Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

Targeting of SUMOylation leads to cBAF complex stabilization and disruption of the SS18::SSX transcriptome in Synovial Sarcoma.

Synovial Sarcoma (SS) is driven by the SS18::SSX fusion oncoprotein. and is ultimately refractory to therapeutic approaches. SS18::SSX alters ATP-dependent chromatin remodeling BAF (mammalian SWI/SNF) complexes, leading to the degradation of canonical (cBAF) complex and amplified presence of an SS18::SSX-containing non-canonical BAF (ncBAF or GBAF) that drives an SS-specific transcription program and tumorigenesis. We demonstrate that SS18::SSX activates the SUMOylation program and SSs are sensitive to the small molecule SAE1/2 inhibitor, TAK-981. Mechanistically, TAK-981 de-SUMOylates the cBAF subunit SMARCE1, stabilizing and restoring cBAF on chromatin, shifting away from SS18::SSX-ncBAF-driven transcription, associated with DNA damage and cell death and resulting in tumor inhibition across both human and mouse SS tumor models. TAK-981 synergized with cytotoxic chemotherapy through increased DNA damage, leading to tumor regression. Targeting the SUMOylation pathway in SS restores cBAF complexes and blocks the SS18::SSX-ncBAF transcriptome, identifying a therapeutic vulnerability in SS, positioning the in-clinic TAK-981 to treat SS.

Commissioning and Assessment of Radiation Field and Dose Inhomogeneity for a Dual X-ray Tube Cabinet Irradiator: To Ensure Accurate Dosimetry in Radiation Biology Experiments.

Purpose: Standardization of x-ray cabinet irradiator dose, geometry, and calibration reporting is an ongoing process. Multi-tube designs have been introduced into the preclinical market and give a theoretical benefit but have not been widely assessed for use in preclinical irradiation conditions. The aim of this study was to report our experience commissioning a dual x-ray source cabinet irradiator (CIXD, Xstrahl Limited, United Kingdom) and assess the dose distribution for various experimental conditions. Methods and Materials: Half-value layer (HVL) measurement, profile measurements, and output calibration were performed using a calibrated ion chamber. Constancy measurements were performed twice daily over 2 weeks to assess output fluctuations. Film measurements were completed using solid water to assess percent depth dose and homogeneity within the field and within variable thicknesses of solid water and phosphate-buffered saline solution. Film measurements were repeated for various arrangements of petri dishes filled with phosphate-buffered saline or water and in a 3D-printed mouse phantom. Results: The x-ray tubes had a measured in-air output of 1.27 Gy/min. The HVL was 1.7 mm Cu. The upper and lower tubes both exhibited the heel effect, but when operated simultaneously, the effect was reduced. Ion chamber measurements revealed a 15% dose inhomogeneity within the tray area (18 × 18 cm2). Film measurements in the petri dishes indicated minor nonuniformities in the arrangements of the experimental apparatus. Measurements from the mouse phantom with film agreed with ion chamber measurements for various phantom placements and orientations. Conclusions: X-ray cell culture and animal irradiation with dual tube cabinet irradiation is efficient and robust when using established dosimetric tools to confirm output and homogeneity. The conditions assumed for calibrations are often not maintained during experiments. We have confirmed that inhomogeneities are present for single-tube use; however, they are reduced with simultaneous tube use. Additional dosimetric monitoring should be performed for each unique irradiation setup.

Lymph node-targeting adjuvant/neoantigen-codelivering vaccines for combination glioblastoma radioimmunotherapy.

Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.

ATM phosphorylates PP2A subunit A resulting in nuclear export and spatiotemporal regulation of the DNA damage response.

