C-myc RNA Research Articles

Association of C-MYC, MYC target gene, and unfolded protein response (UPR) expression with clinical benefit from the oral aurora kinase A (AURKA) inhibitor, alisertib (A), in combination with paclitaxel (P) compared with P alone in patients (Pts) with HER2-negative metastatic breast cancer (MBC).

1037 Background: AURKA is a key regulator of the mitotic spindle, G2/M transition and epithelial-mesenchymal transition. AURKA is amplified and/or overexpressed in breast cancer and is associated with therapy resistance and worse survival. A randomized phase II trial in hormone receptor (HR)-positive, HER2-negative and triple negative (TN) MBC pts showed that addition of A to weekly P significantly improved progression-free survival (PFS) compared with P alone (O’Shaughnessy J et al. JAMA N etwork Open, 2021). Pts’ primary or metastatic disease tissues were analyzed for biomarkers associated with clinical benefit from A. Methods: Retrospective analysis of tumor whole exome and whole transcriptome sequencing of formalin-fixed paraffin embedded pre-treatment tissues from 96 pts (77 HR+, 19 TN) was performed. Clinical benefit from A+P or P was defined as having PFS of at least 6 mos and lack of benefit as PFS less than 6 mos. Enrichment for cancer gene mutations was assessed using Fisher's exact test. Transcriptome data were evaluated for differential expression with DeSeq2 and gene set enrichment analysis (GSEA) and compared to cancer hallmark gene sets. Molecular features were compared between A+P responders and non-responders, P responders and non-responders, and A+P and P responders. P-values <0.05 were considered significant. Results: PIK3CA, TP53, and CDH1 were altered in 41%, 39%, and 13% of tumors, respectively, and these and other known cancer genes were not associated with A+P benefit. Alterations in AKT1, PIK3CA, ERBB2, CDH1, TP53, and KMT2C/ KMT2D, as well as amplifications of CCND1, MYB, and MDM2, did not preclude benefit from A+P. RNA expression of AURKA, or its upstream regulator, FOXM1, was not significantly different based on treatment group or benefit from A+P or P. Increased C-MYC RNA expression (p=0.008), and enrichment for MYC targets and UPR by GSEA were enriched in pts with lack of benefit from P compared to those with benefit from P (p<0.05), but not in pts with lack of benefit from A+P. Wnt/Beta-catenin signaling was enriched in pts with lack of benefit from P compared to those with benefit from P (p=0.03), and with lack of benefit from A+P compared to those with benefit from A+P (p=0.086). Benefit from A+P was associated with enrichment for MYC targets and UPR compared with benefit from P (p=0.024), suggesting activity of A+P, but not P, with high MYC activation and UPR. Conclusions: A added to P improved PFS in HR+ HER2- and TN MBC pts compared with P alone. Pts whose breast cancers had increased C-MYC expression, high MYC activation and increased UPR on RNA expression derived greater clinical benefit from A+P than from P alone. Clinical trial information: NCT02187991 .

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

Satellite Noncoding RNAs (ncRNA) as Cancer Biomarkers? New Insights from FA-SAT ncRNA Molecular and Clinical Profiles in Feline Mammary Tumors.

Satellite noncoding RNAs (ncRNAs) are a new frontier of cancer biology research and biomarkers. While the knowledge on ncRNAs in human cancers is still limited, studies in other species can be informative to guide future translational research and development for cancer molecular targets and diagnostics. In this context, FA-SAT is the major satellite DNA of the cat genome, which is also present in humans, being transcribed in both species. In this study, we report new insights on FA-SAT (DNA and RNA) profile in feline mammary tumors, using disease-free tissues from the same animals as reference. We quantified the FA-SAT DNA and RNA levels (long and small transcripts) by real-time quantitative polymerase chain reaction (qPCR) and RT-qPCR. The comparison of the FA-SAT DNA and RNA levels with clinicopathological parameters revealed several associations, such as (1) the FA-SAT DNA levels' positive relation with lymphovascular invasion, (2) the FA-SAT long RNA negative correlation with Ki-67 index, and its positive association with Estrogen Receptor status, and (3) the FA-SAT small RNA level positive correlation with tumor size and skin ulceration. Also, FA-SAT long RNA is correlated with ERBB2 and c-MYC RNA levels. These data collectively suggest that FA-SAT ncRNA offers prospects as a potential cancer biomarker in cats. Further studies in humans are also needed to decipher the emerging role of ncRNAs in cancer biology and precision medicine fields. This work brings new information on the relation of FA-SAT ncRNAs with the oncogenic process, uncovering a new potential cancer biomarker.

Abstract 2389: LINC00901 regulates glucose metabolism in pancreatic cancer

Abstract Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.

