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Jianjun Wang,1,* Yuchen Li,2,* Binbin Xu,3,* Jiao Dong,1 Haiyan Zhao,1 Dongxia Zhao,1 Yong Wu4 1Department of Pediatric Medicine, Gansu Provincial People’s Hospital, Lanzhou City 730000, Gansu Province, People’s Republic of China; 2Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, People’s Republic of China; 3Department of Ophthalmology, Eye, Ear, Nose, and Throat Hospital, Fudan University, Shanghai, People’s Republic of China; 4Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianjun WangDepartment of Pediatric Medicine, Gansu Provincial People’s Hospital, Lanzhou City 730000, Gansu Province, People’s Republic of ChinaEmail jianjunwang211@163.comYong WuDepartment of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of ChinaEmail edison-1016@163.comBackground: While RNA-binding proteins (RBPs) are known to affect RNA homeostasis during cancer cell initiation and development, their characteristics and biological function in glioblastoma (GBM) remain unclear.Methods: Differences in RBP expression were explored by differential analysis of The Cancer Genome Atlas-GBM and Genotype-Tissue Expression (GTEx) datasets. Real-time PCR was conducted to verify the expressional levels of Aly/REF export factor (ALYREF) in normal brain and GBM tissues. Proliferative assays were performed to investigate molecular functions of ALYREF in GBM cells in vitro and in vivo. Real-time PCR and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the ALYREF downstream signaling pathways. A chromatin immunoprecipitation (ChIP) assay was performed to identify key transcriptional factors that regulate ALYREF expression at RNA level. UV crosslinking, immunoprecipitation (CLIP) and RNA stability assays were conducted to reveal the bound RNAs and their stability regulated by ALYREF.Results: The results showed that ALYREF is frequently increased in GBM tissues, and its mRNA expression is regulated by the MYC proto-oncogene, bHLH transcription factor (MYC). Inhibition of ALYREF expression decreased GBM cell proliferative ability in vitro and tumor formation in vivo. KEGG analysis revealed that high ALYREF expression in GBM tissues was enriched in the upregulation of oncogenic pathways such as the Wnt/β-catenin signaling pathway. The CLIP assay showed that ALYREF drives GBM carcinogenesis by binding to and stabilizing MYC mRNAs. Overexpression of MYC restored the oncogenic property of ALYREF-deficient GBM cells.Conclusion: Our data showed that ALYREF is regulated by MYC at the transcriptional level. ALYREF drives GBM cell proliferation by activating the Wnt/β-catenin signaling pathway and stabilizing MYC mRNA, suggesting that an ALYREF-MYC positive feedback loop might be a potential therapeutic target for treating GBM patients.Keywords: RNA-binding protein, MYC, cell proliferation, GBM

Lung adenocarcinoma (LUAD) is the main histological type of lung cancer, which is the leading cause of cancer-related deaths. Long non-coding RNAs (lncRNAs) were recently revealed to be involved in various cancers. However, the clinical relevance and potential biological roles of most lncRNAs in LUAD remain unclear. Here, we identified a prognosis-related lncRNA ITGB1-DT in LUAD. ITGB1-DT was upregulated in LUAD and high expression of ITGB1-DT was correlated with advanced clinical stages and poor overall survival and disease-free survival. Enhanced expression of ITGB1-DT facilitated LUAD cellular proliferation, migration, and invasion, and also lung metastasis in vivo. Knockdown of ITGB1-DT repressed LUAD cellular proliferation, migration, and invasion. ITGB1-DT interacted with EZH2, repressed the binding of EZH2 to ITGB1 promoter, reduced H3K27me3 levels at ITGB1 promoter region, and therefore activated ITGB1 expression. Through upregulating ITGB1, ITGB1-DT activated Wnt/β-catenin pathway and its downstream target MYC in LUAD. The expressions of ITGB1-DT, ITGB1, and MYC were positively correlated with each other in LUAD tissues. Intriguingly, ITGB1-DT was found as a transcriptional target of MYC. MYC directly transcriptionally activated ITGB1-DT expression. Thus, ITGB1-DT formed a positive feedback loop with ITGB1/Wnt/β-catenin/MYC. The oncogenic roles of ITGB1-DT were reversed by depletion of ITGB1 or inhibition of Wnt/β-catenin pathway. In summary, these findings revealed ITGB1-DT as a prognosis-related and oncogenic lncRNA in LUAD via activating the ITGB1-DT/ITGB1/Wnt/β-catenin/MYC positive feedback loop. These results implicated ITGB1-DT as a potential prognostic biomarker and therapeutic target for LUAD.

MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3-lysine 27-tri-methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high-MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY-MYC, UW228-MYC, CB-MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY-MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.

Objectives The latest revision of lymphoma’s World Health Organization classification describes the new provisional entity “Burkitt-like lymphoma with 11q aberration” (BLL, 11q) as lacking MYC rearrangement, but harboring the specific11q-gain/loss aberration. We report genetic characteristics of 11 lymphoma cases with this aberration. Methods Classical cytogenetics, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism/array comparative genomic hybridization. Results The 11q aberrations were described as duplication, inversion, and deletion. Array comparative genomic hybridization showed two types of duplication: bigger than 50 megabase pairs (Mbp) and smaller than 20 Mbp, which were associated with bulky tumor larger than 20 cm and amplification of the 11q23.3 region, including KMT2A. Six cases revealed a normal FISH status of MYC and were diagnosed as BLL,11q. Five cases showed MYC rearrangement and were diagnosed as Burkitt lymphoma (BL) or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). Conclusions The 11q-gain/loss is not specific for BLL, 11q, but occurs recurrently in MYC-positive BL and MYC-positive HGBL.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Within ALL, precursor B-cell disease predominates and now has survival >90%. Mature B-cell, or Burkitt leukemia/lymphoma, is distinct from ALL and requires short intensive chemotherapy and with the addition of Rituximab, survival rates of >95% are achieved. Its defining characteristic is MYC translocation at 8q24. Patients who have features of both ALL and Burkitt leukemia/lymphoma represent a rare subpopulation of ALL and present a diagnostic and treatment conundrum. We have performed a systematic review on the occurrence of and treatment of MYC positive precursor B-ALL, reported between 1980 and 2016. The review highlighted a lack of data to guide any consensus about how to treat this important group of children and focused research in this area is needed.

Background Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a malignant primary cutaneous T-cell lymphoma that shares significant clinical, histopathologic and immunophenotypic overlap with lupus erythematosus panniculitis (LEP). Methods We performed immunohistochemistry for the MYC oncoprotein on 23 cases of SPTCL (1 CD8 negative) and 12 cases of LEP to evaluate if there are quantitative or qualitative differences in protein expression of this marker in these entities. Results In SPTCL cases, the percentage of all cells that were c-Myc positive ranged from 0.8% to 16%, with a mean of 5.0% and a median of 4.4%. In contrast, in the LEP cases, the percentage of c-Myc-positive cells in the cases ranged from 0.34% to 3.7%, averaged 1.4% and the median was 0.8%. The difference between the means of these 2 diagnostic categories was statistically significant. Fluorescence in situ hybridization performed on 4 cases of SPTCL with a relatively high level of MYC immunohistochemical staining, however, failed to demonstrate evidence of MYC rearrangement or amplification. Conclusions Our work demonstrates that MYC expression levels differ between these 2 histologic mimics and suggests that this important oncoprotein may play a role in the pathogenesis of SPTCL.

