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The Toll-like receptor 5 (TLR5) agonist entolimod, a derivative of Salmonella flagellin, has therapeutic potential for several indications including radioprotection and cancer immunotherapy. However, in Phase 1 human studies, entolimod induced a rapid neutralizing immune response, presumably due to immune memory from prior exposure to flagellated enterobacteria. To enable multi-dose applications, we used structure-guided reengineering to develop a next-generation, substantially deimmunized entolimod variant, GP532. GP532 induces TLR5-dependent NF-κB activation like entolimod but is smaller and has mutations eliminating an inflammasome-activating domain and key B- and T-cell epitopes. GP532 is resistant to human entolimod-neutralizing antibodies and shows reduced de novo immunogenicity. GP532 also has improved bioavailability, a stronger effect on key cytokine biomarkers, and a longer-lasting effect on NF-κB. Like entolimod, GP532 demonstrated potent prophylactic and therapeutic efficacy in mouse models of radiation-induced death and tissue damage. These results establish GP532 as an optimized TLR5 agonist suitable for multi-dose therapies and for patients with high titers of preexisting flagellin-neutralizing antibodies., Mett et al. describe development of GP532, a substantially deimmunized derivative of Toll-like receptor 5 (TLR5) agonist entolimod. GP532 has mutations eliminating key B- and T-cell epitopes and an inflammasome-activating domain yet remains a potent NF-κB activator with biological effects similar to entolimod. Thus, GP532 is suitable for multi-dose TLR5-targeting therapies and patients with high titers of preexisting flagellin-neutralizing antibodies.

Cellular responses to the loss of genomic stability are well-established, while how mammalian cells respond to chromatin destabilization is largely unknown. We previously found that DNA demethylation on p53-deficient background leads to transcription of repetitive heterochromatin elements, followed by an interferon response, a phenomenon we named TRAIN (Transcription of Repeats Activates INterferon). Here, we report that curaxin, an anticancer small molecule, destabilizing nucleosomes via disruption of histone/DNA interactions, also induces TRAIN. Furthermore, curaxin inhibits oncogene-induced transformation and tumor growth in mice in an interferon-dependent manner, suggesting that anticancer activity of curaxin, previously attributed to p53-activation and NF-kappaB-inhibition, may also involve induction of interferon response to epigenetic derepression of the cellular ‘repeatome’. Moreover, we observed that another type of drugs decondensing chromatin, HDAC inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of mammalian cells via elimination of cells with desilenced chromatin.

Pancreatic ductal adenocarcinoma (PDA) continues to be one of the deadliest cancers due to the absence of effective treatment. Curaxins are a class of small molecules with anti-cancer activity demonstrated in different models of cancer in mice. The lead curaxin compound, CBL0137, recently entered Phase I clinical trials. Curaxins modulate several important signaling pathways involved in the pathogenesis of PDA through inhibition of chromatin remodeling complex FACT. FACT is overexpressed in multiple types of tumor, with one of the highest rate of overexpression in PDA (59%). In this study, the efficacy of CBL0137 alone or in combination with current standard of care, gemcitabine, was tested against different models of PDA in vitro and in mouse models. It was found that CBL0137 alone is a potent inducer of apoptosis in pancreatic cancer cell lines and is toxic not only for proliferating bulk tumor cells, but also for pancreatic cancer stem cells. In mice, CBL0137 was effective against several PDA models, including orthotopic gemcitabine resistant PANC-1 model and patient derived xenografts, in which CBL0137 anti-tumor effect correlated with overexpression of FACT. Moreover, we observed synergy of CBL0137 with gemcitabine which may be explained by the ability of CBL0137 to inhibit several transcriptional programs induced by gemcitabine, including NF-kappaB response and expression of ribonucleotide reductase, one of the targets of gemcitabine in cells. This data suggest testing of CBL0137 efficacy in Phase II trial in PDA patients alone and in combination with gemcitabine.

Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy. Amplification of the MYCN oncogene, which occurs in approximately 25% of patients with neuroblastoma, is one of the most powerful independent prognostic markers for this disease. MYCN belongs to the Myc gene family that codes for transcription factors often aberrantly expressed in both solid and liquid malignancies. However, despite their importance in adult and childhood malignancies, Myc oncoproteins have to date proven to be ‘undruggable’. Using a chemical library of 34,000 compounds we aimed to identify novel MYCN inhibitors, and among a number of molecules identified, M606 was found to markedly reduce MYCN protein levels as well as its downstream targets in MYCN-amplified SK-N-BE(2)-C neuroblastoma cells. Bioinformatic analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) demonstrated that this compound inhibits Myc-mediated transcription and simultaneously activates the HIF1α pathway. MYCN/MYC protein and mRNA stability were unaltered whereas their mRNA levels were significantly decreased. Metabolomics analysis suggested that the mechanism by which M606 inhibits MYCN transcription involves its capacity to chelate iron and, in this regard, M606-mediated inhibition of MYCN was reversed by the addition of iron. Progressive deletions of the MYCN promoter in luciferase reporter assays identified a minimal promoter region responsive to M606 that contains two E2F sites. Microarray assays performed on SK-N-BE(2)-C cells after M606 treatment showed significant changes in the expression level of the E2F gene family. Chromatin immune precipitation (ChIP) and gel shift (EMSA) assays revealed a reduction in E2F binding at the MYCN promoter region after M606 treatment. Moreover, western blot analysis showed that the RB protein, which is involved in E2F binding and transcriptional activation, becomes inactive due to hypophosphorylation following M606 treatment. On the other hand, M606 lost its inhibitory activity on a pRB-deficient osteosarcoma cell line SAOS-2, underlying the importance of the RB/E2F axis for M606 function. In conclusion, M606 is a novel Myc inhibitor and iron chelator that directly downregulates MYCN/MYC transcription via the E2F signalling pathway, providing a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins. Citation Format: Emanuele Valli, Chengyuan Xue, Leanna Cheung, Laura Gamble, Ruby Pandher, Simone Di Giacomo, Catherine Burkhart, Natalia Fedtsova, Francesca Ferrucci, Sergei Makarov, Thomas Telfer, Rachel Codd, David Scott, Giovanni Perini, Andrei Osterman, Andrei Gudkov, Murray Norris, Michelle Haber. A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2616.

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.

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Also, current systemic chemotherapies have not been very effective at decreasing tumor burden. Poly(lactic-co-glycolic acid)-based (PLGA) microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their biocompatibility, flexibility in drug encapsulation and extended drug release. The present study investigated whether gemcitabine-loaded microparticles (GMPs), paclitaxel-loaded microparticles (PMPs) or sequential treatment of both, in comparison with blank (no drug) MPs, systemic treatments and no treatment control, are able to promote cancer cell killing effects and modulate drug resistance in vitro and in vivo. We previously showed in vitro studies with PANC-1 cells and we were able to complete the MPs studies with a second human pancreatic cancer cell line, MIAPaCa-2. In both cases, we tested the effect of single and combination treatments on two well-known resistance markers for gemcitabine, ribonucleotide reductase catalytic subunit M1 and cytidine deaminase, as well as on the promotion of cell death measuring cleaved caspase-3 (CC3). When treated with GMPs alone, both markers went up suggesting an increase in resistance against gemcitabine. Interestingly, both cell lines treated with PMPs alone showed an increase in CC3 expression and a significant decrease in the expression of both markers. Subsequently, we tested the in vivo efficacy of MPs by direct injection into established subcutaneous MIAPaCa-2 tumors in nude mice. Following four weeks of treatment, the tumors were excised, biopsied for protein analysis and frozen in OCT to allow visualization of fluorescent MPs and detect apoptosis by immunofluorescence. We also want to evaluate reactive oxygen species production; where we expect to see an increase in the MPs treated groups. This analysis is in progress. In conclusion, we observed a decrease in cell viability and drug resistance proteins in vitro using the drug-loaded microparticles in two grade 3 (or high grade) pancreatic cancer cell lines. The described drug delivery method has the potential to be a more efficient treatment modality than systemic gemcitabine and paclitaxel against early stage and locally advanced pancreatic cancer. Citation Format: Maria Munoz-Sagastibelza, Oluwafeyikemi Okome, Jenny E. Paredes Sanchez, Albert Palileo, Catherine Burkhart, Laura Martello-Rooney. Drug-loaded microparticles as a treatment approach for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3878.

The multidrug resistance–associated protein 1 (MRP1) has been closely linked to poor treatment response in several cancers, most notably neuroblastoma. Homozygous deletion of the MRP1 gene in primary murine neuroblastoma tumors resulted in increased sensitivity to MRP1 substrate drugs (vincristine, etoposide, and doxorubicin) compared with tumors containing both copies of wild-type MRP1, indicating that MRP1 plays a significant role in the drug resistance in this tumor type and defining this multidrug transporter as a target for pharmacologic suppression. A cell-based readout system was created to functionally determine intracellular accumulation of MRP1 substrates using a p53-responsive reporter as an indicator of drug-induced DNA damage. Screening of small-molecule libraries in this readout system revealed pyrazolopyrimidines as a prominent structural class of potent MRP1 inhibitors. Reversan, the lead compound of this class, increased the efficacy of both vincristine and etoposide in murine models of neuroblastoma (syngeneic and human xenografts). As opposed to the majority of inhibitors of multidrug transporters, Reversan was not toxic by itself nor did it increase the toxicity of chemotherapeutic drug exposure in mice. Therefore, Reversan represents a new class of nontoxic MRP1 inhibitor, which may be clinically useful for the treatment of neuroblastoma and other MRP1-overexpressing drug-refractory tumors by increasing their sensitivity to conventional chemotherapy. [Cancer Res 2009;69(16):6573–80]

Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Isolated limb perfusion (ILP) with the chemotherapeutic agent melphalan is an effective treatment option for extremity in-transit melanoma but is toxic and technically challenging to deliver locoregionally. CBL0137 is an experimental clinical drug with broad anticancer activity in animal models, owing to its ability to bind DNA in a nongenotoxic manner and inactivate the FACT chromatin modulator essential for tumor cell viability. Here, we report that CBL0137 delivered by ILP in a murine melanoma model is as efficacious as melphalan, displaying antitumor activity at doses corresponding to only a fraction of the systemic MTD of CBL0137. The ability to bind DNA quickly combined with a favorable safety profile made it possible to substitute CBL0137 in the ILP protocol, using an intra-arterial infusion method, to safely achieve effective tumor suppression. Our findings of a preclinical proof of concept for CBL0137 and its administration via intra-arterial infusion as a superior treatment compared with melphalan ILP allows for locoregional treatment anywhere a catheter can be placed. Cancer Res; 76(22); 6620–30. ©2016 AACR.

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic ( TH-MYCN ) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.

A major impediment to the successful use of Taxol in the treatment of cancer is the development of drug resistance. The major cellular target of Taxol is the microtubule that is comprised of alpha- and beta-tubulin heterodimers. Binding sites for Taxol have been delineated on the beta-tubulin subunit that has six isotypes. We have recently described increased expression of the brain-specific human class III beta-tubulin isotype, encoded by the Hbeta4 gene, in both Taxol-resistant ovarian tumours and non-small-cell lung cancer cell lines. To evaluate directly the role of the class III beta-tubulin isotype in mediating Taxol resistance, antisense phosphorothioate oligodeoxynucleotides (ODN) targeted against various regions of the Hbeta4 gene have been designed and examined for their efficacy in reducing Hbeta4 gene and protein expression. Taxol-resistant lung cancer cells, A549-T24, which are 17-fold resistant to Taxol and display a fourfold increase in Hbeta4 expression compared to the parental A549 cells, were treated with 1 microM antisense ODNs. Two ODNs, AS1 and AS3, were found to reduce mRNA expression by 40-50%, as determined by reverse transcription polymerase chain reaction. A concentration-dependent reduction in Hbeta4 mRNA expression was demonstrated with AS1 ODN. Immunofluorescence staining of cells treated with AS1 ODN revealed a decrease in class III protein expression which corresponded to a 39% increase in sensitivity to Taxol (P < 0.005). These findings support an important role for Hbeta4 (class III) beta-tubulin expression in Taxol resistance and have potential implications for the treatment of Taxol-resistant tumours.

The treatment of advanced ovarian cancer with taxol is hindered by the development of drug resistance. The cellular target for taxol is the microtubule that is stabilized by the drug. Taxol preferentially binds to the beta subunit of tubulin of which there are six distinct isotypes in mammalian cells. We have used highly specific oligonucleotides and polymerase chain reaction to analyze expression of all six beta-tubulin genes. Human lung cancer cells (A549) were selected in 12 and 24 nM taxol resulting in cell lines that were 9- and 17-fold resistant, respectively. These cells displayed an altered ratio of classes I, II, III, and IVa beta-tubulin isotypes. Ovarian tumors, seven untreated primary and four taxol- resistant tumor-bearing ascites, displayed significant increases (P < 0.005) in classes I (3.6-fold), III (4.4-fold), and IVa (7.6-fold) isotypes in the taxol-resistant samples as compared with untreated primary ovarian tumors. The increased expression appears to be related to the resistance phenotype, as the basal levels of the class III and IVa isotypes in the untreated tumors were extremely low. This is the first report of altered expression of specific beta-tubulin genes in taxol-resistant ovarian tumors and we propose that the latter may play a role in clinical resistance to taxol.

Oncoproteins encoded by dominant oncogenes have long been considered as targets for chemotherapeutic intervention. However, oncogenic transcription factors have often been dismissed as "undruggable." Members of the Myc family of transcription factors have been identified as promising targets for cancer chemotherapy in multiple publications reporting the requirement of Myc proteins for maintenance of almost every type of tumor. Here, we describe cell-based approaches to identify c-Myc small molecule inhibitors by screening complex libraries of diverse small molecules based on Myc functionality and specificity.

