Bimodal Expression Research Articles

Expression of COBLL1 encoding novel ROR1 binding partner is robust predictor of survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia is a disease with up-regulated expression of the transmembrane tyrosine-protein kinase ROR1, a member of the Wnt/planar cell polarity pathway. In this study, we identified COBLL1 as a novel interaction partner of ROR1. COBLL1 shows clear bimodal expression with high levels in chronic lymphocytic leukemia patients with mutated IGHV and approximately 30% of chronic lymphocytic leukemia patients with unmutated IGHV. In the remaining 70% of chronic lymphocytic leukemia patients with unmutated IGHV, COBLL1 expression is low. Importantly, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 have an unfavorable disease course with short overall survival and time to second treatment. COBLL1 serves as an independent molecular marker for overall survival in chronic lymphocytic leukemia patients with unmutated IGHV. In addition, chronic lymphocytic leukemia patients with unmutated IGHV and high COBLL1 show impaired motility and chemotaxis towards CCL19 and CXCL12 as well as enhanced B-cell receptor signaling pathway activation demonstrated by increased PLCγ2 and SYK phosphorylation after IgM stimulation. COBLL1 expression also changes during B-cell maturation in non-malignant secondary lymphoid tissue with a higher expression in germinal center B cells than naïve and memory B cells. Our data thus suggest COBLL1 involvement not only in chronic lymphocytic leukemia but also in B-cell development. In summary, we show that expression of COBLL1, encoding novel ROR1-binding partner, defines chronic lymphocytic leukemia subgroups with a distinct response to microenvironmental stimuli, and independently predicts survival of chronic lymphocytic leukemia with unmutated IGHV.

Detection of Mutations Affecting Heterogeneously Expressed Phenotypes by Colony Immunoblot and Dedicated Semi-Automated Image Analysis Pipeline.

To understand how bacteria evolve and adapt to their environment, it can be relevant to monitor phenotypic changes that occur in a population. Single cell level analyses and sorting of mutant cells according to a particular phenotypic readout can constitute efficient strategies. However, when the phenotype of interest is expressed heterogeneously in ancestral isogenic populations of cells, single cell level sorting approaches are not optimal. Phenotypic heterogeneity can for instance make no-expression mutant cells indistinguishable from a subpopulation of wild-type cells transiently not expressing the phenotype. The analysis of clonal populations (e.g., isolated colonies), in which the average phenotype is measured, can circumvent this issue. Indeed, no-expression mutants form negative populations while wild-type clones form populations in which average expression of the phenotype yields a positive signal. We present here an optimized colony immunoblot protocol and a semi-automated image analysis pipeline (ImageJ macro) allowing for rapid detection of clones harboring mutations that affect the heterogeneous (i.e., bimodal) expression of the Type Three Secretion System-1 (TTSS-1) in Salmonella enterica serovar Typhimurium. We show that this protocol can efficiently differentiate clones expressing TTSS-1 at various levels in mixed populations. We were able to detect the emergence of hilC mutants in which the proportion of cells expressing TTSS-1 was reduced compared to the ancestor. We could also follow changes in the frequency of different mutants during long-term infections. This demonstrates that our protocol constitutes a tractable approach to assess semi-quantitatively the evolutionary dynamics of heterogeneous phenotypes, such as the expression of virulence genes, in bacterial populations.

Nonlatching positive feedback enables robust bimodality by decoupling expression noise from the mean

Fundamental to biological decision-making is the ability to generate bimodal expression patterns where 2 alternate expression states simultaneously exist. Here, we use a combination of single-cell analysis and mathematical modeling to examine the sources of bimodality in the transcriptional program controlling HIV’s fate decision between active replication and viral latency. We find that the HIV transactivator of transcription (Tat) protein manipulates the intrinsic toggling of HIV’s promoter, the long terminal repeat (LTR), to generate bimodal ON-OFF expression and that transcriptional positive feedback from Tat shifts and expands the regime of LTR bimodality. This result holds for both minimal synthetic viral circuits and full-length virus. Strikingly, computational analysis indicates that the Tat circuit’s noncooperative “nonlatching” feedback architecture is optimized to slow the promoter’s toggling and generate bimodality by stochastic extinction of Tat. In contrast to the standard Poisson model, theory and experiment show that nonlatching positive feedback substantially dampens the inverse noise-mean relationship to maintain stochastic bimodality despite increasing mean expression levels. Given the rapid evolution of HIV, the presence of a circuit optimized to robustly generate bimodal expression appears consistent with the hypothesis that HIV’s decision between active replication and latency provides a viral fitness advantage. More broadly, the results suggest that positive-feedback circuits may have evolved not only for signal amplification but also for robustly generating bimodality by decoupling expression fluctuations (noise) from mean expression levels.

