MYC mRNA Expression Research Articles

Deciphering Discordance between MYC mRNA and MYC IHC in DLBCL: The Role of MYC Exon 2 Mutations and N11S Polymorphism

Deciphering Discordance between MYC mRNA and MYC IHC in DLBCL: The Role of MYC Exon 2 Mutations and N11S Polymorphism

MYC Activity Inference Captures Diverse Mechanisms of Aberrant MYC Pathway Activation in Human Cancers.

c-MYC (MYC) is deregulated in more than 50% of all cancers. While MYC amplification is the most common MYC-deregulating event, many other alterations can increase MYC activity. We thus systematically investigated MYC pathway activity across different tumor types. Using a logistic regression framework, we established tumor type-specific, transcriptomic-based MYC activity scores that can accurately capture MYC activity. We show that MYC activity scores reflect a variety of MYC-regulating mechanisms, including MYCL and/or MYCN amplification, MYC promoter methylation, MYC mRNA expression, lncRNA PVT1 expression, MYC mutations, and viral integrations near the MYC locus. Our MYC activity score incorporates all of these mechanisms, resulting in better prognostic predictions compared with MYC amplification status, MYC promoter methylation, and MYC mRNA expression in several cancer types. In addition, we show that tumor proliferation and immune evasion are likely contributors to this reduction in survival. Finally, we developed a MYC activity signature for liquid tumors in which MYC translocation is commonly observed, suggesting that our approach can be applied to different types of genomic alterations. In conclusion, we developed a MYC activity score that captures MYC pathway activity and is clinically relevant. IMPLICATIONS: By using cancer type-specific MYC activity profiles, we were able to assess MYC activity across many more tumor types than previously investigated. The range of different MYC-related alterations captured by our MYC activity score can be used to facilitate the application of future MYC inhibitors and aid physicians to preselect patients for targeted therapy.

Dimethoxycurcumin reduces proliferation and induces apoptosis in renal tumor cells more efficiently than demethoxycurcumin and curcumin

Curcumin (Cur), is a pigment with antiproliferative activity but has some pharmacokinetic limitations, which led researchers to look for more effective structure analogs. This work investigated the effects of Cur and compared them with the two analogs, demethoxycurcumin (DeMC) and dimethoxycurcumin (DiMC), to elucidate their mechanisms of action. The cytotoxic, antiproliferative, and genotoxic effects these compounds were correlated based on gene expression analysis in the human renal adenocarcinoma cells (786-O). Cur decreased CYP2D6 expression and exhibited cytotoxic effects, such as inducing monopolar spindle formation and mitotic arrest mediated by the increase in CDKN1A (p21) mRNA. This dysregulation induced cell death through a caspase-independent pathway but was mediated by decrease in MTOR and BCL2 mRNA expression, suggesting that apoptosis occurred by autophagy. DeMC and DiMC had similar effects in that they induced monopolar spindle and mitotic arrest, were genotoxic, and activated GADD45A, an important molecule in repair mechanisms, and CDKN1A. However, the induction of apoptosis by DeMC was delayed and regulated by the decrease of antiapoptotic mRNA BCL.XL and subsequent activation of caspase 9 and caspase 3/7. DiMC treatment increased the expression of CYP1A2, CYP2C19, and CYP3A4 and exhibited higher cytotoxicity compared with other compounds. It induced apoptosis by increasing mRNA expression of BBC3, MYC, and CASP7 and activation of caspase 9 and caspase 3/7. These data revealed that different gene regulation processes are involved in cell death induced by Cur, DeMC, and DiMC. All three can be considered as promising chemotherapy candidates, with DiMC showing the greatest potency.

Mutational Landscape of Virus- and UV-Associated Merkel Cell Carcinoma Cell Lines Is Comparable to Tumor Tissue.

