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DNA double-strand breaks (DSBs) are repaired through homology-directed repair (HDR) or non-homologous end joining (NHEJ). BRCA1/2-deficient cancer cells cannot perform HDR, conferring sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi). However, concomitant loss of the pro-NHEJ factors 53BP1, RIF1, REV7-Shieldin (SHLD1-3) or CST-DNA polymerase alpha (Pol-α) in BRCA1-deficient cells restores HDR and PARPi resistance. Here, we identify the TRIP13 ATPase as a negative regulator of REV7. We show that REV7 exists in active 'closed' and inactive 'open' conformations, and TRIP13 catalyses the inactivating conformational change, thereby dissociating REV7-Shieldin to promote HDR. TRIP13 similarly disassembles the REV7-REV3 translesion synthesis (TLS) complex, a component of the Fanconi anaemia pathway, inhibiting error-prone replicative lesion bypass and interstrand crosslink repair. Importantly, TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARPi resistance and correlates with poor prognosis. Thus, TRIP13 emerges as an important regulator of DNA repair pathway choice-promoting HDR, while suppressing NHEJ and TLS.

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, includingIER5, which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes.IER5is epistatic toPPP2R2A, a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation.

Notch signaling regulates normal squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is a key tumor suppressive pathway. To identify the direct targets of Notch that produce these phenotypes, we introduced a conditional Notch transgene into squamous cell carcinoma cell lines, which respond to Notch activation in 2D culture and in organoid cultures by undergoing differentiation. RNA-seq and ChIP-seq analyses show that in squamous cells Notch activates a context-specific program of gene expression that depends on lineage-specific regulatory elements, most of which lie in long- range enhancers. Among the direct Notch target genes are multiple DNA damage response genes, includingIER5, which is regulated by Notch through several enhancer elements. We show thatIER5is required for Notch-induced differentiation in squamous carcinoma cells and in TERT-immortalized keratinocytes. Its function is epistatic toPPP2R2A, which encodes the B55αsubunit of PP2A, and IER5 interacts with B55αin cells and in purified systems. These results show that Notch and DNA-damage response pathways converge in squamous cells and that some components of these pathways promote differentiation, which may serve to eliminate DNA-damaged cells from the proliferative pool in squamous epithelia. Crosstalk involving Notch and PP2A may enable Notch signaling to be tuned and integrated with other pathways that regulate squamous differentiation. Our work also suggests that squamous cell carcinomas remain responsive to Notch signaling, providing a rationale for reactivation of Notch as a therapeutic strategy in these cancers.ImpactOur findings highlight context-specific crosstalk between Notch, DNA damage response genes, and PP2A, and provide a roadmap for understanding how Notch induces the differentiation of squamous cells.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long distance transport of non-membrane enclosed organelles such as RNA granules is not known. Here we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts directly with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that binding of SFPQ to KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie-Tooth (CMT) Disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations show that non-membrane enclosed organelles can move autonomously and that replacing axonally translated proteins provides a therapeutic approach to axonal degenerative disorders.

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1–DBC1–AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B’’’, striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity., eLife digest Cells maintain a fine balance of signals that promote or counter cell growth and division. Two sets of enzymes – called kinases and phosphatases – contribute to this balance. In general, kinases “switch on” other proteins by tagging them with a phosphate molecule. This process is called phosphorylation. Phosphatases, on the other hand, dephosphorylate these proteins, switching them off. Cancer cells often have mutations that activate kinases to drive cancer growth. The same cells can have mutations that inactivate the phosphatases or reduce their abundance. The roles of phosphatases in cancer are still being studied. One major hurdle in this research is that it is not always clear how they recognize the proteins they dephosphorylate. Protein phosphatase 2A (or PP2A for short) is one of the phosphatases that is often mutated or deleted in human cancers. Even just reduced levels of PP2A can promote cancer. Kim, Berrios, Kim, Schade et al. used an experimental trick to decrease the phosphatase activity of PP2A in human cells growing in a dish. Biochemical analysis of these cells showed that, as expected, many proteins were now in their phosphorylated states. Unexpectedly, however, some proteins were dephosphorylated under these conditions. One of these proteins was called MAP4K4. In the case of MAP4K4, the dephosphorylated state contributes to the growth of the cancer cell. Kim et al. carried out further genetic and biochemical experiments to show that, in these cells, PP2A and MAP4K4 stay physically connected to one another. This connection was enabled by a group of proteins called the STRIPAK complex. The STRIPAK proteins directed the remaining PP2A towards MAP4K4. Low levels or activity of PP2A could, therefore, promote cancer in a different way. Taken together, PP2A is not a single phosphatase that always turns proteins off, but rather is a dual switch that turns off some proteins while turning on others. Future experiments will explore to what extent these findings also apply in tumors. Information about how mutations in PP2A affect human cancers could suggest new targets for cancer drugs.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B’’’, striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.

Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3β, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3β phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment., Regulation of post translational modification of 53BP1 is critical for genome integrity and regulation of DNA damage response. Here the authors reveal a CDK5-PP4 phospho-signaling cascade that leads 53BP1 to sites of DNA damage.

DBC1 plays a critical role in various cellular functions notably cell proliferation, transcription, histone modification and adipogenesis. Current reports about the role of DBC1 in tumorigenesis are paradoxical and designate DBC1 both as a tumor suppressor or an oncogene. Here, using small T antigen of polyoma virus (PyST) as a tool, we have delineated a signaling mechanism that connects LKB1 to AKT1 via DBC1. We report that PyST associates with DBC1 and leads to its down-regulation. Our results also show that PyST expression promotes LKB1 activation which in turn leads to in the downregulation of DBC1 protein. Absence of DBC1 results in transcriptional upregulation and consequently enhanced protein levels of TRB3. TRB3 sequesters AKT1, and consequently the phosphorylation and activity of AKT1 is compromised. This ultimately results in inactivation of pro-survival pathways triggered via AKT1 signaling. Our studies thus provide an insight into a signaling pathway that connects LKB1, DBC1, TRB3 and AKT1.

Comment in :"DNA Methylation and Chromatin: Role(s) of Methyl-CpG-Binding Protein ZBTB38. [Epigenet Insights. 2018]"; International audience; Reactive oxygen species (ROS) are a byproduct of cell metabolism, and can also arise from environmental sources, such as toxins or radiation. Depending on dose and context, ROS have both beneficial and deleterious roles in mammalian development and disease, therefore it is crucial to understand how these molecules are generated, sensed, and detoxified. The question of how oxidative stress connects to the epigenome, in particular, is important yet incompletely understood. Here we show that an epigenetic regulator, the methyl-CpG-binding protein ZBTB38, limits the basal cellular production of ROS, is induced by ROS, and is required to mount a proper response to oxidative stress. Molecularly, these functions depend on a deubiquitinase, USP9X, which interacts with ZBTB38, deubiquitinates it, and stabilizes it. We find that USP9X is itself stabilized by oxidative stress, and is required together with ZBTB38 to limit the basal generation of ROS, as well as the toxicity of an acute oxidative stress. Our data uncover a new nuclear target of USP9X, show that the USP9X/ZBTB38 axis limits, senses and detoxifies ROS, and provide a molecular link between oxidative stress and the epigenome.

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)–mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

Cell migration is essential for embryonic development and tissue formation in all animals. cacn-1 is a conserved gene of unknown molecular function identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode worm Caenorhabditis elegans. In this study we take a proteomics approach to understand CACN-1 function. To isolate CACN-1−interacting proteins, we used an in vivo tandem-affinity purification strategy. Tandem-affinity purification−tagged CACN-1 complexes were isolated from C. elegans lysate, analyzed by mass spectrometry, and characterized bioinformatically. Results suggest significant interaction of CACN-1 with the C. elegans spliceosome. All of the identified interactors were screened for distal tip cell migration phenotypes using RNAi. Depletion of many of these factors led to distal tip cell migration defects, particularly a failure to stop migrating, a phenotype commonly seen in cacn-1 deficient animals. The results of this screen identify eight novel regulators of cell migration and suggest CACN-1 may participate in a protein network dedicated to high-fidelity gonad development. The composition of proteins comprising the CACN-1 network suggests that this critical developmental module may exert its influence through alternative splicing or other post-transcriptional gene regulation.

