Increased Efficacy of Histone Methyltransferase G9a Inhibitors Against <i>MYCN</i>-Amplified Neuroblastoma.

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a pediatric solid tumor with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumors is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638, and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumor suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2, and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumors

Mutant p53 protects ETS2 from non-canonical COP1/DET1 dependent degradation

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2

HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis

Pro-apoptotic protein–protein interactions of the extended N-AChE terminus

The N-terminally extended “synaptic” acetylcholinesterase variant N-AChE-S operates to promote apoptosis; however, the protein partners involved in this function remain unknown. Here, we report that when microinjected to fertilized mouse oocytes, N-AChE-S caused embryonic death as early as the zygotic stage. To identify the putative protein partners involved, we first tried yeast two hybrid screening, but this approach failed, probably because of the N-AChE-S-induced lethality. In contrast, sequence analysis and a corresponding peptide array revealed possible partners, which were validated by co-immunoprecipitation. These include the kinases GSK3, Aurora and GAK, the membrane integrin receptors, and the death receptor FAS. Each of these could potentially modulate N-AChE-S-induced apoptosis with possible therapeutic value for the treatment of Alzheimer’s disease

Kaposi's Sarcoma Herpesvirus Upregulates Aurora A Expression to Promote p53 Phosphorylation and Ubiquitylation

Aberrant expression of Aurora A kinase has been frequently implicated in many cancers and contributes to chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. Previous studies showed that p53 is degraded by Kaposi's sarcoma herpesvirus (KSHV) encoded latency-associated nuclear antigen (LANA) through its SOCS-box (suppressor of cytokine signaling, LANASOCS) motif-mediated recruitment of the EC5S ubiquitin complex. Here we demonstrate that Aurora A transcriptional expression is upregulated by LANA and markedly elevated in both Kaposi's sarcoma tissue and human primary cells infected with KSHV. Moreover, reintroduction of Aurora A dramatically enhances the binding affinity of p53 with LANA and LANASOCS-mediated ubiquitylation of p53 which requires phosphorylation on Ser215 and Ser315. Small hairpin RNA or a dominant negative mutant of Aurora A kinase efficiently disrupts LANA-induced p53 ubiquitylation and degradation, and leads to induction of p53 transcriptional and apoptotic activities. These studies provide new insights into the mechanisms by which LANA can upregulate expression of a cellular oncogene and simultaneously destabilize the activities of the p53 tumor suppressor in KSHV-associated human cancers