CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone mRNA processing.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options, potentiating the importance of uncovering novel drug targets. Here, we target cleavage and polyadenylation specificity factor 3 (CPSF3), the 3' endonuclease that catalyzes mRNA cleavage during polyadenylation and histone mRNA processing. We find that CPSF3 is highly expressed in PDAC and is associated with poor prognosis. CPSF3 knockdown blocks PDAC cell proliferation and colony formation in vitro and tumor growth in vivo. Chemical inhibition of CPSF3 by the small molecule JTE-607 also attenuates PDAC cell proliferation and colony formation, while it has no effect on cell proliferation of nontransformed immortalized control pancreatic cells. Mechanistically, JTE-607 induces transcriptional readthrough in replication-dependent histones, reduces core histone expression, destabilizes chromatin structure, and arrests cells in the S-phase of the cell cycle. Therefore, CPSF3 represents a potential therapeutic target for the treatment of PDAC.

Stimulation of an anti-tumor immune response with "chromatin-damaging" therapy.

Curaxins are small molecules that bind genomic DNA and interfere with DNA-histone interactions leading to the loss of histones and decondensation of chromatin. We named this phenomenon 'chromatin damage'. Curaxins demonstrated anti-cancer activity in multiple pre-clinical tumor models. Here, we present data which reveals, for the first time, a role for the immune system in the anti-cancer effects of curaxins. Using the lead curaxin, CBL0137, we observed elevated expression of several group of genes in CBL0137-treated tumor cells including interferon sensitive genes, MHC molecules, some embryo-specific antigens suggesting that CBL0137 increases tumor cell immunogenicity and improves recognition of tumor cells by the immune system. In support of this, we found that the anti-tumor activity of CBL0137 was reduced in immune deficient SCID mice when compared to immune competent mice. Anti-tumor activity of CBL0137 was abrogated in CD8+ T cell depleted mice but only partially lost when natural killer or CD4+ T cells were depleted. Further support for a key role for the immune system in the anti-tumor activity of CBL0137 is evidenced by an increased antigen-specific effector CD8+ T cell and NK cell response, and an increased ratio of effector T cells to Tregs in the tumor and spleen. CBL0137 also elevated the number of CXCR3-expressing CTLs in the tumor and the level of interferon-γ-inducible protein 10 (IP-10) in serum, suggesting IP-10/CXCR3 controls CBL0137-elicited recruitment of effector CTLs to tumors. Our collective data underscores a previously unrecognized role for both innate and adaptive immunity in the anti-tumor activity of curaxins.

Chromatin Stability as a Target for Cancer Treatment.

In this essay, I propose that DNA-binding anti-cancer drugs work more via chromatin disruption than DNA damage. Success of long-awaited drugs targeting cancer-specific drivers is limited by the heterogeneity of tumors. Therefore, chemotherapy acting via universal targets (e.g., DNA) is still the mainstream treatment for cancer. Nevertheless, the problem with targeting DNA is insufficient efficacy due to high toxicity. I propose that this problem stems from the presumption that DNA damage is critical for the anti-cancer activity of these drugs. DNA in cells exists as chromatin, and many DNA-targeting drugs alter chromatin structure by destabilizing nucleosomes and inducing histone eviction from chromatin. This effect has been largely ignored because DNA damage is seen as the major reason for anti-cancer activity. I discuss how DNA-binding molecules destabilize chromatin, why this effect is more toxic to tumoral than normal cells, and why cells die as a result of chromatin destabilization.

A Translational Hepatic Artery Infusion (HAI) Model for Hepatocellular Carcinoma in Woodchucks.

