Multimodal Therapy Approaches for NUT Carcinoma by Dual Combination of Oncolytic Virus Talimogene Laherparepvec with Small Molecule Inhibitors.

NUT (nuclear-protein-in-testis) carcinoma (NC) is a highly aggressive tumor disease. Given that current treatment regimens offer a median survival of six months only, it is likely that this type of tumor requires an extended multimodal treatment approach to improve prognosis. In an earlier case report, we could show that an oncolytic herpes simplex virus (T-VEC) is functional in NC patients. To identify further combination partners for T-VEC, we have investigated the anti-tumoral effects of T-VEC and five different small molecule inhibitors (SMIs) alone and in combination in human NC cell lines. Dual combinations were found to result in higher rates of tumor cell reductions when compared to the respective monotherapy as demonstrated by viability assays and real-time tumor cell growth monitoring. Interestingly, we found that the combination of T-VEC with SMIs resulted in both stronger and earlier reductions in the expression of c-Myc, a main driver of NC cell proliferation, when compared to T-VEC monotherapy. These results indicate the great potential of combinatorial therapies using oncolytic viruses and SMIs to control the highly aggressive behavior of NC cancers and probably will pave the way for innovative multimodal clinical studies in the near future.

Synthetic lethality: targeting SMARCA2 ATPase in SMARCA4-deficient tumors - a review of patent literature from 2019-30 June 2023.

INTRODUCTION: The multi-subunit SWI/SNF chromatin remodeling complex is a key epigenetic regulator for many cellular processes, and several subunits are found to be mutated in human cancers. The inactivating mutations of SMARCA4, the ATPase subunit of the complex, result in cellular dependency on the paralog SMARCA2 for survival. This observed synthetic lethal relationship posits targeting SMARCA2 in SMARCA4-deficient settings as an attractive therapeutic target in oncology. AREAS COVERED: This review covers patent literature disclosed during the 2019-30 June 2023 period which claim ATPase inhibitors and PROTAC degraders that bind to the ATPase domain of SMARCA2 and/or SMARCA4. A total of 16 documents from 6 applicants are presented. EXPERT OPINION: The demonstration of cellular dependence on SMARCA2 ATPase activity in SMARCA4-deficient settings has prompted substantial research toward SMARCA2-targeting therapies. Although selectively targeting the ATPase domain of SMARCA2 is viewed as challenging, several ATPase inhibitor scaffolds have been disclosed within the last five years. Most early compounds are weakly selective, but these efforts have culminated in the first dual SMARCA2/SMARCA4 ATPase inhibitor to enter clinical trials. Data from the ongoing clinical trials, as well as continued advancement of SMARCA2-selective ATPase inhibitors, are anticipated to significantly impact the field of therapies, targeting SMARCA4-deficient tumors.

Binding Mechanism of Inhibitors to BRD4 and BRD9 Decoded by Multiple Independent Molecular Dynamics Simulations and Deep Learning.

Bromodomain 4 and 9 (BRD4 and BRD9) have been regarded as important targets of drug designs in regard to the treatment of multiple diseases. In our current study, molecular dynamics (MD) simulations, deep learning (DL) and binding free energy calculations are integrated to probe the binding modes of three inhibitors (H1B, JQ1 and TVU) to BRD4 and BRD9. The MD trajectory-based DL successfully identify significant functional function domains, such as BC-loop and ZA-loop. The information from the post-processing analysis of MD simulations indicates that inhibitor binding highly influences the structural flexibility and dynamic behavior of BRD4 and BRD9. The results of the MM-GBSA calculations not only suggest that the binding ability of H1B, JQ1 and TVU to BRD9 are stronger than to BRD4, but they also verify that van der Walls interactions are the primary forces responsible for inhibitor binding. The hot spots of BRD4 and BRD9 revealed by residue-based free energy estimation provide target sites of drug design in regard to BRD4 and BRD9. This work is anticipated to provide useful theoretical aids for the development of selective inhibitors over BRD family members.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

Inhibition of Phlpp1 preserves the mechanical integrity of articular cartilage in a murine model of post-traumatic osteoarthritis.