Ataxia telangiectasia mutated (ATM) is a serine-threonine protein kinase and important regulator of the DNA damage response (DDR). One critical ATM target is the structural subunit A (PR65-S401) of protein phosphatase 2A (PP2A), known to regulate diverse cellular processes such as mitosis and cell growth as well as dephosphorylating many proteins during the recovery from the DDR. We generated mouse embryonic fibroblasts expressing PR65-WT, -S401A (cannot be phosphorylated), and -S401D (phospho-mimetic) transgenes. Significantly, S401 mutants exhibited extensive chromosomal aberrations, impaired DNA double-strand break (DSB) repair and underwent increased mitotic catastrophe after radiation. Both S401A and the S401D cells showed impaired DSB repair (nonhomologous end joining and homologous recombination repair) and exhibited delayed DNA damage recovery, which was reflected in reduced radiation survival. Furthermore, S401D cells displayed increased ERK and AKT signaling resulting in enhanced growth rate further underscoring the multiple roles ATM-PP2A signaling plays in regulating prosurvival responses. Time-lapse video and cellular localization experiments showed that PR65 was exported to the cytoplasm after radiation by CRM1, a nuclear export protein, in line with the very rapid pleiotropic effects observed. A putative nuclear export sequence (NES) close to S401 was identified and when mutated resulted in aberrant PR65 shuttling. Our study demonstrates that the phosphorylation of a single, critical PR65 amino acid (S401) by ATM fundamentally controls the DDR, and balances DSB repair quality, cell survival and growth by spatiotemporal PR65 nuclear-cytoplasmic shuttling mediated by the nuclear export receptor CRM1.

EGFRvIII expression and isocitrate dehydrogenase mutations in patients with glioma.

Molecular pathology and personalized medicine are still being evolved in Saudi Arabia, and genetic testing for the detection of mutations as cancer markers have not been established in the diagnostics laboratories in Saudi Arabia. The aim of the present study was to determine the prevalence of isocitrate dehydrogenase (IDH1 and IDH2) mutations and epidermal growth factor receptor variant (EGFRv)III transcript expression in Saudi Arabian patients with glioma. Out of 117 brain tumors tested by reverse transcription-quantitative PCR for EGFRvIII, 41 cases tested positive. In the glioblastoma (GBM) category, 28/55 tumors were positive, in astrocytoma tumors 5/22, and in oligodendrogliomas 4/13 cases were positive respectively. EGFRvIII transcript was sequenced by capillary electrophoresis to demonstrate the presence of EGFRvIII-specific junction where exons 2-7 were deleted. In the present study 106 tumors were sequenced for IDH1 exon-4 mutations using the capillary sequencing method. The most common substitution missense mutation c.395G>A was found in 16 tumors. In the case of adamantinomatous craniopharyngioma, a novel missense mutation in c.472C>T was detected in IDH2 gene. Using next-generation sequencing (NGS), 74 tumors were sequenced for the IDH1 gene, and a total of 8 missense variants were identified in 36 tumors in a population of Saudi Arabia. The missense mutation (c.395G>A) was detected in 29/36 of tumors. A novel intronic mutation in c.414+9T>A was found in 13 cases in the IDH1 gene. In addition, one case exhibited a novel synonymous mutation in c.369A>G. Eleven tumors were found to have compound mutations in the IDH1 gene. In IDH2 gene, out of a total of 16 variants found in 6 out of 45 tumors, nine were missense, five were synonymous and one was intronic. This is the first report from Saudi Arabian laboratories analyzing glioma tumors for EGFRvIII expression, and the first study from Saudi Arabia to analyze IDH mutations in gliomas using the capillary and NGS methods.

Adamantinomatous Craniopharyngioma in an Adult: A Case Report with NGS Analysis.

