GEP Analysis Research Articles

Financial Development Strategies: Defining Objectives and Priorities

In recent years, scientific and practical interest in the strategic management of countries' financial development has grown significantly. However, an analysis of publications on this topic indicates the absence of a methodology to make strategic decisions on the formation of goals and priorities for financial development. The purpose of this study is to develop a methodology for the strategic management of the financial development of countries. The study uses methods of systemic, comparative, GEP analysis, and a rule-based judgment method. The result of the study is the development of a concept that allows defining strategy, quantified strategic goals and priorities for financial development, and its testing in OECD countries. The novelty of the research lies in the systematic approach to the formation of strategic goals and priorities based on the System of National Accounts, the multi-level system of key indicators of financial development, criteria formalizing alternative strategies, and rules for making strategic decisions. This is the first time such a concept has been proposed. The advantages of the developed concept are high representativeness, objectivity, and a wide range of applications. Its use will improve the quality of strategic management of countries' financial development and ensure transparency in government decisions. Doi: 10.28991/ESJ-2023-07-06-09 Full Text: PDF

Co-enrichment of CD8-positive T cells and macrophages is associated with clinical benefit of tislelizumab in solid tumors

BackgroundActivated immune cells (IC) in the tumor microenvironment (TME) are critical for anti-tumor efficacy. Greater understanding of the dynamic diversity and crosstalk between IC is needed to clarify their association with immune checkpoint inhibitor efficacy.MethodsPatients from three tislelizumab monotherapy trials in solid tumors (NCT02407990, NCT04068519, NCT04004221) were retrospectively divided into subgroups by CD8+ T-cell and macrophage (Mφ) levels, assessed via multiplex immunohistochemistry (mIHC; n = 67) or gene expression profiling (GEP; n = 629).ResultsA trend of longer survival was observed in patients with both high CD8+ T-cell and Mφ levels versus other subgroups in the mIHC analysis (P = 0.11), which was confirmed with greater statistical significance in the GEP analysis (P = 0.0001). Co-existence of CD8+ T cells and Mφ was coupled with elevated CD8+ T-cell cytotoxicity, T-cell trafficking, MHC class I antigen presentation signatures/genes, and enrichment of the pro-inflammatory Mφ polarization pathway. Additionally, a high level of pro-inflammatory CD64+ Mφ density was associated with an immune-activated TME and survival benefit with tislelizumab (15.2 vs. 5.9 months for low density; P = 0.042). Spatial proximity analysis revealed that closer proximity between CD8+ T cells and CD64+ Mφ was associated with a survival benefit with tislelizumab (15.2 vs. 5.3 months for low proximity; P = 0.024).ConclusionsThese findings support the potential role of crosstalk between pro-inflammatory Mφ and cytotoxic T cells in the clinical benefit of tislelizumab.Trial registrationNCT02407990, NCT04068519, NCT04004221.

Targeting Free Light Chain Secretion Via Botulinum Neurotoxin Is a Novel Therapeutic Strategy in AL Amyloidosis By Inducing a Terminal Unfolded Protein Response

BackgroundAL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR).Materials and MethodsGene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs.ResultsIFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention.ConclusionsWe show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability. DisclosuresCzarnecki: Clearview: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bianchi: Karyopharm: Consultancy, Honoraria; MJH: Honoraria; Jacob D. Fuchsberg Law Firm: Consultancy; Pfizer: Consultancy, Honoraria.

MicroRNAs as disease specific diagnostic biomarkers for neoplastic aetiology-related and inflammatory-related pericardial fluid effusion

Abstract Background and aim Malignant involvement of the pericardium is seen in 1 to 20 percent of autopsies in patients with cancer. The most common metastatic tumor involving the pericardium is lung cancer [1]. We aimed to distinguish the origin of the pericardial fluid effusion (i.e. pericarditis vs cancer) based on miRNAs expression in peripheral blood plasma. Method 8 patients hospitalized for collection of pericardial fluid with pericardial effusion of neoplastic aetiology (lung cancer). Control group includes 8 patients with effusion of inflammatory aetiology. Plasma RNA was extracted by mirVANAPARISKit and quality of RNA was assessed by fluorometric assay. GEP analysis was performed using the Clariom D pico chips, analysed on the Affymetrix platform. Statistical analysis by TAC software. Additional analyses were performed in and R using Signal information obtained from the TAC output. We performed the following tests using log2 transformed data and all comparison groups (A-F). Additional FDR correction, logistic regression, Mann-whitney t-test was used depending of the variables. We calculated Area under the curve using ROCp R package. Scores were ranging from 0 to 1. Co-expression analysis to identify genes authentically expressed was performed using Spearman correlation (cutoff = 0.9, Rpval = 0.05). In order to identify the targets of DE miRNAs we used our wizbionet R package and previously developed pipelines [2,3]. We performed target screening using multimiR package, selecting top 20% predictions from all available databases. Results We analyzed targets for all mature versions, and if DE miR was identified as pre-miR we generated -3p and -5p version for it. We also screened DisgeNet database for genes associated with cancer and pericarditis, we identified 2823 and 157 such genes. After identification of the targets of DE miRNAs we performed data aggregation, summarization and obtained information how many targets overall and targets associated with IS are regulated by each DE miRNA. Additionally we identified top targets regulated by the top miRNAs. MiR-5695, miR-4446-5p, miR-572, miR-3131 and miR-4784 were found the most significantly differentially expressed miRNAs in blood plasma for patients with malignancy compared to pericarditis. MiR-22-3p, miR-642a, miR-6771, miR-140-3p, and miR660-5p were found the most significantly differentially expressed miRNAs in pericardial fluid plasma for patients with malignancy compared to pericarditis. Importantly, miR-500b, miR-5188, miR-490, miR-24-3p, miR-383-3p were found the most promising differentially diagnostic biomarkers for malignancy and inflammatory-related pericardial fluid effusion. Conclusions For the first time our results indicate the differentially diagnostic power of miRNAs based on comparison of circulating in peripheral blood and pericardial fluid in patients having excessive pericardial fluid effusion due to different etiologies. Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

MicroRNA-223 might be a predictive biomarker for major adverse cardiovascular events prognosis in patients undergoing transcatheter aortic valve implantation procedure