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent but nucleotide excision repair (NER)-independent manner. More specifically, at UV-stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and postreplicative repair. These BRCA1 functions differ from those required for DSBR.

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.

Excluding 53BP1 from chromatin is required to attenuate the DNA damage response during mitosis, yet the functional relevance and regulation of this exclusion is unclear. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in its well-conserved ubiquitination-dependent recruitment (UDR) motif. Phosphorylating these sites blocks the interaction of the UDR motif with mononuclesomes containing ubiquitinated histone H2A and impedes binding of 53BP1 to mitotic chromatin. Ectopic recruitment of 53BP1- T1609A/S1618A to mitotic DNA lesions was associated with significant mitotic defects that could be reversed by inhibiting non-homologous end joining. We also reveal that protein phosphatase complex, PP4C/R3β dephosphorylates T1609 and S1618 to allow the recruitment of 53BP1 to chromatin in G1 phase. Our results identify key sites of 53BP1 phosphorylation during mitosis, identify the counteracting phosphatase complex that restores the potential for DDR during interphase, and establish the physiological importance of this regulation.

Human translocation liposarcoma (TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) is a fusion oncoprotein found specifically in a malignant tumor of adipose tissue and results from a t(12;16) translocation that fuses the amino-terminal part of TLS to the entire coding region of CHOP. Being that CHOP is a member of the C/EBP transcription factor family, proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were co-expressed along with C/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid liposarcoma. This effect of TLS-CHOP required a functional leucine zipper domain and correlated with its ability to heterodimerize with C/EBPbeta and inhibit C/EBPbeta DNA binding and transactivation activity in situ. In contrast, the TLS-CHOP basic region was dispensable, making it unlikely that the inhibitory effect of TLS-CHOP is attributable to unscheduled gene expression resulting from TLS-CHOP's putative transactivation activity. Another adipogenic transcription factor, PPARgamma2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta-driven adipogenesis and not with other requisite events of the adipocyte differentiation program. Together, the results demonstrate that TLS-CHOP blocks adipocyte differentiation by directly preventing C/EBPbeta from binding to and transactivating its target genes. Moreover, they provide strong support for the thesis that a blockade to normal differentiation is an important aspect of the cancer process.

Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression.

Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for proliferating cell nuclear antigen (PCNA) monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, we show that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair.

The androgen receptor (AR) is an important determinant of normal and malignant prostate growth and its function is regulated by many factors, including molecular chaperones, cochaperones and coregulators. One such factor is the nuclear-resident cochaperone Bag-1L. Overexpression of Bag-1L and the amplification of its gene have been reported in the hormone-refractory and metastatic stages of prostate cancer and in androgen-independent prostate cancer cells. However the exact mechanism of Bag-1L-mediated regulation of AR action in prostate cancer remains unclear. Here we show that Bag-1L and AR directly interact with one another through two distinct domains in both proteins. While the N-terminus of Bag-1L binds to the AR BF-3 domain via a conserved GARRPR motif, the C-terminal Hsp70/Hsc70-binding domain (BAG) of Bag-1L binds to the tau5 region in the intrinsically disordered AR AF1. The latter interaction is of particular significance as tau5 is a region crucial for the ligand-independent transcriptional activity of the receptor. Understanding the details of this interaction will therefore provide new opportunities of controlling AR action for therapeutic purposes outside of targeting the LBD. We have identified the exact amino acids (K231, K232 and K279) in the BAG domain that are required for the interaction with AR. Amino acid substitution of these residues destroy the interaction of Bag-1L and AR but do not affect the binding to Hsp70/Hsc70. We also observed significant reduction in androgen-dependent cell growth when the BAG-domain mutant is overexpressed in prostate cancer cells in culture and in xenograft mouse models. We further tested the consequence of the BAG-domain mutation on AR transactivation by microarray analysis. We observed repressed expression of multiple genes, including the AR coactivator NCoA2. Correspondingly, we observed multiple changes in the Bag-1L interactome when comparing the wild-type with the BAG mutant; this includes the loss of interaction with the heat shock protein Hsp27 for the mutant Bag-1L protein. Combined these results demonstrate the importance of Bag-1L for AR function and the potential of this protein for therapeutic intervention in prostate cancer. Citation Format: Laura Cato, Antje Neeb, Guillaume Adelmant, Scott Ficarro, Thomas Westerling, Jarrod A. Marto, Andrew C. Cato, Myles Brown. Control of androgen receptor function by the genomic action of the cochaperone Bag-1L. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4677. doi:10.1158/1538-7445.AM2015-4677