PURPOSE: Several recent studies have documented CTNNB1 and BRAF mutations which are mutually exclusive for adamantinomatous craniopharyngioma (ACP) and papillary craniopharyngioma (PCP) tumors. This discovery is helpful in the development of novel targeted therapies in successful clinical trials with BRAF mutations in PCP cases. However, no such targeted therapy is available yet for ACP. Here, we report novel mutations, which are not previously reported, in a case of an adult ACP using NGS analysis. RESULTS: Patient DNA was sequenced using Ion PI v3 chip on Ion Proton. A total of 16 variants were identified in this tumor by NGS analysis, out of which four were missense mutations, seven were synonymous mutations, and five were intronic variants. In CTNNB1 gene a known missense mutation in c.101G>T; in TP53 a known missense mutation in c.215C>G; and two known missense variants in PIK3CA, viz., in c.1173A>G; in exon 7, and in c.3128T>C; in exon 21, were found, respectively. Seven synonymous mutations were detected in this tumor, viz., in IDH1 (rs11554137), in FGFR3 (rs7688609), in PDGFRA (rs1873778), in APC (COSM3760869), in EGFR (rs1050171), in MET (rs35775721), and in RET (rs1800861), respectively. Three known, intronic variants were found in genes, such as PIK3CA, KDR, and JAK3, respectively. Also, a 3'-UTR and a splice site acceptor site variant in CSF1R and FLT3 genes were found in this tumor. We have shown allele coverage, allele ratio, and p-value, for all these mutations. The p-values and Phred quality score were significantly high for these variants. CONCLUSION: As reported in previous studies, in ACP tumors we found a CTNNB1 mutation by NGS analysis. The PIK3CA variants we detected were not known previously in ACP tumors. Finding the PIK3CA mutations in the ACP tumors may help develop targeted therapy for a subset of craniopharyngiomas with PIK3CA activating mutations. Clinical trials are in progress with specific PIK3CA inhibitors in advanced stages of many cancers.

PEAMOtecan, a novel chronotherapeutic polymeric drug for brain cancer.

Glioblastoma multiforme (GBM) is an aggressive and difficult to treat form of brain cancer. In this work, we report on a novel chronotherapeutic polymeric drug, PEAMOtecan, for GBM therapy. PEAMOtecan was synthesized by conjugating camptothecin, a topoisomerase I inhibitor, to our proprietary, 'clickable' and modular polyoxetane polymer platform consisting of acetylene-functionalized 3-ethyl-3-(hydroxymethyl)oxetane (EAMO) repeat units (Patent No.: US 9,421,276) via the linker 3,3'-dithiodipropionic acid (DDPA) with a disulfide bond (SS) extended by short-chain polyethylene glycol (PEG). We show that PEAMOtecan is a highly modular polymer nanoformulation that protects covalently bound CPT until slowly being released over extended periods of time dependent on the cleavage of the disulfide and ester linkages. PEAMOtecan kills glioma cells by mitotic catastrophe with p53 mutant/knockdown cells being more sensitive than matched wild type cells potentially providing cancer-specific targeting. To establish proof-of-principle therapeutic effects, we tested PEAMOtecan as monotherapy for efficacy in a mouse orthotopic glioma model. PEAMOtecan was administered by one-time, convection-enhanced delivery (CED) intra-tumorally to achieve superior distribution and extended drug release over time. In addition, the near-infrared (NIR) dye Cy5.5 was coupled to the polymer providing live-animal imaging capability to track tissue distribution and clearance of the injected polymer over time. We show that PEAMOtecan significantly improves the survival of mice harboring intra-cranial tumors (p = .0074 compared to untreated group). Altogether, these results support further development and testing of our nanoconjugate platform.

STING activation in cancer immunotherapy.

Cancer immunotherapy modulates and leverages the host immune system to treat cancer. The past decade has witnessed historical advancement of cancer immunotherapy. A myriad of approaches have been explored to elicit or augment anticancer innate immunity and/or adaptive immunity. Recently, activation of stimulator of interferon (IFN) genes (STING), an intracellular receptor residing in the endoplasmic reticulum, has shown great potential to enhance antitumor immunity through the induction of a variety of pro-inflammatory cytokines and chemokines, including type I IFNs. A number of natural and synthetic STING agonists have been discovered or developed, and tested in preclinical models and in the clinic for the immunotherapy of diseases such as cancer and infectious diseases. Cyclic dinucleotides (CDNs), such as cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), and cyclic GMP-AMP (cGAMP), are a class of STING agonists that can elicit immune responses. However, natural CDNs are hydrophilic small molecules with negative charges and are susceptible to enzymatic degradation, leading to low bioavailability in target tissues yet unwanted toxicities and narrow therapeutic windows. Drug delivery systems, coupled with nucleic acid chemistry, have been exploited to address these challenges. Here, we will discuss the underlying immunological mechanisms and approaches to STING activation, with a focus on the delivery of STING agonists, for cancer immunotherapy.