Abstract Background Transcatheter aortic valve implantation (TAVI) is an emerging invasive therapeutic strategy for patients with severe aortic stenosis (AS) significantly enhancing not only quality-of-life measures but also improving short-and long-term survival rates. Purpose For these purposes we aimed to analyze correlation between expression levels of platelet-derived microRNAs in patients with heart failure (HF) due to AS who underwent TAVI procedure and assess their association with primary major adverse cardiac events (MACE) defined by all cause mortality and secondary MACE defined as cardiovascular mortality, ischemic stroke, and non-fatal myocardial infarction. Methods 61 patients before and after the TAVI procedure were included. PlasmaRNA was extracted by mirVANA PARIS Kit and quality of RNA was assessed by fluorometric assay. GEP analysis was performed using the Clariom D pico chips, analyzed on the Affymetrix platform. RT-PCR was performed in order to validate the miRNAs in 61 patients by using the Taqman advanced protocol. MiRNA related to platelet function/antiplatelet treatment were chosen among those with the most relevant modulation between the groups. Wilcoxon test was performed for miRNAs comparison before and after TAVI. Calculations were performed using SPSS version 22.0, p<0.05 (IBM Corporation, Chicago, USA). Results We have found that miR-223, miR-125b and miR-125a were significantly increased in patients after the TAVI procedure. ROC analysis showed that increased miR-223 expression after the TAVI procedure might slightly have protective value against MACE outcome. Conclusions Our analysis showed alteration of circulating miRNAs after the TAVI procedure and miR-223 might have a predictive value for MACE prognosis in patients with HF due to AS who underwent TAVI procedure. Acknowledgment I-COMET research team Funding Acknowledgement Type of funding sources: None. Figure 1Figure 2

OAB-042: Targeting free light chain secretion via Botulinum Neurotoxin is a novel therapeutic strategy in AL amyloidosis by inducing a terminal unfolded protein response

<h3>Background</h3> AL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR). <h3>Methods</h3> Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. <h3>Results</h3> IFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention. <h3>Conclusions</h3> We show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability.

Bitter Taste Receptors Are Expressed on Acute Myeloid Leukemia Cells and Their Activation Results in Quiescence Induction and Prevention of Migration

Acute Myeloid Leukemia (AML) is a clonal disease sprouting from a rare population of leukemic stem cells (LSCs). Over the past years, increasing interest is gaining the contribution that cell-extrinsic factors have in AML generation and maintenance. In this context, AML cell ability to sense the changes in the microenvironment is important for different cell functions, including the responsiveness to antineoplastic agents. Bitter taste receptors (T2Rs) are typical G-protein coupled receptors and are normally found on the surface of the tongue. Recent studies showed that T2Rs are widely expressed in various parts of human anatomy and have been shown to be involved in physiology of respiratory system, gastrointestinal tract and endocrine system thus suggesting a wider function in “sensing microenvironment”. So far, very few data about T2Rs expression in cancer cells are available. In particular, their expression and function in AML cells has not been investigated. Our aim was to investigate T2Rs expression and role in regulation of leukemic cell functionality.In the present work, for the first time, we showed that leukemia cell lines OCI-AML3, THP-1, KG1 and AML primary cells expressed several T2R subtypes (Fig.1A). The expression of TAS2R was associated with GPR-related downstream targets, including the beta subunit of gustducin and the PLC beta 2 (Fig.2B). Stimulation of leukemia cell lines with denatonium (DEN), a T2Rs agonist, induced intracellular Ca2+ concentration increase, thus demonstrating T2Rs functionality (Fig. 1C). To investigate whether bitter compound could modulate AML cell biological properties, we performed GEP analysis of DEN treated cells. GEP analysis identified a number of genes significantly modulated by denatonium treatment. Enrichment analysis identify significantly affected biological processes, among which, cell cycle/proliferation, apoptosis, migration/adhesion and DNA repair (Fig.1D). Specifically, leukemic cells stimulated with T2R agonist underwent down-regulation of genes involved in positive regulation of cell proliferation, migration, and cell-cycle. Whereas genes involved in cell adhesion and DNA repair were up-regulated. Functional assays confirmed molecular data. Indeed, depending on the extent of stimulation, TAS2R activation inhibited leukemia cell proliferation (Fig.2A) inducing cell cycle arrest in G0/G1 phase (Fig. 2B) or reduced cell viability inducing apoptosis (Fig. 2C), as demonstrated by caspase cascade activation and mitochondrial stress induction (Fig. 2D-E). Of note, a pronounced inhibitory effect of denatonium on leukemia cells motility, both spontaneous and in response to CXCL-12, was observed (Fig. 2F).Overall, our results indicate that in AML cells the activation of fully functional T2Rs is associated with quiescence induction and prevention of migration. These data may suggest a role for microenvironment “bitter” molecules in regulating leukemia cell functions, including lethal sensitivity to antineoplastic agents. [Display omitted] DisclosuresMartinelli:Pfizer: Consultancy; Celgene: Consultancy; Ariad/Incyte: Consultancy; Amgen: Consultancy; Johnson&Johnson: Consultancy; Roche: Consultancy.

Suppression of Cdc37 Induces BTZ Resistance through XBP1s Mediated Plasma Cell Immaturation in Multiple Myeloma