GNB1 encodes a beta subunit (Gβ) of heterotrimeric G proteins, which mediate signals downstream of G protein coupled receptors (GPCRs). We isolated a somatic mutant of GNB1 (K89E) by functional screening of a cDNA library derived from a blastic plasmacytoid dendritic cell neoplasm (BPDCN). A search of cancer genome databases identified recurrent mutations in GNB1 and the highly related protein GNB2. GNB1/2 K89E/T were found in B cell acute lymphoblastic leukemia (B-ALL) (1 case), follicular lymphoma (1) and myelodysplastic syndrome (MDS) (1) as well as BPDCN (1). Interestingly GNB1 K57E/T mutations were found only in myeloid diseases: [acute myeloid leukemia (2), atypical CML (2), polycythemia vera (1) and MDS (6)], while GNB1 I80N/T were found predominantly in B cell diseases [CLL (2), FL (2), DLBCL (1) and MDS (1)]. These mutated codons are all located on the GNB1 protein surface that is critical for interactions between Gβ and alpha subunits (Gα) or downstream effectors. Immunoprecipitation followed by mass spectrometry demonstrated that GNB1 K57E, I80T and K89E mutants failed to bind Gα, including GNAI2/3, GNA11/Q and GNA13 that are normally bound by wild-type (WT) GNB1. All mutations affecting these codons promoted cytokine-independent growth of human TF1 myeloid cells or mouse BaF3 lymphoid cells with activation of MEK/ERK and mTOR/PI3K pathways. Pertussis toxin treatment did not affect GNB1-dependent ERK activation or cell growth, implying a Gα-independent pathway. To investigate the function of GNB1 mutations in vivo, we performed a mouse bone marrow transplantation (BMT) experiment using wild-type and Cdkn2a-deficient donors. Loss of the cell cycle regulator CDKN2A is common in BPDCN, B-ALL, and several other hematologic malignancies. Bone marrow cells were isolated from 5-FU treated donor mice and infected with retrovirus expressing GNB1 WT, K57E, I80T or K89E. Transplantation of GNB1 mutant-expressing Cdkn2a-deficient bone marrow resulted in myeloid dendritic cell neoplasms that were CD11b+, CD11c+, CD19-, B220-, and CD3-. GNB1 mutants did not induce tumors in WT bone marrow after 12 months of observation suggesting that GNB1 requires additional cooperating mutations such as Cdkn2a loss. We performed the same BMT experiment using Cdkn2a-deficient bone morrow cells without 5-FU pretreatment. We found thatGNB1 I80T and K89E mutants induced a progenitor B cell ALL (CD11b-, CD11c-, CD19+, CD3-, TdT+). These data suggest that GNB1 mutations can promote tumorigenesis in more than one cell lineage, as observed in patients. In vivo treatment of the myeloid neoplasm with the dual PI3K/mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, we noted that GNB1 mutations co-occurred with oncogenic kinase alterations, including BCR/ABL, JAK2 V617F and BRAF V600K. Co-expression of patient-derived GNB1 alleles with the mutant kinases resulted in relative resistance to treatment with the corresponding kinase inhibitor in each context. Thus, GNB1 and GNB2 mutations confer transformation and targeted therapy resistance across a range of human tumors and may be targetable with inhibitors of PI3K/mTOR signaling. Disclosures Gotlib: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel Support Other. Deininger:BMS, Novartis, Celgene, Genzyme, Gilead: Research Funding; BMS, ARIAD, Novartis, Incyte, Pfizer: Advisory Board, Advisory Board Other; BMS, ARIAD, Novartis, Incyte, Pfizer: Consultancy. Tyner:Constellation Pharmaceuticals: Research Funding.