A Novel Combination Regimen of BET and FLT3 Inhibition for FLT3-ITD Acute Myeloid Leukemia

Background: Acute myeloid leukemia patients with FLT3-ITD mutations have a high risk of relapse and death. FLT3 tyrosine kinase inhibitors such as quizartinib and gilteritinib improve overall survival in relapsed patients, but their efficacy is limited and most such patients die of the disease. This is because even with potent FLT3 inhibition, the disease persists within the bone marrow microenvironment, mainly due to bone marrow stroma activating parallel signaling pathways that maintain pro-survival factors. BET inhibitors suppress pro-survival factors such as c-Myc and Bcl2, but these drugs thus far have shown only limited single-agent clinical potential. PLX51107 is a novel BET inhibitor designed to inhibit BET activity in intermittent daily fashion to allow for greater tolerability. We investigated whether the addition of PLX51107 to potent FLT3 inhibition with quizartinib could overcome the protective effect of the bone marrow stroma through inhibition of c-Myc expression. Methods: We developed a plasma inhibitory activity assay for c-Myc (c-Myc PIA) to assess in vivo efficacy of the PLX51107 in patients and to identify c-Myc-inhibitory doses of the drug. We tested PLX51107 alone and in combination with quizartinib in murine models of AML and against primary FLT3-ITD AML cells co-cultured with human bone marrow stroma. In addition, we analyzed gene expression patterns in the treatment models to explore the basis of any observed synergistic cytotoxic effect. Results: In a murine xenograft model of AML using MV4-11 cells, PLX51107 alone induced suppression of tumor growth in association with a 90% decrease in c-Myc gene expression. The combination of PLX51107 and quizartinib induced complete tumor regression in 5 out of 7 animals after 14 days of treatment (Figure 1). Animals treated with a 5-day course of quizartinib alone displayed tumor regression persisting until day 26 of treatment, while the addition of PLX51107 resulted in tumor regression until day 39. In patients treated with a dose of 120 mg/day of PLX51107, the c-Myc PIA demonstrated a robust suppression of c-Myc expression for roughly 6 hours, returning to baseline between 7 and 9 hours post-treatment (Figure 2). The mean plasma concentration to achieve this inhibition was 3.3 uM, which, accounting for the difference in protein drug binding between plasma and culture medium with 10% FBS, corresponded to a concentration of 250 nM PLX51107 in culture medium. With this same concentration and schedule (i.e., at concentrations and exposure times equivalent to what human patients would experience taking 120 mg daily of PLX51107 and 60 mg daily of quizartinib), the combination induced synergistic cytotoxicity in a series of 10 different FLT3-ITD AML patient blast samples co-cultured with bone marrow stroma (Figure 3). C-Myc RNA and protein were directly suppressed in these primary samples, and ingenuity pathway analysis of RNA expression confirmed that c-Myc associated genes displayed the highest level of down-regulation. Conclusions: These studies suggest that combination therapy with approximately 120 mg/day PLX51107 and 60 mg/day quizartinib will be a more effective therapy for relapsed FLT3-ITD AML than 60 mg/day of quizartinib alone. The combination of FLT3 inhibition and BET inhibition may represent an attractive therapeutic option for FLT3-ITD AML. Disclosures Hizukuri: Daiichi Sankyo Co, Ltd: Employment. Severson:Plexxikon Inc.: Employment. Powell:Plexxikon Inc.: Employment. Zhang:Plexxikon Inc.: Employment. Ma:Plexxikon Inc: Employment. Narahara:Daiichi Sankyo Co, Ltd: Employment. Sumi:Daiichi Sankyo, Inc.: Employment. Bollag:Plexxikon Inc.: Employment. Levis:FUJIFILM: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Menarini: Consultancy, Honoraria.

Gene expression association study in feline mammary carcinomas.

Works on cancer-related genes expression using feline mammary carcinomas (FMCs) are scarce but crucial, not only to validate these tumours as models for human breast cancer studies but also to improve small animal practice. Here, the expression of the cancer-related genes TP53, CCND1, FUS, YBX1, PTBP1, c-MYC and PKM2 was evaluated by real-time RT-qPCR, in a population of FMCs clinically characterized and compared with the disease-free tissue of the same individual. In most of the FMCs analysed, RNA quantification revealed normal expression levels for TP53, c-MYC, YBX1 and FUS, but overexpression in the genes CCND1, PTBP1 and PKM2. The expression levels of these cancer-related genes are strongly correlated with each other, with exception of c-MYC and PKM2 genes. The integration of clinicopathological data with the transcriptional levels revealed several associations. The oral contraceptive administration showed to be positively related with the TP53, YBX1, CCND1, FUS and PTBP1 RNA levels. Positive associations were found between tumour size and YBX1 RNA, and lymph node metastasis with c-MYC RNA levels. This work allowed to verify that many of these cancer-related genes are associated but may also, indirectly, influence other genes, creating a complex molecular cancer network that in the future can provide new cancer biomarkers.

Abstract 764: MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer

Abstract Although death rates for prostate cancer are down about 50% as a result of improvements in early detection and treatment, it is still the second leading cause of death (26,120 cases) for American men. However, the genetic mechanism underlying the development and progression of prostate cancer remains poorly understood. It is therefore important to identify the genetic risk factors for prostate cancer. Genome-wide association studies (GWAS) showed evidence for an association of prostate cancer risk with independent genetic variants on chromosome 8q24, conferring prostate cancer susceptibility at this locus. Nevertheless, at the locus with these genetic variants, no specific gene has been identified as responsible for the risk of prostate cancer. At 8q24.21, there is a long-range interaction of these genetic variants with c-MYC or long non-coding RNAs (lncRNAs) in a tissue-specific manner, including prostate, suggesting master genetic factors at 8q24.21 that contribute to this genetic risk. The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. Here, the copy numbers and expressions of miRs-1204~1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with expression of miR-1205. Furthermore, functional analysis with a miR-1205 mimic, a miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression but not apoptosis. In these cells, miR-1205 downregulated expression of the Egl-9 family hypoxia inducible factor 3 (EGLN3) gene and targeted a site in its 3’-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Wen Wen, Yicun Wang, Yue Zhang, Xuelian Cui, Xiaoguang M. Liu, Runhua Liu. MicroRNA-1205 contributes to the risk of castration-resistant prostate cancer [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 764.