Backgrounds: Cell division cycle protein 37 (Cdc37) is considered as a key co-chaperone of heat shock protein 90 (Hsp90), guiding the stabilization and activation of client kinases with time specificity and substrate selectivity. Previous studies have demonstrated that Cdc37 is over-expressed in many kinds of cancers, contributing to the tumorigenesis. However, our previous study suggested that some MM cell population with low expression of Cdc37 was correlated with BTZ resistance in clinical MM samples and in vitro study. The purpose of this study is to investigate how Cdc37 contributes to drug resistance in MM.Materials and methods: Nineteen sequential MM samples with gene expression profiling (GSE19554) obtained from published studies were analyzed. The expression of Cdc37 was investigated in MM cell lines and primary CD138+ cells by qRT-PCR. Cdc37 function was then interrupted by Cdc37 shRNA or Hsp90-Cdc37 inhibitor Celastrol in MM cell line. Sensitivity of BTZ was evaluated. Bone marrow MM cells were enriched and gated into subpopulations by CD38 and CD138 status by FACS. CD38+CD138+ plasma cells, CD38+CD138- plasmablasts and CD38-CD138- B cells were examined for Xbp1s and Cdc37 expression by qRT-PCR and IF. Then cellular morphology, supernatant light chain immunoglobulin and makers of plasma cell maturation were checked when MM cells were exposed to Celastrol or underwent Xbp1s overexpression. Finally, the in vivo verification was carried out in 5TGM1 mouse model.Results: The GEP analysis showed that Cdc37 was down-regulated as the progression of MM (base line, pre-1st transplantation, pre-2nd transplantation, post-2nd transplantation) in 13 out of 19 sequential samples. Our results also showed that some relapsed MM patients had a down-regulated Cdc37 expression compared with newly diagnosed cases (0.78±1.13 vs 0.25±1.05, p <0.05). Moreover, Cdc37 was only suppressed in bortezomib (BTZ) resistant cell line ANBL-6. BR compared with their wild type ANBL-6.WT. While there were no significant differences between doxorubicin-, dexamethasone- resistant cell line. Indeed, suppression Cdc37 in MM cell line by Cdc37-shRNA or Celastrol resulted in a lower apoptosis rate after BTZ treatment. These results indicated that Cdc37 may participate in BTZ resistance.To explore the mechanism of Cdc37 mediating BTZ resistance, we tried to determine if Cdc37 suppression might cause immaturity of plasma cells, which is well established to cause MM cells resistant to BTZ. Cdc37 expression was first found to be positively correlated with Xbp1s in primary MM cells (r=0.61, p <0.001). Then CD38+CD138+ plasma cells, CD38+CD138- plasmablasts and CD38-CD138- B cells were examined for Xbp1s and Cdc37 and showed that Cdc37 and Xbp1s were gradually up-regulated during the maturation of plasma cells in 3/5 patients. The results indicated that Cdc37 may induce BTZ resistancethroughXBP1s mediated plasma cell immaturation.To certify our hypothesis, Cdc37-Hsp90 interaction were interrupted with Celastrol, NCI-H929 cells appeared smaller and rounder with larger nucleus, less cytoplasm, resembling plasmablast. ELISA analysis also demonstrated a decreased secretion of free light chain from MM cells. Partial repression of plasma cell maturation markers, such as Xbp1s, Chop, CD49e were also observed when Cdc37 was inhibited in NCI-H929 cells. However, the above changes were reversed when Xbp1s was overexpressed in MM cells. In vivo, the serum IgG2b level was (146.8±4.0) ng/ml and (166.0±13.0) ng/ml (p<0.05) in BTZ group and BTZ+Celastrol group, respectively. Although statistical significance were not reached, the BTZ+Celastrol group showed a shorter overall survival compared with BTZ group.Conclusion: Our results demonstrated that Cdc37 downregulation may result in BTZ resistance through Xbp1s mediated plasma cell immaturation. These findings provide a certain theoretical reference for the proper combination of BTZ and other therapy, forcing BTZ resistant cells differentiation, to fight against BTZ resistance. More importantly, Cdc37 may provide a diagnostic biomarker for predicting BTZ sensitivity prior to treatment. DisclosuresNo relevant conflicts of interest to declare.

Preclinical Validation of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma

Introduction. Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) both promote tumor growth, survival, and drug resistance, as well as induce decreased T/NK effector cell function and immune suppression. Delineation of the mechanism(s) mediating pDCs-MM-T-NK cells interactions may therefore identify novel therapeutic targets to enhance anti-MM immunity. Using gene expression profiling, we show that pDC-MM interactions trigger significant upregulation of the immunosuppressive metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO-1 functions as a glycolytic enzyme and plasminogen receptor which is overexpressed on the surface of tumor cells and MM pDCs. Here, we utilized our coculture models of patient autologous pDC-T-NK-MM cells to examine whether targeting ENO1, either alone or in combination can enhance anti-MM immunity in the BM milieu. Methods Gene expression profiles of MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (&amp;gt;1.5-fold change was considered significant, CI &amp;gt; 95%). MM cells were co-cultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. Cytotoxic T lymphocyte (CTL) and NK cell activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC1:T/NK10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.2 µM) or anti-ENO1 Ab for 5 days; MM pre-stained cells were added for 24h (10T/NK:1MM), followed by quantification of viable MM cells by FACS. Anti-PD-L1 Ab (5 ug/ml) or HDAC 6 selective inhibitor ACY-241 (0.2 uM) were utilized for combination studies with ENO1i. CD107a expression was quantified in a degranulation assay. Results GEP analysis showed that pDCs induce upregulation of ENO1 transcript in MM (1.8-fold vs MM alone; n = 3; CI &amp;gt; 95%). Protein expression analysis showed that ENO1 is expressed in both pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in MM cells (5-6-fold; n=3; p = 0.003). The ENO+ MM cell population is also increased after pDC-MM cell coculture (3-4-fold vs MM; mean ± SD; n = 3; p = 0.005). Blockade of ENO1 with ENO inhibitor (ENOi) activates pDCs, as evidenced by increase in pDCs maturation/activation markers (CD80/CD83/CD86). Importantly, ENOi restores the ability of pDCs to trigger T cell activation and proliferation (n = 3; p = 0.018). Above all, ENOi increases pDC-induced MM-specific CD8+ CTL activity (p = 0.006), as well as NK cell-mediated cytolytic activity against autologous tumor cells (p = 0.005). Moreover, pDC-mediated MM-specific CD8+ CTL activity was effective even against allogeneic HLA-A2+ U266 MM cells (p = 0.008). Consistent with CTL and NK cell activation, ENO1i increases expression of surface CD107a on CD8+ T and NK cells (p = 0.006 and p= 0.0112, respectively; n = 7). The combination of ENO1i and anti-PD-L1 Ab induces more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (% MM lysis: ENO1i: 34%; ENO1i + anti-PD-L1 Ab: 54%; n = 7; p = 0.0013). Finally, the combination of ENOi and ACY-241 also enhances anti-MM immune responses (% MM lysis: ENO1i: 35%; ENO1i + ACY-241: 54%; n = 8; p = 0.0044). Conclusions Our preclinical data provide the basis for novel immune-based therapeutic approaches targeting immunosuppressive metabolic alpha-enolase enzyme ENO1 to restore anti-MM immunity and improve patient outcome. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Calreticulin Ins5 and Del52 Mutations Impair Unfolded Protein and Oxidative Stress Responses in Hematopoietic Cells