Simple SummaryMerkel cell carcinoma (MCC) is an aggressive, rare skin cancer which is caused either by a virus or chronic UV exposure. For both forms, distinct genetic alterations have been described; however, these observations were mostly made in tumor tissue. Since cancer cell lines are frequently used as preclinical models to investigate biological function, we considered it necessary to establish the genomic landscape of MCC cell lines by whole-exome sequencing. We confirmed the presence of UV-induced DNA damage, a high number of mutations and several coding mutations in virus-negative cell lines which were absent in virus-positive cell lines; these, however, harbored characteristic copy number variations, suggesting some virally caused genetic instability. Knowing the genomic features of MCC cell lines validates previous, and facilitates upcoming, experimental studies to discover their biological and translational relevance.Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous malignancy that is either associated with the integration of the Merkel cell polyomavirus or chronic UV exposure. These two types of carcinogenesis are reflected in characteristic mutational features present in MCC tumor lesions. However, the genomic characteristics of MCC cell lines used as preclinical models are not well established. Thus, we analyzed the exomes of three virus-negative and six virus-positive MCC cell lines, all showing a classical neuroendocrine growth pattern. Virus-negative cell lines are characterized by a high tumor mutational burden (TMB), UV-light-induced DNA damage, functionally relevant coding mutations, e.g., in RB1 and TP53, and large amounts of copy number variations (CNVs). In contrast, virus-positive cell lines have a low TMB with few coding mutations and lack prominent mutational signatures, but harbor characteristic CNVs. One of the virus-negative cell lines has a local MYC amplification associated with high MYC mRNA expression. In conclusion, virus-positive and -negative MCC cell lines with a neuroendocrine growth pattern resemble mutational features observed in MCC tissue samples, which strengthens their utility for functional studies.

European society for medical oncology virtual congress: Molecular analysis for precision oncology, October 9th - October 10th, 2020.

European society for medical oncology virtual congress: Molecular analysis for precision oncology, October 9th - October 10th, 2020.

The novel low molecular weight MYC antagonist MYCMI-6 inhibits proliferation and induces apoptosis in breast cancer cells.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as "undruggable" or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC50 values varying form 0.3μM to >10μM. Consistent with its ability to decrease cell growth, MYCMI-6 was found to induce apoptosis in two cell lines in which growth was inhibited but not in two cell lines that were resistant to growth inhibition. Across all breast cancers, MYC was found to be amplified in 15.3% of cases in the TCGA database and 26% in the METABRIC database. Following classification of the breast cancers by their molecular subtypes, MYC was most frequently amplified and exhibited highest expression at both mRNA and protein level in the basal subtype. Conclusions Based on these findings, we conclude that for patients with breast cancer, anti-MYC therapy is likely to be most efficacious in patients with the basal subtype.

High amplification of PVT1 and MYC predict favorable prognosis in early ovarian carcinoma

IntroductionThe objective of this study was to evaluate the status of MYC and PVT1, which are frequently amplified in malignant tumors, and to assess their biological features according to histological subtypes in early-stage epithelial ovarian cancer (EOC). MethodsFormalin-fixed and paraffin-embedded (FFPE) samples of 64 EOC tissues in International Federation of Gynecology and Obstetrics stages I–II and 20 normal ovarian tissues were analyzed for copy number and mRNA expression of MYC and PVT1 by qPCR and for MYC protein expression by immunohistochemistry. MYC protein expression was assessed by western blotting in a PVT1 siRNA-transfected ovarian cancer cell line. MYC and PVT1 was assessed as a prognostic factor using Kaplan–Meier analysis. The median follow-up period was 49.9 months and 17 cases in 64 of EOC recurred during follow-up. ResultsCopy number variations showed significantly higher MYC and PVT1 in EOC than in normal ovaries. The copy number of PVT1 was significantly higher in serous carcinoma than in the other histological types. The mRNA of MYC and PVT1 was also higher in cancer tissues and showed a strong correlation in all histological subtypes. Immunohistochemistry revealed a positive association between the phosphorylated MYC (pMYC) index and high expression of proliferation markers, such as Ki-67 index, and a negative correlation between pMYC protein and the PVT1 copy number. Knockdown of PVT1 in ovarian cancer cell lines resulted in upregulation of MYC protein and pMYC. Kaplan-Meier survival analysis showed that low copy numbers of both MYC and PVT1 were associated with a statistically significantly poor prognosis. ConclusionExpression of pMYC and the Ki-67 index were affected by the PVT1 copy number but not mRNA. A high PVT1 copy number in FFPE samples might suggest favorable prognosis in early ovarian cancers.

PKM2 Expression as Biomarker for Resistance to Oxaliplatin-Based Chemotherapy in Colorectal Cancer.