Time-Dependent Production of the Bioactive Peptides Endolides A and B and the Polyketide Mariline A from the Sponge-Derived Fungus Stachylidium bicolor 293K04

Previous investigations of the sponge-derived fungus Stachylidium bicolor (S. bicolor) 293K04 led to the isolation of the biosynthetically unusual polyketides marilines A-C and the bioactive tetrapeptides endolides A-B, identified as potential neuropathic drug leads. Furthermore, prior extended solid cultivation of S. bicolor 293K04 for 60 days resulted in a significant increase of polyketide yield, and the isolation of seven new polyketides. Due to the interest in endolide activity, unusual biosynthetic diversity, and the late stage polyketide production, we studied the cultivation conditions for determining the production time distribution and yields of these secondary metabolites. Results indicated a first production phase of secondary metabolite dominated by peptides, after 21–23 days. Polyketide mariline A1/A2 only started at day 35 of growth, an unusually late period for secondary metabolite expression. This unusual bimodal sequential expression of different families of secondary metabolites suggests value in exploring extended cultivation times to identify novel bioactive fungal compounds.

Bacterial-Chromatin Structural Proteins Regulate the Bimodal Expression of the Locus of Enterocyte Effacement (LEE) Pathogenicity Island in Enteropathogenic Escherichia coli.

ABSTRACTIn enteropathogenic Escherichia coli (EPEC), the locus of enterocyte effacement (LEE) encodes a type 3 secretion system (T3SS) essential for pathogenesis. This pathogenicity island comprises five major operons (LEE1 to LEE5), with the LEE5 operon encoding T3SS effectors involved in the intimate adherence of bacteria to enterocytes. The first operon, LEE1, encodes Ler (LEE-encoded regulator), an H-NS (nucleoid structuring protein) paralog that alleviates the LEE H-NS silencing. We observed that the LEE5 and LEE1 promoters present a bimodal expression pattern, depending on environmental stimuli. One key regulator of bimodal LEE1 and LEE5 expression is ler expression, which fluctuates in response to different growth conditions. Under conditions in vitro considered to be equivalent to nonoptimal conditions for virulence, the opposing regulatory effects of H-NS and Ler can lead to the emergence of two bacterial subpopulations. H-NS and Ler share nucleation binding sites in the LEE5 promoter region, but H-NS binding results in local DNA structural modifications distinct from those generated through Ler binding, at least in vitro. Thus, we show how two nucleoid-binding proteins can contribute to the epigenetic regulation of bacterial virulence and lead to opposing bacterial fates. This finding implicates for the first time bacterial-chromatin structural proteins in the bimodal regulation of gene expression.

Emotional Intensity Modulates the Integration of Bimodal Angry Expressions: ERP Evidence.

Integration of information from face and voice plays a central role in social interactions. The present study investigated the modulation of emotional intensity on the integration of facial-vocal emotional cues by recording EEG for participants while they were performing emotion identification task on facial, vocal, and bimodal angry expressions varying in emotional intensity. Behavioral results showed the rates of anger and reaction speed increased as emotional intensity across modalities. Critically, the P2 amplitudes were larger for bimodal expressions than for the sum of facial and vocal expressions for low emotional intensity stimuli, but not for middle and high emotional intensity stimuli. These findings suggested that emotional intensity modulates the integration of facial-vocal angry expressions, following the principle of Inverse Effectiveness (IE) in multimodal sensory integration.

RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation.

In the nosocomial opportunistic pathogen Acinetobacter baumannii, RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis-regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo, impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen.IMPORTANCEAcinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis-acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA damage-induced phenotypic variation. Though 5' UTRs are known to provide stability to transcripts in bacteria, this is the first example in which it regulates a bimodal DDR response through recA transcript stabilization, potentially enabling cells to have enough RecA for survival and genetic variability.