Somatic mutations of calreticulin (CALR) have been described in approximately 30-40% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In order to assess whether and how CALR mutations could affect the physiological CALR protein functions in the ER and thus contributing through other mechanisms to the development of MPNs, we decided to study the role of mutated CALR in K562 cells, devoid of MPL expression. To this end, K562 cells stably expressing either wt CALR or the two most common CALR mutated variants CALRdel52 and CALRins5 were generated via retroviral mediated gene transfer.To identify common signalling pathways modulated by CALR mutants, GEP analysis was performed. Ingenuity Pathway analysis performed on Differentially Expressed Gene (DEGs) revealed that the categories “Unfolded protein response”, “Endoplasmic Reticulum Stress Pathway”, and “NRF2-mediated Oxidative Stress Response” were significantly represented in the list of decreased genes in the comparison mutated vs wt K562. Based on these findings, the ability to respond to ER and oxidative stresses were assessed in K562 carrying either wt or mutated CALR. Our data demonstrated that CALR mutants negatively impact on the Unfolded Protein Response (UPR): in particular, CALR mutations appear to reduce the activation of the pro-apoptotic pathway downstream the UPR, therefore allowing the accumulation of misfolded proteins in the ER and conferring resistance to ER stress-induced apoptosis. Moreover, our results showed that CALR mutations also affect the capability to respond to oxidative stress: K562 cells carrying CALR mutants showed decreased SOD activity coupled to increased ROS intracellular levels, suggesting that CALR mutants impair cell ability to counteract ROS accumulation. Furthermore, cells carrying CALR mutants showed increased levels of DNA damage upon oxidative stress exposure and decreased ability to repair the oxidative DNA damage. We also demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR.Altogether our data suggest a novel MPL-independent role for CALR mutations in the development of MPNs. On one side, by affecting the ability to respond to the ER stress, CALR mutants confer resistance to ER stress mediated apoptosis. On the other side, by affecting cell sensitivity to oxidative stress and reducing the capability to respond to oxidative DNA damage, CALR mutants might lead to genomic instability and tendency to accumulate further mutations. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4952: Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer

Abstract Introduction: AKT and MYC are amongst the most prevalent oncogenes in prostate cancer (PCa). MYC cooperates with activated AKT in pre-clinical, genetically engineered in vivo models accelerating disease progression and increasing resistance to therapy. However, the clinical relevance of phosphoAKT/MYC co-expression in human prostate tumors remains unknown. Experimental Procedures: A prospective cohort (Physicians’ Health Study and Health Professionals Follow-up Study) of 578 men with PCa with a mean 13-year follow up was used. Protein expression of MYC and phosphoAKT (Serine 473) was characterized by immunohistochemistry on tissue microarrays. pAKT and MYC expression was analyzed using the Cri Vectra/InForm multispectral analysis platform. The InForm software package was trained to segment PCa regions only (i.e., no stromal regions were included in the analysis). We used quantile normalization to adjust for the potential batch effects of protein expression staining across each of the tissue microarrays using the limma package R. The continuous score for pAKT and MYC were divided into quartiles and assigned a value of 1, 2, 3 or 4 for low (1), moderate (2 or 3) and high staining (4). Spearman correlations between pAKT, MYC and the clinico-pathological data were calculated. Results: High pAKT and High MYC were co-expressed in 10% of patients. (Quartile 4). Also 10% were negative or weakly positive for both pAKT and MYC (Quartile 1). Only 3% of the High pAKT patients had weak or no expression of MYC and 4% of High_MYC patients had Low pAKT expression. The majority of patients were distributed in the Quartiles 2 and 3 (73%). Importantly, only High pAKT and High pMYC co-expressors were associated with tumor stage (pT3 or pT4) (p = 0.03), high Gleason Score (p = 0.021) and presence of metastatic disease (p = 0.011). GEP analysis showed that the same patients exhibited up-regulation of genes involved in cell invasion/migration capacity and stromal remodeling. Conclusions: Co-expression of AKT and MYC is associated poor clinical outcome in PCa patients. Gene expression profiling suggest that concomitant activation of AKT and MYC results in the activation of pathways associated with an invasive phenotype. Citation Format: Nelma Pértega-Gomes, Svitlana Tyekucheva, Kathryn Penney, Giorgia Zadra, Lorelei A. Mucci, Massimo Loda. Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer. [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 4952.

GEP analysis validates high risk MDS and acute myeloid leukemia post MDS mice models and highlights novel dysregulated pathways.

BackgroundIn spite of the recent discovery of genetic mutations in most myelodysplasic (MDS) patients, the pathophysiology of these disorders still remains poorly understood, and only few in vivo models are available to help unravel the disease.MethodsWe performed global specific gene expression profiling and functional pathway analysis in purified Sca1+ cells of two MDS transgenic mouse models that mimic human high-risk MDS (HR-MDS) and acute myeloid leukemia (AML) post MDS, with NRASD12 and BCL2 transgenes under the control of different promoters MRP8NRASD12/tethBCL-2 or MRP8[NRASD12/hBCL-2], respectively.ResultsAnalysis of dysregulated genes that were unique to the diseased HR-MDS and AML post MDS mice and not their founder mice pointed first to pathways that had previously been reported in MDS patients, including DNA replication/damage/repair, cell cycle, apoptosis, immune responses, and canonical Wnt pathways, further validating these models at the gene expression level. Interestingly, pathways not previously reported in MDS were discovered. These included dysregulated genes of noncanonical Wnt pathways and energy and lipid metabolisms. These dysregulated genes were not only confirmed in a different independent set of BM and spleen Sca1+ cells from the MDS mice but also in MDS CD34+ BM patient samples.ConclusionsThese two MDS models may thus provide useful preclinical models to target pathways previously identified in MDS patients and to unravel novel pathways highlighted by this study.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0235-8) contains supplementary material, which is available to authorized users.

CREB Controls C/Ebpd to Impair Myeloid Differentiation Process in Acute Myeloid Leukemia