The proteins of the hedgehog (Hh) family regulate various aspects of development. Recently, members of this family have been shown to regulate skeletal formation in vertebrates and to control both chondrocyte and osteoblast differentiation. In the present study, we analyzed the effect of Sonic hedgehog (Shh) on the osteoblastic and adipocytic commitment/differentiation. Recombinant N-terminal Shh (N-Shh) significantly increased the percentage of both the pluripotent mesenchymal cell lines C3H10T1/2 and ST2 and calvaria cells responding to bone morphogenetic protein 2 (BMP-2), in terms of osteoblast commitment as assessed by measuring alkaline phosphatase (ALP) activity. This synergistic effect was mediated, at least partly, through the positive modulation of the transcriptional output of BMPs via Smad signaling. Furthermore, N-Shh was found to abolish adipocytic differentiation of C3H10T1/2 cells both in the presence or absence of BMP-2. A short treatment with N-Shh was sufficient to dramatically reduce the levels of the adipocytic-related transcription factors C/EBPα and PPARγ in both C3H10T1/2 and calvaria cell cultures. Given the inverse relationship between marrow adipocytes and osteoblasts with aging, agonists of the Hh signaling pathway might constitute potential drugs for preventing and/or treating osteopenic disorders.

Transcription and mRNA processing are coupled events in vivo, but the mechanisms that coordinate these processes are largely unknown. PGC-1 is a transcriptional coactivator that plays a major role in the regulation of adaptive thermogenesis. PGC-1 also has certain motifs characteristic of splicing factors. We demonstrate here that mutations in the serine- and arginine-rich domain and RNA recognition motif of PGC-1 interfere with the ability of PGC-1 to induce mRNAs of target genes. These mutations also disrupt the ability of PGC-1 to co-localize and associate with RNA processing factors. PGC-1 can alter the processing of an mRNA, but only when it is loaded onto the promoter of the gene. These data demonstrate the coordinated regulation of RNA transcription and processing through PGC-1.

The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor that regulates several crucial biological processes such as adipogenesis, glucose homeostasis, and cell growth. It is also the functional receptor for a new class of insulin-sensitizing drugs, the thiazolidinediones, now widely used in the treatment of type 2 diabetes mellitus. Here we report that PPARgamma protein levels are significantly reduced in adipose cells and fibroblasts in response to specific ligands such as thiazolidinediones. Studies with several doses of different ligands illustrate that degradation of PPARgamma correlates well with the ability of ligands to activate this receptor. However, analyses of PPARgamma mutants show that, although degradation does not strictly depend on the transcriptional activity of the receptor, it is dependent upon the ligand-gated activation function 2 (AF2) domain. Proteasome inhibitors inhibited the down-regulation of PPARgamma and ligand activation enhanced the ubiquitination of this receptor. These data indicate that, although ligand binding and activation of the AF2 domain increase the transcriptional function of PPARgamma, these same processes also induce ubiquitination and subsequent degradation of this receptor by the proteasome.

Transcriptional coactivators have been viewed as constitutively active components, using transcription factors mainly to localize their functions. Here, it is shown that PPARγ coactivator–1 (PGC-1) promotes transcription through the assembly of a complex that includes the histone acetyltransferases steroid receptor coactivator–1 (SRC-1) and CREB binding protein (CBP)/p300. PGC-1 has a low inherent transcriptional activity when it is not bound to a transcription factor. The docking of PGC-1 to peroxisome proliferator-activated receptor γ (PPARγ) stimulates an apparent conformational change in PGC-1 that permits binding of SRC-1 and CBP/p300, resulting in a large increase in transcriptional activity. Thus, transcription factor docking switches on the activity of a coactivator protein.