Abstract 3837: JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma cells

Abstract Cholangiocarcinoma (CCA) is an aggressive bile duct neoplasm which is typically not diagnosed until late stage. The current standard of care, resection followed by gemcitabine with or without cisplatin, is relatively ineffective. Additionally, most patients are ineligible for surgical resection, and postoperative chemotherapy rarely prolongs overall survival. No previously identified mutations (KRAS, SMAD4 and p53) have been shown to drive disease progression. We and others have shown that the proto-oncogene c-Myc is expressed at relatively high levels in CCA cells compared to uninvolved normal tissue, suggesting this oncogene as a prospective therapeutic target in CCA. To evaluate whether c-Myc inhibition affected the proliferation of CCA cells, we evaluated the efficacy of the bromodomain and extraterminal domain (BET) inhibitor JQ1 on c-Myc expression and cell proliferation of CCA cells. JQ1 functions as an acetyl lysine (K-Ac) mimetic which binds to the K-Ac binding pocket of BET protein family members (BRD2, BRD3, BRD4 and BRDT). This binding competitively inhibits the association of BET proteins with K-Ac residues of nuclear proteins and inhibits c-Myc expression. Using a patient-derived xenograft (PDX) model of CCA, we showed previously that JQ1 downregulated c-Myc expression and inhibited tumor growth. Therefore, we hypothesized that JQ1 induces CCA cell death through disruption of BET protein binding to the promoter region of the MYC locus, inducing its downregulation. Our data show that JQ1 decreased c-Myc RNA and protein expression as well as the expression c-Myc transcriptional targets CHK1 and BRCA2. JQ1 also decreased CCA cell viability, clonogenic potential and induced apoptosis of KKU-055 CCA cells in vitro. The likely mechanism of JQ1-mediated c-Myc reduction has been postulated to involve competitive inhibition of the K-Ac binding function of the BET protein BRD4. Our current chromatin immunoprecipitation (ChIP) assays indicate that BRD2 binds to the MYC transcriptional start sites (TSS) P1 and P2, while BRD4 primarily binds MYC TSS P1 in KKU-055 cells. JQ1 precludes these BET proteins from binding their respective sites. When exposed to the IC50 of JQ1, ChIP pulldown with an anti-BRD2 antibody displays a near 100% reduction in binding of BRD2 to the MYC TSS while an anti-BRD4 antibody displays a 70% reduction in binding. In addition, stable BRD2 knockdown models of KKU-055 display reduced c-Myc protein expression by 60 to 80%, while BRD4 knockdown models showed no such reduction. The data demonstrate that in addition to BRD4, BRD2 contributes to regulation of expression of c-Myc in CCA cells. Research funded by NIH/NCI R21 CA205501 Citation Format: Samuel C. Fehling, Aubrey L. Miller, Karina J. Yoon. JQ1 induces cell death and disrupts BET protein binding to the MYC locus in cholangiocarcinoma 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 3837.

LncRNA THOR acts as a retinoblastoma promoter through enhancing the combination of c-myc mRNA and IGF2BP1 protein

Long non-coding RNA (lncRNA) THOR is an extremely conserved lncRNA with specifically expressed in testis while widespreadly exist in human multiple cancer tissues. The high expression of it significantly promotes the occurrence and progression of melanoma, non-small cell lung cancer, osteosarcoma and renal cell carcinoma. However, the expression pattern and effects of lncRNA THOR in the progression of retinoblastoma remain unclear. As a result, this study was conducted to discovery the expression and roles of lncRNA THOR in the malignant phenotype transformation of retinoblastoma cells, as well as its underlying mechanism. Our results demonstrated that lncRNA THOR was over-expressed in the retina tissues from retinoblastoma patients and retinoblastoma Y79 and WERI-Rb1 cell lines. Down-regulation of lncRNA THOR with siRNA significantly repressed cell growth, migration and S phase accumulation, while induced cell apoptosis and G1 phase reduction and reduced the expression of c-myc. Besides, knockdown of c-myc promoted cell apoptosis and suppressed cell proliferation. Furthermore, RNA pull down and PIP assays showed that up-regulation of lncRNA THOR enhanced the combination of IGF2BP1 protein and c-myc RNA. And lncRNA THOR up-regulation obviously increased the tumorigenesis of Y79 cells in vivo. In conclusion, this study makes clear that lncRNA THOR is up-regulated in retinoblastoma, and its over-expression significantly enhances the malignant phenotype transformation of retinoblastoma cells through up-regulating c-myc expression via enhancing its combination with TGF2BP1 protein. Overall, our study illustrates that lncRNA THOR/c-myc molecular cascade might be another potent target for retinoblastoma treatment.

Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli.