Purpose. The cAMP response element binding protein (CREB) is a transcription factor documented to be crucial for normal and neoplastic hematopoiesis. Its overexpression has already been demonstrated to impair myelopoiesis and to aberrantly control cell proliferation, apoptosis and cell cycle progression, both in vitro and in vivo. Its protein overexpression has been found in patients with acute myeloid leukemia (AML) contributing to decrease survival. CREB guides the expression of more than 5000 different targets in a tissue/cell-type specific fashion, and to date its main network towards leukemogenesis remains unknown. Here we aim to identify through which genes CREB triggers AML by using a CREB overexpressing transgenic zebrafish which developed AML in adulthood.Patients and methods. We performed gene expression profile of RNA from the kidney marrow of 14-months old CREB-zebrafish (n=5) and control zebrafish (n=5). Principal component analysis was performed using Partek Genomic Suite software to integrate zebrafish AML signature with human signature found in pediatric AML to find common CREB target genes. AML cell lines were used to validate CREB targets in vitro.Results. By GEP analysis and integration data we found 20 differentially expressed genes in both the zebrafish and human leukemia. Several of them were involved in the myeloid differentiation process, such as JUN, FOS and C/EBPδ. We confirmed that C/EBPδ protein levels highy correlated with phosphoCREB levels in zebrafish tumor as well as in a cohort of 66 pediatric patients with AML (r=0.79). We silenced and overexpressed CREB in AML or healthy bone marrow primary cultures respectively, and found that C/EBPδ was controlled by CREB transcriptional activity. We took advantages of a previously created HL60(CREB-) cell line, where CREB protein levels was absent compared to the control cell line called HL60(CREB+), where CREB was overexpressed. HL60(CREB-) cell line expressed lowered C/EBPδ levels compared to HL60(CREB+). To investigate CREB's role on differentiation program, cell lines were treated with all-trans-retinoic acid (ATRA). The evaluation of CD11b expression revealed that HL60(CREB-) differentiated to a higher extent compared to HL60(CREB+).Enhanced myeloid differentiation was also confirmed by cell morphology examination. Then we silenced C/EBPδ gene in HL60(CREB+) cell line and treated with ATRA, confirming that C/EBPδ lowered expression increased CD11b expression, phenocopying the HL60(CREB-) behavior.Conclusion. These results indicated that C/EBPδ is a CREB target crucial for myeloid differentiation. The new role discovered for CREB proto-oncogene in blocking myeloid differentiation process opens for further opportunities with differentiation agents to cure AML. DisclosuresNo relevant conflicts of interest to declare.

Tosedostat Plus Low Dose Cytarabine Induces a High Rate of Responses That Can be Predicted By Genetic Profiling in Elderly AML

Background: Older (age ≥60 years) patients with acute myeloid leukemia (AML) have poor outcomes and intensive induction chemotherapy is frequently unsuitable. Thus, new safe and effective therapies are urgently needed. Tosedostat, a new, orally bioavailable inhibitor of members of the M1 and M17 classes of aminopeptidases, was proven to be effective in both de novo and relapsed AML. We hypothesize that the addition of tosedostat to cytarabine may improve the response rate and remission duration over what is expected with chemotherapy or tosedostat alone.Methods: This was a phase II, prospective, multicenter study, designed according to Fleming's method. Fixing the lowest acceptable rate as 10% and the successful rate as 25%, with a significance level alpha=0.05 and a power 1-beta =0.80, the sample size was estimated in 33 patients. Thirty-three patients (median age 75 years) received Tosedostat 120 milligrams orally once daily until disease progression, coupled with intermittent low-dose cytarabine given subcutaneously at 20 milligrams twice/day for 10 days. Courses of cytarabine were repeated every 4 weeks in the absence of disease progression or unacceptable toxicity, up to 8 cycles. Global gene expression profiling (GEP, Affymetrix Human Gene 2.0 Array) was performed on purified AML blasts of 29 patients from peripheral blood or bone marrow at diagnosis before treatment initiation. Unsupervised clustering was generated using a hierarchical algorithm based on the average-linkage method. Principal component analysis (PCA) and supervised gene expression analysis was performed by using GeneSpring GX 12.0 (Agilent, USA).Results: The characteristic of enrolled patients are listed in table 1 Induction-period mortality was 12%, with 4 deaths occurring in aplasia. According to intent-to-treat, the CR rate was 48.5% (16/33 patients); 2 additional patients obtained a partial response, for an overall response rate of 54.6%. In addition, 4/33 patients remained in stable disease for a median time of 9 months (range: 4-14). Seven patients did not respond and died with progressive disease after having received a median of 2 cycles of cytarabine and 45 days of tosedostat. In responding patients, the median time to best response was 74 days (range 22-145). Responding patients (CR+PR) had a longer median overall survival than non-responders (P=0.018). Six out of 18 (33%) responding patients are still in CR after a median follow-up of 425.5 days (range 208-758); 5 additional patients are alive with stable disease. Twenty-two patients died [while in aplasia (4), in CR (1), due to resistant disease (9) or due to progressive disease (8) after a median CR duration of 192 days (87-535)]. 29 patients had GEP analysis, and a molecular signature associated with the clinical response (CR vs. no CR) was identified. By supervised analysis, 212 genes differentially expressed based on the clinical response (complete remission (CR) vs no CR) were identified (Mann-Whitney, p<0.05; fold change >2). The 212 genes differentially expressed were significantly associated with six relevant biological functions and pathways: β-catenin (βcat); TNFα signaling pathway via NFκB; ERB2; STK33/SKM (serine/threonine kinase 33 expression using the SKM cell line); inflammatory response; and epithelial-mesenchymal transition pathways.Conclusions: The tosedostat and low-dose cytarabine combo produced a CR rate superior to what expected (45.4% versus 25%), and thus met the primary endpoint of study. Further, potential biomarkers were identified by GEP. Specifically, the achievement of CR could be efficiently predicted by the gene expression patterns with an overall accuracy exceeding 90%. A validation analysis is currently being conducted on additional 14 patients in order to confirm the ability of GEP to identify potential responders to TST. The study was registered at European Union Drug Regulating Authorities Clinical Trials (EudraCT) n.2012-000334-19.Acknowledgments: CTI is gratefully acknowledged for providing Tosedostat for the patients. The study was supported in part by AIL Pesaro Onlus.Table 1CharacteristicN = 33Median age, years (range)75 (62-85)Median WBC count, × 109/L (range)3.05 (0.26-24.53)Blasts, % (range)60 (20-96)Cytogenetic risk group*, nNot evaluable3 (9%)Intermediate Karyotype17 (52%)Unfavourable Karyotype13 (39%)AML, nDe novo16 (48%)Secondary, n17 (52%) DisclosuresFanin:Novartis Farma: Speakers Bureau.

