-drug conjugate (ADC) in the presence and absence of dexamethasone. Combination treatment with dexamethasone not only enforced persistently high surface expression of LGR5, but also potentiated efficacy of by Lgr5-MMAE in B-ALL eradication. Conclusion: Lgr5 is critical for normal B cell development and tumor initiation in B-ALL. Lgr5, restricts the levels of nuclear β-catenin and enables B cell survival through negative regulation of Wnt/β-catenin signaling. Moreover, LGR5 represents a promising target for eradicating leukemia initiating cells. Disclosures No relevant conflicts of interest to declare.

Nudix hydrolases with Coenzyme A (CoA) and acyl-CoA pyrophosphatase activities confer growth advantage to Mycobacterium smegmatis.

Nudix hydrolase family proteins hydrolyse toxic by-products of cellular metabolism such as mutagenic nucleoside triphosphates, sugar nucleotides and signalling molecules. We studied the substrate specificities of Nudix hydrolases encoded by rv3672c and rv3040c from Mycobacterium tuberculosis and their respective homologues, msmeg_6185 and msmeg_2327 from M. smegmatis. The rv3672c- and msmeg_6185-encoded proteins (Rv3672 and MSMEG_6185, respectively) showed CoA pyrophosphatase (CoAse) activity that converted acyl-CoA to adenosine-3',5'-diphosphate (3', 5'-ADP) and 4-acyl phosphopantetheine. The efficiencies of Rv3672 and MSMEG_6185 in hydrolysing CoA derivatives were found to be higher than those of the Rv3040 and MSMEG_2327 (encoded by rv3040c and msmeg_2327, respectively). Further, amongst the substrates tested, Rv3672 and MSMEG_6185 used CoA and oxidized CoA as the most preferred substrates. Use of the M. smegmatis model showed that the expression of msmeg_6185 occurs in the log and stationary phases but declines during the late stationary phase and becomes undetectable during hypoxia. The co-culture competition experiments performed between the wild-type and Δmsmeg_6185 strains of M. smegmatis in different carbon sources revealed that the presence of msmeg_6185 provided growth fitness advantage to M. smegmatis, irrespective of the carbon source, implicating its function in regulation for the optimal physiological levels of acyl-CoAs in the cell.

Synthetic modeling reveals HOXB genes are critical for the initiation and maintenance of human leukemia

Mechanistic studies in human cancer have relied heavily on cell lines and mouse models, but are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts; however, these are hampered by variable genetic background, inability to study early events, and practical issues with availability/reproducibility. We report here an efficient, reproducible model of T-cell leukemia in which lentiviral transduction of normal human cord blood yields aggressive leukemia that appears indistinguishable from natural disease. We utilize this synthetic model to uncover a role for oncogene-induced HOXB activation which is operative in leukemia cells-of-origin and persists in established tumors where it defines a novel subset of patients distinct from other known genetic subtypes and with poor clinical outcome. We show further that anterior HOXB genes are specifically activated in human T-ALL by an epigenetic mechanism and confer growth advantage in both pre-leukemia cells and established clones.

A nitric oxide synthase–like protein from Synechococcus produces NO/NO3− from l-arginine and NAPDH in a tetrahydrobiopterin- and Ca2+-dependent manner

