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Significance The cell of origin and the temporal order of oncogenic events in tumors play important roles for disease state. This is of particular interest for PCa due to its highly variable clinical outcome. However, these features are difficult to analyze in tumors. We established an in vitro murine PCa organoid model taking into account the cell of origin and the temporal order of events. We found that TIP5 primes luminal prostate cells for Pten-loss mediated oncogenic transformation whereas it is dispensable once the transformation is established. Cross-species transcriptomic analyses revealed a PTEN-loss gene signature that identified a set of aggressive tumors with PTEN-del, or low PTEN expression, and high-TIP5 expression. This paper provides a powerful tool to elucidate PCa mechanisms., Prostate cancer (PCa) is the second leading cause of cancer death in men. Its clinical and molecular heterogeneities and the lack of in vitro models outline the complexity of PCa in the clinical and research settings. We established an in vitro mouse PCa model based on organoid technology that takes into account the cell of origin and the order of events. Primary PCa with deletion of the tumor suppressor gene PTEN (PTEN-del) can be modeled through Pten-down-regulation in mouse organoids. We used this system to elucidate the contribution of TIP5 in PCa initiation, a chromatin regulator that is implicated in aggressive PCa. High TIP5 expression correlates with primary PTEN-del PCa and this combination strongly associates with reduced prostate-specific antigen (PSA) recurrence-free survival. TIP5 is critical for the initiation of PCa of luminal origin mediated by Pten-loss whereas it is dispensable once Pten-loss mediated transformation is established. Cross-species analyses revealed a PTEN gene signature that identified a group of aggressive primary PCas characterized by PTEN-del, high-TIP5 expression, and a TIP5-regulated gene expression profile. The results highlight the modeling of PCa with organoids as a powerful tool to elucidate the role of genetic alterations found in recent studies in their time orders and cells of origin, thereby providing further optimization for tumor stratification to improve the clinical management of PCa.

Summary Essential E3 ubiquitin ligase HUWE1 (HECT, UBA, and WWE domain containing 1) regulates key factors, such as p53. Although mutations in HUWE1 cause heterogenous neurodevelopmental X-linked intellectual disabilities (XLIDs), the disease mechanisms common to these syndromes remain unknown. In this work, we identify p53 signaling as the central process altered in HUWE1-promoted XLID syndromes. By focusing on Juberg-Marsidi syndrome (JMS), one of the severest XLIDs, we show that increased p53 signaling results from p53 accumulation caused by HUWE1 p.G4310R destabilization. This further alters cell-cycle progression and proliferation in JMS cells. Modeling of JMS neurodevelopment reveals majorly impaired neural differentiation accompanied by increased p53 signaling. The neural differentiation defects can be successfully rescued by reducing p53 levels and restoring the expression of p53 target genes, in particular CDKN1A/p21. In summary, our findings suggest that increased p53 signaling underlies HUWE1-promoted syndromes and impairs XLID JMS neural differentiation., Graphical abstract, Highlights p53 signaling is hyperactivated in HUWE1-promoted intellectual disabilities Neural differentiation is impaired in Juberg-Marsidi syndrome with HUWE1 p.G4310R Juberg-Marsidi brain organoids have reduced size and aberrant cellular organization Restored p53 signaling rescues neural development in Juberg-Marsidi syndrome, Using transcriptome analysis, Aprigliano et al. identify increased p53 signaling as the most significantly deregulated process in HUWE1-promoted XLIDs. Modeling of a severe HUWE1-promoted XLID, Juberg-Marsidi syndrome, reveals impaired neural differentiation capacity, which is successfully rescued by p53 knockdown and restored expression of p53 target genes, such as CDKN1A/p21.

Prostate cancer (PCa) is one of the most prevalent cancers in men. Cancer stem cells are thought to be associated with PCa relapse and represent a target against metastatic PCa. Here we show that BAZ2A (also known as TIP5), a factor previously implicated in aggressive PCa, is required for the dedifferentiation of PCa cells into a cancer stem-like state. We found that BAZ2A genomic occupancy in PCa cells coincides with H3K14ac enriched chromatin regions. This association is mediated by BAZ2A bromodomain (BAZ2A-BRD) that specifically binds to H3K14ac. In PCa cells, BAZ2A-BRD is required for the interaction with a class of inactive enhancers that are marked by H3K14ac and represses transcription of genes implicated in developmental and differentiation processes that are frequently silenced in aggressive and dedifferentiated PCa. BAZ2A-mediated repression of these genes is also linked to the histone acetyltransferase EP300 that acetylates H3K14ac. Mutations of BAZ2A-BRD or treatment with chemical probes that abrogate BAZ2A-BRD association with H3K14ac impair the dedifferentiation of PCa cells into a stem-like state. Furthermore, pharmacological inactivation of BAZ2A-BRD impairs the oncogenic transformation mediated by Pten-loss in prostate organoids. Our findings indicate a role of BAZ2A-BRD in PCa stem cell features and suggest potential epigenetic-reader therapeutic strategies to target BAZ2A in aggressive PCa.