Acute myeloid leukemia (AML) with myelodysplasia-related changes (AML-MRC) is shown to be associated with dismal clinical outcomes and there remains a pressing need for new therapeutic strategies to improve survival outcomes. It is also important to identify targetable markers that could predict an earlier AML transformation from preexisting MDS. Our recent studies demonstrated that high expression level of MYC oncoprotein is associated inferior survival outcome in AML-MRC patients and that MYC plays an oncogenic role by epigenetic regulation of hydroxylation of 5-methylcytosine. However, the role of MYC in MDS to AML progression remain to be answered. Here, we investigated MYC protein expression levels in bone marrow (BM) specimens of patients with preexisting MDS and their subsequent AML-MRC specimens to determine whether increased MYC expression is associated with early AML progression. We retrospectively identified 32 patients with histologically confirmed AML-MRC evolving from MDS and evaluated MYC protein levels in BM biopsy specimens at the time of initial MDS diagnosis and at the time of disease transformation to AML-MRC, respectively. Clinical data including age at diagnosis, gender, CBC with differential count, cytogenetics, somatic mutations common in myeloid disease, blasts count, and IPSS were extracted at the time of MDS and AML-MRC diagnosis. MYC expression was assessed by immunohistochemistry on 4-5µm tissue sections. Deparaffinized slides were stained with anti-MYC antibody (clone Y69, Roche Diagnostics) using a Ventana Benchmark automated system. MYC expression was scored independently by two hematopathologists and categorized as low vs. high as previously described. The cut-off for high MYC expression is ≥5% positive cells. The MDS-to-AML progression free survival (PFS) and overall survival (OS) were calculated from the date of MDS diagnosis and outcomes were estimated with the Kaplan-Meier method and compared using the log-rank test. All statistical analyses were performed using SPSS v24.0. Among 32 patients, 69% (n=22) and 31% (n=10) patients had low and high MYC expression at MDS diagnosis (Table 1). The median age was 67.9 (33.5-78.7) and 61.1 (38.9-73.4) and the % of MYC positive blasts were 1% (range, 0-4.5) and 17.5% (range, 5-27.5) in low and high MYC groups, respectively, at the time of MDS diagnosis. There was no statistical difference in BM blast counts between low vs. high MYC groups (median 5% vs. 7.5%, p=0.4951). IPSS risk assessment in a total of 26 patients (19 in low MYC and 7 in high MYC) showed that 9% (n=2), 27% (n=6), 32% (n=7), and 18% (n=4) in low MYC group and 0% (n=0), 20% (n=2), 20% (n=2), and 30% (n=3) in high MYC groups had low, intermediate-I, intermediate-II, and high risk at the time of MDS diagnosis (Table 1). A total of 84% (86% [n=19] in low MYC and 80% [n=8] in high MYC) patients received treatments for MDS; ESA in 16% (n=5), hypomethylating agents in 81% (n=26), and Revlimid in 16% (n=5) (Table 1). Allogeneic stem cell transplant (allo-SCT) was performed in 12.5% (n=4) patients (n=3, low MYC; n=1, high MYC). Following AML progression, a total of 91% (n=29) patients received treatment for AML including induction chemotherapy (n=18), hypomethylating agents (n=7), and other therapies including trial (n=4) (Table 1). In the univariate analyses, patients with low MYC expression had significantly longer MDS-to-AML PFS (median 15.8 vs. 7.25 months, HR=0.1745, 95%CI=0.0591-0.5157, p=0.0016) (Figure 1), however, there was no OS difference between two groups (4.32 vs. 2.35 years, HR=0.5131, 95%CI=0.1064-2.475, p=0.6909). In a multivariate Cox model (adjusting for IPSS, age, front-line therapy, gender, allo-SCT, and MYC level), high MYC expression (HR=3.939, 95%CI=1.107-14.012, p=0.034) and IPSS risk (HR=1.903, 95%CI=1.139-3.181, p=0.014) were significant factor for MDS-to-AML PFS. In conclusion, high MYC expression at the MDS stage was associated with shorter time to AML progression, indicating that MYC plays an oncogenic role in MDS to AML transformation. Although our findings need further validation by prospective studies with greater sample size, our results support a combination treatment of hypomethylating agents with novel agents targeting MYC or/and its downstream pathways and warrant further study to identify underlying mechanisms of MYC-driven MDS to AML progression. Disclosures Talati: Pfizer: Honoraria; Astellas: Speakers Bureau; Jazz: Speakers Bureau; BMS: Honoraria; AbbVie: Honoraria. Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding. Padron:Novartis: Honoraria; Incyte: Research Funding; Kura: Research Funding; BMS: Research Funding. Komrokji:JAZZ: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Geron: Honoraria; AbbVie: Honoraria; Agios: Honoraria, Speakers Bureau. Lancet:Abbvie: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Astellas Pharma: Consultancy; Celgene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Jazz Pharmaceuticals: Consultancy; Pfizer: Consultancy. Sallman:Celgene, Jazz Pharma: Research Funding; Agios, Bristol Myers Squibb, Celyad Oncology, Incyte, Intellia Therapeutics, Kite Pharma, Novartis, Syndax: Consultancy.

Background Double-expression lymphoma (DEL) is a rare subgroup of diffuse large B-cell lymphoma (DLBCL), which has coexpression of MYC and BCL-2. Coexpression of MYC and BCL-2 is considered a prognostic marker portending poor outcomes. However, the prognostic effect of BCL-2 and BCL-6 expression in DLBCL remains controversial. Materials and Methods Immunohistochemical staining was performed to detect MYC, BCL-2 and BCL-6 expression in 212 patients with newly diagnosed DLBCL and assess the prognostic effects of BCL-2 and BCL-6 expression. The DLBCL patients were treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine [Oncovin], prednisone)–like regimens. Results Retrospective analysis revealed that BCL-2+ and BCL-2+/MYC+ were prognostic factors indicative of poor outcomes. Patients with BCL-2+ and/or MYC+ expression had a poorer prognosis than that of patients with BCL-2− and/or MYC− expression. Patients with BCL-2+/MYC− expression showed a trend toward poorer survival than those with BCL-2−/MYC+ expression, suggesting that BCL-2 plays a more important role than MYC. Also, patients with BCL-6−/MYC+ expression had poorer progression-free survival than those with BCL-6+/MYC+ expression. In addition, patients with BCL-2+/MYC+/BCL-6− expression had the worst prognosis, suggesting that BCL-6− is a prognostic factor for poor outcomes for MYC+ DLBCL patients. Altogether, our findings have shown that BCL-2 is an independent prognostic factor and possibly plays a more important role than MYC in MYC+ DLBCL patients. Furthermore, we found that BCL-6− expression could also be a prognostic factor portending poor outcomes for MYC+ DLBCL patients.

Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

Aims MYC rearrangements with or without BCL2 rearrangements have been shown to be associated with poor prognosis and inferior survival in diffuse large B-cell lymphomas (DLBCL). Most of these cases are still diagnosed by fluorescent in situ hybridisation (FISH) testing, which is expensive, requires expertise and is not routinely available in all laboratories. Immunohistochemistry (IHC) is widely available and has the potential to be used as a screening test to identify cases with increased protein expression and select cases that require confirmatory testing. We correlated the expression of MYC and BCL2 by IHC with FISH studies in an attempt to define a cut-off value, which can be used by laboratories to select cases requiring confirmatory FISH testing. The prevalence of MYC -positive DLBCL and double-hit lymphoma (DHL) has also been studied. Methods 209 cases comprising of 15 cases of Burkitt lymphoma (BL), 13 cases of intermediate BL/DLBCL and 181 cases of DLBCL were included. IHC and FISH for MYC and BCL2 were performed and the results were correlated. Results The prevalence of MYC -positive DLBCL and MYC / BCL2 DHL was 13.4% and 7.4%, respectively, in our study. Germinal-centre subtype was more common in MYC -positive DLBCL and DHL. MYC -positive DLBCL also showed higher median Ki-67 (>90%) and CD10 positivity as compared with MYC -negative cases. Conclusions IHC can be used for screening cases, which require further confirmatory FISH testing. We recommend a cut-off value of ≥30% for MYC by IHC; however, international standardisation of these values is necessary to provide uniformity among laboratories.