Suppression of Cdc37 Induces Bortezomib Resistance through Autophagy Activation and Plasma Cell Immaturation in Multiple Myeloma

Introduction: Cell division cycle protein 37 (Cdc37) is considered as a key co-chaperone for heat shock protein 90 (HSP90) to guide the stabilization and activation of protein kinases, which is critical for cell signaling transduction, proliferation and survival. Our previous study suggested that the population of low expression of cdc37 was correlated with bortezomib resistance in clinical MM samples and in vitro study. The purpose of this study is to investigate how cdc37 contributes to drug resistance in MM.Material and method: Nineteen sequential MM samples with gene expression profiling (GSE19554) obtained from published studies were analyzed. The expression of cdc37 was investigated in MM cell lines and primary purified CD138+ tumor cells by qRT-PCR. Cdc37 was down-regulated by lentivirus in MM cell line NCI-H929. Sensitivity of bortezomib was evaluated by CCK-8 and apoptosis analysis. Autophagosome was detected by electron microscope. Bone marrow MM cells from MM clinical samples were enriched by negative selection for CD2 (T and NK cells) and CD14 (monocytes) using FACS. Tumor cells were gated into subpopulations by CD38 and CD138 status. CD38+ CD138+ plasma cells, CD38+ CD138- plasmablasts and CD38- CD138- B cells were examined for xbp1s and cdc37 protein expression by IF.Results: The GEP analysis showed that cdc37 was down-regulated as the progression of MM (base line, pre-1st transplantation, pre-2nd transplantation, post-2nd transplantation) in 13 out of 19 sequential samples (Data base GEP). Cdc37 was suppressed in bortezomib resistant cell line ANBL-6. BR compared with their wild type ANBL-6.WT. While there were no significant differences between doxorubicin-, dexamethasone- resistant cell line compared with their counterparts. Primary tumor cells from five relapsed MM patients after bortezomib treatment also showed low expression of cdc37 compared with five MM samples that were sensitive to bortezomib treatment. Indeed, suppression cdc37 in MM cell line NC-H929 by shRNA lentivirus (cdc37-shRNA) resulted in a higher IC50 of bortezomib, and lower apoptosis rate after bortezomib treatment compared with scramble. These results indicated that cdc37 may participate in bortezomib resistance in MM samples and in vitro.Autophagy has recently emerged as a new mechanism involved in bortezomib resistance. Our results also showed that autophagy associated proteins Beclin-1 and LC-3b were increased in ANBL-6.BR compared with ANBL-6.WT. Besides, Beclin-1 and LC-3b were up-regulated in NCI-H929- cdc37 shRNA cells. What's more, there were more autophagosomes in NCI-H929- cdc37 shRNA cells detected by electron microscope. Furthermore, combinatorial application of autophagy inhibitor chloroquine and bortezomib would partially rescue bortezomib resistance after cdc37 down-regulation.Moreover, cdc37 expression was also found to be positively correlated with xbp1s in 62 newly diagnosed, 25 relapsed and 18 remission MM patients. Xbp1s, CHOP and BLIMP1, which were essential for the plasma cell differentiation and maturation, were also lower expression in 5 bortezomib resistant primary MM patients compared with the sensitive ones detected by qRT-PCR. Then CD38+ CD138+ plasma cells, CD38+ CD138- plasmablasts and CD38- CD138- B cells were examined for xbp1s and cdc37 protein expression by IF and showed that cdc37 and xbp1s were gradually up-regulated during the maturation of plasma cells in 3/5 patients. The results indicated that cdc37, together with xbp1s, may induce bortezomib resistance through plasma cell immaturation.Conclusion: Our results demonstrated that cdc37 downregulation may result in bortezomib resistance through compensatory increasing of autophagy level and the immaturation of plasma cells. Further study will be focused on how cdc37 interacts with xbp1s to participate in autophagy and promotion plasma cell maturation in MM. DisclosuresNo relevant conflicts of interest to declare.

Gene Expression Profiling of Extramedullary Disease-Related Toward Identification of a Terminal Disease Pathway in Multiple Myeloma

IntroductionExtramedullary disease (EMD) is a primary disease manifestation of MM, which while not seen frequently at presentation increases in incidence at relapse where its incidence seems to be increasing following the introduction of novel agents. Patients with EMD have a shorter overall survival as well as an increased incidence of anemia, thrombocytopenia, elevated serum lactate dehydrogenase, cytogenetic abnormalities, and high-risk features as determined by gene expression profiling. There is also an increased incidence of the high risk MAF subtypes t (14:16 or 14; 20). Understanding the biology of EMD and identifying its present could give important information about how to improve the outcome of this group. In this work we have used GEP analysis of bone marrow derived plasma cells to predict the presence of EMD so that we can identify the genomic risk factors that define the features of a plasma cell clone, which can develop the capacity to metastasize outside the BM.Materials and MethodsWe focused on patients treated on TT protocols, at the UAMS, Myeloma Institute between 1989 - 2010, a total of 1154 patients, of which 46 developed EMD before the start of therapy (EMD-1), and 91 developed EMD after registration to UAMS for MM treatment EMD-2.ResultsWe show that most EMD2 cases (57.14%) develop within 3 years after initiation of therapy at the UAMS with few cases developing after this time.Predicting the risk of EMDCombining patients with EMD1 and EMD2 diagnosis within 3 years gave a total of 98 EMD cases. We used 824 samples from 1017 myeloma patients who never developed EMD and had follow up at least 3 years as a comparator group. The data were divided into training (n=619 with 66 EMD cases and 553 controls) and test sets (n=303 with 32 EMD cases and 271 controls). Using the training set, we identified 5 significant gene probes (with a q value < 0.001) and made a score to predict cases and controls. The sensitivity and specificity turned out to be 74.24% and 77.40% in the training set, and 56.25% and 76.75% in the test set, respectively.Predicting the time to EMD2We tested whether we could predict time to EMD2 based on using baseline GEP samples. In this analysis, all EMD2 cases and controls were included. We divided the data into training (n=743 with 61 EMD2 and 682 controls) and test sets (n=365 with 30 EMD2 and 335 controls). By fitting a uniform Cox regression model to each gene in the training set, we identified 68 gene probes that are associated with time to EMD2 (with a q-value <0.1). We then created a score based on the 68 gene probes and identified an optimal cutoff based on the training set. Applying the optimal cutoff to both training and test sets, we found that the new 68-gene high/low risk model is a good predictor on the cumulative incidence of EMD2 (p value < 0.0001).ConclusionWe show that EMD2 cases mostly occur within 3 years of diagnosis and a 68 gene based risk score that can predict a cumulative incidence of EMD. Of the 68 genes that are used to develop the prognostic score for EMD, 6 genes are also part of the 70-gene risk score developed by our group. GEP studies can help us identify EMD-specific gene signature that can further help develop target agents. [Display omitted] DisclosuresWaheed:University of Arkansas for Medical Sciences: Employment. Wang:Cancer Research and Biostatistics: Employment. Qu:Cancer Research and Biostatistics: Employment. Heuck:Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Celgene: Consultancy. Matin:University of Arkansas for Medical Sciences: Employment. Jethava:University of Arkansas for Medical Sciences: Employment. van Rhee:University of Arkansa for Medical Sciences: Employment. Hoering:Cancer Research and Biostatistics: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Davies:University of Arkansas for Medical Sciences: Employment; Millenium: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Morgan:Weismann Institute: Honoraria; University of Arkansas for Medical Sciences: Employment; CancerNet: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Differentially Expressed Protein Patterns in Chronic Lymphocytic Leukemia (CLL) after Thymosin beta4 (Tb4) and Lenalidomide (Len) Treatment: Two-Dimensional Gel Electrophoresis (2DE) Analysis

