SSRP1 Worsens Malignant Melanoma Progression by Activating MAPKs Pathway.

OBJECTIVE: Structure-specific recognition protein 1 (SSRP1) is highly expressed in a variety of tumors and promotes cell proliferation and migration. Malignant melanoma is a highly malignant skin malignancy with low morbidity and high mortality. The role of SSRP1 in malignant melanoma is still unclear. Thus, this study is intended to investigate the role of SSRP1 in malignant melanoma and reveal the related mechanisms. METHODS: Western blots and immunohistochemistry assays were used to determine the expression of SSRP1 in benign nevi tissues and malignant melanoma tissues. The si-SSRP1 was used to knockdown the expression level of SSRP1 in A375 cells. Cell proliferation was assessed by MTT assay. Wound healing and Transwell assay were performed for detected cell migratory and invasive activities, respectively. Besides, the expression levels of epithelial-mesenchymal transition (EMT) markers and MAPKs signaling pathway were measured by western blot. RESULTS: The results showed that SSRP1 was highly expressed in malignant melanoma tissues and cells, and its expression in metastatic melanoma tissues was significantly higher than that in primary melanoma. Besides, high expression level of SSRP1 was accompanied with poor prognosis in malignant melanoma patients. SSRP1 knockdown inhibited the melanoma cell proliferation, migration, and invasion. Besides, SSRP1 knockdown inhibited the process of EMT by upregulating E-cadherin, and downregulating N-cadherin and vimentin. Further studies revealed that SSRP1 silencing affected MAPK signaling pathway and reduced its phosphorylation activity in melanoma cells. CONCLUSIONS: These results suggested that SSRP1 may promote the proliferation, migration, and invasion of melanoma cells through MAPK signaling pathway. SSRP1 is closely related to the malignancy of melanoma and may be a potential target for its clinical treatment.

Histone chaperone FACT complex inhibitor CBL0137 interferes with DNA damage repair and enhances sensitivity of medulloblastoma to chemotherapy and radiation.

Medulloblastoma (MB) is a malignant pediatric brain tumor with a poor prognosis. Post-surgical radiation and cisplatin-based chemotherapy have been a mainstay of treatment, which often leads to substantial neurocognitive impairments and morbidity, highlighting the need for a novel therapeutic target to enhance the sensitivity of MB tumors to cytotoxic therapies. We performed a comprehensive study using a cohort of 71 MB patients' samples and pediatric MB cell lines and found that MB tumors have elevated levels of nucleosome remodeling FACT (FAcilitates Chromatin Transcription) complex and DNA repair enzyme AP-endonuclease1 (APE1). FACT interacts with APE1 and facilitates recruitment and acetylation of APE1 to promote repair of radiation and cisplatin-induced DNA damage. Further, levels of FACT and acetylated APE1 both are correlate strongly with MB patients' survival. Targeting FACT complex with CBL0137 inhibits DNA repair and alters expression of a subset of genes, and significantly improves the potency of cisplatin and radiation in vitro and in MB xenograft. Notably, combination of CBL0137 and cisplatin significantly suppressed MB tumor growth in an intracranial orthotopic xenograft model. We conclude that FACT complex promotes chemo-radiation resistance in MB, and FACT inhibitor CBL0137 can be used as a chemo-radiation sensitizer to augment treatment efficacy and reduce therapy-related toxicity in high-risk pediatric patients.

Discovery of Selective Small-Molecule Inhibitors for the ENL YEATS Domain.

Eleven-nineteen leukemia (ENL) protein is a histone acetylation reader essential for disease maintenance in acute leukemias, in particular, the mixed-lineage leukemia (MLL)-rearranged leukemia. In this study, we carried out high-throughput screening of a small-molecule library to identify inhibitors for the ENL YEATS domain. Structure-activity relationship studies of the hits and structure-based inhibitor design led to two compounds, 11 and 24, with IC50 values below 100 nM in inhibiting the ENL-acetyl-H3 interaction. Both compounds, and their precursor compound 7, displayed strong selectivity toward the ENL YEATS domain over all other human YEATS domains. Moreover, 7 exhibited on-target inhibition of ENL in cultured cells and a synergistic effect with the bromodomain and extraterminal domain inhibitor JQ1 in killing leukemia cells. Together, we have developed selective chemical probes for the ENL YEATS domain, providing the basis for further medicinal chemistry-based optimization to advance both basic and translational research of ENL.