The purpose of the current study is to investigate the prognostic significance of M2 isoform of pyruvate kinase (PKM2) mRNA expression loss in patients with operable colon cancer (CC). Two hundred sixty-two specimens from patients with stage-III or high-risk stage-II CC (group-A) treated with adjuvant fluoropyrimidine and oxaliplatin chemotherapy (FOLFOX), 118 specimens from metastatic CC patients (group-B) treated with FOLFOX, and 104 metastatic CC patients (group-C) treated with irinotecan-based chemotherapy were analyzed for PKM2, TS, ERCC1, MYC, and NEDD9 mRNA expression, as well as KRAS exon2 and BRAFV600E mutations. High PKM2 mRNA expression was correlated with left-sided located primaries (p = 0.001, group-A; p = 0.003, group-B; p = 0.001, group-C), high-grade tumors (p = 0.001, group-A; p = 0.017, group-B; p = 0.021, group-C), microsatellite-stable tumors (p < 0.001, group-A), pericolic lymph nodes involvement (p = 0.018, group-A), and cMYC mRNA expression (p = 0.002, group-A; p = 0.008, group-B; p = 0.006, group-C). High PKM2 mRNA expression was correlated with significantly lower disease free survival (DFS) (p = 0.002) and overall survival (OS) (p = 0.001) in the group-A. Similarly, PKM2 mRNA expression was associated with significantly decreased progression free survival (PFS) (p = 0.001) and OS (p = 0.001) in group-B. On the contrary, no significant association for the PKM2 mRNA expression has been observed with either PFS (p = 0.612) or OS (p = 0.517) in group-C. To conclude, the current study provides evidence for the prediction of PKM2 mRNA expression oxaliplatin-based treatment resistance.

MYC ASO Impedes Tumorigenesis and Elicits Oncogene Addiction in Autochthonous Transgenic Mouse Models of HCC and RCC

The MYC oncogene is dysregulated in most human cancers and hence is an attractive target for cancer therapy. We and others have shown experimentally in conditional transgenic mouse models that suppression of the MYC oncogene is sufficient to induce rapid and sustained tumor regression, a phenomenon known as oncogene addiction. However, it is unclear whether a therapy that targets the MYC oncogene could similarly elicit oncogene addiction. In this study, we report that using antisense oligonucleotides (ASOs) to target and reduce the expression of MYC impedes tumor progression and phenotypically elicits oncogene addiction in transgenic mouse models of MYC-driven primary hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Quantitative image analysis of MRI was used to demonstrate the inhibition of HCC and RCC progression. After 4 weeks of drug treatment, tumors had regressed histologically. ASOs depleted MYC mRNA and protein expression in primary tumors in vivo, as demonstrated by real-time PCR and immunohistochemistry. Treatment with MYC ASO in vivo, but not with a control ASO, decreased proliferation, induced apoptosis, increased senescence, and remodeled the tumor microenvironment by recruitment of CD4+ T cells. Importantly, although MYC ASO reduced both mouse Myc and transgenic human MYC, the ASO was not associated with significant toxicity. Lastly, we demonstrate that MYC ASO inhibits the growth of human liver cancer xenografts in vivo. Our results illustrate that targeting MYC expression in vivo using ASO can suppress tumorigenesis by phenotypically eliciting both tumor-intrinsic and microenvironment hallmarks of oncogene addiction. Hence, MYC ASO therapy is a promising strategy to treat MYC-driven human cancers.

High-risk breast cancer genes at 8q22-24 and their role in over 5,000 patients evaluated with the 70-gene risk of recurrence assay.