A series of taxol- and taxotere-resistant J774.2 cell lines has been characterized with respect to altered expression of beta-tubulin, the cellular target for these drugs. Vertebrates have six classes of beta-tubulin isotypes, each displaying a distinct pattern of expression. Although the functional significance of multiple beta-tubulins has not been fully defined, there is evidence that the individual isotypes contribute to differences in microtubule dynamics and drug binding. To determine if alterations in the expression of beta-tubulin isotypes play a role in taxol resistance, a PCR-based methodology was developed that permits highly specific amplification of each of the six known murine beta-tubulin isotypes. Two isotypes, M beta 5 and M beta 3, were expressed abundantly in the drug-sensitive parental J774.2 cells. Although expressed at an extremely low level in the parental cells, expression of the M beta 2 isotype was increased 21-fold (0.005) in the cell line most resistant to taxol. These findings suggest that a cell can alter its relative tubulin isotype composition in response to an external stress and specifically imply that altered expression of M beta 2, the class II beta-tubulin isotype, may contribute to the development of high resistance to taxol.

Pancreatic cancer is the fourth leading cause of cancer death in the United States with only 7% of diagnosed patients surviving 5 years. Most pancreatic cancer patients are not surgical candidates due to advanced stage at diagnosis. Current systemic chemotherapies, while exposing patients to the adverse side effects of treatment, have not been very effective at decreasing tumor burden primarily due to poor systemic drug uptake resulting from the dense stromal nature of pancreatic tumors. Poly(lactic-co-glycolic acid)-based (PLGA) microparticles (MPs) are a promising tool for localized drug delivery within the tumor due to their high biocompatibility, flexibility in the encapsulation of different drugs and extended drug release inside the tumor. The present study investigated whether gemcitabine-loaded microparticles (GMPs) in the range of 10-50 microns, in comparison with blank (no drug) MPs (BMPs), saline intraperitoneal injection (SIP) and gemcitabine intraperitoneal injection (GIP) as controls, are able to promote cancer cell killing effects in vivo. In vitro studies with PANC-1 and MIAPaCa-2 human pancreatic adenocarcinoma cell lines treated with different PLGA co-polymer ratios used to encapsulate gemcitabine showed enhanced cell killing and decreased colony formation in the longer release co-polymer ratio after 2 weeks of treatment. Subsequently, the in vivo efficacy of GMPs was tested by direct injection of GMPs into established subcutaneous MIAPaCa-2 tumors in nude mice. Treatment commenced when tumor volume was approximately 250 mm3. Following two weeks of treatment, there was a trending decrease in tumor volume in the GMPs-injected MIAPaCa-2 tumors compared to the BMPs-injected tumors. When comparing the SIP to GIP groups, there was no difference in final tumor volume emphasizing the lack of effective penetration of systemic gemcitabine into the tumor. In addition, we observed less tumor progression in the GMPs group compared to the others. At the endpoint, the tumors were excised, frozen in OCT compound and sectioned to visualize fluorescent MPs and to detect apoptosis by immunofluorescence. Interestingly, we observed a significant increase in apoptosis in the tumors treated with GMPs compared to the BMP tumors (p In conclusion, our data suggest that gemcitabine-loaded MPs could decrease tumor volume and increase local pancreatic tumor cell death. Further studies are needed to optimize the MPs loading and injection to confirm its efficacy. The described drug delivery method has the potential to be a more efficient local treatment modality than systemic gemcitabine against pancreatic cancer. Citation Format: Maria Munoz-Sagastibelza, Vadim Kurbatov, Sophia Dynes, Jennifer Caceres, Michael Chen, Raavi Gupta, Catherine Burkhart, Laura Martello-Rooney. Gemcitabine-loaded microparticles promote cancer cell death in subcutaneous pancreatic cancer xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2070.