Background & AimsGastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP) signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation.MethodsThe H+/K+-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog) in the stomach (H+/K+-Nog mice). Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre) mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice). Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom). Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction.ResultsLgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, and with anti-IF, -CD44, -CD44v9, and -bromodeoxyuridine antibodies.ConclusionsInflammation and inhibition of BMP signaling activate Lgr5+ve cells, which give rise to metaplastic, dysplastic, proliferating lineages that express markers of mucus neck and zymogenic cell differentiation.

Rituximab-Induced CD20-Mediated Signals and Suppression of PI3K-AKT Pathway Cooperates to Inhibit B-Cell Lymphoma Growth By Down-Regulation of Myc.

Rituximab, anti-CD20 monoclonal antibody, shows anti-lymphoma effects by either activating host immune mechanism against lymphoma cells such as ADCC and CDC or suppressing signal(s) important for growth of B-cell lymphoma. Accumulated evidence shows de-regulated PI3K-AKT pathway is important therapeutic target in B-cell lymphoma. Although studies suggested suppression of PI3K-AKT pathway is possibly critical in rituximab-induced anti-lymphoma signals, details of molecular mechanisms covering CD20-mediated signals and PI3K-AKT pathway are still unknown.We analyzed signals in B-cell lymphoma cell line, SD07, which had bi-allelic deletions of both CD20(MS4A1) and PTEN gene. Briefly, SD07 was established from a patient with diffuse large B-cell lymphoma(DLBCL)who showed refractory CD20-negative relapse(Eur.J. Haematol. 2012).We transiently restored protein expression of either or both of CD20 and PTEN by electroporation of expression plasmid having cDNA of those genes in SD07. In this experiment, expression of both proteins were clearly detected by western blot until 72 hours after transfection. CD20 protein was also detectable on cell surface of SD07 cells by flow cytometric(FCM) analysis at comparable levels of those of Daudi cells after transfection on CD20 cDNA.Our previous study showed expression of both c-myc RNA and protein, as judged by FCM, was induced by transient transfection of CD20 cDNA in SD07 cells. Western blot analysis also showed significant increase of myc protein 24hrs after transfection (vector=13.7±0.2, CD20=14.5±0.4, P<0.05, arbitrary density unit) and the increase was not affected by the addition of rituximab(20µg/ml)in culture. In contrast transfection of PTEN cDNA produced significant decrease of myc protein (PTEN=11.2±0.1, P<0.01). Myc protein levels was comparable between cells transfected with CD20 plus PTEN and PTEN alone. However in the presence of rituximab, myc protein expression was markedly downregulated in SD07 cells at both 24 hours(vector=13.7±0.2,CD20+PTEN=8.3±0.1,P=0.0008)and 48hours(vector=12.4±0.3 vs CD20+PTEN=11.2±0.1,P=0.04).24 hours after transfection, de-phosphorylation and down-regulation of retinoblastoma(RB) protein expression was evident only in SD07 cells transfected with both CD20 and PTEN, but not with either alone.Rituximab also augmented down-regulation of RB protein in SD07 cells transfected with both CD20 and PTEN.After transfection, in vitro cell growth of SD07, as evaluated by MTT, was as follows;vector=4.6±0.3, CD20=4.8±0.3(P=NS), PTEN=4.1±0.2(P<0.05), CD20+PTEN=3.6±0.2(P<0.01), CD20+PTEN+rituximab=2.7±0.2(P<0.01),OD570nm, days3)72 hours after transfection, by FCM, SD07 transfected with both CD20 and PTEN showed significant increase of subG1 DNA content potion compared with cells transfected with either alone(vector=2.5±0.2%,CD20=5.3±0.2%,PTEN=8.8 ±0.5%,CD20+PTEN= 16.1±0.6, P<0.01). Furthermore, addition of rituximabsignificantly increase of cells in subG1 in SD07 with transfected with both CD20 and PTEN(18.6±0.5%,P<0.01), suggesting suppression of PI3K-AKT is important for mediateing rituximab-induced apoptotis.Accordance with proliferative effect of CD20 inSD07, transfection of CD20 alone produced significant increase of phosphorylation of AktSer473(vector=11.2±2.3, CD20=13.3±2.3,pAkt/total Akt,P<0.05). Co-transfection of PTEN with CD20 suppress the CD20-induced phosphorylation of Akt in SD07 cells. (CD20+PTEN=12.8±2.3,P=NS).In summary, although data is limited in a cell line, it clearly shows rituximab-induced CD20-mediated signals and suppression of PI3K-AKT pathway by tumor suppressor PTEN cooperatively inhibits growth and induce apoptosis of DLBCL cell line, SD07. Myc protein is critical molecules in this particular cooperative pathways. The present result partly agree studies which showed that inhibition of PI3K-AKT signaling and down-regulation of myc is important in PTEN-induced cytotoxicity in GCB(germinal center B cell)type DLBCL. In addition, we show here myc is not only for PTEN, but also involved in rituximab-induced anti-lymphoma effect. The present result provide a rationale for combination of rituximab and PI3K inhibitor in the treatment of DLBCL. In this context, myc is a candidate of the therapeutic target in rituximab-based immunochemotherapy for B cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

In vitro cardiomyocyte differentiation of umbilical cord blood cells: crucial role for c-kit+ cells