3569 Background: Previous studies have shown that CCNE2 expression is higher in patients’ cancers resistant to CDK4/6 inhibitors. Increased expression of CCNE2, MTDH, or TSPYL5, genes contained within the 70-gene risk of distant recurrence signature (70GS), has also been implicated in breast oncogenesis, poor prognosis, and chemoresistance. These genes are located on chromosome region 8q22.1, one of the most recurrently amplified regions out of all 70GS genes in breast tumors (Fatima et al. 2017). MYC, located on 8q24, is overexpressed in 40% of all breast cancers (BC). Here we examined the expression of CCNE2, MTDH, and TSPYL5 in relation to 70GS risk and the 80-gene molecular subtype signature (80GS), and their correlation with MYC expression in early stage BC patients. Methods: CCNE2, MTDH, TSPYL5, and MYC mRNA expression was measured in 5022 BC samples sent to Agendia (Irvine, CA) for 70GS and 80GS testing, which included FFPE microarray full-transcriptome data. 70GS was used to stratify patients into Ultra Low Risk (UL), Low Risk (LR), High Risk (HR), and Ultra High Risk (UH). Both 70GS and 80GS were used to classify patient samples into Luminal A, Luminal B, HER2, or Basal type. Wilcoxon rank sum test was used to assess expression differences. Results: The expression of CCNE2, MTDH, and TSPYL5 significantly correlated with each other and was higher in HR patients compared to LR patients (p &lt; 0.001) and higher in UH patients compared to HR patients (p &lt; 0.001). Expression of these genes was highest in Basal type tumors, 83% of which were UH, followed by Luminal B type tumors, and lowest in Luminal A type tumors. CCNE2 and MYC expression was elevated in LR compared to UL patients (p &lt; 0.001 and p = 0.0043). There was no difference in MYC expression between HR vs. LR or UH vs. HR. Lastly, there was no association between the expression of 8q22.1 genes and MYC in any 70GS subgroup. Conclusions: Within the 70GS, CCNE2, MTDH, and TSPYL5 have similar expression patterns and when overexpressed may identify an UH cohort of BC. This observation, in addition to their physical proximity at 8q22.1 suggests a possible amplicon in this region. The highest expression of CCNE2, MTDH, and TSPYL5 associated with UH patients and is concordant with previous studies that support the role of these genes in BC metastasis. Furthermore, this analysis suggests MYC may not stratify patients based on metastatic potential. These data may be clinically relevant for stratifying patients in ongoing clinical trials evaluating response and resistance to targeted therapies in early stage BC.

MYC copy number and mRNA expression in chronic lymphocytic leukemia patients exposed to ionizing radiation due to the Chornobyl NPP accident.

To investigate the frequency ofMYCgene copy number amplification in IR-exposed CLL patients and relate the findings to theMYCmRNA levels, the presence of unfavourable prognosis mutations (TP53, SF3B1, NOTCH1), and patient`s outcome. The analysis of MYC copy number was carried out by real-time quantitative polymerase chain reaction (PCR) in 70IR-exposed CLL patients. The MYC mRNA expression was measured by real-time quantitative reverse transcription PCR. Increased MYC gene copy number was present in 5.7% of cases. There was a statistically significant association between increased MYC copy number and increased MYC mRNA (p<0.014). Additionally, somatic deletion in MYC locus was found in one patient. Most of patients (80%) with detected MYC aberrations were previously untreated, suggesting that these lesions might occur early in the course of the disease. The MYC aberrations were found mutually exclusive with high risk TP53and SF3B1mutations, while one case was identified, where MYC amplification and NOTCH1mutation coincided simultaneously. Regarding clinical outcome, the MYC aberrations were associated with a shorter time to first treatment (3vs 25months, p = 0.008) as well as reduced overall survival (60vs 139months). Our data suggest that MYC aberrations might be an early event in IR-related CLL and contribute to aggressive disease development in the absence of high risk TP53and SF3B1mutations.

High MYC mRNA Expression Is More Clinically Relevant than MYC DNA Amplification in Triple-Negative Breast Cancer.

DNA abnormalities are used in inclusion criteria of clinical trials for treatments with specific targeted molecules. MYC is one of the most powerful oncogenes and is known to be associated with triple-negative breast cancer (TNBC). Its DNA amplification is often part of the targeted DNA-sequencing panels under the assumption of reflecting upregulated signaling. However, it remains unclear if MYC DNA amplification is a surrogate of its upregulated signaling. Thus, we investigated the difference between MYC DNA amplification and mRNA high expression in TNBCs utilizing publicly available cohorts. MYC DNA amplified tumors were found to have various mRNA expression levels, suggesting that MYC DNA amplification does not always result in elevated MYC mRNA expression. Compared to other subtypes, both MYC DNA amplification and mRNA high expression were more frequent in the TNBCs. MYC mRNA high expression, but not DNA amplification, was significantly associated with worse overall survival in the TNBCs. The TNBCs with MYC mRNA high expression enriched MYC target genes, cell cycle related genes, and WNT/β-catenin gene sets, whereas none of them were enriched in MYC DNA amplified TNBCs. In conclusion, MYC mRNA high expression, but not DNA amplification, reflects not only its upregulated signaling pathway, but also clinical significance in TNBCs.