Role of lipid inositol phosphatase SHIP-1 in Germinal Center B cell selection and function

Abstract Germinal Centers (GC) are sites within secondary lymphoid organs in which B cells undergo proliferation and differentiation. The PI3K signaling pathway is a central modulator of GC B cells (GCBC) selection and function. The PI3K pathway can be regulated by several enzymes including Src homology 2-containing inositol 5’-phosphatase (SHIP-1). SHIP-1 is known to be hyper-phosphorylated in GCBCs. Upon activation, SHIP-1 hydrolyzes phosphatidyl inositol triphosphate (PIP3) to generate phosphatidyl inositol 3,4 biphosphate (PIP2). PIP2 can bind to signaling proteins that are crucial to BCR signaling such as TAPP1/2 and Akt/PKB. To ascertain the role of SHIP-1 in survival and selection of GCBC we used a tamoxifen inducible Cre system to delete SHIP-1 from B cells during an ongoing GC response to NP-CGG. SHIP-1 deletion led to bi-modal expression of SHIP-1 in GCBC expressing intermediate (SHIP-1 int) and low levels of SHIP-1 (SHIP-1 lo) compared to WT controls (SHIP-1 hi). SHIP-1 int GCBC showed reduced phosphorylation of signaling proteins S6 and increased phosphorylation of Btk upon BCR crosslinking with anti-u antibody. SHIP-1 lo cells were un-responsive to BCR crosslinking and had the highest frequency of dead cells and Caspase 3 positive cells among all 3 groups. This data suggests that SHIP-1 expression and activity is crucial to GCBC signaling and survival. The role of PIP2 in regulating BCR signaling in GCBC remains to be elucidated.

The impact of succinate trace on pWW0 and ortho-cleavage pathway transcription in Pseudomonas putida mt-2 during toluene biodegradation

Toluene is a pollutant catabolised through the interconnected pWW0 (TOL) and ortho-cleavage pathways of Pseudomonas putida mt-2, while upon succinate and toluene mixtures introduction in batch cultures grown on M9 medium, succinate was previously reported as non-repressing. The effect of a 40 times lower succinate concentration, as compared to literature values, was explored through systematic real-time qPCR monitoring of transcriptional kinetics of the key TOL Pu, Pm and ortho-cleavage PbenR, PbenA promoters in mixed-substrate experiments. Even succinate trace inhibited transcription leading to bi-modal promoters expression. Potential carbon catabolite repression mechanisms and novel expression patterns of promoters were unfolded. Lag phase was shortened and biomass growth levels increased compared to sole toluene biodegradation suggesting enhanced pollutant removal efficiency. The study stressed the noticeable effect of a preferred compound’s left-over on the main route of a bioprocess, revealing the beneficiary supply of low preferred substrates concentrations to design optimal bioremediation strategies.

Bimodal expression of yeast GAL genes is controlled by a long non-coding RNA and a bifunctional galactokinase

Bimodality in gene expression can generate phenotypic heterogeneity facilitating fitness and growth of isogenic cell populations in suboptimal environments. We investigated the mechanism by which, in conditions of limiting galactose, yeast cell populations activate GAL genes in a bimodal fashion with a cell fraction expressing GAL genes (ON), while the rest subpopulation is kept at the non-expressing (OFF) state. We show that a long non-coding RNA (GAL10-ncRNA) crossing the bidirectional GAL1-10 promoter, decreases the rate by which single cells commit transition to the ON state without affecting the rate of GAL transcription per se in ON cells. This is accomplished by repressing stochastic expression of the bifunctional Gal1p galactokinase, which besides its enzymatic activity acts as an essential inducer of the system under those conditions. We show that once single cells switch to the ON state, the GAL10-ncRNA effect is overridden by accumulating Gal1p levels sufficient to feedback positively on Gal4p, and not by the active transcription of GAL10 that occurs in opposite direction relative to that of GAL10-ncRNA. Conversely, GAL10-ncRNA does not influence transition of ON cells, where Gal4p is active, back to the OFF state. Our model suggests that the functional interplay between GAL10-ncRNA transcription, stochastic Gal1p expression and Gal1p positive feedback on Gal4p constitutes a novel molecular switch mechanism dictating the commitment of individual cells for either metabolic state.