Inhibition of the FACT Complex Targets Aberrant Hedgehog Signaling and Overcomes Resistance to Smoothened Antagonists.

Hedgehog signaling is aberrantly activated in hematologic malignancies and solid tumors, and targeting it is a promising therapeutic strategy against these cancers. Resistance to clinically available hedgehog-targeted Smoothened inhibitor (SMOi) drugs has become a critical issue in hedgehog-driven cancer treatment. Our previous studies identified inhibition of BET and CDK7 as two epigenetic/transcriptional-targeted therapeutic strategies for overcoming SMOi resistance, providing a promising direction for anti-hedgehog drug development. To uncover additional strategies for inhibiting aberrant hedgehog activity, here we performed CRISPR-Cas9 screening with an single-guide RNA library targeting epigenetic and transcriptional modulators in hedgehog-driven medulloblastoma cells, combined with tumor dataset analyses. Structure specific recognition protein 1 (SSRP1), a subunit of facilitates chromatin transcription (FACT) complex, was identified as a hedgehog-induced essential oncogene and therapeutic target in hedgehog-driven cancer. The FACT inhibitor CBL0137, which has entered clinical trials for cancer, effectively suppressed in vitro and in vivo growth of multiple SMOi-responsive and SMOi-resistant hedgehog-driven cancer models. Mechanistically, CBL0137 exerted anti-hedgehog activity by targeting transcription of GLI1 and GLI2, which are core transcription factors of the hedgehog pathway. SSRP1 bound the promoter regions of GLI1 and GLI2, while CBL0137 treatment substantially disrupted these interactions. Moreover, CBL0137 synergized with BET or CDK7 inhibitors to antagonize aberrant hedgehog pathway and growth of hedgehog-driven cancer models. Taken together, these results identify FACT inhibition as a promising epigenetic/transcriptional-targeted therapeutic strategy for treating hedgehog-driven cancers and overcoming SMOi resistance. SIGNIFICANCE: This study identifies FACT inhibition as an anti-hedgehog therapeutic strategy for overcoming resistance to Smoothened inhibitors and provides preclinical support for initiating clinical trials of FACT-targeted drug CBL0137 against hedgehog-driven cancers.

Effective inhibition of MYC-amplified group 3 medulloblastoma by FACT-targeted curaxin drug CBL0137.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor that can be categorized into four major molecular subgroups. Group 3 MB with MYC amplification (MYCamp-G3-MB) has been shown to be highly aggressive and exhibited worst prognosis, indicating the need for novel effective therapy most urgently. A few epigenetic targeted therapeutic strategies have recently been proven to effectively treat preclinical models of MYCamp-G3-MB, including BET inhibition, HDAC inhibition and SETD8 inhibition, unveiling a promising direction for further investigation. In this study, we carried out systemic bioinformatic analyses of public-available MB datasets as well as functional genomic screening datasets of primary MYCamp-G3-MB lines to search for other potential therapeutic targets within epigenetic modulators. We identified SSRP1, a subunit of histone-chaperone FACT complex, to be the top drug target candidate as it is highly cancer-dependent in whole-genome CRISPR-Cas9 screening across multiple MYCamp-G3-MB lines; significantly upregulated in MYCamp-G3-MB compared to normal cerebellum and most of the rest MB subtypes; its higher expression is correlated with worse prognosis; and it has a blood-brain-barrier penetrable targeted drug that has entered early phase human clinical trials already. Then we utilized RNA-interference approach to verify the cancer-dependency of SSRP1 in multiple MYCamp-G3-MB lines and further confirmed the therapeutic efficacy of FACT-targeted curaxin drug CBL0137 on treating preclinical models of MYCamp-G3-MB in vitro and in vivo, including an orthotopic intracranial xenograft model. Mechanistically, transcriptome analyses showed CBL0137 preferentially suppressed cell-cycle and DNA-repair related biological processes. Moreover, it selectively disrupted transcription of MYC and NEUROD1, two critical oncogenic transcription factors of MYCamp-G3-MB, via depleting FACT complex from their promoter regions. In summary, our study demonstrates FACT-targeted CBL0137 works effectively on treating MYCamp-G3-MB, presenting another promising epigenetic-targeted therapeutic strategy against the most devastating form of MB.

Inhibition of the Super Elongation Complex Suppresses Herpes Simplex Virus Immediate Early Gene Expression, Lytic Infection, and Reactivation from Latency.