FGFR3 alterations (mutations or translocation) are among the most frequent genetic events in bladder carcinoma. They lead to an aberrant activation of FGFR3 signaling, conferring an oncogenic dependence, which we studied here. We discovered a positive feedback loop, in which the activation of p38 and AKT downstream from the altered FGFR3 upregulates MYC mRNA levels and stabilizes MYC protein, respectively, leading to the accumulation of MYC, which directly upregulates FGFR3 expression by binding to active enhancers upstream from FGFR3 . Disruption of this FGFR3/MYC loop in bladder cancer cell lines by treatment with FGFR3, p38, AKT, or BET bromodomain inhibitors (JQ1) preventing MYC transcription decreased cell viability in vitro and tumor growth in vivo . A relevance of this loop to human bladder tumors was supported by the positive correlation between FGFR3 and MYC levels in tumors bearing FGFR3 mutations, and the decrease in FGFR3 and MYC levels following anti‐FGFR treatment in a PDX model bearing an FGFR3 mutation. These findings open up new possibilities for the treatment of bladder tumors displaying aberrant FGFR3 activation.

CTNNB1 mutations and abnormal β-catenin distribution are associated with the pathogenesis of adamantinomatous craniopharyngioma (aCP). We evaluated the expression of the canonical Wnt pathway components in aCPs and its association with CTNNB1 mutations and tumor progression. Tumor samples from 14 aCP patients and normal anterior pituitary samples from eight individuals without pituitary disease were studied. Gene expression of Wnt pathway activator (WNT4), inhibitors (SFRP1, DKK3, AXIN1, and APC), transcriptional activator (TCF7), target genes (MYC, WISP2, and, CDH1), and Wnt modulator (TP53) was evaluated by qPCR. β-Catenin, MYC, and WISP2 expression was determined by immunohistochemistry (IHC). The transcription levels of all genes studied, except APC, were higher in aCPs as compared to controls and TCF7 mRNA levels correlated with CTNNB1 mutation. CDH1 mRNA was overexpressed in tumor samples of patients with disease progression in comparison to those with stable disease. β-Catenin was positive and aberrantly distributed in 11 out of 14 tumor samples. Stronger β-catenin immunostaining associated positively with tumor progression. MYC positive staining was found in 10 out of 14 cases, whereas all aCPs were negative for WISP2. Wnt pathway genes were overexpressed in aCPs harboring CTNNB1 mutations and in patients with progressive disease. Recurrence was associated with stronger staining for β-catenin. These data suggest that Wnt pathway activation contributes to the pathogenesis and prognosis of aCPs.

Noninvasive biomarkers that detect the activity of important oncogenic drivers could significantly improve cancer diagnosis and management of treatment. The goal of this study was to determine whether 68Ga-citrate (which avidly binds to circulating transferrin) can detect MYC-positive prostate cancer tumors, as the transferrin receptor is a direct MYC target gene. PET imaging paired with 68Ga-citrate and molecular analysis of preclinical models, human cell-free DNA (cfDNA), and clinical biopsies were conducted to determine whether 68Ga-citrate can detect MYC-positive prostate cancer. Importantly, 68Ga-citrate detected human prostate cancer models in a MYC-dependent fashion. In patients with castration-resistant prostate cancer, analysis of cfDNA revealed that all patients with 68Ga-citrate avid tumors had a gain of at least one MYC copy number. Moreover, biopsy of two PET avid metastases showed molecular or histologic features characteristic of MYC hyperactivity. These data demonstrate that 68Ga-citrate targets prostate cancer tumors with MYC hyperactivity. A larger prospective study is ongoing to demonstrate the specificity of 68Ga-citrate for tumors with hyperactive MYC. Implications: Noninvasive measurement of MYC activity with quantitative imaging modalities could substantially increase our understanding of the role of MYC signaling in clinical settings for which invasive techniques are challenging to implement or do not characterize the biology of all tumors in a patient. Moreover, measuring MYC activity noninvasively opens the opportunity to study changes in MYC signaling in patients under targeted therapeutic conditions thought to indirectly inhibit MYC. Mol Cancer Res; 15(9); 1221–9. ©2017 AACR.

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted >0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Proto-oncogene Myc, a key transcription factor, is frequently deregulated in human leukemia with aggressive and poor clinical outcome, but the development of MYC inhibitors remains challenging due to MYC helix-loop-helix topology lacking druggable domains. Here we describe a novel oral active small molecule analog of berbamine, tosyl chloride-berbamine (TCB), that efficiently eliminates MYC-positive leukemia in vitro and in vivo. Mechanistically, TCB potently reduced MYC protein by inhibiting CaMKIIγ, a critical enzyme that stabilizes MYC protein, and induces apoptosis of MYC-positive leukemia cells. In vivo, oral administration of TCB markedly eliminated lethal MYC-positive acute lymphoblastic leukemia (ALL) with well tolerability in orthotopic mouse model. Our studies identify CaMKIIγ/Myc axis as a valid target for developing small molecule-based new therapies for treating MYC-mediated leukemia and demonstrate that TCB is an orally active analog of berbamine that kills MYC-positive leukemia cells.

High-grade non-Hodgkin's B cells lymphomas (NHLs) are malignant tumours of the lymphoid system. MYC protein was known correlated with poor prognosis. In addition to promoting cell-cycle progression, MYC plays an important role activation of an alternative pathway of a tumour associated macrophage (TAM). We further determined if MYC protein expression correlated with the high density of TAM. This was a cross-sectional study of 47 specimens of high-grade non-Hodgkin B cell lymphomas. Their immunophenotype was re-evaluated, and stratified into DLBCL and others non-Hodgkin lymphomas. All samples were immunostained with MYC and CD163 antibodies. The expression of MYC and CD163 were scored according to the previous study. Statistical analysis was performed by using SPSS version 22. MYC positive expression was detected in 72,3% of patients. Its found mostly in male patient, age ≤60, and Ki67 >70%. Average density of TAMs was 7,29. An average density of TAM in MYC positive lymphomas was higher rather than MYC negative lymphomas. MYC may facilitate the high density of TAM and contribute to worst prognosis in younger male patient and Ki67 >70%, of high-grade B cell lymphomas.