TDP1 suppresses mis-joining of radiomimetic DNA double-strand breaks and cooperates with Artemis to promote optimal nonhomologous end joining.

The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3'-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3'-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.

The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase-related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.

Orally Bioavailable and Blood-Brain Barrier-Penetrating ATM Inhibitor (AZ32) Radiosensitizes Intracranial Gliomas in Mice.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro AZ32, with enhanced blood-brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637-47. ©2018 AACR.

DNA Damage Response in Human Stem Cells and Neural Descendants.

Glial cells are crucial for the normal function of neurons and are intricately involved in the pathogenesis of neurodegenerative diseases as well as neurologic malignancies. A deeper understanding of the mechanisms by which glial cells influence the development of such pathologies will undoubtedly lead to new and improved therapeutic approaches. Commercially available human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), both of which can be differentiated into neural progenitors (NPs) and various neural cell lineages, have become widely used as sources for producing normal human central nervous system (CNS) cells. A better understanding of the DNA damage response (DDR) that occurs in these cells after therapeutic ionizing radiation (IR) and chemotherapy is essential for assessing the effects on healthy human brain.Neurodegenerative features associated with conditions such as ataxia telangiectasia and Nijmegen breakage syndrome highlight the importance of DNA double strand break (DSB) repair pathways in maintaining genomic integrity in cells of the CNS. Similarly, the development of brain tumors is also intricately linked to DNA repair. The importance of ATM and the other phosphatidylinositol 3-kinase-related kinase (PIKK) family members, ATR and DNA-PKcs, is not fully defined in either CNS developmental or pathological states. While their roles are relatively well established in the DDR of proliferating cells, our recent work has demonstrated that these processes exhibit spatiotemporal evolution during cell differentiation. This chapter discusses and explores various laboratory techniques for investigating the role of ATM in hESCs and differentiated neural cells.

Inhibition of radiation-induced glioblastoma invasion by genetic and pharmacological targeting of MDA-9/Syntenin.

Glioblastoma multiforme (GBM) is an intractable tumor despite therapeutic advances, principally because of its invasive properties. Radiation is a staple in therapeutic regimens, although cells surviving radiation can become more aggressive and invasive. Subtraction hybridization identified melanoma differentiation-associated gene 9 [MDA-9/Syntenin; syndecan-binding protein (SDCBP)] as a differentially regulated gene associated with aggressive cancer phenotypes in melanoma. MDA-9/Syntenin, a highly conserved double-PDZ domain-containing scaffolding protein, is robustly expressed in human-derived GBM cell lines and patient samples, with expression increasing with tumor grade and correlating with shorter survival times and poorer response to radiotherapy. Knockdown of MDA-9/Syntenin sensitizes GBM cells to radiation, reducing postradiation invasion gains. Radiation induces Src and EGFRvIII signaling, which is abrogated through MDA-9/Syntenin down-regulation. A specific inhibitor of MDA-9/Syntenin activity, PDZ1i (113B7), identified through NMR-guided fragment-based drug design, inhibited MDA-9/Syntenin binding to EGFRvIII, which increased following radiation. Both genetic (shmda-9) and pharmacological (PDZ1i) targeting of MDA-9/Syntenin reduced invasion gains in GBM cells following radiation. Although not affecting normal astrocyte survival when combined with radiation, PDZ1i radiosensitized GBM cells. PDZ1i inhibited crucial GBM signaling involving FAK and mutant EGFR, EGFRvIII, and abrogated gains in secreted proteases, MMP-2 and MMP-9, following radiation. In an in vivo glioma model, PDZ1i resulted in smaller, less invasive tumors and enhanced survival. When combined with radiation, survival gains exceeded radiotherapy alone. MDA-9/Syntenin (SDCBP) provides a direct target for therapy of aggressive cancers such as GBM, and defined small-molecule inhibitors such as PDZ1i hold promise to advance targeted brain cancer therapy.