Mice deficient in C/EBP alpha have defective development of adipose tissue, but the precise role of C/EBP alpha has not been defined. Fibroblasts from C/EBP alpha(-/-) mice undergo adipose differentiation through expression and activation of PPAR gamma, though several clear defects are apparent. C/EBP alpha-deficient adipocytes accumulates less lipid, and they do not induce endogenous PPAR gamma, indicating that cross-regulation between C/EBP alpha and PPAR gamma is important in maintaining the differentiated state. The cells also show a complete absence of insulin-stimulated glucose transport, secondary to reduced gene expression and tyrosine phosphorylation for the insulin receptor and IRS-1. These results define multiple roles for C/EBP alpha in adipogenesis and show that cross-regulation between PPAR gamma and C/EBP alpha is a key component of the transcriptional control of this cell lineage.

To identify new oncogene alleles directly from primary tumor specimens, we generate and screen cDNA libraries from patient samples for gain-of-function alterations that can substitute for cytokine signaling in cytokine-dependent cells. Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia of plasmacytoid dendritic cells with a dismal prognosis. No driver oncogenes have been identified in cases of BPDCN. Screening of a cDNA library generated from a BPDCN resulted in multiple cytokine-independent clones that expressed a full-length transcript of GNB1 with a K89E mutation. GNB1 encodes a beta subunit of the heterotrimeric G-protein, a binding complex that transduces signals from G-protein coupled receptors to multiple downstream pathways. Gain-of-function mutations have been reported in alpha subunits of the G-protein, including GNAQ/GNA11 in uveal melanoma and GNAS in pituitary tumors, however, the contributions of beta subunits to cancer remains undefined. To investigate downstream signaling from GNB1 K89E, we performed gene expression profiling and mass spectrometry (MS)-based phosphoproteomics and found significant activation of RAS/MAPK and PI3K/AKT pathways in GNB1 K89E-expressing cells compared to isogenic cells expressing wild-type GNB1. ERK and AKT activation by GNB1 K89E were confirmed by western blotting. To target GNB1 K89E signaling, we screened kinase inhibitors using a multiplex assay of small molecules and found selective sensitivity of GNB1 K89E cells to MEK and pan-PI3-kinase inhibitors. To assay the transforming effects of GNB1 K89E in vivo, we transduced GNB1 (wild-type or K89E) into bone marrow from Cdkn2a-deficient donors after 5-FU treatment and transplanted into wild-type recipients. We opted to utilize Cdkn2a-deficient donors as the loss of CDKN2A is common in cases of BPDCN. Within 4 months after transplantation, all mice (n=10) that received bone marrow transduced with GNB1 K89E developed a lethal malignancy characterized by pancytopenia and massive hepatosplenomegaly. Spleens were infiltrated by large, spindly cells with extensive dendritic projections, as well as extensive fibrosis that completely effaced the normal splenic architecture. The cells were negative for T-cell (CD2, CD3) and B-cell (CD19, B220) markers but positive for the dendritic cell/macrophage markers MAC-2 and MAC-3. Further characterization by flow cytometry demonstrated that the cells infiltrating the spleen were CD8, CD103, MHC class II, CD26, FLT3 and CD11c positive, consistent with neoplastic dendritic cells. Serial transplantation of splenic cells from five different GNB1 K89E-transplanted mice into secondary wild-type recipients resulted in 100% fatality within 50 days. We searched published datasets from exome, transcriptome and whole genome sequencing of hematologic malignancies for GNB1 mutations. We identified one case of K89E in B-cell acute lymphoblastic leukemia (ALL), four cases with I80T/N in chronic lymphocytic leukemia or B-cell lymphomas, six cases with K57E/T in myeloid neoplasms, and D76G in T-cell ALL. Expression of any of these alleles but not wild-type GNB1 was sufficient to promote cytokine-independent growth of human TF1 cells. The published structure of GNB1 (Ford et al. Science 1998) reported a small number of residues, including K57, I80 and K89 that mediate interactions with both G-alpha subunits and effector proteins. In fact, affinity purification followed by MS using tagged GNB1 (wild-type, I80T and K89E) demonstrated that, unlike wild-type GNB1, the GNB1 mutants fail to bind distinct Gα subunits. The repertoire of protein interactors, which includes potential G protein effectors, also differed between different GNB1 alleles. Thus, gain-of-function mutations in GNB1 occur across a broad range of hematologic malignancies, modify essential interaction G-protein subunit interactions, can drive in vivo transformation, and activate targetable downstream kinases. Disclosures: Tyner: Incyte Corporation: Research Funding.