Heart failure is the leading cause of death and disability. Stimulating endogenous heart regeneration is a relatively new and tantalizing approach to combat heart failure. However, current endogenous regenerative approaches have very limited cytokinesis and little new cardiomyocyte(CM) mass. Here

Purpose Diffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and Methods We determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. Results In the training cohort (n = 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P < .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P < .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P < .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. Conclusion Assessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

Introduction; The prognosis of relapsed and refractory DLBCL (RR-DLBCL) after R-CHOP therapy is poor and the more than half of these cases had no cure. Autologous peripheral blood stem cell transplantation (auto-PBSCT) raises the survival rate about 20%, however many patients cannot reach the transplantation. The poor prognostic factors in RR-DLBCL have not been cleared yet. MYC/IgH translocations induced poor prognosis of DLBCL, especially double hit DLBCL is independent category in WHO 2016 criteria. However, in RR-DLBCL, the impact of MYC/IgH translocation had not been cleared yet. In this study, we studied the expression of MYC/IgH translocation in RR-DLBCL cases and analyzed the relationships with the prognosis by retrospectively. Methods; We studied the translocations of MYC and BCL-2 by FISH analysis RR-DLBCL cases from 2006 to May 2017 in our institute. The cells were obtained from the biopsy samples at the first diagnosis and relapse or refractory status. The cells have been fixed by carnoy solution. After hybridizing with each probe, the 100 cells or more were analyzed by In Cell Analyzer 6000 and the positive levels were determined as 5% or more in both MYC and BCL-2. Additionally pathological review and clinical status were studied among these cases, retrospectively. All cases are treated by R-CHOP at diagnosis and followed by salvage therapy after relapse with or without auto-PBSCT. This study protocol was approved in our institutional ethical review board. Results; Within 46 RR-DLBCL, 24 cases were negative for MYC translocation and 22 were positive. All MYC-positive cases were translocated with IgH except one case and the 9 cases (47.8%) were BCL2-positive. The median age was 62.4 (40-83) in MYC-negative and 62.6 (44-82) in MYC-positive cases. GC type was 11 (45%) and 9 (40.9%), and High IPI cases were 8 (33.3%) and 12 (54.5%) in MYC-negative and positive cases respectively. In these RR-DLBCL, the 5-year Overall survival (OS) was inferior in MYC-positive group (79.4%, 95% CI; 53.5-91.8%) compared to MYC-negative group (56.2%, 95%CI; 32.1-74.7%) (P=0.0414). There was no significant difference in progression free survival (PFS). There were no statistically significant differences in overall response rate (ORR), OS and PFS after second therapy between MYC-positive and negative group. The death cases were 6 (25%) and 11 (50%), respectively (P=0.079). By the pathological analysis, the transformed DLBCL cases from follicular or indolent lymphoma were significantly more in MYC negative group (12; 50%) than in MYC positive group (4; 18.1%) (P=0.024). In the transformed DLBCL, at the first relapse, 2nd biopsy detected heterogeneous pattern, such as the only indolent lymphoma, DLBCL or contamination of both type. In such cases, at the 2nd relapse, the pathological diagnoses had changed again. OS and PFS were significant superior in transformed DLBCL than de-novo DLBCL after second therapy. 2-year OS were 59.0%(95%CI 37.9-75.1%) and 92.9%(59.1-99.0%) (P=0.00474) and PFS were 16.13 and 49.43 moths (95% CI was 10.4 to 20.1 months and 16.1 to 63.7 months; P=0.00063), in transformed FL and de novo-DLBCL, respectively. The multivariate analysis identified transformed DLBCL as independent prognostic factor for both PFS and OS. Also, in MYC positive group, transformed DLBCL cases were superior to de-novo DLBCL for both OS (17.5 months vs. not yet reached, P=0.0353) and PFS (13.3 vs. 63.3moths, P= 0.0353) respectively. Discussion and conclusion; From our results, it was suggested MYC-positive is still poor prognostic factor of overall survival in RR-DLBCL. However, RR-DLBCL contaminated transformed DLBCL especially in the MYC-negative group. After 2nd therapy, the transformed DLBCL were related to good prognoses compared to de-novo-DLBCL, in also MYC-positive group. We can consider that treatment strategy of transformed DLBCL were should be separated from de-novo DLBCL at the relapse, then MYC-positive transformed DLBCL still possesses for indolent character. Disclosures Mishima: Chugai pharmaceutical inc, Roche: Other: commissioned work. Yokoyama:Chugai pharmaceutical inc, Roche: Other: commissioned work. Nishimura:Chugai pharmaceutical inc, Roche: Other: commissioned work. Terui:Celgene: Honoraria; Janssen Pharmaceutical KK: Honoraria; Takeda pharmaceutical: Honoraria; Novartis pharma: Honoraria; Bristol myers Squib: Honoraria.

Diffuse large B-cell lymphoma (DLBCL) overexpressing MYC and BCL-2 have a poor outcome with standard therapies irrespective of the cell of origin (COO), representing an unmet medical need. Furthermore, precision therapy approaches specifically designed for MYC/BCL-2 positive DLBCL are lacking. Recently we reported that MYC positive DLBCL harbor high levels of inherent DNA damage and display constitutive activation of the DNA damage response (DDR) pathways, these alterations being correlated with poor outcome (Derenzini et al. Oncotarget 2015). In an effort to design specific therapies for MYC/BCL-2 positive DLBCL, we integrated nanostring-based gene expression profiling and functional characterization of replicative and oxidative stress biomarkers in 2 independent DLBCL cohorts treated with chemoimmunotherapy: 69 patients from the DLC04 study (Chiappella et al. Lancet Oncol 2017), and 66 patients treated in real life clinical practice (total 135 patients). We used a digital multiplex gene expression profiling (Nanostring technology) panel containing 28 genes: 20 genes used to assign COO subtype according to LST algorithm (Scott et al. Blood 2014), and 8 additional genes employed to assess the MYC and BCL-2 status and to detect potential therapeutic targets (STAT3, TP53, NFKBIA, PTEN, PIK3CA, CD68). Immunohistochemistry (IHC) profiling for MYC, BCL-2, the phosphorylated form of H2AX at S139 (γH2AX: a biomarker of DNA damage and DDR activation) and for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (an oxidative DNA damage marker) was available in all patients. MYC/BCL-2 status evaluated by nanostring outperformed IHC for prognostic stratification in both cohorts (patients were stratified in high and low MYC or BCL-2 expressors based on the median normalized MYC and BCL-2 mRNA levels in the respective cohorts). The 2 cohorts displayed similar patterns of γH2AX and 8-OHdG expression (cut off value 50% of positive cells). 8-OHdG negative cases showed very low levels of γH2AX whereas high 8-OHDG expressors displayed significantly higher levels of γH2AX phosphorylation. In line with this, γH2AX positivity was significantly associated with increased 8-OHdG levels in both cohorts (Figure 1A). These data suggest that oxidative stress could be a major source of inherent DNA damage contributing to constitutive DDR activation in DLBCL. Dual positivity for γH2AX and 8-OhDG was significantly associated with adverse outcome in both cohorts. However, this difference was even more pronounced when focusing the analysis on MYC/BCL-2 positive DLBCL (evaluated by nanostring): in fact dual positivity for H2AX and 8-OHdG identified a subgroup of MYC/BCL-2 positive cases (MYC/BCL-2ox) characterized by a dismal outcome. Interestingly MYC/BCL-2 positive cases without oxidative DNA damage had a very favorable outcome, with similar overall survival (OS) compared to MYC/BCL-2 negative cases. Similar trends were observed in both cohorts and a cumulative analysis confirmed a significantly worse OS for the MYC/BCL-2ox subgroup, compared to all other subgroups (Figure 1B). We found an increased proportion of low NFKBIA expressors in the MYC/BCL-2ox subgroup (with no differences in the COO subtyping and in the fraction of double hit cases). Since NFKBIA is an endogenous inhibitor of NF-kB signaling, these data could indicate a compensatory NF-kB activation in MYC/BCL-2ox patients, in line with the role of NF-kB signaling in driving antioxidant responses. These findings suggest a model of oncogenic cooperation where constitutive NF-kB activation and BCL-2 overexpression could counteract the deleterious effects of inherent oxidative DNA damage driven by MYC. To test this hypothesis and to determine the therapeutic implications of these findings we run a combinatorial high-throughput drug screening (HTS) investigating combinations based on 2 different inhibitors of the DDR (the CHK1/2 inhibitors AZD7762 and MK8776) in a large panel of DLBCL cell lines of ABC and GCB origin (n=10) (Figure 1C). Both CHK1/2 inhibitors showed synergistic interactions with BCL-2 inhibitors (venetoclax) and with drugs inhibiting NF-KB pathway such as PI3K, HDAC, and BET inhibitors. Taken together these data suggest synthetic lethal interactions between MYC, BCL-2, NF-kB and the DDR in DLBCL, providing the rationale for targeted therapeutic interventions in poor prognosis MYC/BCL-2 positive subgroups. Disclosures Chiappella: Nanostring: Other: lecture fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: lecture fees; Roche: Other: lecture fees; Teva: Other: lecture fees; Amgen: Other: lecture fees. Zinzani:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; SERVIER: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; PFIZER: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; TG Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vitolo:Takeda: Speakers Bureau; Sandoz: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Approximately 10 % of cases of diffuse large B cell lymphoma (DLBCL) harbor a MYC rearrangement and this has been associated with an inferior outcome following standard therapy across many different studies. Double-hit and triple-hit lymphomas harbor concurrent rearrangements of MYC and BCL2 and/or BCL6 and are also associated with a very aggressive course and poor clinical outcome. It is unclear and there is lack of consensus on how these diseases should be approached therapeutically. They are characterized typically by high tumor proliferation and likely require Burkitt lymphoma-type strategies and several retrospective studies suggest that more intensive approaches than rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) may be beneficial. One challenge in this respect is that most patients with these diseases are older than 60 years and generally have poor tolerability of regimens typically used in Burkitt lymphoma. Dose-adjusted EPOCH-R is an alternative effective immunochemotherapy platform for DLBCL and is effective in Burkitt lymphoma, and retrospective studies suggest that it is effective and feasible in patients with DLBCL that harbors a MYC rearrangement with or without a BCL-2 translocation (double-hit). A multicenter study of this approach in MYC-rearranged DLBCL is ongoing and preliminary results are very encouraging. There is a lack of consensus on the role of consolidation stem cell transplantation in patients who achieve a good response to initial therapy but at this point in time, no (retrospective) studies have demonstrated any benefit. These diseases are also associated with a high rate of CNS involvement and progression and checking for cerebrospinal fluid by cytology and flow cytometry at initial diagnosis should be considered. In summary, based on retrospective data and preliminary prospective data (as more mature data is awaited), while Burkitt-type regimens may be feasible in young patients, DA-EPOCH-R is a reasonable approach for patients with these diseases, particularly for those over the age of 60 years. CNS prophylaxis is a reasonable consideration (depending on clinical characteristics) given that the rate of CNS progression is high. Many exciting new small molecule inhibitors such as BCL-2 and MYC inhibitors are in development and are exciting for these diseases and attempts are underway to combine them with effective immunochemotherapy platforms.