BACKGROUND: Woodchucks (Marmota monax) are a well-accepted animal model for the investigation of spontaneous hepatocellular carcinoma (HCC). As HCC tumors obtain nutrient blood supply exclusively from the hepatic artery, hepatic artery infusion (HAI) has been applied to HCC. However, there is a scarcity of experimental animal models to standardize drug regimens and examine novel agents. The purpose of this study was to establish an HAI model in woodchucks. MATERIALS AND METHODS: HAI ports were placed in the gastroduodenal artery (GDA) of 11 woodchucks. The ports were infused with either a vehicle (dextrose 5% in water) or an experimental drug, CBL0137, once a week for 3 wk. Technical success rates, anatomical variation, morbidity and mortality, and tumor responses between groups were analyzed. RESULTS: The GDA access was feasible and reproducible in all woodchucks (11/11). The average operation time was 95 ± 20 min with no increase in the levels of liver enzymes detected from either infusate. The most common morbidity of CBL0137 therapy was anorexia after surgery. One woodchuck died due to hemorrhage at the gallbladder removal site from hepatic coagulopathy. Significantly higher CBL0137 concentrations were measured in the liver compared with blood after each HAI. Tumor growth was suppressed after multiple CBL0137 HAI treatments which corresponded to greater T cell infiltration and increased tumor cell apoptosis. CONCLUSIONS: HAI via GDA was a feasible and reproducible approach with low morbidity and mortality in woodchucks. The described techniques serve as a reliable platform for the identification and characterization of therapeutics for HCC.

Inhibition of the FACT Complex Reduces Transcription from the Human Cytomegalovirus Major Immediate Early Promoter in Models of Lytic and Latent Replication.

The successful colonization of the majority of the population by human cytomegalovirus is a direct result of the virus's ability to establish and, more specifically, reactivate from latency. The underlying cellular factors involved in viral reactivation remain unknown. Here, we show that the host complexfacilitateschromatintranscription (FACT) binds to the major immediate early promoter (MIEP) and that inhibition of this complex reduces MIEP transactivation, thus inhibiting viral reactivation.

Small-molecule xenomycins inhibit all stages of the Plasmodium life cycle.

Widespread resistance to most antimalaria drugs in use has prompted the search for novel candidate compounds with activity against Plasmodium asexual blood stages to be developed for treatment. In addition, the current malaria eradication programs require the development of drugs that are effective against all stages of the parasite life cycle. We have analyzed the antimalarial properties of xenomycins, a novel subclass of small molecule compounds initially isolated for anticancer activity and similarity to quinacrine in biological effects on mammalian cells. In vitro studies show potent activity of Xenomycins against Plasmodium falciparum. Oral administration of xenomycins in mouse models result in effective clearance of liver and blood asexual and sexual stages, as well as effective inhibition of transmission to mosquitoes. These characteristics position xenomycins as antimalarial candidates with potential activity in prevention, treatment and elimination of this disease.

Alkaloid-rich fraction of Ervatamia coronaria sensitizes colorectal cancer through modulating AMPK and mTOR signalling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ervatamia coronaria, a popular garden plant in India and some other parts of the world is known traditionally for its anti-inflammatory and anti-cancer properties. The molecular bases of these functions remain poorly understood. AIM OF THE STUDY: Efficacies of the existing therapies for colorectal cancer (CRC) are limited by their life-threatening side effects and unaffordability. Therefore, identifying a safer, efficient, and affordable therapeutic is urgent. We studied the anti-CRC activity of an alkaloid-rich fraction of E. coronaria leaf extracts (AFE) and associated underlying mechanism. MATERIALS AND METHODS: Activity guided solvant fractionation was adopted to identify the activity in AFE. Different cell lines, and tumor grown in syngeneic mice were used to understand the anti-CRC effect. Methodologies such as LCMS, MTT, RT-qPCR, immunoblot, immunohistochemistry were employed to understand the molecular basis of its activity. RESULTS: We showed that AFE, which carries about six major compounds, is highly toxic to colorectal cancer (CRC) cells. AFE induced cell cycle arrest at G1 phase and p21 and p27 genes, while those of CDK2, CDK-4, cyclin-D, and cyclin-E genes were downregulated in HCT116 cells. It predominantly induced apoptosis in HCT116p53+/+ cells while the HCT116p53-/- cells under the same treatment condition died by autophagy. Notably, AFE induced upregulation of AMPK phosphorylation, and inhibition of both of the mTOR complexes as indicated by inhibition of phosphorylation of S6K1, 4EBP1, and AKT. Furthermore, AFE inhibited mTOR-driven conversion of cells from reversible cell cycle arrest to senescence (geroconversion) as well as ERK activity. AFE activity was independent of ROS produced, and did not primarily target the cellular DNA or cytoskeleton. AFE also efficiently regressed CT26-derived solid tumor in Balb/c mice acting alone or in synergy with 5FU through inducing autophagy as a major mechanism of action as indicated by upregulation of Beclin 1 and phospho-AMPK, and inhibition of phospho-S6K1 levels in the tumor tissue lysates. CONCLUSION: AFE induced CRC death through activation of both apoptotic and autophagy pathways without affecting the normal cells. This study provided a logical basis for consideration of AFE in future therapy regimen to overcome the limitations associated with existing anti-CRC chemotherapy.

