HGG-43. INTERROGATING THE ROLE OF PEA3 ONCOGENIC TRANSCRIPTION FACTORS IN PEDIATRIC HIGH-GRADE K27M GLIOMAS

Pediatric high-grade gliomas (HGGs) are an aggressive form of pediatric brain tumors which pose a grim five-year survival with little advancement in therapeutic efficacy, often requiring a multimodal therapeutic combination of chemotherapy, resection, and radiation. We have previously shown that proper function of ETS transcription factors is necessary for gliomagenesis in Ras/MAPK-driven pediatric gliomas. It is our hypothesis that ETS transcription factors are necessary for tumor initiation in HGGs by promoting the necessary glial cell fates in glioma. Further, we hypothesize that functional inhibition of ETS proteins following tumor formation will improve survival and outcome in HGG. Functional inhibition of ETS proteins using a competitive dominant-negative mutant was shown to completely rescue neural stem cell depletion, tumor formation and tumor-free survival in two rodent models of HGGs. Mechanistically, we show evidence that Pea3 factors may induce glial-cell fate by promoting Olig2 expression and activation of glial transcriptional programs. Indeed, transcriptomic analysis of ETS-perturbed HGG tumors revealed that Sox9 and Olig2 transcription factor networks were dependent on proper ETS function. Further, we show evidence that Etv5 can directly interact with promoter regions of glial fate master regulators in human primary glioma cell lines. To empirically determine the effect of Pea3 proteins on tumorigenesis, we have created a novel methodology for inducible gain- and loss-of-function genetic interrogation of these factors in vivo. Our survival results and combined single-cell RNA-sequencing of individual groups show that inhibition of the Pea3 family leads to a marked increase in survival in K27M glioma by regulating key features of glioblasts. All in all, our group provides evidence that the ETS family of transcription factors is necessary for glial specification of tumor cells and induce pro-glial transcriptional programs by activating OPC- and astrocyte-specific genes in K27M-driven tumors.

Ubiquitin-Mediated Control of ETS Transcription Factors: Roles in Cancer and Development

Genome expansion, whole genome and gene duplication events during metazoan evolution produced an extensive family of ETS genes whose members express transcription factors with a conserved winged helix-turn-helix DNA-binding domain. Unravelling their biological roles has proved challenging with functional redundancy manifest in overlapping expression patterns, a common consensus DNA-binding motif and responsiveness to mitogen-activated protein kinase signalling. Key determinants of the cellular repertoire of ETS proteins are their stability and turnover, controlled largely by the actions of selective E3 ubiquitin ligases and deubiquitinases. Here we discuss the known relationships between ETS proteins and enzymes that determine their ubiquitin status, their integration with other developmental signal transduction pathways and how suppression of ETS protein ubiquitination contributes to the malignant cell phenotype in multiple cancers.

Gene Regulatory Network of ETS Domain Transcription Factors in Different Stages of Glioma

The ETS domain family of transcription factors is involved in a number of biological processes, and is commonly misregulated in various forms of cancer. Using microarray datasets from patients with different grades of glioma, we have analyzed the expression profiles of various ETS genes, and have identified ETV1, ELK3, ETV4, ELF4, and ETV6 as novel biomarkers for the identification of different glioma grades. We have further analyzed the gene regulatory networks of ETS transcription factors and compared them to previous microarray studies, where Elk-1-VP16 or PEA3-VP16 were overexpressed in neuroblastoma cell lines, and we identify unique and common regulatory networks for these ETS proteins.

MRTFA augments megakaryocyte maturation by enhancing the SRF regulatory axis

Serum response factor (SRF) is a ubiquitously expressed transcription factor that binds DNA at CArG (CC[A/T]6GG) domains in association with myocardin-family proteins (eg, myocardin-related transcription factor A [MRTFA]) or the ternary complex factor family of E26 transformation-specific (ETS) proteins. In primary hematopoietic cells, knockout of either SRF or MRTFA decreases megakaryocyte (Mk) maturation causing thrombocytopenia. The human erythroleukemia (HEL) cell line mimics the effects of MRTFA on Mk maturation, and MRTFA overexpression (MRTFAOE) in HEL cells enhances megakaryopoiesis. To identify the mechanisms underlying these effects, we performed integrated analyses of anti-SRF chromatin immunoprecipitation (ChIP) and RNA-sequencing data from noninduced and phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])–induced HEL cells, with and without MRTFAOE. We found that 11% of genes were upregulated with TPA induction, which was enhanced by MRTFAOE, resulting in an upregulation of 25% of genes. MRTFAOE increased binding of SRF to genomic sites and enhanced TPA-induced expression of SRF target genes. The TPA-induced genes are predicted to be regulated by SRF and ETS factors, whereas those upregulated by TPA plus MRTFAOE lack ETS binding motifs, and MRTFAOE skews SRF binding to genomic regions with CArG sites in regions relatively lacking in ETS binding motifs. Finally, ChIP–polymerase chain reaction using HEL cells and primary human CD34+ cell–derived subpopulations confirms that both SRF and MRTFA have increased binding during megakaryopoiesis at upregulated target genes (eg, CORO1A). We show for the first time that MRTFA increases both the genomic association and activity of SRF and upregulates genes that enhance primary human megakaryopoiesis.