Background: Diffuse large B cell lymphoma (DLBCL) is the most common type of non Hodgkin lymphoma (NHL).Nearly 50% of high-risk DLBCL patients are not cured with standard rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (RCHOP). High risk DLBCL may be defined as double hit lymphoma (DHL, translocation of MYC and BCL2 or BCL6), double expressor lymphoma (DEL, over expression of MYC and BCL2), high risk international prognostic index (IPI) of 3-5, high Ki-67, and non-germinal center subtype (non-GCB). The majority of DHL cases occur in the GCB subtype, as opposed to the majority of DEL cases which occur in non-GCB. Hence we sought to compare different high risk subsets treated with dose-adjusted etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone and rituximab (DA) EPOCH-R. In single arm phase II clinical trials, dose adjusted (DA) EPOCH-R has shown promising results, with potential greater efficacy in the GCB subtype in subset analyses (Wilson et al, Hematologica 2012). A randomized phase III study comparing RCHOP with (DA) EPOCH-R in newly diagnosed DLBCL has completed accrual, with highly anticipated results due in late 2016. Methods: We conducted a retrospective reviewof all consecutive, newly diagnosed DLBCL patients treated with (DA) EPOCH-R at MD Anderson Cancer Center from 2010 to 2014. Eligible patients were 18 years or greater, had high-risk DLBCL as determined by the treating physician, and had available data of treatment and response. The cell of origin subtype was determined by immunohistochemistry using Hans algorithm, and MYC and BCL2 positivity were defined as BCL2 positive in at least 70% and MYC positive in at least 40% of cells. DHL was defined as rearrangement of MYC and BCL2 or BCL6 by fluorescent in situ hybridization. The objectives were to analyze demographic, prognostic, and treatment variables in comparison with clinical response and survival outcomes in three sub groups which included 1. DHL (GCB) 2. DLBCL without MYC and BCL2 expression (GCB), and 3. DEL (GCB and non GCB). Complete response (CR), overall survival (OS) and progression free survival (PFS) were calculated using standard methods. Statistical analysis was done using Fishers exact test or Chi-square test / Kruskal-Wallis test. Kaplan-Meier method was used for time-to-event analysis including overall survival and progression free survival. The Log-rank test was used to evaluate the difference in time-to-event endpoints between patient groups. Results: We identified 233 high risk DLBCL patients treated with (DA) EPOCH-R. After filtering the data to identify patients which were included in our three groups, we identified 22 patients with DHL (GCB), 46 patients with non DEL (GCB), and 16 with DEL. The demographic features and outcomes are mentioned in the table 1 below. The DHL group had more frequent bone marrow (BM) involvement, and the DHL and DEL groups were more frequently age >60 years and high IPI in comparison to the non DEL GCB group. The CR rate, OS and PFS at 1 year were not significantly different between these three groups. Figure 1 highlights the OS (A) and PFS (B) results of each group. Conclusions: (DA) EPOCH-R is highly effective in patients with subsets of patients with high-risk DLBCL and may be able to overcome prognostic factors which have been shown to be adverse with RCHOP therapy. The results of this retrospective study suggest that OS in DHL, DEL and non DEL (GCB) are not statistically different. Hence, intensive chemotherapy with (DA) EPOCH-R could be considered as a frontline treatment option for patients with high risk DLBCL, pending further confirmation in randomized trials. Disclosures Oki: Novartis: Research Funding. Fowler:Infinity: Consultancy, Research Funding; Roche: Consultancy, Research Funding; TG Therapeutics: Consultancy; Celgene: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Gilead: Research Funding. Wang:Pharmacyclics: Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; BeiGene: Research Funding; Kite Pharma: Research Funding; Onyx: Research Funding; Asana BioSciences: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fayad:Seattle Genetics: Consultancy, Research Funding. Westin:ProNAi: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees.

7563Background: Several studies on single and double protein expressing DLBCL (DPL), defined as BCL2 positive in > 70% and MYC positive in > 40%, have identified inferior clinical outcomes when tre...