Nitric oxide synthases (NOSs) are heme-based monooxygenases that convert l-Arg to l-citrulline and nitric oxide (NO), a key signaling molecule and cytotoxic agent in mammals. Bacteria also contain NOS proteins, but the role of NO production within these organisms, where understood, differs considerably from that of mammals. For example, a NOS protein in the marine cyanobacterium Synechococcus sp. PCC 7335 (syNOS) has recently been proposed to function in nitrogen assimilation from l-Arg. syNOS retains the oxygenase (NOSox) and reductase (NOSred) domains present in mammalian NOS enzymes (mNOSs), but also contains an N-terminal globin domain (NOSg) homologous to bacterial flavohemoglobin proteins. Herein, we show that syNOS functions as a dimer and produces NO from l-Arg and NADPH in a tetrahydrobiopterin (H4B)-dependent manner at levels similar to those produced by other NOSs but does not require Ca2+-calmodulin, which regulates NOSred-mediated NOSox reduction in mNOSs. Unlike other bacterial NOSs, syNOS cannot function with tetrahydrofolate and requires high Ca2+ levels (>200 μm) for its activation. NOSg converts NO to NO3- in the presence of O2 and NADPH; however, NOSg did not protect Escherichia coli strains against nitrosative stress, even in a mutant devoid of NO-protective flavohemoglobin. We also found that syNOS does not have NOS activity in E. coli (which lacks H4B) and that the recombinant protein does not confer growth advantages on l-Arg as a nitrogen source. Our findings indicate that syNOS has both NOS and NO oxygenase activities, requires H4B, and may play a role in Ca2+-mediated signaling.

Abstract 821: DNA hypermethylation of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells

Abstract Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to the loss of key cellular pathways in tumorigenesis. Methyl-CpG targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA demethylating agents. Previous study using microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in pancreatic cancer cell lines. In this study, IRX4 (Iroquois homeobox 4) was focused on because IRX4 was located at chromosome 5p15.33 where one of pancreatic cancer susceptibility loci has been identified through genome-wide association study (GWAS). IRX4 has also been identified as a candidate tumor suppressor gene in prostate cancer. IRX4 was greatly downregulated in all of the analyzed 12 pancreatic cancer cell lines by promoter hypermethylation. In addition, IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected pancreatic cancers (15/22: 68%); corresponding normal pancreatic tissues were unmethylated. To examine whether IRX4 has the growth inhibitory effect on pancreatic cancer cell lines, colony formation assay using PK-1 and PK-9 cell lines has been performed; compared with empty vector-transfected cells, IRX4 expression vector-transfected cells formed significantly reduced number of colonies. Furthermore, tetracycline-inducible IRX4 expressing vector system was constructed and a pancreatic cancer cell line PK-1 was analyzed for further functional investigation; induction of IRX4 suppressed cell growth in PK-1. Our results suggest that DNA methylation-mediated silencing of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells and MeTA may be a powerful tool to search critical hypermethylated genes in tumorigenesis. Citation Format: Kanchan Chakma, Zhaodi Gu, Fuyuhiko Motoi, Michiaki Unno, Akira Horii, Shinichi Fukushige. DNA hypermethylation of IRX4 is a frequent event that may confer growth advantage to pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 821.

2-Oxoglutarate cooperativity and biphasic ammonium saturation of Aspergillus niger NADP-glutamate dehydrogenase are structurally coupled

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized. The C-terminal sequence (residues 315–460, named the D-segment) was implicated in the AnGDH cooperativity. The D-segment residues largely contribute to the monomer-monomer interface of each trimer in the native hexamer and are far removed from the enzyme active site. The D-segment appears to be a part of the allosteric network responsible for 2-oxoglutarate homotropic interactions in AnGDH. AnGDH and its C415S mutant, but not AtGDH, also showed atypical, biphasic ammonium saturation, particularly at sub-saturating 2-oxoglutarate concentrations. We found that the sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response are tightly coupled; the analysis of AnGDH-AtGDH chimeras ascribes the two features to the AnGDH D-segment. The two non-Michaelis-Menten substrate saturations of AnGDH were influenced by ionic strength. Increase in ionic strength reduced the nH of 2-oxoglutarate saturation as well as abolished the biphasic response, suggesting that polar/ionic interactions determine the allosteric, inter-subunit communications. The biochemical analysis in the context of available structural data implicates the D-segment of AnGDH in the allosteric feature of this enzyme. The coupling of sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response could possibly confer growth advantage to A. niger experiencing carbon and/or nitrogen limitation.