Background aimsAlthough bone marrow c-kit+ progenitor cells support myocardial regeneration, the cardiomyocyte differentiation potential of umbilical cord blood (UCB) c-kit+ cells is unknown. MethodsUCB mononuclear cells (MNCs) and c-kit+ cells purified by use of immunomagnetic beads were used. Cardiomyocyte differentiation was induced with (i) α-minimum essential medium (MEM) with cyclosporine A, (ii) α-MEM with bone morphogenic protein 4 (BMP-4) and transforming growth factor-β (TGF-β) or (iii) MEM with dexamethasone. The expression of cardiac markers (GATA4, GATA6, β-myosin heavy chain, α-sarcomeric actin and cardiac Troponin T) was investigated, and whole-cell current and voltage-clamp recordings were performed. ResultsAlthough c-kit+ cells revealed an immature gene profile, with high expression of CD34, CD133, aldehyde dehydrogenase-A1 and c-myc RNAs, purified c-kit+ cells did not succeed in differentiating into cardiomyocyte-like cells in culture. In contrast, MNCs (either in α-MEM plus cyclosporine A or in α-MEM plus BMP-4 and TGF-β) produced large, adherent cells expressing several cardiac genes and exhibiting an excitable phenotype. Cardiomyocyte-like cell formation was prevented by removing the c-kit+ cell fraction from MNCs. Furthermore, after co-culturing carboxyfluorescein diacetate succynimidyl ester (CFSE)-tracked c-kit+ cells together with c-kit– cells, we found that cardiac Troponin T–expressing cells were also CFSE+. ConclusionsWe show that UCB contains progenitors endowed with differentiation potential into cardiomyocytes: these cells reside in the c-kit+ fraction and require the presence of abundant accessory cells to accomplish the differentiation. These preliminary observations provide the basis for consider the storage of autologous UCB in patients with prenatal diagnosis of congenital heart diseases potentially amenable by myocardial regenerative approaches.

Abstract 1779: Bromodomain inhibitor JQ1 inhibits cholangiocarcinoma tumor growth in patient-derived xenograft models

Abstract Cholangiocarcinoma (CCA) is a lethal malignancy of the biliary epithelium that can arise in any part of the biliary tree. Surgery is the only curative treatment for CCA, but only ∼30% of patients present with resectable disease. The remaining 70% of patients present with advanced or metastatic disease and, if eligible, undergo systemic chemotherapy with the first-line combination of gemcitabine and cisplatin. This combination was shown in a phase II clinical trial to significantly increase median survival from 8.1 to 11.7 months, compared to gemcitabine alone. In order to improve on current treatment, pre-clinical evaluation of novel therapeutics is essential to improving outcome. Unfortunately, the paucity of data describing characteristics common to CCA make development of targeted therapy difficult. However, as is true for other types of solid tumors, c-Myc expression likely contributes to CCA phenotype: c-Myc expression has been observed in 95% of CCA tumors, and experimental down-regulation of c-Myc decreases the invasive potential of CCA cells in vitro. Recently it has become possible to inhibit expression of c-Myc using BET inhibitors. Therefore, we evaluated the efficacy of the bromodomain (BET) inhibitor JQ1 using in vivo models of CCA. The five patient-derived xenograft (PDX) models of CCA that we developed are the first such models to be reported. These models retain the heterogeneity, architecture and specific genetic characteristics of the primary tumors from which they were derived. We used three of these models to examine whether the BET inhibitor JQ1 inhibited CCA tumor growth and generated expression profiles of vehicle- and drug-treated tumors. We administered 50 mg/kg of JQ1 i.p. daily for 20 days and monitored tumor growth. This treatment regimen was well tolerated by tumor-bearing mice, without apparent toxicity. Our data demonstrate that JQ1 suppressed tumor growth in two of the three models, compared to vehicle control treated mice. The data also showed that JQ1-treated tumors had lower levels of c-Myc RNA (↓5-fold) and protein and of RNA encoding multiple transcriptional targets downstream of this oncogenic transcription factor. We conclude that BET inhibitors such as JQ1 warrant further investigation as potentially effective drugs for the treatment of CCA. Citation Format: Patrick L. Garcia, Aubrey L. Miller, Kelly Kreitzburg, Tracy L. Gamblin, Leona N. Council, John D. Christein, Pablo Arnoletti, Marty Heslin, Sushanth Reddy, Joseph H. Richardson, Eddy S. Yang, Jun Qi, James E. Bradner, Karina J. Yoon. Bromodomain inhibitor JQ1 inhibits cholangiocarcinoma tumor growth in patient-derived xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2015-1779

Bromodomain Inhibition By OTX015 Regulates c-MYC and HEXIM1 in a Panel of Human Acute Leukemia Cell Lines