Evidence suggests that the carcinogenic process guided by Helicobacter pylori is related to the expression of cell cycle and apoptosis proteins as BCL-2, BAX, and MYC. However, the literature is conflicting regarding the expression frequency in the histological subtypes and did not consider cagA gene presence. To investigate the expression of these proteins considering the histological subtypes of gastric cancer associated with H. pylori (cagA), a total of 89 cases were used. H. pylori infection and cagA status were determined by PCR. Immunodetection was performed for MYC, BCL-2, and BAX proteins. H. pylori was found in 95.5% of the patients, among them, 65.8% were cagA(+). Nuclear MYC was detected in 36.4%, BAX in 55.7%, while BCl-2 in just 5%. Nuclear MYC staining was significantly lower in the intestinal than diffuse subtype (p = 0.008) and was related with the presence of H. pylori cagA(+). Additionally, most of the few cases cytoplasmic MYC positive were in the intestinal subtype. In diffuse tumors, although most nuclear MYC positive cases were cagA(+), it was not significant. No difference was observed between BCL-2 or BAX expression considering the presence of cagA gene in the histological subtypes. It seems that MYC could be relevant for the diffuse tumorigenic pathway associated with H. pylori and possibly influenced by the presence of cagA gene, while in intestinal tumors, the tumorigenic pathway does not occur through the MYC expression.

MicroRNAs (miRNAs) are small, noncoding RNAs that play an important role in many key biological processes, including development, cell differentiation, the cell cycle and apoptosis, as central post-transcriptional regulators of gene expression. Recent studies have shown that miRNAs can act as oncogenes and tumor suppressors depending on the context. The present work focuses on the physiological significance of miRNAs and their role in regulating the switching behavior. We illustrate an abstract model of the Myc/E2F/miR-17-92 network presented by Aguda et al. (2008), which is composed of coupling between the E2F/Myc positive feedback loops and the E2F/Myc/miR-17-92 negative feedback loop. By systematically analyzing the network in close association with plausible experimental parameters, we show that, in the presence of miRNAs, the system bistability emerges from the system, with a bistable switch and a one-way switch presented by Aguda et al. instead of a single one-way switch. Moreover, the miRNAs can optimize the switching process. The model produces a diverse array of response-signal behaviors in response to various potential regulating scenarios. The model predicts that this transition exists, one from cell death or the cancerous phenotype directly to cell quiescence, due to the existence of miRNAs. It was also found that the network involving miR-17-92 exhibits high noise sensitivity due to a positive feedback loop and also maintains resistance to noise from a negative feedback loop.

Background Both BL and DLBCL are prevalent in HIV/AIDS patients and the correct classification of these two subtypes of Non-Hodgkin's lymphomas (NHL) particularly between DLCBL with high proliferation and BL, is critical to chemotherapy choice. Transcriptional gene expression profiling of sporadic and endemic BL by Stein et al (Berlin) identified a molecular signature for BL that included a down regulation of CD44 expression in a group of lymphomas from Africa. Immunophenotyping of formalin-fixed paraffin-embedded (FFPE) BL tissues by this group demonstrated difficulties in correctly classifying BL using morphology, BCL-2 (immunohistochemistry – IHC) presence and MYC (fluorescent in situ hybridization – FISH) absence. The differential expression of CD44, albeit nonmembranous, suggests an intricate regulatory circuit that allows for low expression, supporting the possibility that CD44 plays a role in BL/BLL dissemination. CD44 (IHC) could be a valuable marker to aid classification of tumors whose morphologic and phenotypic overlap is among BL, BLL and DLBCL. CD44 absence clearly separates MYC+ BL/BLL and MYC-DLBCL. DLBCL, Hodgkin's lymphoma (HL) and normal lymphocytes from lymphoid mantle/ marginal cell and interfollicular T-cell-rich zones should be strongly CD44 positive. Materials and methods We evaluated 128 FFPE lymphoma tissue biopsy collected at St. Mary's Lacor Hospital, Gulu, Uganda, Africa, from 1994 to 2007 from tissue microarray (TMA) blocks constructed on site using the UNITMA Quick-Ray System (Seoul, South Korea). The completed TMA blocks were sectioned and stained for HE in situ hybridization (ISH) for EBV (EBER) and kappa and lambda light chains and FISH break apart assay for CMYC translocation.

The transcription factors E2F and Myc participate in the control of cell proliferation and apoptosis, and can act as oncogenes or tumor suppressors depending on their levels of expression. Positive feedback loops in the regulation of these factors are predicted—and recently shown experimentally—to lead to bistability, which is a phenomenon characterized by the existence of low and high protein levels (“off” and “on” levels, respectively), with sharp transitions between levels being inducible by, for example, changes in growth factor concentrations. E2F and Myc are inhibited at the posttranscriptional step by members of a cluster of microRNAs (miRs) called miR-17-92. In return, E2F and Myc induce the transcription of miR-17-92, thus forming a negative feedback loop in the interaction network. The consequences of the coupling between the E2F/Myc positive feedback loops and the E2F/Myc/miR-17-92 negative feedback loop are analyzed using a mathematical model. The model predicts that miR-17-92 plays a critical role in regulating the position of the off–on switch in E2F/Myc protein levels, and in determining the on levels of these proteins. The model also predicts large-amplitude protein oscillations that coexist with the off steady state levels. Using the concept and model prediction of a “cancer zone,” the oncogenic and tumor suppressor properties of miR-17-92 is demonstrated to parallel the same properties of E2F and Myc.

Background Though the majority of patients (pts) with newly diagnosed diffuse large B-cell lymphoma (DLBCL) are curable with R-CHOP chemotherapy, a significant proportion will relapse or have refractory disease. The most commonly used clinical tool is the international prognostic index (IPI), though this cannot fully capture the heterogeneity of cases seen in practice. In recent years biomarkers such as MYC are entering clinical use. Pts with lymphomas demonstrating dual abnormalities of MYC in association with BCL2 and/or BCL6-known as 'double-hit' lymphomas are consistently shown to have poorer disease free and overall survival. While Fluorescence in-situ hybridization (FISH) for MYC translocation is the gold-standard, immunohistochemistry (IHC) is faster and significantly cheaper. Studies in recent years have confirmed the prognostic significance of increased MYC expression by IHC. Due to significant inter-laboratory variability however, internal validation is required. Methods Tissue samples of pts treated at Royal North Shore Hospital in Sydney, Australia between 2003-2012 were retrospectively assessed. Pts were included if they were transplant eligible (age A significant cut-off for MYC staining was determined using X-Tile (Rimmlab, New Haven, CT) and confirmed with the log-rank test. Estimation of OS and EFS was performed using the Kaplan-Meier method. Pt characteristics were compared using Chi-squared test. Statistical analysis was performed using MedCalc 15.4 (MedCalc software, Ostend, Belgium). Ethics approval was received for a retrospective study. Results 105 patients met study criteria. The 5 year OS and EFS was 86% and 77% respectively (Figure 1 and 2). The optimal cut-off for positive MYC IHC was >70%. This was seen in 23% of samples. From the 13 cases with MYC FISH results, the positive and negative predictive values of positive MYC IHC were 50% and 92% respectively. There was no significant difference between the MYC positive and negative groups with respect to demographics or IPI score (Table 1). Significantly more patients with MYC positivity received intensive treatment (37% versus 16%, p=0.047). Despite this, 5 year OS was significantly poorer at 51% versus 87% at median follow-up of 40 months (P=0.0025, Figure 3). There was a trend towards worse EFS at 61% versus 75% though this did not reach statistical significance (P=0.242). On multivariate analysis, MYC IHC and IPI score were the only independent prognostic factors. Based on the relative odds ratio, a combined scoring system was designed, attributing 1 point for positive MYC IHC and/ or IPI intermediate-high risk, and 2 points for IPI high risk. This resulted in 4 risk groups with significantly different 5 year OS of 94%, 78%, 45% and 0% (P Discussion MYC IHC is of independent prognostic significance. Due to significant variability between laboratories, local validation is required. A composite score combining IPI and MYC IHC is simple to calculate and may provide better prognostic utility than either factor alone. Ongoing prospective studies investigating the role of clinical risk stratification and newer biomarkers are required to identify patients likely to need more intensive or novel therapies. Figure 1. Figure 1. Disclosures Imlay-Gillespie: Novartis: Honoraria. Arthur:Amgen: Honoraria; Novartis: Honoraria; BMS: Honoraria. Mackinlay:Roche: Research Funding; Sanofi Aventis: Research Funding. Mulligan:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi Aventis: Research Funding.