The Combination of Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Delays KMT2A-Rearranged Leukemia Progression.

Rearrangements of the Mixed Lineage Leukemia (MLL/KMT2A) gene are present in approximately 10% of acute leukemias and characteristically define disease with poor outcome. Driven by the unmet need to develop better therapies for KMT2A-rearranged leukemia, we previously discovered that the novel anti-cancer agent, curaxin CBL0137, induces decondensation of chromatin in cancer cells, delays leukemia progression and potentiates standard of care chemotherapies in preclinical KMT2A-rearranged leukemia models. Based on the promising potential of histone deacetylase (HDAC) inhibitors as targeted anti-cancer agents for KMT2A-rearranged leukemia and the fact that HDAC inhibitors also decondense chromatin via an alternate mechanism, we investigated whether CBL0137 could potentiate the efficacy of the HDAC inhibitor panobinostat in KMT2A-rearranged leukemia models. The combination of CBL0137 and panobinostat rapidly killed KMT2A-rearranged leukemia cells by apoptosis and significantly delayed leukemia progression and extended survival in an aggressive model of MLL-AF9 (KMT2A:MLLT3) driven murine acute myeloid leukemia. The drug combination also exerted a strong anti-leukemia response in a rapidly progressing xenograft model derived from an infant with KMT2A-rearranged acute lymphoblastic leukemia, significantly extending survival compared to either monotherapy. The therapeutic enhancement between CBL0137 and panobinostat in KMT2A-r leukemia cells does not appear to be mediated through cooperative effects of the drugs on KMT2A rearrangement-associated histone modifications. Our data has identified the CBL0137/panobinostat combination as a potential novel targeted therapeutic approach to improve outcome for KMT2A-rearranged leukemia.

Inhibition of encephalomyocarditis virus and poliovirus replication by quinacrine: implications for the design and discovery of novel antiviral drugs.

The 9-aminoacridine (9AA) derivative quinacrine (QC) has a long history of safe human use as an antiprotozoal and antirheumatic agent. QC intercalates into DNA and RNA and can inhibit DNA replication, RNA transcription, and protein synthesis. The extent of QC intercalation into RNA depends on the complexity of its secondary and tertiary structure. Internal ribosome entry sites (IRESs) that are required for initiation of translation of some viral and cellular mRNAs typically have complex structures. Recent work has shown that some intercalating drugs, including QC, are capable of inhibiting hepatitis C virus IRES-mediated translation in a cell-free system. Here, we show that QC suppresses translation directed by the encephalomyocarditis virus (EMCV) and poliovirus IRESs in a cell-free system and in virus-infected HeLa cells. In contrast, IRESs present in the mammalian p53 transcript that are predicted to have less-complex structures were not sensitive to QC. Inhibition of IRES-mediated translation by QC correlated with the affinity of binding between QC and the particular IRES. Expression of viral capsid proteins, replication of viral RNAs, and production of virus were all strongly inhibited by QC (and 9AA). These results suggest that QC and similar intercalating drugs could potentially be used for treatment of viral infections.

Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

The 3D Genome as a Target for Anticancer Therapy.

The role of 3D genome organization in the precise regulation of gene expression is well established. Accordingly, the mechanistic connections between 3D genome alterations and disease development are becoming increasingly apparent. This opinion article provides a snapshot of our current understanding of the 3D genome alterations associated with cancers. We discuss potential connections of the 3D genome and cancer transcriptional addiction phenomenon as well as molecular mechanisms of action of 3D genome-disrupting drugs. Finally, we highlight issues and perspectives raised by the discovery of the first pharmaceutical strongly affecting 3D genome organization.

Prevention of Chromatin Destabilization by FACT Is Crucial for Malignant Transformation.