Proteomic approaches are commonly secondary to genetic studies but are essential in the multi-disciplinary field of hematological research. As opposed to mRNA microarray data, proteomics provides a better understanding of which proteins are actually expressed, although the identification of specific proteins remains challenging (Unwin et al Blood Rev 2014; Boyd J Proteomics 2010). In neoplastic hematology such as CLL, protein studies have contributed to the elucidation of disease mechanisms, defined prognostic or therapeutic biomarkers (Boyd J Proteomics 2010). In this study we used proteomics and 2DE analysis to evaluate differential protein expression patterns after treatment with Len. Len can improve immune dysfunction in CLL by repairing F-actin polymerization and signaling at the immunological synapse (Ramsay et al 2008 J Clin Invest). Our previous data obtained from MALDI-TOF analysis identified Tβ4, a G-actin sequestering protein involved in the regeneration of injured tissues and cell migration, as a downregulated protein in CLL patients, also confirmed by an independent GEP analysis comparing B-cell from CLL cases (n=80) and normal controls (n=6), supported by Tβ4 mRNA down-regulation in CLL (3604±1244 vs 5715±1004, respectively; mean±SD; p=0.001). Here, we investigated whether purified B-CLL cells respond differently to the chemoattractant SDF1a and whether different protein expression patterns can be identified after exogenous Tβ4 and Len treatment using 2DE analysis.Highly purified B-CLL lymphocytes were isolated from untreated Binet stage A CLL patients prospectively enrolled from diagnosis (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540) and healthy controls. Tb4 was identified by MALDI TOF using 100 patient samples. Next, cells were pre-treated with Len (5uM) and then treated with Tb4 (100nM) for 30min. Cells were plated in transwells using 5.0 um pores with SDF1a as chemoattractant for migration assays. Protein was extracted from CLL cell pellets by RIPA buffer and quantified. Sample preparation and 2DE was performed as described by Scielzo et al (J Clin Inves, 2005). Protein samples (100 ug) were applied to 7-cm IPG strips, pH 3-11NL (Amersham Biosciences), respectively, by in-gel rehydration. Isoelectric focusing was performed with a Protean i12 IEF system (Biorad). Strips were equilibrated and loaded onto 9-16% gradient acrylamide SDS-PAGE gels for the second dimension separation. Silver nitrate staining (Sinha P et al Proteomic, 2001) was used to visualize proteins and images were digitally acquired (ChemiDoc MP, Biorad) and spots were analyzed using PDQuest basic 2D Gel Analysis Software (Biorad).CLL samples with the lowest Tβ4 expression (n=12) also had higher F-actin levels as evaluated by FC analysis than normal controls. B-CLL strongly responded to the migratory stimulus SDF-1a, which was further increased (by 20%) in presence of Len treatment, likely due to an alteration in actin remodeling and changes in the expression of unknown proteins. Purified CD19+CD5+ leukemic cells were lysed and proteins resolved on 2DE and visualized by silver nitrate staining. The protein profile analysis on silver-stained gels showed a number of protein spots ranging from 18 to 60kDa that were differentially expressed with respect to untreated cells. Our preliminary qualitative analysis suggests that there are groups of proteins with a lower expression in the presence of Tβ4 or Len, which are more strongly inhibited following exposure to their combination. Conversely, an opposite pattern of protein expression was observed whereby an additive effect on protein expression was observed by combined exposure to Tβ4 and Len. This approach allowed us to identify an altered protein expression pattern after treatment with Len and Tβ4, and may be useful to identify changes in expression profiles of CLL proteins, which may translate into functional differences in the malignant clone. DisclosuresNo relevant conflicts of interest to declare.

The Outcomes and Next Landscapes of Project HOPE (High-Tech Omics-Based Patient Evaluation) for Hepatocellular Carcinoma

Introduction: Project HOPE (High-tech Omics-based Patient Evaluation) aims to provide the multiomics data of cancer patients for the promotion of individualized medicine and the practice of prophylaxis. To reveal the relationship between the clinical data and genetic expression, genetic analyses based on the results of project HOPE were performed for 24 patients who underwent hepatectomy for hepatocellular carcinoma (HCC). Methods: Fresh surgical specimens obtained from HCC patients who underwent surgery at the Shizuoka Cancer Center Hospital, were analyzed by whole-exon sequencing (WES, Life Technologies) and gene expression profiling (GEP, Agilent Technologies). As of April 1, 2015, 24 patients have been analyzed, and the aberrant genes were identified from the results of the WES and GEP analyses. Results: The present study included 17 males and 7 females, with a median age of 67 years (range: 42-81 years). Six of the patients were positive for hepatitis B surface antigen, 9 were positive for hepatitis C antibody and 9 patients were negative for hepatitis virus infection. The WES analysis showed that specific single nucleotide variants (SNVs) were identified in the genes of CTNNB1 and TP53 at a frequency of 15% and 11%, respectively. The GEP analysis revealed the upregulation of the HIST1H3B (59%), EZH2 (33%) and MYCN (22%) genes in tumor tissue (in comparison to normal tissue), while the expressions of CDH1 (11%) and SOCS1 (11%) were found to be downregulated in tumor tissue. In the analysis of the association between the clinical data and gene expression, the rate of advanced tumor staging (UICC stage > 2) was significantly higher in the patients with upregulated HIST1H3B expression in tumor tissue than in those with normal HIST1H3B expression (57.1% vs. 10%, P=0.033). Moreover, the relapse-free survival rate of the patients with upregulated HIST1H3B expression of tumor tissue was significantly lower than that of patients with normal HIST1H3B expression (estimated median relapse-free survival time: 5.8 vs. 7.7 months; P=0.009). Conclusion: Our genetic analysis based on the results of Project HOPE revealed HIST1H3B to be novel cancer-related gene and a predictive factor for HCC. In the future, we plan to accumulate the results of 150 HCC patients, and it is hoped that the additional novel cancer-related genes will be identified.