MYC and BCL2 translocations as well as TP53 deletion/mutation are known risk factors in diffuse large B-cell lymphoma (DLBCL) but their interplay is not well understood.In this retrospective cohort study, we evaluated the combined prognostic impact of TP53 deletion and mutation status, MYC and BCL2 genomic breaks in tumor samples of 101 DLBCL patients. The cohort included 53 cases with MYC rearrangements (MYC+).TP53 deletions/mutations (TP53+) were found in 32 of 101 lymphomas and were equally distributed between MYC+ and MYC- cases (35.8% vs. 27.1%). TP53+ lymphomas had lower responses to treatment than TP53- (complete remission 34.4% vs. 60.9%; P = 0.01). TP53 alteration was the dominant independent prognostic factor in multivariate analysis (P = 0.01). Overall survival (OS) varied considerably between subgroups with different genomic alterations: Patients with sole MYC translocation, and interestingly, with triple MYC+/BCL2+/TP53+ aberration had favorable outcomes (median OS not reached) similar to patients without genomic alterations (median OS 65 months). In contrast, patients with MYC+/BCL2+/TP53- double-hit lymphomas (DHL) (28 months), MYC+/BCL2-/TP53+ lymphomas (10 months) or sole TP53 mutation/deletion (12 months) had a poor median OS. Our findings demonstrate differences in OS of DLBCL patients depending on absence or presence of single or combined genetic alterations of MYC, BCL2, and TP53. Cooccurrence of TP53 and BCL2 aberrations ameliorated the poor prognostic impact of single TP53+ or BCL2+ in MYC positive patients.This pilot study generates evidence for the complex interplay between the alterations of genetic pathways in DLBCL, which goes beyond the concept of DHL. The variable survival of DLBCL patients dependent on single or combined alterations in the TP53, MYC, and BCL2 genes indicates the need for comprehensive genomic diagnosis.

Germinal centers (GC)s are sites of intense B-cell proliferation, central for T-cell dependent antibody responses. Although much is known of GC B-cell physiology, the mechanisms of cellular proliferation have remained elusive. Specifically, the role of MYC, a key cell cycle regulator, in GC B-cell proliferation has been questioned. We have identified MYC positive B-cell subpopulations in both immature and mature GCs, and shown through genetic ablation of Myc at these stages that they play indispensable roles in GC formation and maintenance, respectively. The identification of functionally critical MYC-expressing GC B-cell subsets has important implications for B-cell lymphomagenesis, which mostly originates from GC cells and frequently involves MYC deregulation through chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may represent B-cells with a particularly high risk for malignant transformation. Citation Format: Dinis Pedro Calado. MYC in germinal center B cell physiology and pathology. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA24.

Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Our recent studies revealed that Myc-dependent ATP depletion activates the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. AMPK-stabilized tumor suppressor protein p53 then accumulates in the mitochondria and interacts with the protein complex comprised of Bak and Bcl-xL, to sensitize cells to apoptosis. These results reveal an unexpected pro-apoptotic function of AMPK in context of Myc transformed cells. We have explored possibilities to therapeutically exploit the Myc-AMPK facilitated apoptosis pathway. We show that a BH3-mimetic small molecule ABT-737, which neutralizes Bcl-2 and Bcl-xL, sensitizes to Myc-dependent apoptosis ex vivo and in vivo WAP-Myc tumours. ABT-737 also reduced WAP-Myc driven primary tumor growth and lung metastases in transgenic and syngrafted mice. Nevertheless, ABT-737 only delayed but did not prevent the formation of Wap-Myc tumors. In a drug combination screen designed to identify small molecules that potentiate the efficacy of ABT-737, we found several compounds influencing the status of AMPK activity. The synergistic apoptotic action between ABT-737 and AMPK modulating agents has been validated in explant 3D cultures from patient derived Myc positive breast cancers. The mechanisms behind the proapoptotic AMPK function will be discussed. Our results provide evidence that AMPK has a context-dependent pro-apoptotic function that can be therapeutically exploited as a tumour cell-specific vulnerability. Citation Format: Heidi M. Haikala, Elsa Marques, Vilja Eskelinen, Anni Viheriäranta-Nieminen, Sirkku Saarikoski, Liina Nevalaita, Panu Kovanen, Päivi Heikkilä, Marjut Leidenius, Daniel Laajala, Tero Aittokallio, Pauliina Munne, Juha Klefström. Pro-apoptotic functions of AMPK revealed by Myc. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B32.

Introduction MYC translocations involving chromosome 8q24 can occur in a wide variety of B-cell lymphomas other than Burkitt’s lymphoma (BL), especially diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with intermediate features between DLBCL and BL (BCLU). These lymphomas can harbor additional translocations of BCL2 and/or BCL6, that have been referred to as double and triple-hit lymphomas. MYC positive lymphomas other than BL are not well characterized and the standard treatment has to be established. Aim The objective was to study the clinic-biological characteristics and prognosis of a series of lymphomas with MYC-translocation other than BL. Methods Retrospective study of patients with MYC-translocation other than BL treated in three hospitals of Spain between 2003 and 2012. Cases with diagnosis of BL, Burkitt’s-like lymphoma and any other with MYC-translocation were reviewed and classified according to the WHO 2008 criteria. The status of MYC, BCL2 and BCL6 genes were evaluated in all cases by fluorescent in situ hybridization (FISH) using dual-colour break-apart commercial probes (LSI MYC DC BA, LSI BCL6 DC BA and LSI BCL2 DC BA; Abbot Molecular, Abbot Park, IL, USA) on whole tissue sections of formalin-fixed paraffin-embedded tissue. Main clinical and biological data were collected from the records. Results Between 2003 and 2012, 34 patients with a median follow-up of 1.9 years (range 0.7-9.7) were included. Median age was 59.5 years (range 36-83) and 21 (62%) were male. ECOG score at diagnosis was ≥2 in 14 patients (42%), 16 (49%) had ≥2 extranodal sites involved, serum LDH was elevated in 24 out of 32 (75%), Ann Arbor stage III/IV in 24 (73%) and B symptoms were present in 18 (56%). IPI was high or intermediate/high in 19 out of 32 (59%). Eighteen patients (53%) presented with a mass (13 abdominal location). Twenty-four cases were diagnosed with DLBCL and 10 with BCLU. Conventional cytogenetics showed a complex karyotype in the 9 studied cases. MYC rearrangement alone without BCL2 or BCL6 rearrangements was observed in 13 (38%) cases, 15 were double-hit, and 6 triple-hit lymphomas. There were no differences between patients with MYC translocation alone and patients with double or triple-hit, regarding the clinical and biological characteristics. Moreover, there were no clinical and biological differences between patients with DLBCL and those with BCLU. Twenty-one cases were treated with R-CHOP (19 DLBCL and 2 BCLU), and 13 with a specific treatment for BL (Burkimab) (8 BCLU and 5 DLBCL) (P=0.002). Complete response (CR) was achieved in 14 out of 32 (44%) cases (9 out of 21 [45%] treated with R-CHOP and 5 out of 13 [42%] with Burkimab). Two patients died during chemotherapy. Six out of the 9 patients in CR to R-CHOP relapsed, but none of the 5 patients in CR to Burkimab did it. The overall survival (OS) probability and progression free survival (PFS) at 2 years, (95% CI) for the whole series were 32% (14%, 50%) and 21% (6%, 36%) respectively. The two-year OS and PFS probabilities were not significantly different between patients treated with Burkimab and those treated with R-CHOP: 62% (95% CI: 36%, 88%) versus 28% (95% CI: 8%, 48%) for OS (P=0.67); and 46% (95% CI: 19%, 73%) versus 15% (95% CI: 0%, 31%) for PFS (P=0.549), respectively. Conclusions Lymphomas with MYC translocation other than BL present aggressive characteristics at diagnosis and have poor response to immunochemotherapy. Supported in part by grants EC11-041 and RD12/0036/0029 RTICC from Instituto Carlos III, Spain Disclosures: Lopez-Guillermo: Roche: Membership on an entity’s Board of Directors or advisory committees.

