The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma—a highly chemotherapy-resistant disease—TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I–IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

Highlights d Cohesion loss is a common feature of cancer cells d DNA replication stress induces cohesion loss d The cohesin remover WAPL is essential in replication stress conditions d WAPL promotes repair and restart of a broken replication fork

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.

ranscription of protein-coding and noncoding genes requires RNA polymerase II (RNAPII), which synthesizes RNA transcripts complementary to the DNA template strand. The presence of DNA lesions in the template strand causes stalling of elongating RNAPII (RNAPIIo), which leads to genome-wide transcriptional arrest 1-3 . It is essential that cells overcome this arrest and restore transcription. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions through the proteins CSB, CSA and UVSSA [4][5][6] . Inactivating mutations in CSB and CSA cause Cockayne syndrome (CS), which is characterized by severe neurological dysfunction related to persistent RNAPII arrest at DNA lesions 2,7 .The sequential and cooperative actions of CSB, CSA and UVSSA recruit TFIIH to DNA damage-stalled RNAPII to initiate DNA repair 6 . In addition to protein-protein contacts, efficient transfer of TFIIH onto RNAPII requires ubiquitylation of a single lysine on the largest subunit of RNAPII (RPB1-K1268), which is essential for efficient TCR 2 . This DNA damage-induced modification of RNAPII is dependent on cullin-ring type E3-ligases (CRLs) and is strongly decreased in CSA-deficient cells 2 , which indicates that the CRL4 CSA E3 ligase complex drives RNAPII ubiquitylation.CSB binds to DNA upstream of RNAPII 8 (Extended Data Fig. 1a) and recruits the CRL4 CSA complex through an evolutionarily conserved motif in its carboxy terminus 6 . However, how the activity of CRL4 CSA ubiquitin ligase is specifically directed towards the K1268 site remains to be elucidated. Results A CRISPR screen identifies ELOF1 as a putative TCR gene.To identify unknown TCR genes, we performed a genome-wide CRISPR screen in the presence of the compound illudin S, which induces transcription-blocking DNA lesions that are eliminated by TCR 9 . RPE1-iCas9 cells were transduced with the pLCKO-TKOv3 library, which contains 70,948 single guide RNAs (sgRNAs) targeting open reading frames 10 , and cultured for 12 population doublings, after which sgRNA contents were analysed (Extended Data Fig. 1b).Using a false-discovery rate (FDR) cut-off of 0.01, we found 104 sensitizer hits and 18 hits conferring resistance to illudin S. The strongest resistance was conferred by guide RNAs (gRNAs) targeting PTGR1, which is in line with its known role in bioactivating illudin S 11 (Fig. 1a and Extended Data Fig. 1c). Nine known core TCR genes, including CSB, CSA and UVSSA, but also genes connected to transcription recovery after UV irradiation (HIRA 12 , DOT1L 13 and STK19 (ref. 14 ) (Fig. 1a,b), were required for illudin S tolerance. Consistent with known effects of illudin S on replication 9 , we found the 9-1-1 complex, translesion synthesis and sister-chromatid cohesion components (Fig. 1b). Our screen also identified the ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Yana van der Weegen 1,10 , Klaas de Lint 2,10 , Diana van den Heuvel

Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers.

The p53 regulatory network is critically involved in preventing the initiation of cancer. In unstressed cells, p53 is maintained at low levels and is largely inactive, mainly through the action of its two essential negative regulators, HDM2 and HDMX. p53 abundance and activity are up-regulated in response to various stresses, including DNA damage and oncogene activation. Active p53 initiates transcriptional and transcription-independent programs that result in cell cycle arrest, cellular senescence, or apoptosis. p53 also activates transcription of HDM2, which initially leads to the degradation of HDMX, creating a positive feedback loop to obtain maximal activation of p53. Subsequently, when stress-induced post-translational modifications start to decline, HDM2 becomes effective in targeting p53 for degradation, thus attenuating the p53 response. To date, no clear function for HDMX in this critical attenuation phase has been demonstrated experimentally. Like HDM2, the HDMX gene contains a promoter (P2) in its first intron that is potentially inducible by p53. We show that p53 activation in response to a plethora of p53-activating agents induces the transcription of a novel HDMX mRNA transcript from the HDMX-P2 promoter. This mRNA is more efficiently translated than that expressed from the constitutive HDMX-P1 promoter, and it encodes a long form of HDMX protein, HDMX-L. Importantly, we demonstrate that HDMX-L cooperates with HDM2 to promote the ubiquitination of p53 and that p53-induced HDMX transcription from the P2 promoter can play a key role in the attenuation phase of the p53 response, to effectively diminish p53 abundance as cells recover from stress.The tumor suppressor protein p53 functions primarily as a stress-inducible transcriptional activator of genes that promote cell cycle arrest and apoptosis (1). Stress-induced p53 activation can form a rate-limiting barrier to tumorigenesis (2, 3), and the manipulation of p53 function is key to the mechanism of action of many cancer chemotherapeutic strategies (4, 5). In unstressed cells, p53 is maintained at low levels and inactive, largely through the action of several p53-inducible negative feedback pathways, the most extensively studied of which involves the oncoproteins HDM2 and HDMX (also called MDM4) (MDM2 and MDMX/MDM4 in mice) (6, 7). Considerable research effort has been applied to understanding the mechanisms whereby these two proteins regulate p53 function. HDM2 and HDMX both contain an N-terminal pocket that binds to the primary transactivation domain of p53; they can, therefore, function independently of each other to repress p53-dependent transcription (8 -10). HDM2 also forms both HDM2-HDM2 homodimers and HDM2-HDMX heterodimers. These function as E3 ubiquitin ligases for p53; monoubiquitination of p53 by HDM2 inhibits p53 activity by both inhibiting acetylation and promoting nuclear export, whereas polyubiquitination promotes proteasome-mediated p53 degradation and is largely responsible for the rapid turnover of p53 protein that occurs in proli...

The prognosis of patients with uveal melanoma is poor. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. Because p53 mutations are uncommon in uveal melanoma, reactivation of p53 may be used to achieve tumor regression. We investigated the use of combination therapies for intraocular melanoma, based on the p53 activators Nutlin-3 and reactivation of p53 and induction of tumor cell apoptosis (RITA) and the topoisomerase I inhibitor Topotecan. Nutlin-3 treatment induced p53-dependent growth inhibition in human uveal melanoma cell lines. The sensitivity to Nutlin-3 of the investigated cell lines did not correlate with basal Hdm2 or Hdmx levels. Nutlin-3 synergized with RITA and Topotecan to induce apoptosis in uveal melanoma cell lines and short-term cultures. Drug synergy correlated with enhanced induction of p53-Ser46 phosphorylation, which was attenuated by ATM inhibition. Nutlin-3 and Topotecan also significantly delayed tumor growth in vivo in a murine B16F10 model for ocular melanoma. Combination treatment appeared to inhibit tumor growth slightly more efficient than either drug alone. Nutlin-3, RITA and Topotecan lead to comparable p53 activation and growth inhibition under normoxia and hypoxia. Treatment with Nutlin-3 or RITA had no effect on HIF-1a induction by hypoxia, whereas the combination of these two drugs did inhibit hypoxia-induced HIF-1a. Also Topotecan, alone or in combination with Nutlin-3, reduced HIF-1a protein levels, suggesting that a certain level of DNA damage response is required for p53-mediated downregulation of HIF-1a. In conclusion, combination treatments based on small-molecule-induced p53 activation may have clinical potential for uveal melanoma.

Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 -HdmX and Wip1, leading to efficient elimination of tumour cells.