Amplification of the transcription factor MYCN is associated with poor outcome and a multidrug-resistant phenotype in neuroblastoma. N-Myc regulates the expression of several ATP-binding cassette (ABC) transporter genes, thus affecting global drug efflux. Because these transporters do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, we explored the prognostic significance and transcriptional regulation of the phase II detoxifying enzyme, glutathione S-transferase P1 (GSTP1). Using quantitative real-time PCR, GSTP1 gene expression was assessed in a retrospective cohort of 51 patients and subsequently in a cohort of 207 prospectively accrued primary neuroblastomas. These data along with GSTP1 expression data from an independent microarray study of 251 neuroblastoma samples were correlated with established prognostic indicators and disease outcome. High levels of GSTP1 were associated with decreased event-free and overall survival in all three cohorts. Multivariable analyses, including age at diagnosis, tumor stage, and MYCN amplification status, were conducted on the two larger cohorts, independently showing the prognostic significance of GSTP1 expression levels in this setting. Mechanistic investigations revealed that GSTP1 is a direct transcriptional target of N-Myc in neuroblastoma cells. Together, our findings reveal that N-Myc regulates GSTP1 along with ABC transporters that act to control drug metabolism and efflux. Furthermore, they imply that strategies to jointly alter these key multidrug resistance mechanisms may have therapeutic implications to manage neuroblastomas and other malignancies driven by amplified Myc family genes. Cancer Res; 72(4); 845–53. ©2011 AACR.

Effective eradication of cancer requires treatment directed against multiple targets. The p53 and nuclear factor κB (NF-κB) pathways are dysregulated in nearly all tumors, making them attractive targets for therapeutic activation and inhibition, respectively. We have isolated and structurally optimized small molecules, curaxins, that simultaneously activate p53 and inhibit NF-κB without causing detectable genotoxicity. Curaxins demonstrated anticancer activity against all tested human tumor xenografts grown in mice. We report here that the effects of curaxins on p53 and NF-κB, as well as their toxicity to cancer cells, result from "chromatin trapping" of the FACT (facilitates chromatin transcription) complex. This FACT inaccessibility leads to phosphorylation of the p53 Ser(392) by casein kinase 2 and inhibition of NF-κB-dependent transcription, which requires FACT activity at the elongation stage. These results identify FACT as a prospective anticancer target enabling simultaneous modulation of several pathways frequently dysregulated in cancer without induction of DNA damage. Curaxins have the potential to be developed into effective and safe anticancer drugs.

Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children's cancers still have dismal outcomes. Included here are high risk neuroblastomas, Diffuse Intrinsic Pontine Gliomas (DIPG), and infant leukemias with MLL translocations. New treatments for these aggressive childhood cancers are urgently needed. Evidence is emerging of the importance of alterations in chromatin modifier genes in pediatric cancers. In this regard, CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl Med, 2011). We have examined FACT expression in neuroblastoma, DIPG and MLL leukemia, as well as the efficacy of CBL0137 in preclinical models of these diseases. Methods: Expression of the FACT subunits, SSRP1 and SPT16, was examined in neuroblastoma, DIPG and MLL leukemia cells using RT-PCR and Western analysis. The clinical significance of SSRP1 and SPT16 was also analysed using expression array data on 650 primary untreated neuroblastomas. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs. Cohorts of neuroblastoma, DIPG and MLL leukemia xenografted mice, as well as neuroblastoma-prone TH-MYCN mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs. Results: High levels of SSRP1 and SPT16 expression were observed in all three types of child cancer. In addition, in neuroblastoma, the two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor outcome (p Conclusions: Targeting FACT offers a highly promising novel therapeutic approach for aggressive childhood cancers. The results for CBL0137 are as good or better than any chemotherapy regimens we have tested in our preclinical models, and a Phase I COG trial of this nongenotoxic agent in refractory pediatric cancer patients is currently being planned. Citation Format: Michelle Haber, Jayne Murray, Laura Gamble, Ashleigh Carnegie-Clark, Hannah Webber, Michelle Ruhle, Michelle J. Henderson, Shiloh Middlemass, Daniel Carter, Maria Tsoli, Anahid Ehteda, Sandy Simon, Andre Oberthuer, Matthias Fischer, Katerina Gurova, Catherine Burkhart, Andrei Purmal, Richard B. Lock, David Ziegler, Glenn M. Marshall, Andrei V. Gudkov, Murray D. Norris. The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2015-1611