OBJECTIVE: Phlpp1 inhibition is a potential therapeutic strategy for cartilage regeneration and prevention of post-traumatic osteoarthritis (PTOA). To understand how Phlpp1 loss affects cartilage structure, cartilage elastic modulus was measured with atomic force microscopy (AFM) in male and female mice after injury. METHODS: Osteoarthritis was induced in male and female Wildtype (WT) and Phlpp1-/- mice by destabilization of the medial meniscus (DMM). At various timepoints post-injury, activity was measured, and knee joints examined with AFM and histology. In another cohort of WT mice, the PHLPP inhibitor NSC117079 was intra-articularly injected 4 weeks after injury. RESULTS: Male WT mice showed decreased activity and histological signs of cartilage damage at 12 but not 6-weeks post-DMM. Female mice showed a less severe response to DMM by comparison, with no histological changes seen at any time point. In both sexes the elastic modulus of medial condylar cartilage was decreased in WT mice but not Phlpp1-/- mice after DMM as measured by AFM. By 6-weeks, cartilage modulus had decreased from 2 MPa to 1 MPa in WT mice. Phlpp1-/- mice showed no change in modulus at 6-weeks and only a 25% decrease at 12-weeks. The PHLPP inhibitor NSC117079 protected cartilage structure and prevented signs of OA 6-weeks post-injury. CONCLUSIONS: AFM is a sensitive method for detecting early changes in articular cartilage post-injury. Phlpp1 suppression, either through genetic deletion or pharmacological inhibition, protects cartilage degradation in a model of PTOA, validating Phlpp1 as a therapeutic target for PTOA.

BRG1/BRM inhibitor targets AML stem cells and exerts superior preclinical efficacy combined with BET or menin inhibitor.

ABSTRACT: BRG1 (SMARCA4) and BRM (SMARCA2) are the mutually exclusive core ATPases of the chromatin remodeling BAF (BRG1/BRM-associated factor) complexes. They enable transcription factors/cofactors to access enhancers/promoter and modulate gene expressions responsible for cell growth and differentiation of acute myeloid leukemia (AML) stem/progenitor cells. In AML with MLL1 rearrangement (MLL1r) or mutant NPM1 (mtNPM1), although menin inhibitor (MI) treatment induces clinical remissions, most patients either fail to respond or relapse, some harboring menin mutations. FHD-286 is an orally bioavailable, selective inhibitor of BRG1/BRM under clinical development in AML. Present studies show that FHD-286 induces differentiation and lethality in AML cells with MLL1r or mtNPM1, concomitantly causing perturbed chromatin accessibility and repression of c-Myc, PU.1, and CDK4/6. Cotreatment with FHD-286 and decitabine, BET inhibitor (BETi) or MI, or venetoclax synergistically induced in vitro lethality in AML cells with MLL1r or mtNPM1. In models of xenografts derived from patients with AML with MLL1r or mtNPM1, FHD-286 treatment reduced AML burden, improved survival, and attenuated AML-initiating potential of stem-progenitor cells. Compared with each drug, cotreatment with FHD-286 and BETi, MI, decitabine, or venetoclax significantly reduced AML burden and improved survival, without inducing significant toxicity. These findings highlight the FHD-286-based combinations as a promising therapy for AML with MLL1r or mtNPM1.

Design and development of a novel series of oral bivalent BET inhibitors with potent anticancer activities.

Bromodomain and extraterminal domain (BET) subfamily members are intriguing targets for cancer treatment. Most of the reported BET inhibitors were monovalent inhibitors. Recently, some bivalent inhibitors were disclosed, which bound to two bromodomains simultaneously. They had good activities, however, most of them also showed unsatisfactory pharmacokinetic properties, which were caused by long chain linkers. Based on our previous work on monovalent BRD4 inhibitors, we designed and synthesized a series of novel bivalent inhibitors with short and hydrophilic linkers. These compounds exhibited better activities than the corresponding monovalent inhibitors and good pharmacokinetic properties. Compound 21 showed excellent in vitro activities. And it also demonstrated potent in vivo antitumor efficacy under oral administration and was well tolerated in in vivo tests.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

DTL Is a Prognostic Biomarker and Promotes Bladder Cancer Progression through Regulating the AKT/mTOR axis.