In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells

BackgroundAcute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be “undrugable” because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription.MethodsThis study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt’s lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents.ResultsGQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin.ConclusionThese results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

MYC and BCL2 mRNA Expression As Determined By NGS Predicts Survival in DLBCL in GCB but Not in ABC Subgroup

Introduction: RNA expression profiling using Next Generation Sequencing (NGS) provides important and reproducible information on expression levels of various genes. We studied the expression levels of MYC and BCL2 in patients with diffuse large B-Cell lymphoma (DLBCL) using NGS targeted RNA sequencing. We correlated levels of expression of these genes with outcome. Methods: RNA extracted from 441 FFPE samples with DLBC lymphoma and sequenced targeting 1408 genes. The RNA sequencing is based on hybrid capture and the number of reads ranged from 5 to 10 million. RNA quantification was performed using Cufflinks. The RNA levels were normalized to PAX5 mRNA levels. Of these cases, 380 were subclassified as ABC or GCB using expression profiling. The rest were classified as "undetermined", therefore were not included in further analysis. All patients were treated with R-CHOP. Results: The expression of MYC and BCL2 mRNA was slightly higher in ABC as compared with GCB (P=0.01 and P=0.02, respectively). However, in the GCB subtype, patients with MYC expression above the median showed significantly shorter survival as compared with those below the median (P=0.0007, Log-rank test). In contrast, there was no significant difference in survival using median of MYC expression as cutoff in patients classified as ABC subtype (P=0.38). Using upper 15% cut-off point for BCL2 mRNA expression, GCB patients with high BCL2 expression had significantly shorter survival (P=0.005). In contrast, there was no significant difference in survival between high and low BCL2 expression groups in the ABC subtype (P=0.1). When both MYC and BCL2 are considered, patients with high expression of both BCL2 and MYC (double-RNA expression) had significantly shorter survival as compared with patients with low expression of both MYC and BCL2 (P=0.0009) when both GCB and ABC groups are considered. Patients with high BCL2 expression also had poor survival similar to those double-RNA expression. Considering only patients with GCB, high expressor of both MYC and BCL2 had significantly worse outcome (P=0.0015) as compared with low expressors of both MYC and BCL2, but patients with high BCL2 also had significantly poor outcome as compared to low expressor of both MYC and BCL2 (P=0.0005). In contrast, there was no difference in survival for high or low MYC and BCL2 expressor in the ABC group. Conclusion: The data support the concept that in DLBCL, MYC and BCL2 mRNA expression levels are clinically relevant in GCB, but not ABC subtype. Furthermore, targeted RNA sequencing might provide a reliable and practical objective approach for the subclassification of DLBCL and determining double-RNA expression lymphoma. Figure Disclosures Albitar: Genomic Testing Cooperative: Employment, Equity Ownership. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&amp;Abbvie Biotherapeutics Inc: Patents &amp; Royalties: US8,603,477 B2; Jazz: Consultancy. Piris:Calgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding. Kantarjian:Immunogen: Research Funding; BMS: Research Funding; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Ariad: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; AbbVie: Honoraria, Research Funding; Astex: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Research Funding.

LINC01116 accelerates nasopharyngeal carcinoma progression based on its enhancement on MYC transcription activity.

Long noncoding RNAs (lncRNAs) have been demonstrated to be frequently involved in the development of cancers, whereas only a few of them was investigated in nasopharyngeal carcinoma (NPC). Here, we found that LINC01116 was highly expressed in NPC cell lines, and inhibition of LINC01116 notably restrained cell viability, proliferation, and migration in NPC cells. Besides, we unveiled that LINC01116 was mainly distributed in the cytoplasm of NPC cells. Surprisingly, the cytoplasmic LINC01116 could directly interact with the 5′UTR of MYC mRNA, whereas such interaction had no influence on MYC mRNA expression, but facilitated MYC mRNA translation so as to enhance MYC protein level in NPC cells. Moreover, LINC01116 per se had no impact on the transcription of MYC targets but affected their expression through MYC‐dependent manner. Furthermore, MYC overexpression offset the suppression of LINC01116 silence on NPC development. In turn, we discovered that MYC could also serve as the transcriptional activator of LINC01116 in NPC cells. By and large, our findings elucidated a LINC01116/MYC feedback loop in accelerating the tumorigenesis of NPC, revealing a promising target to establish novel biomarkers for NPC patients.

