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Supplementary Table 1, Supplementary Figures 1-5 from Small-Molecule Multidrug Resistance–Associated Protein 1 Inhibitor Reversan Increases the Therapeutic Index of Chemotherapy in Mouse Models of Neuroblastoma

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.

CBL0137 is a lead drug for human African trypanosomiasis, caused by Trypanosoma brucei. Herein, we use a four-step strategy to 1) identify physiologic targets and 2) determine modes of molecular action of CBL0137 in the trypanosome. First, we identified fourteen CBL0137-binding proteins using affinity chromatography. Second, we developed hypotheses of molecular modes of action, using predicted functions of CBL0137-binding proteins as guides. Third, we documented effects of CBL0137 on molecular pathways in the trypanosome. Fourth, we identified physiologic targets of the drug by knocking down genes encoding CBL0137-binding proteins and comparing their molecular effects to those obtained when trypanosomes were treated with CBL0137. CBL0137-binding proteins included glycolysis enzymes (aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, phosphoglycerate kinase) and DNA-binding proteins [universal minicircle sequence binding protein 2, replication protein A1 (RPA1), replication protein A2 (RPA2)]. In chemical biology studies, CBL0137 did not reduce ATP level in the trypanosome, ruling out glycolysis enzymes as crucial targets for the drug. Thus, many CBL0137-binding proteins are not physiologic targets of the drug. CBL0137 inhibited 1) nucleus mitosis, 2) nuclear DNA replication, and 3) polypeptide synthesis as the first carbazole inhibitor of eukaryote translation. RNA interference (RNAi) against RPA1 inhibited both DNA synthesis and mitosis, whereas RPA2 knockdown inhibited mitosis, consistent with both proteins being physiologic targets of CBL0137. Principles used here to distinguish drug-binding proteins from physiologic targets of CBL0137 can be deployed with different drugs in other biologic systems. SIGNIFICANCE STATEMENT: To distinguish drug-binding proteins from physiologic targets in the African trypanosome, we devised and executed a multidisciplinary approach involving biochemical, genetic, cell, and chemical biology experiments. The strategy we employed can be used for drugs in other biological systems.

A cure for human immunodeficiency virus type-1 (HIV-1) has been hampered by the limitation of current combination antiretroviral therapy (cART) to address the latent reservoirs in HIV-1 patients. One strategy proposed to eradicate these reservoirs is the "shock and kill" approach, where latency-reversing agents (LRAs) are used to reactivate and promote viral cell death and/or immune killing of reactivated cells. Here, we report that curaxin CBL0137, an antitumor compound, can potentiate tumor necrosis factor-α-mediated reactivation of latently infected HIV-1cell lines. Additionally, the single use of CBL0137 is sufficient to reactivate HIV-1 latent reservoirs in peripheral mononuclear cells (PBMCs) isolated from HIV-1 positive, cART-treated, aviremic patients. Thus, CBL0137 possesses capabilities as a LRA and could be considered for the "shock and kill" approach.

The class of carbazoles includes compounds with high biological activities and broad spectra of action. PLX01107 and PLX01008 are xenomycins, a new subclass of antimicrobial carbazole derivatives demonstrating strong antifungal activity in vitro. We performed three tests, a bacterial reverse mutation assay (Ames test), in vitro cytokinesis-block micronucleus assay, and chromosome aberration test in mouse bone marrow cells, to investigate the possible genotoxicity of these compounds. Despite their structural similarity, the two compounds had different genotoxicity profiles. PLX01008 showed positive effects in all assays. PLX01107 showed no mutagenicity in the Ames test but demonstrated strong cytogenetic activity in vitro and in vivo. PLX01107 was also tested in the in vivo alkaline comet assay, where a weak but statistically significant increase in DNA damage was seen in liver cells 24h after treatment. Significantly increased levels of formamidopyrimidine DNA glycosylase (FPG)-sensitive sites were found in bone marrow cells of PLX01107-treated mice (FPG-modified comet assay), suggesting induction of oxidative or alkylation damage to DNA.

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/β-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.

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.

The protozoan parasite Trypanosoma brucei causes the fatal illness human African trypanosomiasis (HAT). Standard of care medications currently used to treat HAT have severe limitations, and there is a need to find new chemical entities that are active against infections of T. brucei. Following a “drug repurposing” approach, we tested anti-trypanosomal effects of carbazole-derived compounds called “Curaxins”. In vitro screening of 26 compounds revealed 22 with nanomolar potency against axenically cultured bloodstream trypanosomes. In a murine model of HAT, oral administration of compound 1 cured the disease. These studies established 1 as a lead for development of drugs against HAT. Pharmacological time-course studies revealed the primary effect of 1 to be concurrent inhibition of mitosis coupled with aberrant licensing of S-phase entry. Consequently, polyploid trypanosomes containing 8C equivalent of DNA per nucleus and three or four kinetoplasts were produced. These effects of 1 on the trypanosome are reminiscent of “mitotic slippage” or endoreplication observed in some other eukaryotes.