Background: The bromodomain-containing protein 4 (BRD4) activates the transcription elongation factor b (P-TEFb) which regulates RNA polymerase II. Conversely, hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) inactivates P-TEFb. BRD4/HEXIM1 interplay influences cell cycle progression and tumorigenesis. It has been widely demonstrated that BRD4 knockdown or inhibition by JQ1 is associated with c-MYC downregulation and antileukemic activity. We recently reported that the small molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland), currently in clinical development, mimics the effects of JQ1 (Braun et al, ASH 2013). We evaluated the effect of OTX015 on c-MYC, BRD2/3/4, and HEXIM1 in human in vitro leukemic models.Methods: c-MYC, BRD2/3/4 and HEXIM1 expression was assessed in six acute myeloid leukemia (AML; K562, HL-60, NB4, NOMO-1, KG1, OCI-AML3) and two acute lymphoid leukemia (ALL; JURKAT and RS4-11) cell lines after exposure to 500 nM OTX015. Quantitative RT-PCR and Western blotting were performed at different time points (24-72h). A heatmap was computed with R-software.Results: c-MYC RNA levels were ubiquitously downregulated in all AML and ALL cell lines after 24h exposure to OTX015 (Figure 1). c-MYC protein levels decreased to a variable extent at 24-72h in all cell lines evaluated other than KG1. BRD2, BRD3 and BRD4 mRNA expression was significantly decreased in K562 cells (known to be OTX015-resistant) after 48h exposure to OTX015 but was increased in HL60 and NOMO-1 cells, while minimal to no increases were observed in other cell lines. OTX015 induced a decrease in BRD2 protein expression in most cell lines, but not in K562 cells. In contrast, decreased BRD4 protein expression was only seen in the OCI-AML3, NB4 and K562 cell lines. BRD3 protein levels were not modified after OTX015 exposure in all cell lines evaluated other than KG1. HEXIM1 mRNA expression increased after 24h exposure to 500 nM OTX015 in all cell lines except OTX015-resistant K562 cells in which the increase was considered insignificant (less than two-fold). Increases in HEXIM1 protein levels were observed in OCI-AML3, JURKAT and RS4-11 cell lines at 24-72h but not in K562 cells.Conclusion: Taken together, these results show that BRD inhibition by OTX015 modulates HEXIM1 gene and protein expression, in addition to c-MYC decrease and BRD variations. HEXIM1 upregulation seems to be restricted to OTX015-sensitive cell lines and was not significantly affected in OTX015-resistant K562 cells. Further studies are needed to clarify the role of HEXIM1 in antileukemic activity of BRD inhibitors. [Display omitted] DisclosuresRiveiro:OTD: Employment. Herait:OncoEthix: Employment. Dombret:OncoEthix: Research Funding.

Ultrasmall gold nanoparticles as carriers for nucleus-based gene therapy due to size-dependent nuclear entry.

The aim of this study was to determine the size-dependent penetration ability of gold nanoparticles and the potential application of ultrasmall gold nanoparticles for intranucleus delivery and therapy. We synthesized gold nanoparticles with diameters of 2, 6, 10, and 16 nm and compared their intracellular distribution in MCF-7 breast cancer cells. Nanoparticles smaller than 10 nm (2 and 6 nm) could enter the nucleus, whereas larger ones (10 and 16 nm) were found only in the cytoplasm. We then investigated the possibility of using ultrasmall 2 nm nanoparticles as carriers for nuclear delivery of a triplex-forming oligonucleotide (TFO) that binds to the c-myc promoter. Compared to free TFO, the nanoparticle-conjugated TFO was more effective at reducing c-myc RNA and c-myc protein, which resulted in reduced cell viability. Our result demonstrated that the entry of gold nanoparticles into the cell nucleus is critically dependent on the size of the nanoparticles. We developed a strategy for regulating gene expression, by directly delivering TFOs into the nucleus using ultrasmall gold nanoparticles. More importantly, guidelines were provided to choose appropriate nanocarriers for different biomedical purposes.

Altered Endoribonuclease Activity of Apurinic/Apyrimidinic Endonuclease 1 Variants Identified in the Human Population

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. APE1 also has 3′-5′ DNA exonuclease and 3′ DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76–85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3′ of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60–80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.

Cyclin dependent kinase-9 mediated transcriptional de-regulation of cMYC as a critical determinant of endocrine-therapy resistance in breast cancers

Endocrine therapy resistance in estrogen receptor alpha positive (ERα+) breast cancers remains a major obstacle for maintaining efficacy of targeted therapies. We investigated the significance and the mechanisms involved in cMYC over-expression in a MCF7 derived panel of ERα+ breast cancer cells which can proliferate in the absence of estrogen with different sensitivities to anti-hormone therapies. We show that all the resistant cell lines tested over-express cMYC as compared to parental MCF7 cells and its inhibition lead to the differential blocking of estrogen-independent proliferation in resistant cells. Further investigation of the resistant cell line, MCF7:5C, suggested transcriptional de-regulation of cMYC gene was responsible for its over-expression. Chromatin immuno-precipitation assay revealed markedly higher recruitment of phosphorylated serine-2 carboxy-terminal domain (CTD) of RNA polymerase-II at the proximal promoter of cMYC gene, which is responsible for transcriptional elongation of the cMYC RNA. The level of CDK9, a factor responsible for the phosphorylation of serine-2 of RNA polymerase II CTD, was found to be elevated in all the resistant cell lines. Pharmacological inhibition of CDK9 not only reduced the transcripts and the protein levels of cMYC in MCF7:5C cells but also selectively inhibited the estrogen-independent growth of all the resistant cell lines. This study describes the up-stream molecular events involved in the transcriptional over-expression of cMYC gene in breast cancer cells proliferating estrogen-independently and identifies CDK9 as a potential novel drug target for therapeutic intervention in endocrine-resistant breast cancers.

