Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

Guided Direct Time-of-Flight Lidar Using Stereo Cameras for Enhanced Laser Power Efficiency.

Self-driving vehicles demand efficient and reliable depth-sensing technologies. Lidar, with its capability for long-distance, high-precision measurement, is a crucial component in this pursuit. However, conventional mechanical scanning implementations suffer from reliability, cost, and frame rate limitations. Solid-state lidar solutions have emerged as a promising alternative, but the vast amount of photon data processed and stored using conventional direct time-of-flight (dToF) prevents long-distance sensing unless power-intensive partial histogram approaches are used. In this paper, we introduce a groundbreaking 'guided' dToF approach, harnessing external guidance from other onboard sensors to narrow down the depth search space for a power and data-efficient solution. This approach centers around a dToF sensor in which the exposed time window of independent pixels can be dynamically adjusted. We utilize a 64-by-32 macropixel dToF sensor and a pair of vision cameras to provide the guiding depth estimates. Our demonstrator captures a dynamic outdoor scene at 3 fps with distances up to 75 m. Compared to a conventional full histogram approach, on-chip data is reduced by over twenty times, while the total laser cycles in each frame are reduced by at least six times compared to any partial histogram approach. The capability of guided dToF to mitigate multipath reflections is also demonstrated. For self-driving vehicles where a wealth of sensor data is already available, guided dToF opens new possibilities for efficient solid-state lidar.

ZEB1 promotes non-homologous end joining double-strand break repair.

Repair of DSB induced by IR is primarily carried out by Non-Homologous End Joining (NHEJ), a pathway in which 53BP1 plays a key role. We have discovered that the EMT-inducing transcriptional repressor ZEB1 (i) interacts with 53BP1 and that this interaction occurs rapidly and is significantly amplified following exposure of cells to IR; (ii) is required for the localization of 53BP1 to a subset of double-stranded breaks, and for physiological DSB repair; (iii) co-localizes with 53BP1 at IR-induced foci (IRIF); (iv) promotes NHEJ and inhibits Homologous Recombination (HR); (v) depletion increases resection at DSBs and (vi) confers PARP inhibitor (PARPi) sensitivity on BRCA1-deficient cells. Lastly, ZEB1's effects on repair pathway choice, resection, and PARPi sensitivity all rely on its homeodomain. In contrast to the well-characterized therapeutic resistance of high ZEB1-expressing cancer cells, the novel ZEB1-53BP1-shieldin resection axis described here exposes a therapeutic vulnerability: ZEB1 levels in BRCA1-deficient tumors may serve as a predictive biomarker of response to PARPis.

Induction of PARP7 Creates a Vulnerability for Growth Inhibition by RBN2397 in Prostate Cancer Cells.

The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397, to study the effects of PARP7 inhibition in androgen receptor (AR)-positive and AR-negative prostate cancer cells. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the AR. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the AR, or the aryl hydrocarbon receptor, and induce PARP7 expression. We show that the growth-inhibitory effects of RBN2397 are distinct from its enhancement of IFN signaling recently shown to promote tumor immunogenicity. RBN2397 treatment also induces trapping of PARP7 in a detergent-resistant fraction within the nucleus, which is reminiscent of how inhibitors such as talazoparib affect PARP1 compartmentalization. Because PARP7 is expressed in AR-negative metastatic tumors and RBN2397 can affect cancer cells through multiple mechanisms, PARP7 may be an actionable target in advanced prostate cancer. Significance: RBN2397 is a potent and selective inhibitor of PARP7 that reduces the growth of prostate cancer cells, including a model for treatment-emergent neuroendocrine prostate cancer. RBN2397 induces PARP7 trapping on chromatin, suggesting its mechanism of action might be similar to clinically used PARP1 inhibitors.

A Step Toward Personalized Surgical Decision Making: Machine Learning Predicts 1 Versus Numerous Melanoma Lymph Node Metastases Using RNA-sequencing.

OBJECTIVE: Develop a predictive model to identify patients with 1 pathologic lymph node (pLN) versus >1 pLN using machine learning applied to gene expression profiles and clinical data as input variables. BACKGROUND: Standard management for clinically detected melanoma lymph node metastases is complete therapeutic LN dissection (TLND). However, >40% of patients with a clinically detected melanoma lymph node will only have 1 pLN on final review. Recent data suggest that targeted excision of just the single enlarged LN may provide excellent regional control, with less morbidity than TLND. The selection of patients for less morbid surgery requires accurate identification of those with only 1 pLN. METHODS: The Cancer Genome Atlas database was used to identify patients who underwent TLND for melanoma. Pathology reports in The Cancer Genome Atlas were reviewed to identify the number of pLNs. Patients were included for machine learning analyses if RNA sequencing data were available from a pLN. After feature selection, the top 20 gene expression and clinical input features were used to train a ridge logistic regression model to predict patients with 1 pLN versus >1 pLN using 10-fold cross-validation on 80% of samples. The model was then tested on the remaining holdout samples. RESULTS: A total of 153 patients met inclusion criteria: 64 with one pLN (42%) and 89 with >1 pLNs (58%). Feature selection identified 1 clinical (extranodal extension) and 19 gene expression variables used to predict patients with 1 pLN versus >1 pLN. The ridge logistic regression model identified patient groups with an accuracy of 90% and an area under the receiver operating characteristic curve of 0.97. CONCLUSIONS: Gene expression profiles together with clinical variables can distinguish melanoma metastasis patients with 1 pLN versus >1 pLN. Future models trained using positron emission tomography/computed tomography imaging, gene expression, and relevant clinical variables may further improve accuracy and may predict patients who can be managed with a targeted LN excision rather than a complete TLND.

MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.

Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly.

Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

The Role of Ubiquitination and SUMOylation in DNA Replication.

DNA replication is a tightly regulated conserved process that ensures the faithful transmission of genetic material to define heritable phenotypic traits. Perturbations in this process result in genomic instability, mutagenesis, and diseases, including malignancy. Proteins involved in the initiation, progression, and termination of DNA replication are subject to a plethora of reversible post-translational modifications (PTMs) to provide a proper temporal and spatial control of replication. Among these, modifications involving the covalent attachment of the small protein ubiquitin or the small ubiquitin-like modifier (SUMO) to replication and replication-associated proteins are particularly important for the proper regulation of DNA replication as well as for optimal cellular responses to replication stress. In this article, we describe how the ubiquitination and SUMOylation processes impact DNA replication in eukaryotes and highlight the consequences of deregulated signals emanating from these two versatile regulatory pathways on cellular activities.

A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

Intrinsically disordered protein RBM14 plays a role in generation of RNA:DNA hybrids at double-strand break sites.

Accumulating evidence suggests participation of RNA-binding proteins with intrinsically disordered domains (IDPs) in the DNA damage response (DDR). These IDPs form liquid compartments at DNA damage sites in a poly(ADP ribose) (PAR)-dependent manner. However, it is greatly unknown how the IDPs are involved in DDR. We have shown previously that one of the IDPs RBM14 is required for the canonical nonhomologous end joining (cNHEJ). Here we show that RBM14 is recruited to DNA damage sites in a PARP- and RNA polymerase II (RNAPII)-dependent manner. Both KU and RBM14 are required for RNAPII-dependent generation of RNA:DNA hybrids at DNA damage sites. In fact, RBM14 binds to RNA:DNA hybrids. Furthermore, RNA:DNA hybrids and RNAPII are detected at gene-coding as well as at intergenic areas when double-strand breaks (DSBs) are induced. We propose that the cNHEJ pathway utilizes damage-induced transcription and intrinsically disordered protein RBM14 for efficient repair of DSBs.

The Barrier Molecules Junction Plakoglobin, Filaggrin, and Dystonin Play Roles in Melanoma Growth and Angiogenesis.

OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Chemotherapy-Induced Distal Enhancers Drive Transcriptional Programs to Maintain the Chemoresistant State in Ovarian Cancer.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. SIGNIFICANCE: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Combined c-Met/Trk Inhibition Overcomes Resistance to CDK4/6 Inhibitors in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain malignancy and carries an extremely poor prognosis. Recent molecular studies revealed the CDK4/6-Rb-E2F axis and receptor tyrosine kinase (RTK) signaling to be deregulated in most GBM, creating an opportunity to develop more effective therapies by targeting both pathways. Using a phospho-RTK protein array, we found that both c-Met and TrkA-B pathways were significantly activated upon CDK4/6 inhibition in GBM cells. We therefore investigated the efficacy of combined CDK4/6 and c-Met/TrkA-B inhibition against GBM. We show that both c-Met and TrkA-B pathways transactivate each other, and targeting both pathways simultaneously results in more efficient pathway suppression. Mechanistically, inhibition of CDK4/6 drove NF-κB-mediated upregulation of hepatocyte growth factor, brain-derived neurotrophic factor, and nerve growth factor that in turn activated both c-Met and TrkA-B pathways. Combining the CDK4/6 inhibitor abemaciclib with the c-Met/Trk inhibitor altiratinib or the corresponding siRNAs induced apoptosis, leading to significant synergy against GBM. Collectively, these findings demonstrate that the activation of c-Met/TrkA-B pathways is a novel mechanism involved in therapeutic resistance of GBM to CDK4/6 inhibition and that dual inhibition of c-Met/Trk with CDK4/6 should be considered in future clinical trials.Significance: CDK4/6 inhibition in glioblastoma activates the c-Met and TrkA-B pathways mediated by NF-κB and can be reversed by a dual c-Met/Trk inhibitor. Cancer Res; 78(15); 4360-9. ©2018 AACR.

High Dynamic Range Imaging at the Quantum Limit with Single Photon Avalanche Diode-Based Image Sensors.