In silico learning of tumor evolution through mutational time series

Cancer arises through the accumulation of somatic mutations over time. Understanding the sequence of mutation occurrence during cancer progression can assist early and accurate diagnosis and improve clinical decision-making. Here we employ long short-term memory (LSTM) networks, a class of recurrent neural network, to learn the evolution of a tumor through an ordered sequence of mutations. We demonstrate the capacity of LSTMs to learn complex dynamics of the mutational time series governing tumor progression, allowing accurate prediction of the mutational burden and the occurrence of mutations in the sequence. Using the probabilities learned by the LSTM, we simulate mutational data and show that the simulation results are statistically indistinguishable from the empirical data. We identify passenger mutations that are significantly associated with established cancer drivers in the sequence and demonstrate that the genes carrying these mutations are substantially enriched in interactions with the corresponding driver genes. Breaking the network into modules consisting of driver genes and their interactors, we show that these interactions are associated with poor patient prognosis, thus likely conferring growth advantage for tumor progression. Thus, application of LSTM provides for prediction of numerous additional conditional drivers and reveals hitherto unknown aspects of cancer evolution.

Comparison of different functional prediction scores using a gene-based permutation model for identifying cancer driver genes

BackgroundIdentifying cancer driver genes (CDG) is a crucial step in cancer genomic toward the advancement of precision medicine. However, driver gene discovery is a very challenging task because we are not only dealing with huge amount of data; but we are also faced with the complexity of the disease including the heterogeneity of background somatic mutation rate in each cancer patient. It is generally accepted that CDG harbor variants conferring growth advantage in the malignant cell and they are positively selected, which are critical to cancer development; whereas, non-driver genes harbor random mutations with no functional consequence on cancer. Based on this fact, function prediction based approaches for identifying CDG have been proposed to interrogate the distribution of functional predictions among mutations in cancer genomes (eLS 1–16, 2016). Assuming most of the observed mutations are passenger mutations and given the quantitative predictions for the functional impact of the mutations, genes enriched of functional or deleterious mutations are more likely to be drivers. The promises of these methods have been continually refined and can therefore be applied to increase accuracy in detecting new candidate CDGs. However, current function prediction based approaches only focus on coding mutations and lack a systematic way to pick the best mutation deleteriousness prediction algorithms for usage.ResultsIn this study, we propose a new function prediction based approach to discover CDGs through a gene-based permutation approach. Our method not only covers both coding and non-coding regions of the genes; but it also accounts for the heterogeneous mutational context in cohort of cancer patients. The permutation model was implemented independently using seven popular deleteriousness prediction scores covering splicing regions (SPIDEX), coding regions (MetaLR, and VEST3) and pan-genome (CADD, DANN, Fathmm-MKL coding and Fathmm-MKL noncoding). We applied this new approach to somatic single nucleotide variants (SNVs) from whole-genome sequences of 119 breast and 24 lung cancer patients and compared the seven deleteriousness prediction scores for their performance in this study.ConclusionThe new function prediction based approach not only predicted known cancer genes listed in the Cancer Gene Census (CGC), but also new candidate CDGs that are worth further investigation. The results showed the advantage of utilizing pan-genome deleteriousness prediction scores in function prediction based methods. Although VEST3 score, a deleteriousness prediction score for missense mutations, has the best performance in breast cancer, it was topped by CADD and Fathmm-MKL coding, two pan-genome deleteriousness prediction scores, in lung cancer.

Integrative PDGF/PDGFR and focal adhesion pathways are downregulated in ERCC1-defective non-small cell lung cancer undergoing sodium glycididazole-sensitized cisplatin treatment