P11.54 Identification of PDGFRA and MYC(N) as somatic driver genes in Glioblastoma

Abstract BACKGROUND Somatic oncogene amplification happens frequently in glioblastoma (GBM). The second most frequently amplified gene encoding receptor tyrosine kinases in GBMs is platelet derived growth factor alpha (PDGFRA) (15%). In contrast, MYC and MYCN amplification occurs in 1.6% and 2.9%, respectively. Our goal was to characterize the role of PDGFRɑ and Myc in GBM. MATERIAL AND METHODS Neurosphere cultures were implanted in cohorts of 10–15 nude mice. 5 PDX lines, presenting median survival of 29–59 days were classified as short survivors, and 5 lines with median survival between 104–134 days classified as long survivors. Total RNA was extracted from PDX terminal tumors (3 biological replicates) and sequenced in a paired-end read format. Mouse reads were filtered out using Xenome. MYC and PDGFRA expression patterns were analyzed in tissue microarrays representing duplicated samples from 40 glioma neurosphere-derived PDX lines by IHC (1 anaplastic oligodendroglioma, 8 recurrent GBM with 2 newly diagnosed/recurrent pairs). Normalized staining intensity (MI) and area (A) were quantified using Fiji/ImageJ. RESULTS PDGFRA, MYC, MYCN gene amplifications were represented in a molecularly diverse panel of GBM patient-derived cancer stem-like cells (CSC) and orthotopic mouse xenografts (PDX). Transforming to a normal distribution (log10), 4/13 of cell lines had a PDGFRA mRNA expression (RPKM) higher than 1.5. Similarly, one PDX line had a staining index of greater than 10, 11 (27.5%) had an index between 5–10. The range of intra-tumoral variance, represented by standard deviation, was 0.09–24.25 highlighting the heterogeneity of PDGFRɑ expression. PDGFRɑ phosphorylation (Y754) did not differ between 8 cell lines cultured in NMGF, but deviated in alternate medias without growth factors, supplemented with FBS. In comparison, MYC(N) mRNA expression is only elevated in the context of a known amplification. Furthermore, a a MYC activity signature consisting of 18 target genes was only evident in the 5 amplified CSC lines. Taking advantage of genomic heterogeneity, we have isolated subclones lacking PDGFRA amplification from a PDGFRA amplified GBM CSC. The absence of PDGFRA amplification reduced the self-renewal potential to 37% of the PDGFRA amplified cell population (p=0.001) in clone 1 and 57% in clone 2 (p=0.013). Pertaining to determinants of in vivo survival, MYC was altered in 80% of short survivors (2/5 MYC, 2/5 MYCN amplification) and in 0% of long survivors. Myc signature was highly correlated with in vivo survival (Pearsons’ corr. = -0.77) and MYC gene expression was correlated with in vivo TMZ resistance (corr. = 0.7). CONCLUSION These results suggest that PDGFRɑ expression and activity can occur in the absence of gene amplification, while Myc activity is dependent on gene amplification. Both oncogenes drive oncogenic pathways that should be explored as therapeutic targets.

Abstract 2967: Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells

Abstract Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that can occur when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state and proliferate. Ongoing efforts have focused on developing therapies that specifically target the protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A type of therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes, which are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the activity of G-quadruplex stabilizing small molecule GQC-05 that has been shown to down-regulate MYC, which is commonly misregulated in AML. Treatment of MYC-expressing AML cell lines KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with the effect more pronounced in KG-1a cells. GQC-05 treatment of the AML cells decreased cell viability, while increasing apoptosis and DNA damage. Combinational drug screening was performed to identify compounds that potentiated the anti-proliferative effects of GQC-05. Results from the screen identified the BCL-2 inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed synergistic effect on cell viability of AML cells as determined by Chou-Talalay analysis. Furthermore, co-treatment with Navitoclax did not affect the downregulation of MYC by GQC-05, but did increase apoptosis and DNA damage leading to rapid cytotoxicity. These results indicate that the G-quadruplex stabilizing small molecule GQC-05 may function more as a DNA damaging agent in AML cells and that combining GQC-05 with a Bcl-2/Bcl-XLinhibitor such as Navitoclax can result in increased cytotoxic activity. Citation Format: Justin J. Montoya, Megan A. Turnidge, Daniel H. Wai, David W. Lee, Apruvi Patel, Vijay Gokhale, Laurence J. Hurley, David O. Azorsa. Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2967.