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.

The nearly universal recurrence of glioblastoma (GBM) is driven in part by a treatment-resistant subpopulation of GBM stem cells (GSC). To identify improved therapeutic possibilities, we combined the EGFR/HER2 inhibitor lapatinib with a novel small molecule, CBL0137, which inhibits FACT (facilitates chromatin transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Lapatinib and CBL0137 synergistically inhibited the proliferation of patient-derived GBM cells. Compared with non–stem tumor cells (NSTC) enriched from the same specimens, the GSCs were extremely sensitive to CBL0137 monotherapy or FACT knockdown. FACT expression was elevated in GSCs compared with matched NSTCs and decreased in GSCs upon differentiation. Acute exposure of GSCs to CBL0137 increased asymmetric cell division, decreased GSC marker expression, and decreased the capacity of GSCs to form tumor spheres in vitro and to initiate tumors in vivo. Oral administration of CBL0137 to mice bearing orthotopic GBM prolonged their survival. Knockdown of FACT reduced the expression of genes encoding several core stem cell transcription factors (SOX2, OCT4, NANOG, and OLIG2), and FACT occupied the promoters of these genes. FACT expression was elevated in GBM tumors compared with non-neoplastic brain tissues, portended a worse prognosis, and positively correlated with GSC markers and stem cell gene expression signatures. Preferential targeting of GSCs by CBL0137 and synergy with EGFR inhibitors support the development of clinical trials combining these two agents in GBM. Cancer Res; 76(8); 2432–42. ©2016 AACR.

To date, there are no safe and effective drugs available for protection against ionizing radiation damage. Therefore, a great need exists to identify and develop non-toxic agents that will be useful as radioprotectors or postirradiation therapies under a variety of operational scenarios. We have developed a new pharmacological agent, CBLB613 (a naturally occurring Mycoplasma-derived lipopeptide ligand for Toll-like receptor 2/6), as a novel radiation countermeasure. Using CD2F1 mice, we investigated CBLB613 for toxicity, immunogenicity, radioprotection, radiomitigation and pharmacokinetics. We also evaluated CBLB613 for its effects on cytokine induction and radiation-induced cytopenia in unirradiated and irradiated mice. The no-observable-adverse-effect level of CBLB613 was 1.79 mg/kg and 1 mg/kg for single and repeated doses, respectively. CBLB613 significantly protected mice against a lethal dose of (60)Co γ radiation. The dose reduction factor of CBLB613 as a radioprotector was 1.25. CBLB613 also mitigated the effects of (60)Co γ radiation on survival in mice. In both irradiated and unirradiated mice, the drug stimulated induction of interleukin-1β (IL-1β), IL-6, IL-10, IL-12, keratinocyte-derived chemokine, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-1α. CBLB613 also reduced radiation-induced cytopenia and increased bone marrow cellularity in irradiated mice. Our immunogenicity study demonstrated that CBLB613 is not immunogenic in mice, indicating that it could be developed as a radioprotector and radiomitigator for humans against the potentially lethal effects of radiation exposure.

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]

The realization, that the androgen receptor (AR) is essential for prostate cancer (PC) even after relapse following androgen deprivation therapy motivated the search for novel types of AR inhibitors. We proposed that targeting AR expression versus its function would work in cells having either wild type or mutant AR as well as be independent of androgen synthesis pathways. Previously, using a phenotypic screen in androgen-independent PC cells we identified a small molecule inhibitor of AR, ARTIK-52. Treatment with ARTIK-52 caused the loss of AR protein and death of AR-positive, but not AR-negative, PC cells. Here we present data that ARTIK-52 induces degradation of AR mRNA through a mechanism that we were unable to establish. However, we found that ARTIK-52 is toxic to breast cancer (BC) cells expressing AR, although they were not sensitive to AR knockdown, suggesting an AR-independent mechanism of toxicity. Using different approaches we detected that ARTIK-52 induces replication-dependent double strand DNA breaks exclusively in cancer cells of prostate and breast origin, while not causing DNA damage, or any toxicity, in normal cells, as well as in non-PC and non-BC tumor cells, independent of their proliferation status. This amazing specificity, combined with such a basic mechanism of toxicity, makes ARTIK-52 a potentially useful tool to discover novel attractive targets for the treatment of BC and PC. Thus, phenotypic screening allowed us to identify a compound, whose properties cannot be predicted based on existing knowledge and moreover, uncover a barely known link between AR and DNA damage response in PC and BC epithelial cells.

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.