BACKGROUND: Denticleless E3 ubiquitin protein ligase homolog (DTL) has been reported to be an important regulator for tumorigenesis and progression. Nonetheless, the biological functions and molecular mechanisms of DTL in BCa remain elusive. METHODS: We implemented integrative bioinformatics analysis to explore the diagnostic and prognostic values of DTL based on The Cancer Genome Atlas (TCGA), ArrayExpress, and Gene Expression Omnibus (GEO) databases. Then, we utilized qRT-PCR and immunohistochemistry to verify the clinical significance of DTL expression according to clinical specimens and tissue microarray (TMA). Moreover, the biological functions and underlying mechanisms of DTL in BCa were investigated through in vitro and in vivo experiments. RESULTS: Integrative bioinformatics analysis revealed that DTL was a key gene associated with BCa progression, and increased DTL expression was correlated with malignant biological behavior and poor prognosis. Experiments on clinical specimens and tissue microarray (TMA) further confirmed our findings. Bioinformatics analysis demonstrated that DTL could be associated with cell cycle- and DNA replication-associated pathways in BCa. The suppression of DTL inhibited BCa cell proliferation, migration, and invasion in vivo and in vitro. Mechanistically, DTL may promote BCa progression through the AKT/mTOR pathway. CONCLUSIONS: Increased DTL expression was correlated with malignant biological behavior and poor prognosis of BCa patients, and it may promote BCa progression through the AKT/mTOR pathway. Our research provided a potential predictor and therapeutic target for BCa.

Hypoxia Tumor Microenvironment Activates GLI2 through HIF-1α and TGF-ß2 to Promote Chemotherapy Resistance of Colorectal Cancer.

BACKGROUND: A majority of relapse cases have been reported in colorectal cancer patients due to cancer stem cell progenitors. The factors responsible for chemoresistance have yet to be discovered and investigated as CSCs have reported escaping from chemotherapy's killing action. OBJECTIVE: In this study, we have investigated the effects of HIF-1α and TGF-ß2 in hypoxia conditions on the expression of GLI2, which is a potential factor for causing chemoresistance. Material and Methods. Colorectal samples of treated patients were collected from the Hospital Biological Sample Library. Culture of patient-derived TSs and fibroblasts was performed. The collected patient samples and cells were used for immunohistochemistry, quantitative PCR, and western blotting studies which were performed. RESULTS: It was reported that HIF-1α (hypoxia-inducible factor) and TGF-ß2 secreted from cancer-associated fibroblasts (CAFs) synergistically work to express GLI2 in cancer stem cells. Hence, it increased the stemness as well as resistance to chemotherapy. CONCLUSION: The HIF-1α/TGF-ß2-mediated GLI2 signaling was responsible for causing chemoresistance in the hypoxia environment. High expressions of HIF1α/TGF-ß2/GLI2 cause the relapsing of colorectal cancer, thus making this a potential biomarker for identifying the relapse and resistance in patients. The study uncovers the mechanism involved in sternness and chemotherapy resistance which will help in targeted treatment.

BRD4 Inhibition Attenuates Inflammatory Pain by Ameliorating NLRP3 Inflammasome-Induced Pyroptosis.

Chronic pain, such as persistent inflammatory pain, remains a public health problem that has no effective treatment at present. Bromodomain-containing protein 4 (BRD4) inhibition, induced by JQ1 injection or BRD4 knockdown, has been used to attenuate inflammatory pain; However, it remains elusive whether BRD4 aggravates inflammatory pain by regulating inflammasome. Western blot and immunofluorescence staining showed that BRD4 expression increased after administration of complete Freund's adjuvant (CFA) and reached its peak on day 3. Immunofluorescence staining showed that BRD4 was mainly colocalized with NeuN-positive neurons in the spinal cord, which was accompanied by upregulation of inflammasome component proteins, such as NLRP3, gasdermin D, and caspase-1. JQ1 was intrathecally injected into mice 1 h before CFA administration, and the mechanical and thermal hyperalgesia levels were measured on days 1, 3, and 7 after CFA administration. CFA-induced inflammatory pain, paw inflammation, and swelling were attenuated by pre-treatment with JQ1. To our knowledge, this study was the first to prove that NLRP3 inflammasome-induced neuronal pyroptosis participates in inflammatory pain. BRD4 inhibition decreased the expression of pyroptosis-related proteins by inhibiting the activation of NF-κB signaling pathway, both in vivo and in vitro. Taken together, BRD4 inhibition exerted analgesic and anti-inflammatory effects against inflammatory pain by inhibiting NF-κB and inflammasome activation, which protected neural cells from pyroptosis.

lncRNA TMPO antisense RNA 1 promotes the malignancy of cholangiocarcinoma cells by regulating let-7g-5p/ high-mobility group A1 axis.