Click synthesis of a polyamidoamine dendrimer-based camptothecin prodrug.

In the present work we report on the click synthesis of a new camptothecin (CPT) prodrug based on anionic polyamidoamine (PAMAM) dendrimer intended for cancer therapy. We applied 'click' chemistry to improve polymer-drug coupling reaction efficiency. Specifically, CPT was functionalized with a spacer, 1-azido-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (APO), via EDC/DMAP coupling reaction. In parallel, propargylamine (PPA) and methoxypoly(ethylene glycol) amine were conjugated to PAMAM dendrimer G4.5 in sequence using an effective coupling agent 4-(4,6-dimethoxy-(1,3,5)triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM). CPT-APO was then coupled to PEGylated PAMAM dendrimer G4.5-PPA via a click reaction using copper bromide/2,2'-bipyridine/ dimethyl sulfoxide (catalyst/ligand/solvent). Human glioma cells were exposed to the CPT-conjugate to determine toxicity and cell cycle effects using WST-1 assay and flow cytometry. The CPT-conjugate displayed a dose-dependent toxicity with an IC50 of 5 µM, a 185-fold increase relative to free CPT, presumably as a result of slow release. As expected, conjugated CPT resulted in G2/M arrest and cell death while the dendrimer itself had little to no toxicity. Altogether, highly efficient click chemistry allows for the synthesis of multifunctional dendrimers for sustained drug delivery.

Mutation of the BRCA1 SQ-cluster results in aberrant mitosis, reduced homologous recombination, and a compensatory increase in non-homologous end joining.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

BRCA1 functions as a novel transcriptional cofactor in HIV-1 infection.

BACKGROUND: Viruses have naturally evolved elegant strategies to manipulate the host's cellular machinery, including ways to hijack cellular DNA repair proteins to aid in their own replication. Retroviruses induce DNA damage through integration of their genome into host DNA. DNA damage signaling proteins including ATR, ATM and BRCA1 contribute to multiple steps in the HIV-1 life cycle, including integration and Vpr-induced G2/M arrest. However, there have been no studies to date regarding the role of BRCA1 in HIV-1 transcription. METHODS: Here we performed various transcriptional analyses to assess the role of BRCA1 in HIV-1 transcription by overexpression, selective depletion, and treatment with small molecule inhibitors. We examined association of Tat and BRCA1 through in vitro binding assays, as well as BRCA1-LTR association by chromatin immunoprecipitation. RESULTS: BRCA1 was found to be important for viral transcription as cells that lack BRCA1 displayed severely reduced HIV-1 Tat-dependent transcription, and gain or loss-of-function studies resulted in enhanced or decreased transcription. Moreover, Tat was detected in complex with BRCA1 aa504-802. Small molecule inhibition of BRCA1 phosphorylation effector kinases, ATR and ATM, decreased Tat-dependent transcription, whereas a Chk2 inhibitor showed no effect. Furthermore, BRCA1 was found at the viral promoter and treatment with curcumin and ATM inhibitors decreased BRCA1 LTR occupancy. Importantly, these findings were validated in a highly relevant model of HIV infection and are indicative of BRCA1 phosphorylation affecting Tat-dependent transcription. CONCLUSIONS: BRCA1 presence at the HIV-1 promoter highlights a novel function of the multifaceted protein in HIV-1 infection. The BRCA1 pathway or enzymes that phosphorylate BRCA1 could potentially be used as complementary host-based treatment for combined antiretroviral therapy, as there are multiple potent ATM inhibitors in development as chemotherapeutics.

Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation.