To help expand substantiate that DNA binding is a prerequisite for the proteasomal degradation of CIC we fused a known SRY DNA binding domain name (DBD) construct to CIC wild type or CIC(R1515H) mutant resulting in DNA binding (Supplementary Determine?3S) and robust ubiquitylation of both the wild type and mutant CIC (Supplementary Physique?3S). We find that CIC protein levels are negligible in GBM due to continuous proteasome-mediated degradation, which is usually mediated by the E3 ligase PJA1 and show that this occurs through binding of CIC to its DNA target and phosphorylation on residue S173. PJA1 knockdown increased CIC stability and extended survival using in-vivo models of GBM. Deletion of the ERK binding site resulted in stabilization of CIC and increased therapeutic efficacy of ERK inhibition in GBM models. Our results provide a rationale to target CIC degradation in Ras/ERK-driven tumors, including GBM, to increase efficacy of ERK inhibitors. Introduction Glioblastoma (GBM) is the most common and malignant main neuroepithelial tumor and remains incurable despite aggressive therapy. Molecular alterations of various signaling pathways potentiate receptor tyrosine kinase (RTK) MLN120B activation, such as MMP10 the frequent EGFR amplifications or variant III mutations (EGFRvIII) that are linked with the aggressive behavior of GBMs1C3. Regrettably, results from clinical trials targeting Ras/Raf/MEK/ERK signaling downstream of RTK have only experienced limited success4, indicating a need for increasing understanding of the mechanisms regulating this pathway in GBM. The high-mobility group (HMG)-box transcriptional repressor capicua (and mammals5. In unstimulated cells, CIC represses EGFR/Ras pathway-responsive genes. Following EGFR activation, CIC repression is usually relieved, allowing for the expression of target genes. The best-characterized CIC targets in mammalian cells are the oncogenic transcription factors ETV1, ETV4, and ETV55, which mediate cell proliferation, motility and invasion downstream of Ras6. Much has been learned from studies in was first described to be involved in developmental patterning and cell fate modulated through EGFR activation7C10, in a manner termed default repression. While CICs function is usually less well-understood in vertebrate organisms, the importance of CIC protein in maintaining cellular homeostasis downstream of EGFR/Ras/ERK signaling has recently become obvious in humans11C13, but the molecular mechanisms governing CIC MLN120B functions in normal cells and in malignancy are lacking. Investigation into the molecular function of CIC in malignancy and GBM in particular, is further merited by recent findings connecting CICs downstream target ETV1 in GBM14. is not mutated in GBM, but mutations of this gene, located on chromosome 19q, occur in 70% of 1p19q-co-deleted oligodendrogliomas15C18. Decreased CIC expression is usually correlated with poorer end result in these tumors19. Two CIC isoforms exist that differ in size, the short (CIC-S) and the long (CIC-L), and in their N-terminal region20. Given that the disease-associated mutations map to the CIC-S isoform of the protein, which suggests that this CIC-S isoform may be more important in tumorigenesis, we focus on the CIC-S isoform in the current study referred to as CIC throughout the manuscript21. In addition to loss-of-function mutations in oligodendrogliomas, and other tumor types, translocation events MLN120B resulting in gene fusions of with either or has been shown in round cell sarcomas22,23. Additionally, CIC has most recently been shown to suppress invasion and metastasis in lung malignancy, through an effector identified as MMP2412. In addition, germline CIC inactivation in adult mice was shown to induce T-cell acute lymphoblastic lymphoma24. Despite obvious genetic evidence of its connection to one of the most important pathways in malignancy, molecular mechanisms governing CIC regulation by Ras/ERK signaling and its potential involvement in GBM remain unknown. In this study, we present data to establish a role for in GBM. We find that activation of Ras/ERK signaling mediates ubiquitylation and degradation of CIC by a nuclear E3 ligase PRAJA1 (PJA1) to drive GBM growth. We provide mechanistic insights into regulation of CIC downstream of EGFR activation via serine/threonine phosphorylation. Importantly, a degradation-resistant CIC mutant, insensitive to the effects of ERK activation, resulted in suppression of GBM growth and sensitized tumors to the effects of ERK inhibition, a potential therapeutic opportunity for further study in this aggressive neoplasm. Results CIC protein levels are low in GBM despite strong mRNA levels Information is lacking regarding the mechanism by which Ras/ERK signaling regulates CIC to alleviate target gene repression. In particular, it is not established whether CIC is as an important signaling regulator in GBM. The ETS family of oncogenic transcription factors, ETV1, ETV4, and ETV5 downstream of RTK/Ras/ERK activation have been shown to MLN120B mediate gliomagenesis14,25, yet the role of CIC, a well-established repressor of these genes21, is unknown. To explore this, we first examined the expression of CIC protein in human newly diagnosed GBM human tumors. Interestingly, in 30/30 GBM patient tumor samples, CIC protein level was substantially reduced or absent compared to lysates derived from non-neoplastic brain tissue (Physique?1a and Supplementary Physique?1A and C). By contrast, mRNA expression was readily detected.