Cholangiocarcinoma (CHOL) is often diagnosed at an advanced stage; therefore, exploring its key regulatory factors is important for earlier diagnosis and treatment. This study aimed to identify the mechanisms of long non-coding RNA (lncRNA) TMPO Antisense RNA 1 (TMPO-AS1), microRNA let-7 g-5p, and high-mobility group A1 (HMGA1) proteins in CHOL. Our results, through quantitative real-time PCR and Western blot detection, showed that TMPO-AS1 and HMGA1 were overexpressed while let-7 g-5p was underexpressed in CHOL. Cell function experiments in CHOL cells revealed that TMPO-AS1 knockdown inhibited cell proliferation, colony formation, and cell migration, but induced apoptosis. TMPO-AS1 knockdown also suppressed tumor growth in vivo. Together with luciferase assay and Western blotting, we found that TMPO-AS1 could sponge let-7 g-5p to promote HMGA1 expression. Moreover, HMGA1 overexpression attenuated the effect of TMPO-AS1 downregulation in CHOL cells. Overall, our findings identified the oncogenic effect of TMPO-AS1 on CHOL cells, which may put forward a novel methodology for CHOL diagnosis and therapy.

Discovery, X-ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site.

Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.

The transcript ENST00000444125 of lncRNA LINC01503 promotes cancer stem cell properties of glioblastoma cells via reducing FBXW1 mediated GLI2 degradation.

LINC010503 is a novel oncogenic lncRNA in multiple cancers. In this study, we further explored the expression of LINC010503 transcripts and their regulations on the glioblastoma (GBM) stem cell (GSC) properties. LINC01503 transcription patterns in GBM and normal brain tissues were compared using RNA-seq data from Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA)-GBM. GBM cell lines (U251 and U87) were used as in vitro cell models for cellular and molecular studies. The results showed that ENST00000444125 was the dominant transcript of LINC01503 in both normal and tumor tissues. Its expression was significantly elevated in the tumor group and associated with poor survival outcomes. LINC01503 had both cytoplasmic and nuclear distribution. It positively modulated the expression of multiple GSC markers, including CD133, SOX2, NESTIN, ALDH1A1, and MSI1, and tumorsphere formation in U251 and U87 cells. RNA pull-down and RIP-qPCR assay confirmed an interaction between ENST00000444125 and GLI2. ENST00000444125 positively regulated the half-life of the GLI2 protein in GBM cells. ENST00000444125 overexpression reduced GLI2 ubiquitination and partially attenuated FBXW1 overexpression induced GLI2 ubiquitination. ENST00000444125 overexpression could activate Wnt/ß-catenin signaling in GBM cells. However, these activating effects were remarkedly hampered when GLI2 was knocked down. In conclusion, this study revealed that LINC01503 might have isoform-specific dysregulation in GBM. Among the two major transcripts expressed in GBM cells, ENST00000444125 might be the major functional transcript. Its upregulation might enhance the GSC properties of GBM cells via reducing FBXW1-mediated proteasomal degradation of GLI2.

BET Inhibition Enhances TNF-Mediated Antitumor Immunity.

Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.

Structure-activity relationship studies of allosteric inhibitors of EYA2 tyrosine phosphatase.

Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg2+ , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors. A series of analogues were synthesized to improve potency of the inhibitors and to elucidate structure-activity relationships. Two co-crystal structures confirm the binding modes of this class of inhibitors. Our medicinal chemical, structural, biochemical, and biophysical studies provide insight into the molecular interactions of EYA2 with these allosteric inhibitors. The compounds derived from this study are useful for exploring the function of the Tyr phosphatase activity of EYA2 in normal and cancerous cells and serve as reference compounds for screening or developing allosteric phosphatase inhibitors. Finally, the co-crystal structures reported in this study will aid in structure-based drug discovery against EYA2.

Simultaneous administration of bromodomain and histone deacetylase I inhibitors alleviates cognition deficit in Alzheimer's model of rats.