PF245 METFORMIN INHIBITS THE GROWTH OF MYELOID LEUKEMIA CELLS PARTLY DUE TO SUPPRESSION OF TYRO3 EXPRESSION

Background:Metformin, an anti‐diabetic drug, is known to inhibit leukemia cell growth mainly through activation of AMP‐activated protein kinase (AMPK), resulting in inhibition of mTORC1 signaling. Meanwhile, TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in leukemia cell growth have been reported by us and others, the roles of TYRO3 have not been well elucidated.Aims:We examined the involvement of TYRO3 in anti‐leukemia effects of metformin.Methods:Four myeloid leukemia cell lines (OCI‐AML2, OCI‐AML3, AA, and K562) expressing TYRO3 protein were used. AA is a human cell line derived from pure erythroid leukemia. All cell lines were cultured with increasing concentrations of metformin. To knockdown TYRO3 expression, small interfering RNA (siRNA) against TYRO3 or control siRNA was transfected using an electroporation system. Cell growth was assessed with a colorimetric assay after culturing for three days. Expression levels and the activation of various signaling proteins in lysates from cultured cells were examined using immunoblotting. Expression levels of mRNA were examined by quantitative RT‐PCR.Results:Treatment with metformin suppressed the growth of these four cell lines in a dose‐dependent manner. Immunoblotting revealed that metformin increased phosphorylated AMPK while decreased phosphorylation of mTOR and the downstream proteins such as 4E‐BP1 and P70S6K without affecting expression levels of those proteins in general. Moreover, metformin treatment decreased expression levels of TYRO3 and c‐MYC and phosphorylation of TYRO3, STAT3, and ERK1/2. In K562 cells treated with metformin, decrease in phosphorylated TYRO3 was more evident than decrease in TYRO3 expression levels. In OCI‐AML2, OCI‐AML3, and K562 cells, which express AXL, metformin treatment also decreased expression levels and phosphorylation of AXL. Quantitative RT‐PCR analysis showed that expression of TYRO3 and MYC mRNA decreased by metformin treatment. Next, siRNA experiments showed that TYRO3 knockdown suppressed the cell growth to approximately 50% of the control. It also suppressed phosphorylation of STAT3 and ERK1/2, which are downstream molecules of TYRO3 signaling, while it did not obviously suppress the phosphorylation of mTOR.Summary/Conclusion:We revealed that metformin treatment suppressed TYRO3 expression in myeloid leukemia cells for the first time. We also found that TYRO3 is involved in leukemia cell growth because TYRO3 knockdown suppressed the growth. From these findings, we showed that growth inhibition by metformin might be partly due to its suppression of TYRO3. In leukemia cells expressing AXL, suppression of AXL expression might also be involved in growth inhibition by metformin. It is widely speculated that growth inhibition by metformin is mainly due to inhibition of mTORC1 signaling through AMPK activation and some AMPK‐independent mechanisms. We showed here that suppression of TYRO3 and/or AXL by metformin might also be involved in growth inhibition, although the individual effects of AMPK‐mTORC1 and TYRO3/AXL pathways are not clearly distinguished because mTOR is one of the downstream molecules of TYRO3/AXL pathway. In clinical settings in the future, metformin treatment might be effective for patients with leukemia cells expressing TYRO3 and/or AXL proteins as well as mTOR. Detection of expression and activation of these proteins can be done to select the appropriate patients for administration of metformin.image

SAT-353 The Synthetic Vitamin D Analog EB1089 Restricts Adrenocortical Tumor Proliferation in NCI-H295 Xenograft Model