A significant proportion of high-grade serous ovarian carcinomas arise from the secretory cells of the fallopian tube. Exocytosis, the process through which cells release vesicles containing various growth factors and signaling molecules is important for cell-cell and cell-microenvironment communications. Importantly, defects in regulated-exocytosis have been shown to be involved in tumorigenesis. Understanding the molecular determinants of exocytosis in secretory cells has immense potential for developing new cancer biomarkers and therapeutic agents. Using HE4, a secreted glycoprotein and a biomarker of ovarian cancer as a surrogate marker in our in silico approach, we recently identified Tumor Necrosis Factor alpha-induced protein 2 (TNFAIP2; also known as B94) as a potential regulator of exocytosis. TNFAIP2 is a retinoic acid pathway target gene that is induced by tumor necrosis factor alpha (TNFa), interleukin 1-beta (IL1-beta), and lipopolysaccharides. TNFAIP2 is evolutionarily conserved, yet little is known about its biological function and its role in cancer. Its only structural domain is a Sec6 domain which is found in proteins of the exocyst complex and in those involved with cytokinesis. Analysis of high-density tissue microarrays (TMAs) of all major cancer histotypes showed that TNFAIP2 is most highly expressed in ovarian serous carcinomas and certain lymphomas. Gene Ontology and Ingenuity Pathway Analyses of TNFAIP2 expression and co-expressed genes suggest a possible connection to tumors of the reproductive tract and a role in cell proliferation and migration. We interrogated the role of TNFAIP2 in these processes using RNA interference. Robust knock-down of TNFAIP2 expression using siRNA led to decrease in proliferation and migration of ovarian cancer cells. Analysis of sub-cellular localization shows that TNFAIP2 is localized to cell membrane and regions of the cytoplasm. To further understand the mechanism of TNFAIP2 function, we cloned full length TNFAIP2 and expressed it as a FLAG-StrepTactin tagged fusion protein, and the TNFAIP2 complex was tandem-affinity purified. The associated proteins were identified by mass spectrometry. TNFAIP2 interacts with various component of adherens junctions and vesicular trafficking, including E-cadherin, integrins, alpha, beta, and delta-catenins, and Rab proteins. Interestingly, TNFAIP2 also interacts with a group of receptor tyrosine kinases (RTKs), including AXL. Our results show that TNFAIP2 is an important mediator of endosome recycling and that it regulates the stability of certain receptor tyrosine kinases, including AXL and MER, by enhancing their processing through recycling-endosomes. Structure-function studying are ongoing as well as studies aimed at targeting TNFAIP2 in the setting of RTK activation. Citation Format: Sekhar Duraisamy, Keren Levanon, Guillaume Adelmant, Huiying Piao, Joyce Liu, Ursula Matulonis, Michelle Hirsch, Jarrod A. Marto, Ronny Drapkin. TNFAIP2 is required for growth and migration of ovarian cancer cells and functions to regulate trafficking of receptor tyrosine kinases. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A80.

Abstract 2455 Activating mutations in the small GTPase N-RAS occur in about 10% of acute myeloid leukemia (AML) cases. Active N-RAS is thought to drive the disease and is therefore a potential target for drug development. There have been numerous unsuccessful efforts to target RAS itself with small molecules, and blocking post-translational modifications of RAS proteins, such as through inhibition of farnesyl transferase, has similarly not proven useful. In addition, efficacy of targeting critical downstream effectors has been limited by the complexity of RAS signaling, such as redundancy of signaling pathways and feedback mechanisms. While targeting RAS is challenging, it was our hypothesis that inhibiting the right combination of downstream pathways in a particular lineage with small molecules could be effective. Initially, we created a Ba/F3 cell line that was completely dependent on oncogenic N-RAS-G12D for growth and survival. Growth was suppressed >99% by shRNA for N-RAS, but could be rescued entirely by interleukin-3 (IL-3), which does not require N-RAS signaling in these cells. Using this cell line, we performed a high-throughput chemical screen with a large library of multi-targeted kinase inhibitors. The lead compound (NRAS1) showed a 70-fold difference in the EC50 for growth inhibition between BaF3-NRAS G12D cells cultured in the absence (0.01μM) or presence (0.77μM) of IL-3. Importantly, this compound showed selectivity towards several leukemia cell lines that were shown to be dependent on mutant N-RAS by shRNA compared to cells expressing wild-type N-RAS (p=0.02). Also, in a xenotransplant model using NRAS-G12D+ OCI-AML3 cells, this compound significantly reduced tumor burden (P=0.005) and prolonged survival (P=0.002) compared to controls. Next, we sought to identify the targets of NRAS1, Interestingly, the compound did not suppress MEK or ERK, which are classical targets of RAS signaling in epithelial cells. NRAS1 profoundly reduced AKT and RPS6 phosphorylation. Kinase selectivity profiling of this compound (1μM) in OCI-AML3 cells (EC50: 0.3μM) identified 13 major binding partners with more than 85% efficacy. The targets consisted mainly of SRC family proteins (SRC, FGR, and LYN etc.) and MAPK family proteins (MAP4K2, 3, 5, and p38 etc.) and others (ZAK and BTK etc), but not MEK and ERK, and AKT was not detected in this assay. In preliminary studies, most of these target kinases were knocked-down by shRNA and, as expected, no single kinase was found to be responsible for mediating growth inhibition. Using a phospho-antibody microarray, the most significantly de-phosphorylated kinases were p38, AKT and SRC, which supports our preliminary findings. To validate the significance of these results, we treated Ba/F3-N-RAS cells with combinations of kinase inhibitors. Combining the AKT inhibitor MK2206 and Dasatinib (SRC family inhibitor) revealed marked synergy, while neither had activity individually. Also, the combination of MK2206 and a cleaner SRC family inhibitor, AZD0530, also synergized, although to lesser extent. In both examples, however, the inhibition of N-RAS transformed cells by NRAS1 proved superior, suggesting that one or more additional targets are required for inhibition of NRAS signaling. To identify additional critical targets of our compound we generated several derivatives with different potency. In particular, one less potent analog of NRAS1 (analog 6, 1% EC50 of original compound) showed a loss of binding activity towards the MAP4K family of proteins, especially MAP4K2. Observed synergy between the selective MAP4K2 inhibitor NG25 and selective inhibitors of MK2206 and Dasatinib in Ba/F3-NRAS G12D cells further points toward MAP4K2 as being of additional significance for oncogenic RAS signaling. Together with the previous data, we propose AKT and MAP4K2 as critical targets of NRAS1. In conclusion, we have identified a novel and selective kinase inhibitor of the N-RAS signaling pathway by chemical screen using Ba/F3-N-RAS G12D cells. By combination of signaling study, kinase selectivity profiling and phosphoproteomics, the main functional targets were found to be AKT, and MAP4K2, and additional functional targets will be elucidated. Our approach also could be applied for other type of oncogenes, and it could help to find therapeutic compound and also help to decipher signaling mechanisms of the oncogenes which are thus far undruggable. Disclosures: No relevant conflicts of interest to declare.

Mitochondrial number and function are altered in response to external stimuli in eukaryotes. While several transcription/replication factors directly regulate mitochondrial genes, the coordination of these factors into a program responsive to the environment is not understood. We show here that PGC-1, a cold-inducible coactivator of nuclear receptors, stimulates mitochondrial biogenesis and respiration in muscle cells through an induction of uncoupling protein 2 (UCP-2) and through regulation of the nuclear respiratory factors (NRFs). PGC-1 stimulates a powerful induction of NRF-1 and NRF-2 gene expression; in addition, PGC-1 binds to and coactivates the transcriptional function of NRF-1 on the promoter for mitochondrial transcription factor A (mtTFA), a direct regulator of mitochondrial DNA replication/transcription. These data elucidate a pathway that directly links external physiological stimuli to the regulation of mitochondrial biogenesis and function.