Abstract P6-10-02: MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates

Abstract Background: Lymphocyte-activation gene 3 (LAG-3) is a T-cell checkpoint regulator and a current target in immunotherapy trials. LAG-3's main ligand is MHC class II (MHC-II), to which it binds with higher affinity than CD4. Binding of LAG3 to MHC-II antigen-presenting cells negatively regulates cellular proliferation, activation, and homeostasis of T cells, similarly to CTLA-4 and PD-1, suggesting that antibodies targeting LAG-3 may demonstrate similar anti-tumor immune effects. Hypothesis: We recently reported an association of MHC-II on tumor cells and its involvement in mediating sensitivity to PD-1/PD-L1 monoclonal antibodies. MHC-II demonstrates a strong bimodal expression pattern on tumor cells from a variety of tissues, including those of the breast. In breast cancer patients, tumor-specific MHC-II expression on TNBCs is correlated with a 'hot' immune environment. We hypothesized that 1) MHC-II expression may drive potent anti-tumor immune responses and 2) MHC-II-positive tumors that generate immunotolerance may develop a specific immune checkpoint dependency on LAG-3, since LAG-3 is the inhibitory receptor for MHC-II-mediated antigen presentation. Methods: To determine the functionality of MHC-II in driving anti-tumor immune responses, we constitutively expressed the MHC-II master regulator CIITA in MMTV-neu mouse tumor cells and determined their ability to form tumors in immunocompetent syngeneic hosts. To evaluate the association of MHC-II+ tumors with LAG-3 expression, we evaluated LAG-3-positivity by immunohistochemistry (IHC) in lymphocytic infiltrates in a series of 111 post-NAC TNBC specimens from patients with residual disease remaining after presurgical chemotherapy. Tumor-infiltrating lymphocytes (TILs) were scored by H&E, PD-L1 and MHC-II (HLA-DR) were scored in the stroma and tumor compartments using automated quantitative immunofluorescence (AQUA). Results: Enforced expression of MHC-II via constitutive expression of CIITA caused rejection in 60% of mice, while only 11% of mice rejected MMTV-neu tumors expressing the vector control (Fisher's exact p=0.04). All rejecting mice were immune to rechallenge with parental (non-CIITA-expressing) MMTV-neu cells, suggesting a memory effector response. Clinically, 11/102 patients (10.8%) had LAG-3+ immune cells in their tumor. LAG-3+ tumors were strongly correlated with MHC-II positivity in tumor cells (p<0.0001). Presence of LAG-3+ cells also correlated strongly with overall TILs (p<0.0001), and PD-L1 expression on TILs (p<0.02). Since the likelihood of identifying LAG3+ lymphocytes is confounded by the inclusion of poorly-infiltrated tumors, we performed a subset analysis on only those tumors with substantial TILs (>20%). When this subset was analyzed, LAG-3 positivity retained its association with tumor MHC-II expression (p=0.0001), while the association of LAG-3 with stromal PD-L1 was reduced below the level of significance (p=0.052). Conclusions: MHC-II expression causes increased immune activation in breast cancers, consistent with our previous findings. MHC-II positivity in breast tumors may identify a population with preferential dependence on the LAG-3 checkpoint, which may be important for future immunotherapy trials. Citation Format: Balko JM, Loi S, Giltnane JM, Combs S, Estrada MV, Sanchez V, Rimm D, Sanders ME, Salgado R, Gomez H, Johnson DB. MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-10-02.

An Immature Immunophenotype (CD34pos, CD38dim, nTdTdim) on Malignant B-Cell Precursor Blasts at Diagnosis Predicts High Minimal Residual Disease in BCP-ALL