Histone chaperone FACT is commonly expressed and essential for the viability of transformed but not normal cells, and its expression levels correlate with poor prognosis in patients with cancer. FACT binds several components of nucleosomes and has been viewed as a factor destabilizing nucleosomes to facilitate RNA polymerase passage. To connect FACT's role in transcription with the viability of tumor cells, we analyzed genome-wide FACT binding to chromatin in conjunction with transcription in mouse and human cells with different degrees of FACT dependence. Genomic distribution and density of FACT correlated with the intensity of transcription. However, FACT knockout or knockdown was unexpectedly accompanied by the elevation, rather than suppression, of transcription and with the destabilization of chromatin in transformed, but not normal cells. These data suggest that FACT stabilizes and reassembles nucleosomes disturbed by transcription. This function is vital for tumor cells because malignant transformation is accompanied by chromatin destabilization.

Prevention of Colorectal Carcinogenesis by DNA-Binding Small-Molecule Curaxin CBL0137 Involves Suppression of Wnt Signaling.

Chemoprevention is considered a valid approach to reduce the incidence of colorectal cancer, one of the most common malignancies worldwide. Here, we investigated the tumor-preventive activity of curaxin CBL0137. This compound represents a new class of nonmutagenic DNA-binding small molecules that alter chromatin stability and inhibit the function of the histone chaperone FACT. Among downstream effects of CBL0137 treatment are activation of p53 and type I interferons and inhibition of NFκB, HSF1, and MYC. In addition, our data show that in both human and mouse colorectal cancer cells in vitro, CBL0137 inhibits the APC/WNT/ß-catenin signaling pathway, which plays a key role in colon carcinogenesis. Using quantitative RT-PCR and microarray hybridization, we have demonstrated decreased expression of multiple components and downstream targets of the WNT pathway in colon cancer cells treated with CBL0137. At the same time, CBL0137 induced expression of WNT antagonists. Inhibition of WNT signaling activity by CBL0137 was also confirmed by luciferase reporter assay. Tumor-preventive activity of CBL0137 in vivo was tested in a murine model of colorectal carcinogenesis induced by 1,2-dimethylhydrazine (DMH), which is known to involve WNT pathway dysregulation. After DMH subcutaneous treatment, mice were administered CBL0137 in drinking water. Efficacy of CBL0137 in suppressing development of colorectal cancer in this model was evidenced by reduced incidence of adenocarcinomas and adenomas in both males and females and decrease in tumor multiplicity. These data support the prospective use of CBL0137 in chemoprevention of colorectal cancer as well as of other malignances associated with activated WNT signaling.

Small-molecule inhibitor which reactivates p53 in human T-cell leukemia virus type 1-transformed cells.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of the aggressive and fatal disease adult T-cell leukemia. Previous studies have demonstrated that the HTLV-1-encoded Tax protein inhibits the function of tumor suppressor p53 through a Tax-induced NF-kappaB pathway. Given these attributes, we were interested in the activity of small-molecule inhibitor 9-aminoacridine (9AA), an anticancer drug that targets two important stress response pathways, NF-kappaB and p53. In the present study, we have examined the effects of 9AA on HTLV-1-transformed cells. Treatment of HTLV-1-transformed cells with 9AA resulted in a dramatic decrease in cell viability. Consistent with these results, we observed an increase in the percentage of cells in sub-G(1) and an increase in the number of cells positive by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay following treatment of HTLV-1-transformed cells with 9AA. In each assay, HTLV-1-transformed cells C8166, Hut102, and MT2 were more sensitive to treatment with 9AA than control CEM and peripheral blood mononuclear cells. Analyzing p53 function, we demonstrate that treatment of HTLV-1-transformed cells with 9AA resulted in an increase in p53 protein and activation of p53 transcription activity. Of significance, 9AA-induced cell death could be blocked by introduction of a p53 small interfering RNA, linking p53 activity and cell death. These results suggest that Tax-repressed p53 function in HTLV-1-transformed cells is "druggable" and can be restored by treatment with 9AA. The fact that 9AA induces p53 and inhibits NF-kappaB suggests a promising strategy for the treatment of HTLV-1-transformed cells.

Histone Chaperone FACT and Curaxins: Effects on Genome Structure and Function.

The histone chaperone FACT plays important roles in essentially every chromatin-associated process and is an important indirect target of the curaxin class of anti-cancer drugs. Curaxins are aromatiс compounds that intercalate into DNA and can trap FACT in bulk chromatin, thus interfering with its distribution and its functions in cancer cells. Recent studies have provided mechanistic insight into how FACT and curaxins cooperate to promote unfolding of nucleosomes and chromatin fibers, resulting in genome-wide disruption of contact chromatin domain boundaries, perturbation of higher order chromatin organization, and global disregulation of gene expression. Here, we discuss the implications of these insights for cancer biology.