Background: Human MYCN (hMYCN) oncogene amplification is a powerful predictor of treatment failure in childhood neuroblastoma, and dysregulation of hMYCN protein expression appears to be critically involved in the pathogenesis of this disease. We used hMYCN antisense (AS) oligonucleotides to investigate, both in vitro and in vivo, the therapeutic potential of inhibiting hMYCN expression. Methods: We transiently transfected human neuroblastoma IMR-32 cells, which have an amplified hMYCN gene, with fluorescently labeled hMYCN AS or scrambled (SCR) control oligonucleotides and used fluorescence-activated cell sorting to enrich for cell populations containing different levels of the oligonucleotides. We used fluorescence immunocytochemistry or reverse transcription polymerase chain reaction to assay gene expression levels and trypan blue exclusion to assay growth inhibition in the cell populations. We examined the effects of continuous treatment for 6 weeks with AS or SCR oligonucleotides via subcutaneously implanted microosmotic pumps on tumor growth in a transgenic mouse model of hMYCN-induced neuroblastoma (n = 20 mice per group). All statistical tests were two-sided. Results: IMR-32 cells treated with AS oligonucleotides had approximately half as much hMYCN protein and cell proliferation as either SCR oligonucleotide–transfected or mock-transfected controls; the differences were statistically significant. Transgenic mice treated with AS oligonucleotides had lower tumor incidence and statistically significantly lower tumor mass than SCRtreated or untreated control mice. Compared with control treatments, AS oligonucleotide treatment in vitro and in vivo was associated with decreased expression of hMYCN and putative hMYCN target genes but not with that of closely related genes. Several AS oligonucleotide–treated mice developed tumors contralateral to the site of oligonucleotide administration, whereas SCR oligonucleotide–treated or untreated mice displayed bilateral tumor growth. Conclusions: Decreased expression of hMYCN protein is achievable with the use of AS oligonucleotide treatment, even in the presence of hMYCN oncogene amplification. Antisense strategies targeting the hMYCN oncogene in vivo decrease mouse neuroblastoma tumorigenesis. Investigation of their clinical effect in children with neuroblastoma is warranted. [J Natl Cancer Inst 2003;95:1394–1403]

Primary hepatocellular carcinoma (HCC) is the 6th most common malignancy diagnosed worldwide with an incidence that continues to rise. Even with the use of the current standard of care drug sorafenib, survival of patients remains at ∼10-11 months and sorafenib toxicity results in dose interruptions and delays. Therefore, tolerable, effective therapies against HCC are greatly needed. CBL0137 represents a novel class of small molecules that simultaneously activate p53 and inhibit cancer-associated stress response pathways, such as NF-κB and HSF-1, and has demonstrated broad range antitumor activity against pre-clinical tumor models. The effects of CBL0137 are mediated by the inhibition of the chromatin remodelling factor FACT (FAcilitates Chromatin Transcription) comprised of SSRP1 and SPT16 subunits. FACT is expressed in undifferentiated progenitors and stem cells of adult tissues, but is almost undetectable in differentiated cells. It is expressed in many tumor types, including HCC, and is correlated with high grade and worse overall survival. Thus, FACT represents a potentially important target for cancer therapy. We investigated the effect of CBL0137 on HCC using HepG2 and Hep3B xenograft models. Treatment of tumor bearing mice intravenously with 60-70 mg/kg CBL0137 every 4th day resulted in ∼88-94% tumor growth inhibition in both models, including tumor regression in 60-80% of tumors during the course of treatment. Furthermore, combination of suboptimal doses of CBL0137 with sorafenib had greater efficacy compared to either drug alone (tumor growth inhibition: HepG2- 87.9% combination vs 73.6% CBL0137 only (P=0.0014), 75.7% sorafenib only (P=0.0006); Hep3B- 79.6% combination vs 38.2% CBL0137 only (P=0.003), 51.4% sorafenib only (P=0.032)). Although the mechanism resulting in the combination effect is unknown, in vitro analysis of markers of CBL0137 and sorafenib activity revealed that both drugs caused a marked decrease in the basal and induced expression of NF-κB target genes (IL-8 and TNF). Furthermore, the combination of CBL0137 and sorafenib almost completely abolished the expression of these genes in both HCC cell lines, suggesting that the combination effect may be in part mediated through inhibition of the NF-κB pathway. Together, these data indicate that CBL0137 may provide a clinical benefit for the treatment of HCC both alone and in combination with sorafenib. Citation Format: Catherine Burkhart, Rachael Kohrn, Brittany Walker, Katerina Gurova, Andrei Purmal, Andrei Gudkov. CBL0137 demonstrates significant antitumor activity against hepatocellular carcinoma alone and in combination with sorafenib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 815. doi:10.1158/1538-7445.AM2014-815