Abstract Background Flow cytometry is widely used for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) diagnosis, and the detection of Minimal Residual Disease (MRD) by flow cytometry is an established stratification tool after induction therapy. Early risk stratification in BCP-ALL allows for individualized treatment strategies, improving prognosis and lowering treatment related toxicity. The Leukemia-Associated ImmunoPhenotype (LAIP) at time of diagnosis might comprise an early and easily accessible risk stratification tool. However, immunophenotypically distinct subpopulations are common in BCP-ALL (Øbro et al, Leukemia, 2011), yet current scoring methods do not account for this heterogeneity. This could make classification less accurate and hereby underestimate the prognostic value of the LAIP, urging the need for detailed LAIP characterization. Aim To systematically characterize the LAIP including immunophenotypic subpopulations at time of diagnosis in order to assess the prognostic potential of the LAIP in patients with BCP-ALL Methods We retrospectively evaluated flow cytometry data from time of diagnosis in 200 patients diagnosed with BCP-ALL between October 2009 and June 2015 and treated according to the NOPHO ALL 2008 protocol. Age, white blood cell count (WBC), cytogenetics and day 0 treatment stratification were registered. The expression of intracellular and surface B-lineage markers (CD19, CD20, CD22, nTdT, cyCD79a, and cyCD22), non-lineage (CD45, CD34, CD38, CD10, and nTdT) and cross-lineage expressed markers (CD123, CD66c, CD133, CD13, CD33, and CD15) was scored as negative or positive (dim/normal/bright), and broad or bimodal expression was registered. For bimodal expression the overall score was assigned according to that of the dominant subpopulation. For reference intervals, five non-ALL bone marrow samples with unaffected B-lymphopoiesis were used. The CD34, CD38, nTdT and CD45 median fluorescence intensity was assessed (data will be presented). All samples were run on a FACS Canto and analyzed in DIVA 6.0 software. End-of-induction (day 29) MRD by flow cytometry was used as primary outcome. The best combination of markers was found using a forward selection procedure in a multiple regression analysis. Gene chip analyses were performed in 171 of the 200 patients (human gene 1.0 ST array, Affymetrix) (data will be presented) Results Immunophenotypically distinct subpopulations were common at time of diagnosis (54%) with 19 patients (9.5%) having three or more subpopulations. CD34 was the most commonly bimodal marker (bimodal in 32.7%). We examined the association of the LAIP with MRD, accounting for the frequent bimodal marker expressions in LAIP scoring. Of all markers, CD34 expression had the strongest association with MRD (p<0.0001, linear regression. p=0.0034 adjusting for age, cytogenetic group, treatment arm and WBC). A combination of CD34 (p=0.0005), CD38 (p=0.0288) and nTdT (p=0.0093) (multiple regression) predicted MRD best with a CD34pos/bright, CD38 neg/dim, nTdT neg/dim phenotype associating with higher MRD. In order to exclude marker expression differences related to cytogenetic groups, we further examined patients with no cytogenetic aberrations (n=73). The association of the three markers with MRD was confirmed in this group. We examined whether high CD34 expression was enriched among patients who later relapsed (n=7), and this was the case: CD34 was brightly expressed in 42% vs. 5% in non-relapse patients, further suggesting an association between CD34 expression and prognosis. Discussion By applying a detailed LAIP characterization accounting for subpopulations, we showed that malignant BCP blasts expressing an immature phenotype (CD34pos, CD38dim, nTdTdim), resembling the pre-pro B cell-like phenotype (Sanz et al, PNAS, 2010), seem to be associated with poor treatment response in BCP-ALL. The LAIP might be a useful tool for early treatment stratification in the future. Disclosures No relevant conflicts of interest to declare.

Genetic Analysis of Distinct Phenotypic Subsets within MM1.S Multiple Myeloma Cell Line Reveals the Pre-Existence of MM.1R-like Glucocorticoid Resistance and a Sub-Clone with an Activating PI3-Kinase Delta Mutation That Is Preferentially Sensitive to the Selective PI3-Kinase Inhibitor, Idelalisib