SUMMARYEssential E3 ubiquitin ligase HUWE1 (HECT, UBA and WWE domain containing 1) regulates key factors, as p53. Mutations in HUWE1 have been associated with neurodevelopmental X-linked intellectual disabilities (XLIDs), however the pathomechanism at the onset of heterogenous XLIDs remains unknown. In this work, we identify p53 signaling as the process hyperactivated in lymphoblastoid cells from patients with HUWE1-promoted XLIDs. The hiPSCs-based modeling of the severe HUWE1-promoted XLID, the Juberg Marsidi syndrome (JMS), reviled majorly impaired neural differentiation, accompanied by increased p53 signaling. The impaired differentiation results in loss of cortical patterning and overall undergrowth of XLID JMS patient-specific cerebral organoids, thus closely recapitulating key symptoms, as microcephaly. Importantly, the neurodevelopmental potential of JMS hiPSCs is successfully rescued by restoring p53 signaling, upon reduction of p53 levels. In summary, our findings indicate that increased p53 signaling leads to impaired neural differentiation and is the common cause of neurodevelopmental HUWE1-promoted XLIDs.

Endogenous DNA damage is causally associated with the functional decline and transformation of stem cells that characterize aging. DNA lesions that have escaped DNA repair can induce replication stress and genomic breaks that induce senescence and apoptosis. It is not clear how stem and proliferating cells cope with accumulating endogenous DNA lesions and how these ultimately affect the physiology of cells and tissues. Here we have addressed these questions by investigating the hematopoietic system of mice deficient for Rev1, a core factor in DNA translesion synthesis (TLS), the postreplicative bypass of damaged nucleotides. Rev1 hematopoietic stem and progenitor cells displayed compromised proliferation, and replication stress that could be rescued with an antioxidant. The additional disruption of Xpc, essential for global-genome nucleotide excision repair (ggNER) of helix-distorting nucleotide lesions, resulted in the perinatal loss of hematopoietic stem cells, progressive loss of bone marrow, and fatal aplastic anemia between 3 and 4 months of age. This was associated with replication stress, genomic breaks, DNA damage signaling, senescence, and apoptosis in bone marrow. Surprisingly, the collapse of the Rev1Xpc bone marrow was associated with progressive mitochondrial dysfunction and consequent exacerbation of oxidative stress. These data reveal that, to protect its genomic and functional integrity, the hematopoietic system critically depends on the combined activities of repair and replication of helix-distorting oxidative nucleotide lesions by ggNER and Rev1-dependent TLS, respectively. The error-prone nature of TLS may provide mechanistic understanding of the accumulation of mutations in the hematopoietic system upon aging.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID.

Mutations in the HECT, UBA and WWE domain-containing 1 (HUWE1) E3 ubiquitin ligase cause neurodevelopmental disorder X-linked intellectual disability (XLID). HUWE1 regulates essential processes such as genome integrity maintenance. Alterations in the genome integrity and accumulation of mutations have been tightly associated with the onset of neurodevelopmental disorders. Though HUWE1 mutations are clearly implicated in XLID and HUWE1 regulatory functions well explored, currently much is unknown about the molecular basis of HUWE1-promoted XLID. Here we showed that the HUWE1 expression is altered and mutation frequency increased in three different XLID individual (HUWE1 p.R2981H, p.R4187C and HUWE1 duplication) cell lines. The effect was most prominent in HUWE1 p.R4187C XLID cells and was accompanied with decreased DNA repair capacity and hypersensitivity to oxidative stress. Analysis of HUWE1 substrates revealed XLID-specific down-regulation of oxidative stress response DNA polymerase (Pol) λ caused by hyperactive HUWE1 p.R4187C. The subsequent restoration of Polλ levels counteracted the oxidative hypersensitivity. The observed alterations in the genome integrity maintenance may be particularly relevant in the cortical progenitor zones of human brain, as suggested by HUWE1 immunofluorescence analysis of cerebral organoids. These results provide evidence that impairments of the fundamental cellular processes, like genome integrity maintenance, characterize HUWE1-promoted XLID. © The Author(s) 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Prostate cancer (PCa) is one of the most prevalent cancers in men. Cancer stem cells are thought to be associated with PCa relapse. Here, we show that BAZ2A is required for PCa cells with a cancer stem‐like state. BAZ2A genomic occupancy in PCa cells coincides with H3K14ac‐enriched chromatin regions. This association is mediated by BAZ2A‐bromodomain (BAZ2A‐BRD) that specifically binds H3K14ac. BAZ2A associates with inactive enhancers marked by H3K14ac and repressing transcription of genes frequently silenced in aggressive and poorly differentiated PCa. BAZ2A‐mediated repression is also linked to EP300 that acetylates H3K14ac. BAZ2A‐BRD mutations or treatment with inhibitors abrogating BAZ2A‐BRD/H3K14ac interaction impair PCa stem cells. Furthermore, pharmacological inactivation of BAZ2A‐BRD impairs Pten‐loss oncogenic transformation of prostate organoids. Our findings indicate a role of BAZ2A‐BRD in PCa stem cell features and suggest potential epigenetic‐reader therapeutic strategies to target BAZ2A in aggressive PCa., BAZ2A is an epigenetic reader of H3K14ac and regulates prostate cancer stem cells though its association with inactive enhancers. Inactivation of BAZ2A‐bromodomain impairs prostate cancer stem cells and PTEN‐loss mediated oncogenic transformation.