Induction of herpes simplex virus (HSV) immediate early (IE) gene transcription promotes the initiation of lytic infection and reactivation from latency in sensory neurons. IE genes are transcribed by the cellular RNA polymerase II (RNAPII) and regulated by multiple transcription factors and coactivators. The HCF-1 cellular coactivator plays a central role in driving IE expression at multiple stages through interactions with transcription factors, chromatin modulation complexes, and transcription elongation components, including the active super elongation complex/P-TEFb (SEC-P-TEFb). Here, we demonstrate that the SEC occupies the promoters of HSV IE genes during the initiation of lytic infection and during reactivation from latency. Specific inhibitors of the SEC suppress viral IE expression and block the spread of HSV infection. Significantly, these inhibitors also block the initiation of viral reactivation from latency in sensory ganglia. The potent suppression of IE gene expression by SEC inhibitors indicates that transcriptional elongation represents a determining rate-limiting stage in HSV IE gene transcription and that the SEC plays a critical role in driving productive elongation during both phases of the viral life cycle. Most importantly, this supports the model that signal-mediated induction of SEC-P-TEFb levels can promote reactivation of a population of poised latent genomes.IMPORTANCE HSV infections can cause pathologies ranging from recurrent lesions to significant ocular disease. Initiation of lytic infection and reactivation from latency in sensory neurons are dependent on the induced expression of the viral immediate early genes. Transcription of these genes is controlled at multiple levels, including modulation of the chromatin state of the viral genome and appropriate recruitment of transcription factors and coactivators. Following initiation of transcription, IE genes are subject to a key regulatory stage in which transcriptional elongation rates are controlled by the activity of the super elongation complex. Inhibition of the SEC blocks both lytic infection and reactivation from latency in sensory neurons. In addition to providing insights into the mechanisms controlling viral infection and reactivation, inhibitors of critical components such as the SEC may represent novel antivirals.

Cell-Based Ligand Discovery for the ENL YEATS Domain.

ENL is a transcriptional coactivator that recruits elongation machinery to active cis-regulatory elements upon binding of its YEATS domain-a chromatin reader module-to acylated lysine side chains. Discovery chemistry for the ENL YEATS domain is highly motivated by its significance in acute leukemia pathophysiology, but cell-based assays able to support large-scale screening or hit validation efforts do not presently exist. Here, we report on the discovery of a target engagement assay that allows for high-throughput ligand discovery in living cells. This assay is based on the cellular thermal shift assay (CETSA) but does not require exposing cells to elevated temperatures, as small-molecule ligands are able to stabilize the ENL YEATS domain at 37 °C. By eliminating temperature shifts, we developed a simplified target engagement assay that requires just two steps: drug treatment and luminescence detection. To demonstrate its value for higher throughput applications, we miniaturized the assay to a 1536-well format and screened 37 120 small molecules, ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain inhibitor.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Targeting Histone Chaperone FACT Complex Overcomes 5-Fluorouracil Resistance in Colon Cancer.

Fluorouracil (5-FU) remains a first-line chemotherapeutic agent for colorectal cancer. However, a subset of colorectal cancer patients who have defective mismatch-repair (dMMR) pathway show resistance to 5-FU. Here, we demonstrate that the efficacy of 5-FU in dMMR colorectal cancer cells is largely dependent on the DNA base excision repair (BER) pathway. Downregulation of APE1, a key enzyme in the BER pathway, decreases IC50 of 5-FU in dMMR colorectal cancer cells by 10-fold. Furthermore, we discover that the facilitates chromatin transcription (FACT) complex facilitates 5-FU repair in DNA via promoting the recruitment and acetylation of APE1 (AcAPE1) to damage sites in chromatin. Downregulation of FACT affects 5-FU damage repair in DNA and sensitizes dMMR colorectal cancer cells to 5-FU. Targeting the FACT complex with curaxins, a class of small molecules, significantly improves the 5-FU efficacy in dMMR colorectal cancer in vitro (∼50-fold decrease in IC50) and in vivo xenograft models. We show that primary tumor tissues of colorectal cancer patients have higher FACT and AcAPE1 levels compared with adjacent nontumor tissues. Additionally, there is a strong clinical correlation of FACT and AcAPE1 levels with colorectal cancer patients' response to chemotherapy. Together, our study demonstrates that targeting FACT with curaxins is a promising strategy to overcome 5-FU resistance in dMMR colorectal cancer patients.

Targeting Spt5-Pol II by Small-Molecule Inhibitors Uncouples Distinct Activities and Reveals Additional Regulatory Roles.

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.