CBL0137 represents a novel class of small molecules that simultaneously activate p53 and inhibit cancer-associated stress response pathways, such as NF-κB and HSF-1. The effects of CBL0137, culminating in tumor cell death, are mediated by the inhibition of FACT, a chromatin remodelling factor complex composed of SSRP1 and SPT16 subunits, that is involved in the transcription of genes with highly ordered chromatin structure, replication, and mitosis. FACT is expressed during early embryogenesis and in undifferentiated progenitors and stem cells of adult tissues while protein levels of both FACT subunits are almost undetectable in differentiated cells and tissues. FACT is expressed in several tumor types compared to equivalent normal tissues. In particular, SSRP1 is expressed in a high proportion of lung and pancreatic cancers (∼45-63%). FACT positive tumors are associated with an aggressive malignant phenotype (high grade, metastatic disease, worse overall survival). Therefore, FACT represents a potentially important target for cancer therapy. We investigated the effect of CBL0137 and its combination with gemcitabine a nucleoside analog used in treatment of non-small lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDA). We found that CBL0137 had significant antitumor activity against a H1975 NSCLC xenograft model (89.6% growth inhibition) as well as a patient-derived PDA (PDA#2) model (70.4% growth inhibition). Furthermore, CBL0137 acted synergistically with gemcitabine against both tumor types as evidenced by increased median survival time compared to each drug administered as a single agent (increase in survival time: H1975- >47 days combination vs 24 days CBL0137 only, 32.5 days gemcitabine only; PDA#2- >42 days combination vs 28 days CBL0137 only, 30.5 days gemcitabine only). Preliminary investigation into the mechanism underlying the synergy of this combination suggest that CBL0137 may enhance gemcitabine activity in part by abrogating the expression of modulators of gemcitabine response, such as cytidine deaminase and ribonucleotide reductase. Together, these data indicate that CBL0137 may provide a clinical benefit for the treatment of both NSCLC and PDA when combined with standard agent gemcitabine. Citation Format: Catherine Burkhart, Rachael Kohrn, Brittany Walker, David Meyer, Katerina Gurova, Andrei Gudkov. Synergistic effects of CBL0137 and gemcitabine against non-small cell lung and pancreatic cancer xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 800. doi:10.1158/1538-7445.AM2014-800

Background: We have reported that the anti-malarial drug, quinacrine (QC), has significant in vivo anti-tumor activity (Neznanov et al, Cell Cycle, 2009, 8: 1-11). We have since identified curaxins, a more potent class of compounds with similar mechanism of action, whose anti-cancer activity results from chromatin-trapping of the Facilitates Chromatin Transcription (FACT) complex (Gasparian et al, Science Transl. Med, 2011, 3, 95ra74). These novel nongenotoxic agents, like QC, cause simultaneous p53 activation and NF-kB inhibition and tumor cell death. We have now studied the effects of QC and one of the most active curaxins, CBL0137, in the TH-MYCN mouse model of neuroblastoma as well as a nude mouse xenograft model of the disease. Methods: Cohorts of homozygous TH-MYCN mice with small palpable tumors or of human MYCN-amplified BE(2)-C neuroblastoma tumor-bearing nude mice (n=10) were treated with QC (TH-MYCN mice only) or CBL0137, alone or combined with chemotherapeutic drugs. Results: QC alone had no significant effect on tumor progression and did not enhance cisplatin or VP16 activity. However, strong anti-tumor activity was observed when QC was combined with cyclophosphamide (CPM) (4/10 QC/CPM-treated vs 0/10 CPM-treated long-term survivors, P Conclusions: These are the most impressive results that we have ever observed for any therapeutic protocol in either the TH-MYCN homozygous or BE(2)-C xenograft mouse models. The combination of CPM/TOPO with iv CBL0137 appears to be a highly promising new treatment approach for refractory neuroblastoma. Safety and efficacy of CBL0137 is currently being investigated in a Phase I clinical trial in advanced cancer patients. Citation Format: Michelle Haber, Jayne Murray, Ashleigh Carnegie-Clark, Hannah Webber, Glenn M. Marshall, Katerina V. Gurova, Catherine A. Burkhart, Andrei Purmal, Andrei V. Gudkov, Murray D. Norris. Anticancer compound curaxin CBL0137, that simultaneously suppresses NF-κB and activates p53, is highly effective in two independent mouse models of neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2759. doi:10.1158/1538-7445.AM2013-2759

Multidrug resistance is one of the major causes of treatment failure in cancer therapy. While multidrug transporter proteins are known for their contributions to chemoresistance and efflux of anti-cancer drugs from cancer cells, a significant body of evidence points to their fundamental roles in tumour biology (Fletcher et al, Nature Rev Cancer, 10:147-156, 2010). We have investigated the ABCC subfamily of multidrug transporter genes in the highly malignant and aggressive childhood solid tumour, neuroblastoma and found that high levels of ABCC1 and ABCC4 are powerful independent prognostic indicators of clinical outcome. However, the agents used to treat the patients described in these studies are not known substrates of ABCC4 and we have shown that siRNA-mediated knockdown of ABCC4 results in reduced proliferation and enhanced morphological differentiation of neuroblastoma cells, in the absence of any cytotoxic drug treatment (Henderson et al, JNCI, 103;1-16, 2011). The aim of this study is to develop a potential new approach for the treatment of neuroblastoma through the inhibition of ABCC4 using small molecule inhibitors. A cell-based screen of a library of diverse chemical small molecules was used to isolate potent small molecule inhibitors of ABCC4 that were able to sensitize HEK293 cells over-expressing MRP4 to a cytotoxic MRP4 substrate, 6-mercaptopurine (6MP). Filtering of compounds based upon their ability to cause 6MP accumulation, together with focused library screening, have resulted in the identification of several chemical structures able to specifically block MRP4. In addition to reversing drug resistance to 6MP, these compounds cause morphological differentiation and inhibition of cell growth, as seen with MRP4 siRNA, in the BE2C neuroblastoma cell line. Importantly, these compounds significantly potentiate morphological differentiation caused by the established differentiation agent, all-trans retinoic acid. We further show that these inhibitors have potency in other MRP4 expressing cancer cell lines beyond neuroblastoma. These pharmacological inhibitors offer clinical potential for the treatment of neuroblastoma and other MRP4 overexpressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1834. doi:1538-7445.AM2012-1834