Medulloblastoma is the most common malignant brain tumor in children. Currently, treatment consists of surgical resection, chemotherapy, and whole brain and spine radiation, and is associated with high morbidity. Medulloblastoma is divided into four subgroups with different associated mutations and clinical prognoses. Group 3 medulloblastoma tumors are associated with elevated MYC levels and have the worst clinical prognosis of all four subtypes. Because MYC over-expression can lead to increased glutamine metabolism and glutamine addiction in other cancer types, we hypothesized that MYC-driven medulloblastoma would exhibit increased glutamine metabolism and be sensitive to inhibitors of glutamine metabolism. In medulloblastoma cell lines, MYC expression positively correlates with increased expression of enzymes involved in glutamine metabolism as measured by western blot and qPCR. Also, forced MYC expression in the non-MYC driven medulloblastoma cell lines UW228 and DAOY leads to corresponding increases in glutaminase expression as measured by western blot. To investigate the hypothesis that inhibition of glutamine metabolism would preferentially target MYC driven medulloblastoma, we tested the effects of two glutamine analogs—acivicin and 6-diazo-5-L-norleucine (DON)—that inhibit enzymes in the glutamine metabolism pathway. Acivicin decreased the growth of the high-MYC medulloblastoma cell lines D425 and D283 by 50% and 75% respectively compared to vehicle treated cells, but did not significantly affect UW228, a MYC-negative cell line. Acivicin treatment caused a 36% decrease in growth in UW228 cells forced to express MYC compared to a 5% decrease in growth in parental UW228 cells (p=.00029). We next tested the effect of acivicin on cell proliferation as measured by BrdU incorporation in neurosphere models of MYC and non-MYC driven medulloblastoma. MYC-expressing cells had a 30% decrease in proliferation following acivicin treatment, while MYC negative cells only showed a 7% decrease in proliferation (p=0.02). Treatment of MYC expressing cells with acivicin lead to increased apoptosis as measured by flow cytometry. MYC positive neurospheres treated with acivicin had a 65% increase in the percentage of cells in the sub2N population, while MYC-negative neurospheres did not show an increase in the sub2N population. Our laboratory is currently investigating the in vivo efficacy of glutamine metabolism inhibitors in orthotopic xenograft experiments using MYC-driven medulloblastoma cell lines. Taken together, our data suggests that glutamine metabolism may be a therapeutic target in MYC-driven medulloblastoma and that glutamine analogs may be useful therapeutic agents. Citation Format: Allison R. Hanaford, Charles G. Eberhart, Eric H. Raabe. Targeting MYC-driven medulloblastoma using inhibitors of glutamine metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1872. doi:10.1158/1538-7445.AM2013-1872

Abstract 1552 Background: Multifunctional MYC oncogene overexpression resulting from genomic rearrangement plays a critical role in lymphomagenesis and lymphoma manifestation, particularly in aggressive B-cell lymphomas such as Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL including double hit or triple hit lymphomas (DHL/THL). Accurate detection of MYC gene rearrangement, its presence or absence, has become increasingly important with its diagnostic and consequent therapeutic implications. In practice, FISH is the best test for MYC alterations. However, this is technically challenging, requiring knowledgeable and skillful cytogeneticists to design probe panels, correctly analyze and interpret atypical as well as typical signal patterns. For instance, up to 10% of patients with otherwise typical BL can be MYC rearrangement negative by FISH and, gene amplification and miRNA deregulation have been reported as possible reasons in occasional cases, although some of the FISH negative patients could be detected by better application of probes and interpretation of FISH findings. Here we retrospectively analyze FISH results from 879 consecutive MYC positive cases and share our experience with other FISH labs and physicians who are involved in diagnosis, differential diagnosis and treatment of aggressive lymphomas. Results: A MYC/IGH dual fusion translocation probe and a MYC break apart (ba) probe (Vysis) were applied to paraffin embedded tissue, lymph node biopsy, bone marrow and peripheral blood specimens. Of the 879 cases, MYC ba was positive in 258, MYC ba and MYC-IGH both positive in 331, MYC ba positive/MYC-IGH negative in 276, and MYC-IGH positive/MYC ba negative in 14 cases. Two subsets of cases with significant atypical signal patterns were observed. Firstly, in 14 cases with both probes tested, MYC ba was negative but MYC/IGH positive, including 6 BLs, 3 DLBCLs, 1 MCL, 1 Burkitt leukemia transformed from BL, 1 subtype unknown lymphoma and 2 DHLs with history of Hodgkin lymphoma and Burkitt-like lymphoma respectively and with concomitant BCL6 gene rearrangement in both. Eleven of these cases showed a single fusion signal pattern suggesting an insertion of IGH sequences into MYC or vise versa. This single insertion fusion pattern can also be resulted from complex chromosome changes as seen cytogenetically in 2 of the 11 cases. Secondly, 26 cases with various diagnoses including 4 DHLs and 2 THLs showed MYC rearrangement with a concomitant partial deletion of the MYC ba probe with 17 cases for 3' and 9 for 5' deletion. Three of these cases were also positive for concomitant probe deletion and amplification, two with 3' deletion and 5' amplification and one with 5' deletion and 3' amplification. Conclusion and discussion: MYC gene rearrangement detection by FISH in aggressive B-cell lymphomas is widely used. However, FISH labs should realize the technical limitations of each probe/panel based on underlying mechanisms involving MYC rearrangement and minimize false negative results. From our experience and previous studies by other groups, it is recommended that MYC ba probe should not be tested alone but together with a MYC/IGH t(8;14) translocation probe to detect cryptic insertions and variant translocations which often present as MYC ba negative yet MYC/IGH positive with a single fusion signal pattern inserting IGH promoter/enhancer elements into MYC or part or all of the MYC probe into IGH locus. Another observation that can also be misinterpreted is that the MYC ba probe often shows a deletion of either 5' or 3' flanking sequence. This is not because of tissue sectioning but indicative of MYC rearrangement with a concomitant loss of the DNA sequences adjacent to the breakpoints. A flanking sequence deletion revealed at translocation breakpoints using a FISH ba probe is a common finding. As well documented, BCR/ABL translocation results in an ASS deletion upstream of ABL in ∼15% of CMLs, and a 5' probe deletion in ∼30% ALK rearrangement positive NSCLC cases. Three of 26 such cases in our study also showed simultaneous retained MYC probe amplification making FISH interpretation even more difficult. As a diagnostic, prognostic and predictive biomarker, MYC gene plays a pivotal role in aggressive B-cell lymphomas, and its accurate detection will help improve disease risk stratification and therapy selection. Disclosures: No relevant conflicts of interest to declare.