Ell3 functions as a critical decision maker at the crossroad between stem cell senescence and apoptosis.

BACKGROUND: Ell3 is a RNA polymerase II elongation factor that has various cell type-dependent functions, such as regulating the differentiation efficiency of embryonic stem cells and sensitizing cancer cells to anticancer drugs. However, there has been little research on the role of Ell3 on the regulation of senescence and apoptosis of stem cells. METHODS: We analyzed the senescence of Ell3-suppressed stem cells by mitochondrial activity, ß-gal (+) cells, and lineage differentiation efficiency. The apoptosis of Ell3-overexpressing stem cells was analyzed by Annexin V staining, Immunoblot, and Live&dead assay. In addition, chromatin immunoprecipitation and luciferase assays were used to demonstrate p53 functions as a direct transcriptional activator of Ell3. RESULTS: Suppression of Ell3 expression induced senescence in stem cells by increasing Bcl-2 expression. Unlike the effect of Ell3 suppression, the ectopic expression of Ell3 induces apoptosis of stem cells and induces apoptosis of adjacent cells. In addition, p53 functions as a direct transcriptional activator of Ell3 during the stem cell apoptosis. CONCLUSIONS: We suggest that the function of Ell3 is associated with the p53-Bcl2 axis in both senescent and apoptotic ADSCs.

The histone chaperone complex FACT promotes proliferative switch of G0 cancer cells.

Cancer cell repopulation through cell cycle re-entry by quiescent (G0 ) cell is thought to be an important mechanism behind treatment failure and cancer recurrence. Facilitates Chromatin Transcription (FACT) is involved in DNA repair, replication and transcription by eviction of histones or loosening their contact with DNA. While FACT expression is known to be high in a range of cancers, the biological significance of the aberrant increase is not clear. We found that in prostate and lung cancer cells FACT mRNA and protein levels were low at G0 compared to the proliferating state but replenished upon cell cycle re-entry. Silencing of FACT with Dox-inducible shRNA hindered cell cycle re-entry by G0 cancer cells, which could be rescued by ectopic expression of FACT. An increase in SKP2, c-MYC and PIRH2 and a decrease in p27 protein levels seen upon cell cycle re-entry were prevented or diminished when FACT was silenced. Further, using mVenus-p27K- infected cancer cells to measure p27 degradation capacity, we confirm that inhibition of FACT at release from quiescence suppressed the p27 degradation capacity resulting in an increased mVenus-p27K- signal. In conclusion, FACT plays an important role in promoting the transition from G0 to the proliferative state and can be a potential therapeutic target to prevent prostate and lung cancer from progression and recurrence.

Discovery of a Selective Inhibitor for the YEATS Domains of ENL/AF9.

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain-histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL ( Kd = 745 ± 45 nM) and its paralog AF9 ( Kd = 523 ± 53 nM) and performed "SAR by catalog" with the aim of starting the development of a chemical probe for ENL.

Structure-Based Approach toward Identification of Inhibitory Fragments for Eleven-Nineteen-Leukemia Protein (ENL).

Lysine acetylation is an epigenetic mark that is principally recognized by bromodomains, and recently structurally diverse YEATS domains also emerged as readers of lysine acetyl/acylations. Here we present a crystallography-based strategy and the discovery of fragments binding to the ENL YEATS domain, a potential drug target. Crystal structures combined with synthetic efforts led to the identification of a submicromolar binder, providing first starting points for the development of chemical probes for this reader domain family.

Structure-guided development of YEATS domain inhibitors by targeting π-π-π stacking.

Chemical probes of epigenetic 'readers' of histone post-translational modifications (PTMs) have become powerful tools for mechanistic and functional studies of their target proteins in normal physiology and disease pathogenesis. Here we report the development of the first class of chemical probes of YEATS domains, newly identified 'readers' of histone lysine acetylation (Kac) and crotonylation (Kcr). Guided by the structural analysis of a YEATS-Kcr complex, we developed a series of peptide-based inhibitors of YEATS domains by targeting a unique π-π-π stacking interaction at the proteins' Kcr recognition site. Further structure optimization resulted in the selective inhibitors preferentially binding to individual YEATS-containing proteins including AF9 and ENL with submicromolar affinities. We demonstrate that one of the ENL YEATS-selective inhibitors, XL-13m, engages with endogenous ENL, perturbs the recruitment of ENL onto chromatin, and synergizes the BET and DOT1L inhibition-induced downregulation of oncogenes in MLL-rearranged acute leukemia.