BackgroundChemoresistance to cisplatin in lung cancer treatment remains prevalent. Since targeting excision repair cross-complementing 1 (ERCC1) may be a viable strategy to reestablish the therapeutic sensitivity to platinum-based agents in chemoresistant cases, and low ERCC1 level is beneficial to metastatic lung cancer patients undergoing platinum-based chemotherapy, we hypothesized that metastasis-associated process is involved in ERCC1-dependent cisplatin-resistance in lung adenocarcinoma. MethodsWe performed an RNA-Sequencing (RNA-Seq) analysis to identify differentially expressed genes in ERCC1-deficient cells co-treated with cisplatin and sodium glycididazole (CMNa). Differentially expressed genes and the hub genes in the cisplatin/CMNa-treated cells were identified by systematic network analysis. Oncomine expression analysis was also performed to evaluate the clinical implication of the identified hub gene. ResultsPlatelet-derived growth factor receptor (PDGF/PDGFR) and focal adhesion genes were downregulated in ERCC1-defective non-small cell lung cancer (NSCLC) cells undergoing combined cisplatin/CMNa treatment. Consistent with the finding, cell migration was reduced in these cells. PDGFRB was identified as the hub gene in the process by differential expressed gene network analysis. Importantly, elevated PDGFRB level was observed in advanced lung adenocarcinoma patients with metastases. ConclusionPDGF/PDGFR and focal adhesion signaling may serve as another mechanism in addition to ERCC1-mediated cisplatin-resistance in lung adenocarcinoma. Multiple pro-invasion/pro-migration/proliferation and DNA damage repair pathways may be integrated to confer growth advantages on tumor cells.

Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease.The in vitro component of this synthetic system affords direct access to “pre-leukemia” cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the “HOXB high” subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and “unknown” transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models.The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly “evolved” samples. DisclosuresSteidl:Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

Potential IL6R-ADAR1-STAT3 Interplay Promotes Oncogenicity in 1q21(amp) Multiple Myeloma

1q21 amplification (1q21(amp)) is an important prognostic marker in multiple myeloma (MM) and the critical genes within the minimally amplified region has not been fully characterized. Survival of MM cells is highly dependent on the bone marrow (BM) microenvironment, which is enriched with an array of growth factors secreted by the BM stromal cells. Amongst these growth factors, interleukin-6 (IL6) is a potent driver for MM disease progression, mechanistically through the activation of IL6R and the downstream STAT3 signaling. ADAR1, an RNA editing enzyme, exists in two distinct isoforms, namely the P110 and P150, of which its aberrant expression and activity have been reported in various cancer types, including MM. However, the knowledge on the upstream regulators of ADAR1 and its mechanism of actions have remained elusive. In view of the close proximity of IL6R and ADAR1 on chromosome 1q21, we postulated that concomitant gain of both genes could lead to a more aggressive disease nature in the 1q21(amp) patients and seek to elucidate the potential driving mechanisms.Herein, we analyzed the publicly available myeloma clinical datasets and identified that both IL6R and ADAR1 are overexpressed in the 1q21(amp) patients. With respect to ADAR1, we observed that among the 1q21(amp) MM cell lines, the IL6-responsive and -dependent cells lines demonstrated more intense expression of P150 isoform but the P110 did not differ much, indicating that P150 expression is associated with IL6 regulation. Besides potently activating the IL6R-STAT3 signaling, IL6 stimulation could indeed result in an increased expression of ADAR1 P150 isoform, at both the protein and mRNA level, with minimal effects seen on the P110 isoform, suggesting a transcriptional regulation of P150 by STAT3. Downstream validations with ChIP-qPCR and Luciferase Reporter Assay revealed that ADAR1 P150 is a direct target of STAT3 transcription factor. Specific STAT3 inhibition caused a downregulation of P150 and its oncogenic properties. Likewise, ectopic introduction of P150 confers growth advantage to the cells. Importantly, we also observed that IL6-induced-P150 expression could in turn mediate the activity of STAT3 signaling, in which high P150 cells had a more rapid IL6-induced-STAT3 activation. In summary, we identified that 1q21(amp) and IL6R-STAT3 hyperactivity render a double blow to the regulation of ADAR1-P150, leading to its overexpression, which in turn formed a regulatory feedback loop in further stimulating the STAT3 pathway, conferring disease aggressiveness in the 1q21(amp) patients. The potential interplay between IL6R, ADAR1 and STAT3 may represent a novel mechanism by which IL6 confers oncogenicity in 1q21(amp) MM. DisclosuresChng:Aslan: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Merck: Research Funding; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.