BACKGROUND: Histone deacetylases (HDACs) target various genes responsible for cognitive functions. However, chromatin readers, particularly bromodomain-containing protein 4 (BRD4), are capable to change the final products of genes. The objective of this study was to evaluate the simultaneous effects of inhibition of HDACs and BRD4 on spatial and aversive memories impaired by amyloid ß (Aß) in a rat model of Alzheimer's disease (AD) considering CREB and TNF-α signaling. METHODS: Forty male Wistar rats aged 3 months were randomly divided into five groups: saline +DMSO, Aß+saline+DMSO, Aß+JQ1, Aß+MS-275, Aß+JQ1+MS-275, and received the related treatments. MS-275, is the second generation of HDACs inhibitor, and JQ1 is a potent inhibitor of the BET family of bromodomain proteins in mammals. After the treatments, cognitive function was assessed by Morris water maze (MWM) and passive avoidance learning (PAL). The hippocampal level of mRNA for CREB and TNF-α, and also phosphorylated CREB were measured using real-time PCR and western blotting respectively. RESULTS: Administration of JQ1 and MS-275, either separately or simultaneously, improved acquisition and retrieval of spatial and aversive memories as it was evident by decreased escape latency and increased time spent in the target quadrant (TTS) in Morris water maze (MWM), together with increase in step-through latency, but reduced time spent in the dark zone time in passive avoidance learning (PAL) compared with Aß+saline+DMSO. Furthermore, there was a significant rise in the hippocampal level of CREB mRNA and phosphorylated CREB, but a reduction in TNF-α expression in comparison with Aß + Saline. CONCLUSION: Simultaneous administration of JQ1 and MS-275 improves acquisition and retrieval of both spatial and aversive memories partly via CREB and TNF-α signaling with no superiority to monotherapy.

Dihydropyridine Lactam Analogs Targeting BET Bromodomains.

Inhibitors of Bromodomain and Extra Terminal (BET) proteins are investigated for various therapeutic indications, but selectivity for BRD2, BRD3, BRD4, BRDT and their respective tandem bromodomains BD1 and BD2 remains suboptimal. Here we report selectivity-focused structural modifications of previously reported dihydropyridine lactam 6 by changing linker length and linker type of the lactam side chain in efforts to engage the unique arginine 54 (R54) residue in BRDT-BD1 to achieve BRDT-selective affinity. We found that the analogs were highly selective for BET bromodomains, and generally more selective for the first (BD1) and second (BD2) bromodomains of BRD4 rather than for those of BRDT. Based on AlphaScreen and BromoScan results and on crystallographic data for analog 10 j, we concluded that the lack of selectivity for BRDT is most likely due to the high flexibility of the protein and the unfavorable trajectory of the lactam side chain that do not allow interaction with R54. A 15-fold preference for BD2 over BD1 in BRDT was observed for analogs 10 h and 10 m, which was supported by protein-based 19 F NMR experiments with a BRDT tandem bromodomain protein construct.

Novel structural-related analogs of PFI-3 (SRAPs) that target the BRG1 catalytic subunit of the SWI/SNF complex increase the activity of temozolomide in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and treatment-refractory malignant adult brain cancer. After standard of care therapy, the overall median survival for GBM is only ∼6 months with a 5-year survival <10%. Although some patients initially respond to the DNA alkylating agent temozolomide (TMZ), unfortunately most patients become resistant to therapy and brain tumors eventually recur. We previously found that knockout of BRG1 or treatment with PFI-3, a small molecule inhibitor of the BRG1 bromodomain, enhances sensitivity of GBM cells to temozolomide in vitro and in vivo GBM animal models. Those results demonstrated that the BRG1 catalytic subunit of the SWI/SNF chromatin remodeling complex appears to play a critical role in regulating TMZ-sensitivity. In the present study we designed and synthesized Structurally Related Analogs of PFI-3 (SRAPs) and tested their bioactivity in vitro. Among of the SRAPs, 9f and 11d show better efficacy than PFI-3 in sensitizing GBM cells to the antiproliferative and cell death inducing effects of temozolomide in vitro, as well as enhancing the inhibitor effect of temozolomide on the growth of subcutaneous GBM tumors.

MED12 and BRD4 cooperate to sustain cancer growth upon loss of mediator kinase.

Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.