The FACT inhibitor CBL0137 Synergizes with Cisplatin in Small-Cell Lung Cancer by Increasing NOTCH1 Expression and Targeting Tumor-Initiating Cells.

Traditional treatments of small-cell lung cancer (SCLC) with cisplatin, a standard-of-care therapy, spare the tumor-initiating cells (TIC) that mediate drug resistance. Here we report a novel therapeutic strategy that preferentially targets TICs in SCLC, in which cisplatin is combined with CBL0137, an inhibitor of the histone chaperone facilitates chromatin transcription (FACT), which is highly expressed in TICs. Combination of cisplatin and CBL0137 killed patient-derived and murine SCLC cell lines synergistically. In response to CBL0137 alone, TICs were more sensitive than non-TICs, in part, because CBL0137 increased expression of the tumor suppressor NOTCH1 by abrogating the binding of negative regulator SP3 to the NOTCH1 promoter, and in part because treatment decreased the high expression of stem cell transcription factors. The combination of cisplatin and CBL0137 greatly reduced the growth of a patient-derived xenograft in mice and also the growth of a syngeneic mouse SCLC tumor. Thus, CBL0137 can be a highly effective drug against SCLC, especially in combination with cisplatin.Significance: These findings reveal a novel therapeutic regimen for SCLC, combining cisplatin with an inhibitor that preferentially targets tumor-initiating cells. Cancer Res; 78(9); 2396-406. ©2018 AACR.

CDK9 and SPT5 proteins are specifically required for expression of herpes simplex virus 1 replication-dependent late genes.

DNA replication greatly enhances expression of the herpes simplex virus 1 (HSV-1) γ2 late genes by still unknown mechanisms. Here, we demonstrate that 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole (DRB), an inhibitor of CDK9, suppresses expression of γ2 late genes with an IC50 of 5 µm, which is at least 10 times lower than the IC50 value required for inhibition of expression of early genes. The effect of DRB could not be explained by inhibition of DNA replication per se or loading of RNA polymerase II to late promoters and subsequent reduction of transcription. Instead, DRB reduces accumulation of γ2 late mRNA in the cytoplasm. In addition, we show that siRNA-mediated knockdown of the transcription factor SPT5, but not NELF-E, also gives rise to a specific inhibition of HSV-1 late gene expression. Finally, addition of DRB reduces co-immunoprecipitation of ICP27 using an anti-SPT5 antibody. Our results suggest that efficient expression of replication-dependent γ2 late genes is, at least in part, regulated by CDK9 dependent co- and/or post-transcriptional events involving SPT5 and ICP27.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma.

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

Anticancer drug candidate CBL0137, which inhibits histone chaperone FACT, is efficacious in preclinical orthotopic models of temozolomide-responsive and -resistant glioblastoma.

Background: The survival rate for patients with glioblastoma (GBM) remains dismal. New therapies targeting molecular pathways dysregulated in GBM are needed. One such clinical-stage drug candidate, CBL0137, is a curaxin, small molecules which simultaneously downregulate nuclear factor-kappaB (NF-ĸB) and activate p53 by inactivating the chromatin remodeling complex, Facilitates Chromatin Transcription (FACT). Methods: We used publicly available databases to establish levels of FACT subunit expression in GBM. In vitro, we evaluated the toxicity and effect of CBL0137 on FACT, p53, and NF-ĸB on U87MG and A1207 human GBM cells. In vivo, we implanted the cells orthotopically in nude mice and administered CBL0137 in various dosing regimens to assess brain and tumor accumulation of CBL0137, its effect on tumor cell proliferation and apoptosis, and on survival of mice with and without temozolomide (TMZ). Results: FACT subunit expression was elevated in GBM compared with normal brain. CBL0137 induced loss of chromatin-unbound FACT, activated p53, inhibited NF-ĸB-dependent transcription, and was toxic to GBM cells. The drug penetrated the blood-brain barrier and accumulated in orthotopic tumors significantly more than normal brain tissue. It increased apoptosis and suppressed proliferation in both U87MG and A1207 tumors. Intravenous administration of CBL0137 significantly increased survival in models of early- through late-stage TMZ-responsive and -resistant GBM, with a trend toward significantly increasing the effect of TMZ in TMZ-responsive U87MG tumors. Conclusion: CBL0137 targets GBM according to its proposed mechanism of action, crosses the blood-brain barrier, and is efficacious in both TMZ-responsive and -resistant orthotopic models, making it an attractive new therapy for GBM.