This paper examines methods to best exploit the High Dynamic Range (HDR) of the single photon avalanche diode (SPAD) in a high fill-factor HDR photon counting pixel that is scalable to megapixel arrays. The proposed method combines multi-exposure HDR with temporal oversampling in-pixel. We present a silicon demonstration IC with 96 × 40 array of 8.25 µm pitch 66% fill-factor SPAD-based pixels achieving >100 dB dynamic range with 3 back-to-back exposures (short, mid, long). Each pixel sums 15 bit-planes or binary field images internally to constitute one frame providing 3.75× data compression, hence the 1k frames per second (FPS) output off-chip represents 45,000 individual field images per second on chip. Two future projections of this work are described: scaling SPAD-based image sensors to HDR 1 MPixel formats and shrinking the pixel pitch to 1-3 µm.

Impact of Sex Disparities on Outcomes of Living-Donor Kidney Transplant in Egypt: Data of 979 Patients.

OBJECTIVES: Renal transplant is the criterion standard for treatment of end-stage renal disease. The effects of disparities between men and women on renal transplant outcomes have been evaluated in many studies but with debatable results. It has been suggested that female kidney donors have poor outcomes after transplant compared with male kidney donors, especially when implanted in a male recipient. The aim of the study was to evaluate the effects of sex on living-donor kidney transplant outcome. MATERIALS AND METHODS: The data of 979 patients who underwent living-donor kidney transplant from January 2000 to December 2010 at a single center were reviewed retrospectively. The patients were divided into 4 groups according to recipient and donor sex: male donor-to-male recipient (n = 307), male donor-to-female recipient (n = 132), female donor-to-male recipient (n = 411), and female donor-to-female recipient (n = 129). We compared the demographic characteristics, posttransplant rejection and complications, and graft and patient survival rates among the groups. RESULTS: Male recipients were older than female recipients, whereas male donors were younger than female donors (P < .001). No statistically significant differences were shown regarding recipient body mass index, ischemia time and time to diuresis, and acute and chronic rejection rates between the groups. Graft (P = .947) and patient (P = .421) survival rates were comparable between groups. CONCLUSIONS: Donor and recipient sex had no significant effect on outcomes of living-donor renal allograft recipients.

The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors.

The cullin RING E3 ubiquitin ligase 4 (CRL4) with its substrate receptor CDT2 (CRL4-CDT2) is emerging as a critical regulator of DNA replication through targeting CDT1, SET8, and p21 for ubiquitin-dependent proteolysis. The aberrant increased stability of these proteins in cells with inactivated CRL4-CDT2 results in DNA rereplication, which is deleterious to cells due to the accumulation of replication intermediates and stalled replication forks. Here, we demonstrate that CDT2 is overexpressed in head and neck squamous cell carcinoma (HNSCC), and its depletion by siRNA inhibits the proliferation of human papilloma virus-negative (HPV-ve) HNSCC cells primarily through the induction of rereplication. Treatment of HNSCC with the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924), which inhibits all cullin-based ligases, induces significant rereplication and inhibits HNSCC cell proliferation in culture and HNSCC xenografts in mice. Pevonedistat additionally sensitizes HNSCC cells to ionizing radiation (IR) and enhances IR-induced suppression of xenografts in mice. Induction of rereplication via CDT2 depletion, or via the stabilization or activation of CDT1, also radiosensitizes HNSCC cells. Collectively, these results demonstrate that induction of rereplication represents a novel approach to treating radioresistant HNSCC tumors and suggest that pevonedistat may be considered as an adjuvant for IR-based treatments. Mol Cancer Ther; 17(2); 368-80. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Combined CDK4/6 and mTOR Inhibition Is Synergistic against Glioblastoma via Multiple Mechanisms.

Purpose: Glioblastoma (GBM) is a deadly brain tumor marked by dysregulated signaling and aberrant cell-cycle control. Molecular analyses have identified that the CDK4/6-Rb-E2F axis is dysregulated in about 80% of GBMs. Single-agent CDK4/6 inhibitors have failed to provide durable responses in GBM, suggesting a need to combine them with other agents. We investigate the efficacy of the combination of CDK4/6 inhibition and mTOR inhibition against GBM.Experimental Design: Preclinical in vitro and in vivo assays using primary GBM cell lines were performed.Results: We show that the CDK4/6 inhibitor palbociclib suppresses the activity of downstream mediators of the mTOR pathway, leading to rebound mTOR activation that can be blocked by the mTOR inhibitor everolimus. We further show that mTOR inhibition with everolimus leads to activation of the Ras mediator Erk that is reversible with palbociclib. The combined treatment strongly disrupts GBM metabolism, resulting in significant apoptosis. Further increasing the utility of the combination for brain cancers, everolimus significantly increases the brain concentration of palbociclib.Conclusions: Our findings demonstrate that the combination of CDK4/6 and mTOR inhibition has therapeutic potential against GBM and suggest it should be evaluated in a clinical trial. Clin Cancer Res; 23(22); 6958-68. ©2017 AACR.

Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9.

ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.