Background: Understanding the pathology of Multiple Myeloma and the testing of therapeutic options has relied heavily on isogeneic cell lines due to the inability to sustain myeloma plasma cells in long-term in vitro culture. The cell lines MM.1S and MM.1R are well recognised in the field of myeloma research, providing a model of glucocorticoid drug resistance, primarily believed to be through variable expression of the glucocorticoid receptor NR3C1 but here we found no evidence of a genetic basis for this. Here we set out to examine the phenotype, function and genotype of the MM.1S and MM.1R cell lines in order to explore the origins of glucocorticoid drug resistance manifested by MM.1R cells and establish whether exome analysis could identify sub-clones with preferential sensitivity to molecular targeted inhibitors.Methods: MM.1S and MM.1R cell lines were purchased from ATCC. A 10-colour flow cytometry panel (CD38, CD138, CD19, CD45, CD56, CD49d, CXCR4, MMP-9, Ki-67, IL-6) was analysed on a BD LSR Fortessa flow cytometer and MM.1S subsets were sorted using a FACS Aria III. Telomere length was assessed using Single Telomere Length Analysis (STELA) and drug toxicity assays using Annexin V-FITC/PI staining. Bioinformatics of whole exome sequencing was carried out on the GATK platform and gene list analysis using Enrichr. PI3K isoforms were analysed by quantitative PCR and immunoblotting.Results: The MM.1S cell line demonstrated bimodal CD38 expression, with a 1.5 log difference in CD38 expression (p<0.0001) between the two populations (termed MM.1Sdim and MM.1Sbright). In contrast the MM.1R cell line was uniformly CD38bright, with expression a further 0.5 log higher than MM.1Sbright. When cell sorted subsets of MM.1S cells were subjected to increasing concentrations of Dexamethasone, the MM.1Sbright cells had a significantly higher LC50 than the MM.1Sdim cells (62nM v 29nM respectively; p<0.0001). In contrast, these subsets showed no significant difference in sensitivity to bortezomib (p=0.84. Furthermore, MM.1Sbright cells had a shorter doubling time than both MM.1Sdim (p=0.0001) and MM.1R (p=0.048). This was underscored by an increased proportion of MM.1Sbright cells in S-phase coupled with shorter mean telomere length when compared with MM.1Sdim and MM.1R (2.58 Kb v 3.29Kb v 3.2Kb respectively). We next subjected purified MM.1Sbright, MM.1Sdim and MM.1R cells to whole exome sequencing. The common clonal origin of the three cell lines was evident from the analysis but each line possessed unique genetic lesions. For example, MM.1Sbright had a FIP1L1-PDGFRA fusion mutation that was not present in the MM.1Sdim cells. This was associated with increased expression of the p110d isoform in MM.1Sdim cells. We therefore analysed the effects of the PI3Kd inhibitor, Idelalisib, on the two cell lines and showed that MM.1Sdim cells were more sensitive (p=0.003) to the effects of this agent. The specific nature of this response was confirmed by the fact that the pan p13K inhibitor PKI-402, was equipotent in both MM.1Sbright and MM.1Sdim cells (p=0.89).Conclusion: Analysis of two phenotypically distinct subsets within the MM.1S cell line revealed differences in function and genetics thereby confirming the sub-clonal architecture within this cell line. Intriguingly, our data point to the pre-existence of a dexamethasone resistant sub-clone the MM.1Sbright (CD38+) population. The subsequent production of the dexamethasone resistant cell line (MM.1R) allowed us to perform comparative genomics thereby identifying the genetic origins of dexamethasone resistance (selection) in MM.1Sbright cells and to track the subsequent clonal evolution (induction) in the MM.1R cells. Furthermore, we showed the potential for developing bespoke treatment plans based on the identification of cell signalling pathway mutations via genomic sequencing. By selective targeting of of these genetic lesions it may be possible to remove multiple sub-clones thereby diminishing the potential for clonal tiding and the development of drug resistance. In theory this could result in longer time to relapse and ultimately improved overall survival. DisclosuresFegan:Roche: Honoraria; Gilead Sciences: Honoraria; AbbVie: Honoraria.

Juxtaposed genes in 7q21-22 amplicon contribute for two major gastric cancer sub-Types by mutual exclusive expression.

Genomic Copy Number Variations (CNV) and the associated gene signatures are useful for cancer prognosis, diagnosis, and targeted therapeutics. Earlier, 7q21-22 region was reported for frequent amplification in gastric cancer and potential candidate genes were identified. An analysis of the expression pattern of the 159 genes located in this amplicon revealed the consistent elevated expression of 21 genes in gastric tumors. These genes are closely arranged within the 20 Mb region, and they showed a bimodal expression pattern. SHFM1 and 14 other genes are expressed in intestinal type gastric tumors. COL1A2 and PCOLCE genes of this region are expressed in diffuse type gastric tumors. Similarly, genome-wide expression neighbors of SHFM1 and COL1A2 also showed mutually exclusive expression pattern, and stratify intestinal and diffuse type gastric tumors. The expression of COL1A2 gene-set is associated with poor prognosis, whereas the SHFM1 gene-set is associated with better prognosis among the gastric cancer patients. Despite being physical neighbors, the SHFM1 and COL1A2 genes express differentially in the two major clinical sub-types of gastric cancer in a mutually exclusive manner. The tight gene regulations operating between these juxtaposed genes deserve investigation to understand the molecular regulatory switch defining the determinants of the gastric cancer sub-types. © 2016 Wiley Periodicals, Inc.

Prognostic outlier genes for enhanced prostate cancer treatment.