Abstract 5308: Distinct expression, activity, regulation and gene expression signature of NF-κB subunit c-Rel and the prognostic impact of crosstalk between p53, p63 and c-Rel in different subsets of diffuse large B-cell lymphoma

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous aggressive non-Hodgkin lymphoma and can be classified into GCB or ABC DLBCL. Aberrant activation of NF-κB has been associated with tumor proliferation and survival ABC-DLBCL. The “canonical” pathway of NF-κB activation involves activities of IκB kinase IKK2, which lead to degradation of NF-κB inhibitors IκBα/β/ϵ and nuclear translocation of p50/p65 and p50/c-Rel dimers from the cytoplasm. Objective: To investigate the common mechanisms for c-Rel activation and function, and to evaluate the prognostic significance of c-Rel in a large group of DLBCL patients (n = 460). Results: Nuclear c-Rel expression (c-Rel+) was observed in 29.6% of DLBCL patients and correlated with higher expression levels of p50 (P = 0.12), p52 (P = 0.724E-8), p65 (P = 0.073), RelB (P = 0.013). Coimmunoprecipitation analysis using nuclear extract purified from primary GCB and ABC cells showed that in addition to the known p50 to form dimers with c-Rel, p65, p52 and RelB could also form dimers with c-Rel in some cell lines. A unique function of c-Rel in terminating NF-κB as negative feedback of IKK2 activation was suggested by the significantly upregulated genes encoding IκBα/β/ϵ, A20 and TNIP1 at the mRNA level in either GCB- or ABC-DLBCL. The B-cell receptor signaling (BCR) appeared most relevant for c-Rel activation in GCB-DLBCL, with upreglated B-cell receptors (CD19, CD20, CD79A) and downstream LYN, SYK, CARD11, MALT1, BLNK, TAK1. The role of c-Rel in cytokine production was validated in our study cohort, showing significant correlation between c-Rel+ and mRNA levels of IL1, IL3, IL6, IL10, IL12, IL21 and chemokine receptor CXCR4, mostly in GCB-DLBCL. GEP analysis showed enrichment in cytokine, growth factors, inflammation, gene expression, metabolism, actin, collagen, cell morphology and adhesion. c-Rel did not correlate with prognosis in overall DLBCL. However, c-Rel+ patients with MUT-p53 had significantly worse survival compared with other MUT-p53 DLBCL patients, especially in in ABC-DLBCL, in which c-Rel+ was associated with upregulated MUT-TP53 transcription (P = 0.0087). Conversely, MUT-p53 expression was associated significantly with upregulated REL mRNA expression (P = 0.0021), predominantly in the ABC-DLBCL subtype (P = 0.0047). Furthermore, the lack of c-Rel prognostic impact is likely due to the antagonism of p63, whose expression was correlated with c-Rel nuclear expression, and significantly better OS and PFS in ABC-DLBCL. Conclusions: Unique function and upstream activation regulation of c-Rel was suggested by analysis in 460 DLBCLs. TP53 mutation status appears to a critical determinant for c-Rel prognostic impact. p63 is another tumor suppressor for the c-Rel oncogenic function. Citation Format: Zijun Y. Xu-Monette, Ken H. Young. Distinct expression, activity, regulation and gene expression signature of NF-κB subunit c-Rel and the prognostic impact of crosstalk between p53, p63 and c-Rel in different subsets of diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5308. doi:10.1158/1538-7445.AM2015-5308

PRIMA-1 (p53 Reactivation and Induction of Massive Apoptosis) Mediates Anti-Myeloma Effect through the ER Stress Pathway

It is well established that 17p13(del) and p53 mutations represent poor prognosis in MM patients. We have previously shown that p53 is a haploinsufficient tumor suppressor in myeloma and p53 expression level is correlated with functional status of the pathway. In addition, p53 expression levels do not always correlate with p53 genotype. The absence of p53 expression in myeloma cells severely abolishes the integrity of its downstream signaling response, thus, driving disease aggressiveness and drug resistance. There is a pressing need for a more effective treatment strategy for high-risk patients harbouring p53 abnormalities. PRIMA-1 is a first-in-class drug that was identified to specifically kill cancer cells harboring mutant p53. It has shown substantial efficacy in various solid tumors with mutant as well as non-mutant p53. However, the discovery of the anti-myeloma activity of PRIMA-1 is still at its infancy. By utilizing human myeloma cell lines (HMCLs) of different p53 status as our study model, we aim to identify the efficacy of PRIMA-1 in MM and to dissect the mechanisms behind PRIMA-1-induced toxicity. Despite its known ability to reactivate mutant-p53 function, we did not observe any restoration of mutant p53 transcriptional activities. The expression levels of the p53-direct-targets, p21, MDM2 and PUMA, were unchanged upon treatment in the p53-mutant-HMCLs. In fact, PRIMA-1 was found to be able to decrease the cell viability of all the HMCLs, irrespective of their p53 status (Mutant, WT, Null), strongly indicating a p53-independent response. Importantly, PRIMA-1 showed the highest cytotoxicity in a p53-deficient background (IC50 of 16uM in -/-, 55uM in WT/WT and 70uM in mutant HMCLs). This finding was validated when p53 silencing in a WT HMCL significantly enhanced its sensitivity to PRIMA-1. GEP analysis on the two most sensitive cell lines (JJN3 and KMS11) demonstrated significant enrichment of genes related to unfolded (p=1.90E-06 in JJN3 and 6.12E-08 in KMS11) and misfolded protein (p=2.99E-06 in JJN3 and 8.21E-08 in KMS11) responses. Subsequent validation by Western blot and qPCR confirmed the activation of ER stress pathway in response to PRIMA-1, evidenced by the up-regulation of ER stress markers such as GRP78, GADD34 and CHOP. Further investigations then revealed that the PERK/CHOP axis was most important as CHOP knockdown saw an attenuation of MM cells sensitivity to PRIMA-1-induced apoptosis. Recently, it was reported that cells with increased ER stress are more sensitive to bortezomib. Indeed, we showed that PRIMA-1 synergizes with bortezomib, and was able to rescue bortezomib resistance when both compounds were co-incubated in a bortezomib-resistant-HMCL. In conclusion, we identified a novel pathway by which PRIMA-1 exerts its toxicity in MM through induction of ER stress. This may be a novel therapeutic strategy to induce ER stress to sensitize cells to proteasome inhibitor. Owing to the versatility of the drug in targeting myeloma cells with or without p53 abnormalities; PRIMA-1 can potentially be used alone or in combination with other drugs such as bortezomib in a broad range of MM including those with high-risk disease. DisclosuresNo relevant conflicts of interest to declare.