Preclinical Study Of The Bromodomain Inhibitor OTX015 In Acute Myeloid (AML) and Lymphoid (ALL) Leukemias

BackgroundBromodomain and extra-terminal (BET) proteins, including the ubiquitous BRD2/3/4 proteins, are epigenetic readers implicated in c-MYC transcription, cellular proliferation, cell-cycle progression, RNA elongation and DNA damage response. Using shRNA screening and BRD inhibitors, BRD4 has been established as a promising therapeutic target in acute leukemia (Zuber, Nature 2011). In the present study, we investigated the in vitro anti-leukemic effects of the small-molecule BRD2/3/4 inhibitor OTX015 (Oncoethix, Lausanne, Switzerland). MethodsExpression of BRD2/3/4 and c-MYC was assessed by RQ-PCR in 5 myeloid (HL60, KG1, KG1a, K562, NOMO1) and 4 lymphoid (Jurkat, RS4-11, BV173, TOM1) leukemia cell lines and by Western blotting (WB) using commercial antibodies in the HL60, K562, Jurkat and RS4-11 lines. Nineteen AML and ten ALL patient banked leukemic cells were assayed by RQ-PCR only. Cell viability and IC50 values were assessed in cell lines by MTT assays after exposure to OTX015 (0.1nM-10µM) for 72h. Cell-cycle distribution was determined by cytofluorometric analysis detecting nuclear propidium iodide (PI) intercalation. Induction of apoptosis was evaluated in cell lines and patient cells by outer membrane phosphatidylserine exposure and PI incorporation at 72 hours with increasing doses of OTX015 (25nM-500nM). Caspase-3 activation and mitochondrial cytochrome c release were studied by immunofluorescence (IF). Maturation was assessed by morphological studies after MGG staining and detection of CD11b by FACS analysis. Modulation of BRD2/3/4 proteins was investigated by WB. ResultsOTX015 IC50 values were in the submicromolar range for KG1 and the MLL-driven NOMO1 cell lines (198.3 and 229.1nM, respectively), while K562 was the most resistant myeloid line, with an IC50 of 11.3µM. In contrast, in lymphoid cell lines tested, IC50 values ranged from 34.2 to 249.7nM, with the MLL-driven cell line RS4-11 being the most sensitive. Cell cycle arrest in subG1/G1 to S transition was observed in 8/9 cell lines and was most pronounced in RS4-11 and BV173. Significant apoptosis (up to 88% Annexin V positive cells) was only observed in KG1a and NOMO1 among myeloid cell lines, while OTX015 induced apoptosis in all lymphoid cell lines tested, ranging from 57% in RS4-11 to 90% in the BCR-ABL+ TOM1 cells. Similarly, OTX015 triggered caspase-dependent cell death, as NOMO1 and RS4-11 displayed significant caspase-3 activation and cytochrome c release, when compared to the resistant K562 cell line. Seven primary patient fresh samples (5 AML, 2ALL) were also analyzed. Ex vivo treatment induced apoptosis ranged from 35% to 87% in 6/7 patients. Exposure to OTX015 at 500nM for 7 days induced maturation in 51% and 65% of HL60 cells as detected by CD11b expression and morphology, respectively. Baseline expression of BRD2/3/4 varied among cell lines or patient samples, lower BRD2/3/4 expression levels were observed in the BCR-ABL+ K562 and BV173 cell lines, as well as in the 4 BCR-ABL+ ALL samples analyzed. Upon OTX015 exposure, down-regulation of the BRD4 target gene c-MYC was observed in all cell lines, without clear correlation with the proliferation inhibition rate and/or the intensity of induced apoptosis while no consistent BRD2/3/4 mRNAs down-regulation was seen. Interestingly, BRD2 protein was down-regulated in HL60, Jurkat and RS4-11 cell lines, but not in the K562 cell line. ConclusionOTX015 affects cell viability, induces cell cycle arrest in G1/S phase, and is able to induce significant apoptosis in leukemic cell lines and fresh AML and ALL samples at submicromolar drug concentrations. These concentrations were achieved in the serum of healthy volunteers after safe administration of the drug. With such characteristics, OTX015 appears to be an attractive anti-leukemic therapy, currently under early evaluation in a Phase Ib dose-escalation trial conducted in relapsed/refractory AML/ALL patients. Disclosures:Riveiro:Oncology Therapeutic Development: Employment. Herait:Oncoethix: Employment. Dombret:Oncoethix: Research Funding.

The Lin28/let-7a/c-Myc pathway plays a role in non-muscle invasive bladder cancer

We investigate the role of the Lin28/let-7a/c-Myc pathway in non-muscle invasive bladder cancer (NMIBC). Using RT-PCR, western blot and immunohistochemistry techniques, the levels of pre-let-7a, let-7a, Lin28 and c-Myc RNA and/or proteins were determined in samples of normal bladder tissue and bladder cancer. Expression of pre-let-7a was found to be negatively correlated with the pathological grade of bladder cancer, while let-7a showed a positive correlation with bladder cancer pathological grade. Expression of Lin28 RNA and protein was not significantly different between normal bladder tissue and low-grade transitional cell carcinoma of bladder (TCC) but the expression levels in high-grade TCC were remarkably increased. Expression of c-Myc RNA and protein was significantly higher in bladder cancer samples in comparison to normal bladder tissue without correlation with cancer differentiation. Expression of all the above RNAs and proteins showed no significant difference in Ta and T1 stages. The Lin28/let-7a/c-Myc pathway plays an important role in NMIBC. In particular, expression levels of let-7a correlate with the degree of cancer differentiation but not cancer stage.