To review the current landscape of outlier genes in the field of prostate cancer. A comprehensive review was performed. Prostate cancer continues to be a significant worldwide health issue. In the era of personalized medicine, more emphasis is being placed on the ability to determine the timing, intensity and type of treatment, according to each patient's unique disease. Several commercial tests are available to determine the risk of aggressive prostate cancer based on genomic biomarkers and gene expression. Outlier genes represent a form of cancer classification that focuses on bimodal expression of a gene in a specific subset of patients. Outlier genes identified in prostate cancer include TMPRSS2-ERG, SPINK1, ScHLAP1, NVL, SMC4 and SQLE. Classifying patient prostate cancers by outlier genes may allow for individualized cancer therapies and improved cancer therapy outcomes.

Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation

Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies.

A Novel mTORC1-Dependent, Akt-Independent Pathway Differentiates the Gut Tropism of Regulatory and Conventional CD4 T Cells.

The vitamin A metabolite all-trans retinoic acid (ATRA) induces a gut-homing phenotype in activated CD4(+) conventional T cells (Tconv) by upregulating the integrin α4β7 and the chemokine receptor CCR9. We report that, in contrast to mouse Tconv, only ∼50% of regulatory T cells (Treg) upregulate CCR9 when stimulated by physiological levels of ATRA, even though Tconv and Treg express similar levels of the retinoic acid receptor (RAR). The resulting bimodal CCR9 expression is not associated with differences in the extent of their proliferation, level of Foxp3 expression, or affiliation with naturally occurring Treg or induced Treg in the circulating Treg pool. Furthermore, we find that exposure of Treg to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin suppresses upregulation of both CCR9 and α4β7, an effect that is not evident with Tconv. This suggests that in Treg, ATRA-induced upregulation of CCR9 and α4β7 is dependent on activation of a mTOR signaling pathway. The involvement of mTOR is independent of Akt activity, because specific inhibition of Akt, pyruvate dehydrogenase kinase-1, or its downstream target glycogen synthase kinase-3 did not prevent CCR9 expression. Additionally, Rictor (mTOR complex [mTORC]2)-deficient Treg showed unaltered ability to express CCR9, whereas Raptor (mTORC1)-deficient Treg were unable to upregulate CCR9, suggesting the selective participation of mTORC1. These findings reveal a novel difference between ATRA signaling and chemokine receptor induction in Treg versus Tconv and provide a framework via which the migratory behavior of Treg versus Tconv might be regulated differentially for therapeutic purposes.

Regulation of SATB1 during thymocyte development by TCR signaling.

T lymphocyte development and differentiation is a multi-step process that begins in the thymus and completed in the periphery. Sequential development of thymocytes is dependent on T cell receptor (TCR) signaling and an array of transcription factors. In this study we show that special AT-rich binding protein 1 (SATB1), a T lineage-enriched chromatin organizer and regulator, is induced in response to TCR signaling during early thymocyte development. SATB1 expression profile coincides with T lineage commitment and upregulation of SATB1 correlates with positive selection of thymocytes. CD4 thymocytes exhibit a characteristic bimodal expression pattern that corresponds to immature and mature CD4 thymocytes. We also demonstrate that GATA3, the key transcriptional regulator of αβ T cells positively regulates SATB1 expression in thymocytes suggesting an important role for SATB1 during T cell development.

Abstract 4076: Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells

Abstract Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis. Though functional CSCs can be identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in a dish-based format. Tumor-stromal interaction is another key regulator of cancer micro-environments, affecting tumorigenesis and metastasis. To enable tumorsphere assays that incorporate interaction with stromal cells, we developed a microfluidic dual adherent/suspension co-culture device. This device combined a suspension environment for single-cell tumorsphere assay and an adherent environment for co-culture of stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allowed the use of small samples (&amp;lt;100 cells), affording the potential to study rare cells such as circulating tumor cells (CTCs). As a proof-of-concept, we co-cultured single T47D breast cancer cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-cultured T47D cells, co-cultured T47D have significantly higher tumorigenic potential (sphere formation rate) by three times and proliferation rates (larger sphere size). In addition to the phenotypic observation, the platform allows the retrieval of mono-cultured and co-cultured spheres for downstream analyses. Multiplexed single-cell gene expression analyses using C1 and BioMark HD platforms were performed to compare the expression of 96 target genes in co-cultured and mono-cultured T47D cells. Co-cultured cells expressed significantly higher MKI67, which suggests a possible mechanism for the elevated proliferation rate. Additionally, lower BAX (pro-apoptotic marker) expression matches well with higher sphere formation rate in the co-cultured cells. Interestingly, we noticed the down-regulation of CDH1, KRT8, and KRT18 in the co-cultured cells, indicating possible epithelial-mesenchymal transition induced by CAF. Single cell resolution, while to comparing between groups, also allows examination of cellular heterogeneity within one population. While cells in the mono-cultured group were more homogeneous co-cultured cells displayed clear bimodal gene expression distribution including, ANXA3, MTOR, MKI67, BAX, and PGR. This is suggestive of variable responses to the same stimuli at the single cell level. Combining the dual adherent/suspension co-culture platform presented here with the single cell gene expression analysis, we may successfully identify functional CSCs, investigate the phenotypic and transcriptional effects induced by tumor-stromal interactions, and study cellular heterogeneity in greater detail. Citation Format: Yu-Chih Chen, Shamileh Fouladdel, Zhixiong Zhang, Ebrahim Azizi, Euisik Yoon, Max S. Wicha. Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4076.