The vitamin D (VD) receptor (VDR) is underexpressed in adult and pediatric adrenocortical tumors (ACT), especially in carcinomas (ACC). VDR activation by VD represses B-catenin signaling in different types of tumors that like ACT, present aberrant B-catenin expression. EB1089 is a potent synthetic VD analog that presents lower calcemic effects and is well tolerated at therapeutic doses. Since there is no effective adjuvant target-therapy for patients with ACT, we evaluated the effects of EB1089 in adrenocortical tumor proliferation. Methods:In vitro, we treated NCI-H295 (B-catenin mutant/activated) and Y1 (normal B-catenin) ACC cells with EB1089 (10-7M) and evaluated VDR and B-catenin signaling (qPCR) and cell proliferation (MTS). In vivo, we bilaterally inoculated subcutaneously 10 female NGS mice (8 week-old) with NCI-H295 cells (2.5x106 / 100 mcL). We assessed body weight (BW) and tumor volume (TuV; d2D/2) weekly. Once the TuV reached 100 mm3, we randomized the animals for EB1089 (n=5; 0.8mcg/kg BW) or vehicle [Control group (CG) n=5; 80% propylene glycol-20% PBS] treatment (sc injection; 5 days/week during 5 weeks). At the end of the protocol, mice were sacrificed and each tumor excised, weighted and fixed for pathological evaluation (H&E). Blood samples were taken for serum calcium (colorimetry), cortisol and testosterone (RIA) assessment. Data were presented as percentage or mean ± SEM. Results: In NCI-H295 cells the treatment with EB1089 (48h) increased VDR mRNA expression (47%; p=0.01) and activated its target gene CYP24A1 (p<0.0001). B-catenin expression and signaling decreased, as shown by reduced CTNNB1 (-24%; p=0.001), MYC (-40%; p=0.0001), CCND1 (-38%; p=0.02), and DKK3 (-40%; p=0.02) mRNA expression. Cell proliferation reduced after 96h (-15%; p=0.001). None of these observations were present in Y1 cells. In vivo, EB1089 significantly restricted tumor growth starting after 2 weeks of treatment (TuV: 284 ± 52 vs. 156 ± 20 mm3; p=0.05). At the end of the experiment TuV was ~50% lower in EB1089 xenografts (683 ± 345 vs. 368 ± 211 mm3; p=0.03). TuV and tumor weight correlated positively (r=0.88; p<0.0001). Both, EB1089 and CG tumors presented highly proliferative areas (pleomorphism, mitosis and few apoptotic bodies). However, EB1089 xenografts presented large necrotic areas. BW (p=0.98) and calcium levels (9.96 ± 0.4 vs. 11.3 ± 0.3 mg/dL; p=0.06) did not differ between groups. EB1089 mice presented reduced serum testosterone (76.7 ± 11.6 vs. 39 ± 5.6 ng/dL; p=0.02) and undetectable cortisol levels (CG: 2 ± 1.2 mcg/dL). Conclusion: The activation of VDR signaling by EB1089 inhibited B-catenin activated ACT proliferation. Furthermore, in vivo, EB1089 reduced tumor steroid secretion and did not result in important VD side effects like significant hypercalcemia and weight loss. VD analogs have antitumor activity and may emerge as an adjuvant therapy for patients with ACT.

Loss of LKB1 Protein Expression Correlates with Increased Risk of Recurrence and Death in Patients with Resected, Stage II or III Colon Cancer.

PurposeThe purpose of this study was to investigate the prognostic significance of liver kinase b1 (LKB1) loss in patients with operable colon cancer (CC).Materials and MethodsTwo hundred sixty-two specimens from consecutive patients with stage III or high-risk stage II CC, who underwent surgical resection with curative intent and received adjuvant chemotherapy with fluoropyrimidine and oxaliplatin, were analyzed for LKB1 protein expression loss, by immunohistochemistry as well as for KRAS exon 2 and BRAFV600E mutations by Sanger sequencing and TS, ERCC1, MYC, and NEDD9 mRNA expression by real-time quantitative reverse transcription polymerase chain reaction.ResultsLKB1 expression loss was observed in 117 patients (44.7%) patients and correlated with right-sided located primaries (p=0.032), and pericolic lymph nodes involvement (p=0.003), BRAFV600E mutations (p=0.024), and TS mRNA expression (p=0.041). Patients with LKB1 expression loss experienced significantly lower disease-free survival (DFS) (hazard ratio [HR], 1.287; 95% confidence interval [CI], 1.093 to 1.654; p=0.021) and overall survival (OS) (HR, 1.541; 95% CI, 1.197 to 1.932; p=0.002), compared to patients with LKB1 expressing expressing tumors. Multivariate analysis revealed LKB1 expression loss as independent prognostic factor for both decreased DFS (HR, 1.217; 95% CI, 1.074 to 1.812; p=0.034) and decreased OS (HR, 1.467; 95% CI, 1.226 to 2.122; p=0.019).ConclusionLoss of tumoral LKB1 protein expression, constitutes an adverse prognostic factor in patients with operable CC.