Reduced C/EBPα Expression Favors Monopoiesis Over Granulopoiesis

Abstract 1230C/EBPα is required for the formation of the granulocyte-monocyte progenitor; however, its role in subsequent lineage specification remains uncertain. We previously compared G-CSF with M-CSF signaling in murine marrow, finding that G-CSF more potently induces phosphorylation of SHP2 whereas M-CSF more potently activates ERK. Knockdown (KD) of SHP2 impaired granulopoiesis relative to monopoiesis and unexpectedly led to reduced C/EBPα RNA. A. Cantor and colleagues recently reported that SHP2 dephosphorylates Src kinase-modified RUNX1 and increases the ability of RUNX1 to induce megakaryopoiesis, and we demonstrated that in myeloid cells RUNX1 binds and activates the CEBPA gene, via its promoter and via a conserved 450 bp enhancer located at +37 kb containing 4 RUNX1-binding sites. Thus, G-CSF or other cytokines potentially direct SHP2 to activate RUNX1 which then increases C/EBPα transcription in myeloid progenitors to favor granulopoiesis. As a test of this model, we have now transduced lineage-depleted murine marrow cells, cultured in SCF, TPO, and Flt3L to minimize myeloid differentiation, with shRNAs targeting CEBPA or with a control lentiviral vector. Western blotting indicated 2-fold KD by shRNAs B9 or B11 after 2 days of transduction followed by 2 days of puromycin selection. As this analysis was done on a mixed population, greater KD may occur in rare progenitors. Upon transfer to IL3, IL6, and SCF to favor myeloid maturation, vector-transduced cells produced 4-fold more CFU-G than CFU-M in methylcellulose, whereas CEBPA KD resulted in 4- or 5-fold more CFU-M than CFU-G. FACS analysis of pooled CFUs demonstrated significant reduction of Mac-1+Gr-1+ granulocytes and an increase in Mac-1+Gr-1− monocytes, and examination of cell morphology after Wright’s-Giemsa staining further confirmed a shift towards monopoiesis in response to reduced C/EBPα. Similar findings were obtained for cells placed in liquid culture. In addition, on day 3 of liquid culture Q-RTPCR analysis showed that CEBPA shRNAs induced 2- or 3-fold CEBPA RNA KD, reduced granulocytic MPO, NE, PR3, and GCSFR RNAs, increased monocytic MCSFR and CD14 RNAs, and increased expression of EGR1, IRF8, and c-Jun RNAs, encoding transcription factors that help direct monopoiesis. RUNX1 RNA was unchanged, and PU.1 RNA was reduced <2-fold. To determine the effect of CEBPA KD in vivo, transduced, puromycin-selected CD45.2+ marrow cells were transplanted into lethally irradiated, syngeneic CD45.1+ recipients together with CD45.1+ carrier cells. At day 28, FACS analysis indicated a shift towards monopoiesis amongst shRNA B9- or B11-transduced marrow cells, and CFU assay of sorted CD45.2+ cells demonstrated significantly reduced CFU-G with increased CFU-M. Besides affecting myeloid lineage determination, CEBPA KD increased the rate of cell proliferation in liquid culture, associated with blasts evident upon morphologic analysis and an increase in Mac-1−Gr-1− cells, which mainly consisted of c-Kit+Sca-1− myeloid progenitors. In addition, CEBPA KD by shRNA B9, which impaired granulopoiesis more effectively than shRNA B11, increased CFU replating to 8 generations, compared with 4 generations for control cells. Analysis on day 3 of liquid culture did not reveal an alteration in the proportion of cells in G1, S, or G2/M or increased apoptosis, and c-Myc and Bcl-2 RNA levels were unaffected. We propose that CEBPA KD not only strongly impairs granulopoiesis, but also modestly impedes monopoiesis to produce a transient accumulation of cells with blast morphology and the ability to proliferate. Overall, our findings suggest that relative to monopoiesis, granulopoiesis requires increased level or increased activity of C/EBPα. Offering a potential mechanism, we previously found that C/EBPα :AP-1 leucine zipper heterodimers are more abundant than C/EBPα homodimers during monopoiesis compared with granulopoiesis, in part due to c-Fos, c-Jun, and JunB induction, and that C/EBPα :AP-1 heterodimers can direct marrow monopoiesis. Perhaps higher levels of C/EBPα favor homodimer formation, with reduced levels still able to heterodimerize with AP-1 proteins. ERK modification of C/EBPα on S21, as occurs downstream of M-CSF, reduces its ability to direct granulopoiesis; phospho-C/EBPα (S21) potentially retains the ability to direct AP-1 proteins to hybrid DNA elements recently identified in numerous monocytic enhancers. Disclosures:No relevant conflicts of interest to declare.