The anti-cancer drugs curaxins target spatial genome organization.

Recently we characterized a class of anti-cancer agents (curaxins) that disturbs DNA/histone interactions within nucleosomes. Here, using a combination of genomic and in vitro approaches, we demonstrate that curaxins strongly affect spatial genome organization and compromise enhancer-promoter communication, which is necessary for the expression of several oncogenes, including MYC. We further show that curaxins selectively inhibit enhancer-regulated transcription of chromatinized templates in cell-free conditions. Genomic studies also suggest that curaxins induce partial depletion of CTCF from its binding sites, which contributes to the observed changes in genome topology. Thus, curaxins can be classified as epigenetic drugs that target the 3D genome organization.

Uncovering the fine print of the CreERT2-LoxP system while generating a conditional knockout mouse model of Ssrp1 gene.

FAcilitates Chromatin Transcription (FACT) is a complex of SSRP1 and SPT16 that is involved in chromatin remodeling during transcription, replication, and DNA repair. FACT has been mostly studied in cell-free or single cell model systems because general FACT knockout (KO) is embryonically lethal (E3.5). FACT levels are limited to the early stages of development and stem cell niches of adult tissues. FACT is upregulated in poorly differentiated aggressive tumors. Importantly, FACT inhibition (RNAi) is lethal for tumors but not normal cells, making FACT a lucrative target for anticancer therapy. To develop a better understanding of FACT function in the context of the mammalian organism under normal physiological conditions and in disease, we aimed to generate a conditional FACT KO mouse model. Because SPT16 stability is dependent on the SSRP1-SPT16 association and the presence of SSRP1 mRNA, we targeted the Ssrp1 gene using a CreERT2- LoxP approach to generate the FACT KO model. Here, we highlight the limitations of the CreERT2-LoxP (Rosa26) system that we encountered during the generation of this model. In vitro studies showed an inefficient excision rate of ectopically expressed CreERT2 (retroviral CreERT2) in fibroblasts with homozygous floxed Ssrp1. In vitro and in vivo studies showed that the excision efficiency could only be increased with germline expression of two alleles of Rosa26CreERT2. The expression of one germline Rosa26CreERT2 allele led to the incomplete excision of Ssrp1. The limited efficiency of the CreERT2-LoxP system may be sufficient for studies involving the deletion of genes that interfere with cell growth or viability due to the positive selection of the phenotype. However, it may not be sufficient for studies that involve the deletion of genes supporting growth, or those crucial for development. Although CreERT2-LoxP is broadly used, it has limitations that have not been widely discussed. This paper aims to encourage such discussions.

Role of Chromatin Damage and Chromatin Trapping of FACT in Mediating the Anticancer Cytotoxicity of DNA-Binding Small-Molecule Drugs.

Precisely how DNA-targeting chemotherapeutic drugs trigger cancer cell death remains unclear, as it is difficult to separate direct DNA damage from other effects in cells. Recent work on curaxins, a class of small-molecule drugs with broad anticancer activity, shows that they interfere with histone-DNA interactions and destabilize nucleosomes without causing detectable DNA damage. Chromatin damage caused by curaxins is sensed by the histone chaperone FACT, which binds unfolded nucleosomes becoming trapped in chromatin. In this study, we investigated whether classical DNA-targeting chemotherapeutic drugs also similarly disturbed chromatin to cause chromatin trapping of FACT (c-trapping). Drugs that directly bound DNA induced both chromatin damage and c-trapping. However, chromatin damage occurred irrespective of direct DNA damage and was dependent on how a drug bound DNA, specifically, in the way it bound chromatinized DNA in cells. FACT was sensitive to a plethora of nucleosome perturbations induced by DNA-binding small molecules, including displacement of the linker histone, eviction of core histones, and accumulation of negative supercoiling. Strikingly, we found that the cytotoxicity of DNA-binding small molecules correlated with their ability to cause chromatin damage, not DNA damage. Our results suggest implications for the development of chromatin-damaging agents as selective anticancer drugs.Significance: These provocative results suggest that the anticancer efficacy of traditional DNA-targeting chemotherapeutic drugs may be based in large part on chromatin damage rather than direct DNA damage. Cancer Res; 78(6); 1431-43. ©2018 AACR.

Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins.

Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).

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.