NKX2-5 mutations causative for congenital heart disease retain functionality and are directed to hundreds of targets

We take a functional genomics approach to congenital heart disease mechanism. We used DamID to establish a robust set of target genes for NKX2-5 wild type and disease associated NKX2-5 mutations to model loss-of-function in gene regulatory networks. NKX2-5 mutants, including those with a crippled homeodomain, bound hundreds of targets including NKX2-5 wild type targets and a unique set of "off-targets", and retained partial functionality. NKXΔHD, which lacks the homeodomain completely, could heterodimerize with NKX2-5 wild type and its cofactors, including E26 transformation-specific (ETS) family members, through a tyrosine-rich homophilic interaction domain (YRD). Off-targets of NKX2-5 mutants, but not those of an NKX2-5 YRD mutant, showed overrepresentation of ETS binding sites and were occupied by ETS proteins, as determined by DamID. Analysis of kernel transcription factor and ETS targets show that ETS proteins are highly embedded within the cardiac gene regulatory network. Our study reveals binding and activities of NKX2-5 mutations on WT target and off-targets, guided by interactions with their normal cardiac and general cofactors, and suggest a novel type of gain-of-function in congenital heart disease.

Novel Function Of Chromosome 7 Open Reading Frame 41 Gene To Promote Leukemic Megakaryocyte Differentiation By Modulating TPA-Induced MAPK/ERK, SAPK/JNK, and NF-κB Signaling

Phorbol 12-myristate 13-acetate (TPA) primarily activates PKC and subsequently leads to activation of downstream signaling pathways including MAPK/ERK, SAPK/JNK, and NF-κB, which causes gene expression alteration and leukemic cell differentiation. How these TPA-induced genes may contribute to leukemic cell differentiation remains to be addressed. We noticed that a novel gene chromosome 7 open reading frame 41 (C7ORF41) was one of TPA-induced genes without any known functions. Differential expression of C7ORF41 has been identified in human embryo development and predicted to function in hematopoiesis based on hierarchical clustering analysis. To support this, we found high expression level of C7ORF41 in bone marrow. By using K562 cell as a model, we discovered that ectopic expression of C7ORF41 significantly promoted TPA-induced megakaryocyte differentiation evidenced by an increase of CD61 expression. Consistently, two types of transcription factors critical for megakaryopoiesis, RUNX1 and ETS proteins, were simultaneously upregulated by C7ORF41. Furthermore, cytoplasmic distribution of C7ORF41 suggests that it may act as a signaling molecule. As expected, C7ORF41 overexpression enhanced ERK and JNK phosphorylation. In contrast, C7ORF41 knockdown led to an opposite phenotype: impaired megakaryocyte differentiation, attenuated signaling, and reduced transcription factor expression. These observations suggest that C7ORF41 may promote megakaryocyte differentiation by enhancing ERK and JNK signaling that subsequently leads to upregulation of RUNX1 and ETS proteins. Indeed, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by PD98059, a potent ERK inhibitor, while JNK inhibition abrogated the effect of C7ORF41 on upregulation of ETS proteins. In addition, C7ORF41 was highly conserved in evolution and several tyrosine residues including Y34 were strictly preserved, suggesting the importance of tyrosine phosphorylation in C7ORF41 function. In fact, mutant C7ORF41 with Y34 substitution by phenylalanine functioned to inhibit megakaryocyte differentiation. Finally, NF-κB appeared to be the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536. Taken together, we have identified novel function of a new gene C7ORF41 that may promote leukemic megakaryocyte differentiation through a novel mechanism in which C7ORF41 forms a well-balanced regulatory network in TPA-induced signaling. In this network, initial TPA treatment primes downstream signaling including MAPK/ERK, SAPK/JNK, and NF-kB. TPA-induced NF-κB activation further upregulates C7ORF41 that may serve to amplify TPA-induced ERK and JNK signaling to ensure megakaryocyte differentiation. On the other hand, C7ORF41 upregulation also serves as a negative regulator of NF-κB activity that may quench TPA-indcued NF-κB signaling. In addition, enhanced ERK signaling feeds back to damp C7ORF41 upregulation that may tune TPA-induced signaling under controllable level. Our findings shed light on understanding forced differentiation in leukemic cells and may provide useful information for rational differentiation therapy. Disclosures: No relevant conflicts of interest to declare.