Abstract 3870: SLFN11 is a biomarker of sensitivity to PARP inhibition and chemotherapy in small cell lung cancer (SCLC)

Abstract Small cell lung cancer (SCLC) is an aggressive disease that accounts for 14% of lung cancers. Very little progress has been made towards the treatment of SCLC in the past four decades and there are no established biomarkers to predict effective therapies for patients. In other cancers, SLFN11 plays an important role in sensitizing cancer cells to topoisomerase inhibitors, DNA synthesis inhibitors and alkylating agents. Previously, our lab identified an increase in poly (ADP-Ribose) polymerase 1 (PARP1), an enzyme involved in DNA damage repair, in SCLC patients and cell lines. PARP inhibitors demonstrate significant anti-tumor activity in cell line and animal models of SCLC and are currently being tested in clinical trials for SCLC patients. In Ewing sarcoma (EWS), SLFN11 has been proposed as a biomarker of PARP inhibitor response. Because both EWS and SCLC have high PARP levels and respond favorably to PARP inhibition, we hypothesized that SLFN11 may also be a biomarker for drug sensitivity in SCLC. Using SCLC patient tumors and a large panel of molecularly profiled SCLC cell lines, we investigated the expression of SLFN11 in SCLC and its association with in vitro sensitivity to PARP inhibition (olaparib) and chemotherapy. SLFN11 mRNA levels are significantly higher in SCLC patient tumors relative to normal lung tissue (P = 0.005). In a panel of 51 SCLC cell lines, higher SLFN11 protein expression correlates with both cisplatin (P&amp;lt;0.001) and olaparib (PARP inhibitor; P = 0.05) sensitivity. In fact, SLFN11 is the top protein biomarker of cisplatin sensitivity in SCLC cell lines (of 171 proteins measured). Consistent with what has been shown for other cancer types, SLFN11 mRNA expression is also correlated with sensitivity to irinotecan (P = 0.005) and topotecan (P = 0.05) in SCLC, two drugs frequently used to treat this disease. Expression of other SLFN family members does not correlate with drug sensitivity. SLFN11 protein and mRNA levels are concordant (P&amp;lt;0.001) and both demonstrate a bimodal expression pattern in SCLC cell lines which corresponds to cisplatin (protein, P&amp;lt;0.001; mRNA, P = 0.005) and olaparib (mRNA, P = 0.05) sensitive (SLFN11 high) versus resistant (SLFN11 low) groups. In EWS, leukemia, colon, breast and prostate cancers, SLFN11 functions as an ETS transcription factor response gene. However, a panel of 26 ETS transcription factors did not correlate well with SLFN11 or PARP1 mRNA expression or cisplatin sensitivity, suggesting that SLFN11 expression may be regulated in a different manner in SCLC. In summary, SLFN11 is a predictor of sensitivity to chemotherapies routinely used in the treatment of SCLC, including alkylating agents (cisplatin) and topoisomerase inhibitors (irinotecan, topotecan). Moreover, SLFN11 predicts in vitro response to PARP inhibition, which may have important clinical implications given the ongoing clinical trials investigating PARP as a novel therapeutic target for SCLC. Citation Format: C. Allison Stewart, Pan Tong, Robert Cardnell, Triparna Sen, Fatemah Mina Masrorpour, Youhong Fan, Jing Wang, Lauren Averett Byers. SLFN11 is a biomarker of sensitivity to PARP inhibition and chemotherapy in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3870.