In childhood neuroblastoma, amplification of the transcription factor MycN is associated with poor outcome and a multidrug resistant phenotype. We have previously shown that MycN regulates the expression of several ATP-binding cassette (ABC) transporter genes, and that these genes represent powerful prognostic markers in neuroblastoma (1,2,3). Although these transporters are able to efflux a wide range of cytotoxic drugs, they do not confer resistance to several important cytotoxic agents used to treat neuroblastoma, including cisplatin and cyclophosphamide, hence we explored the prognostic significance and transcriptional regulation of the phase II detoxifying enzyme, glutathione S-transferase P1 (GSTP1). Using quantitative real-time PCR, GSTP1 gene expression was assessed in a retrospective cohort of 51 patients, and subsequently in a cohort of 207 prospectively accrued primary neuroblastomas. These data, and GSTP1 expression data from an independent microarray study of 251 neuroblastoma samples, were correlated with established prognostic indicators and disease outcome. High levels of GSTP1 were associated with decreased event-free and overall survival in all three cohorts. Multivariate analyses, including age at diagnosis, tumor stage and MYCN amplification status, were conducted on the two larger cohorts and demonstrated independent prognostic significance of GSTP1 expression levels. We also explored the regulation of GSTP1 by MycN using chromatin immunoprecipitation (ChIP) and luciferase reporter assays in neuroblastoma cell lines and found that GSTP1 is a direct transcriptional target of MycN. We conclude that N-Myc regulates multiple components of drug metabolism and efflux pathways, including GSTP1 and transporters of the ABC superfamily. The regulation of these key multidrug resistance mechanisms may have particular significance for malignancies with amplification of Myc family genes. 1) J Biol Chem, 285:19532-19543, 2010 2) J Natl Cancer Institute, 103:1236-51, 2011 4) Nat Rev Cancer, 10:147-156, 2010 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 789. doi:1538-7445.AM2012-789

Aberrant expression of Myc oncoproteins is a major causal factor in a large proportion of human cancer. In particular, MYCN oncogene amplification is one of the most powerful prognostic markers identified in the childhood cancer neuroblastoma and, as such, represents a valuable therapeutic target for the development of novel treatment approaches. A diverse chemical library of small molecules was screened using a cell-based assay to identify inhibitors of MYCN. Among a number of molecules identified as potential Myc inhibitors, M606 was found to reduce protein expression of MYCN and its downstream target genes in the MYCN-amplified neuroblastoma cell line BE(2)-C. A similar effect was also observed in a number of c-Myc over-expressing tumor cell lines. Analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc.) indicated that this small molecule inhibited Myc mediated transcription in a dose-dependent manner. Interestingly, hypoxia inducible factor 1 alpha (HIF1A) was induced ∼100 fold. Consistently, we observed HIF1A protein accumulation and nuclear translocation post-M606 treatment under normoxic conditions accompanied by activation of transcription of the HIF1A target, VEGF. siRNA-mediated knockdown of c-Myc or HIF1A in HepG2 cells followed by M606 treatment demonstrated that downregulation of c-Myc and upregulation of HIF1A by M606 are two independent events. Furthermore, inhibition of HIF1A prolyl hydroxylases by dimethyloxalylglycine (DMOG) resulted in downregulation of c-Myc protein independent of any HIFA upregulation. These results suggest that the same molecular mechanism, targeted by M606 and presumably involving inhibition of prolyl hydroxylases, can affect both HIF1A and Myc but in opposite ways. Further development of this compound may be useful in the treatment of cancers in which Myc oncoproteins are overexpressed as well as in the treatment or prevention of ischemic injury since recent studies suggest HIF1A activation may protect against ischemia-reperfusion injury. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 617. doi:10.1158/1538-7445.AM2011-617