BACKGROUND: The transmission of parasites, including Babesia, plasmodia, and Trypanosoma cruzi, via transfusions is an important public health concern. INACTINE technology is a pathogen-reduction process that utilizes PEN110, an electrophilic agent that inac-tivates a wide range of pathogens by disrupting nucleic acid replication. The present study investigated the effect of PEN110 treatment on the viability of protozoa in RBCs. STUDY DESIGN AND METHODS:B. microti-parasitized RBCs from infected hamsters were treated with PEN110 and inoculated to naive animals. Parasitemia was detected by blood smears and PCR. Human RBCs infected with P. falciparum were treated with PEN110 and incubated with fresh RBCs. P. falciparum multiplication was detected by blood smears. Human RBCs spiked with T. cruzi and treated with PEN110 were analyzed for the presence of live parasites using in-vitro infectivity assay or by inoculating susceptible mice. RESULTS: Treatment of RBCs infected with B. microti or P. falciparum with 0.01 to 0.1 percent (vol/vol) PEN110 resulted in parasite inactivation to below the limit of detection during 24 hours. T. cruzi inoculated into human RBCs was inactivated below the limit of detection by 0.1 percent PEN110 after 3 hours. CONCLUSION: The study demonstrates that treatment of blood with PEN110 is highly effective in eradicating transfusion-transmitted protozoan parasites.

BACKGROUND: Mycoplasmas have been associated with multiple acute and chronic diseases. Mycoplasma genome is found in the blood of 10 to 15 percent of subjectively healthy individuals. If blood borne and viable in donated blood, mycoplasmas could potentially be transfusion transmissible. The INACTINE PEN110 technology is a pathogen reduction process that is in Phase 3 clinical studies. The present study investigated the ability of this process to eradicate mycoplasmas in human blood. STUDY DESIGN AND METHODS: Identical whole blood or RBC units inoculated with Mycoplasma arthritidis or M. pneumoniae were incubated with PEN110 (inactivating agent) for 24 hours at 23°C. Sham controls were treated with buffer under the same conditions. 4°C controls were put on storage immediately after the spike. RESULTS: No viable microorganisms were detected in PEN110-treated units after 24 hours of incubation. Sham controls showed no changes to mycoplasma titers during the incubation. In 4°C controls, minor decrease of mycoplasma titers was observed during the storage. CONCLUSION: The INACTINE process inactivates more than 107 mycoplasma CFU per mL in whole blood and RBCs. This study is the first demonstration of susceptibility of mycoplasmas to pathogen reduction. The data provide further support for the ability of INACTINE technology to address microbial safety issues that are not well characterized.

BACKGROUND: Development and characterization of methods for preventing transfusion-associated GVHD have utilized in vitro studies with human WBCs and in vivo studies in animal models. The limitation of these assays is that the in vivo GVHD response of treated human WBCs has not been tested directly. STUDY DESIGN AND METHODS: PBMNCs isolated from nonleukoreduced RBC units exposed to gamma irradiation, treated with PEN110 or PBS, were tested for their ability to induce xenogeneic GVHD when injected into severe combined immunodeficient (SCID) mice. RESULTS: These studies showed that the SCID mice injected with PBS-treated PBMNCs developed serum levels of human immunoglobulin that were followed by weight loss and display of ruffled fur characteristic of xenogeneic GVHD in these mice. In contrast, SCID mice injected with PEN110-treated or gamma-irradiated PBMNCs did not exhibit any of these responses. CONCLUSIONS: In these studies PEN110 treatment and gamma irradiation were equally effective at preventing in vivo GVHD responses when the treated cells were injected into SCID recipients. These results are consistent with previous results obtained when these two treatment methods were compared with in vitro studies with PBMNCs and in vivo studies in mouse models.

BACKGROUND : Yersinia enterocolitica, Pseudomonas fluorescens, and P. putida are responsible for a significant amount of the bacterial sepsis cases attributed to RBC transfusions. INACTINE is a pathogen-reduction process for RBCs, which consists of incubation of RBCs with PEN110 (proprietary compound) followed by automated washing of the RBCs. INACTINE is an electrophilic agent, which inactivates a wide range of viruses and WBCs by disruption of nucleic acid replication. The present study investigated the effect of the PEN110 process on Y. enterocolitica, P. fluorescens, and P. putida. STUDY DESIGN AND METHODS : Identical units of reduced CPD/ADSOL additive solution (AS-1) or CP2D/Nutricel additive solution (AS-3) RBCs were inoculated with 10 to 100 CFU per mL of either> Y. enterocolitica, P. fluorescens, or P. putida. The control units were put on storage immediately after the bacterial spike. The test units were subjected to the PEN110 process and then stored. Sham control units were processed the same way as test units without addition of PEN110. Bacterial titer in all units was monitored during the 6-week storage period. RESULTS : No bacteria were detected in any of the RBC units (n = 9 for each microorganism) prepared using the PEN110 process throughout 6 weeks of storage. Substantial bacterial growth occurred in all control and in a majority of sham control units (11 out of 15 experiments). The bacterial inactivation by the INACTINE process was found to be equally effective in CPD/AS-1 and CP2D/AS-3 RBC units. CONCLUSION : The INACTINE process effectively prevented the outgrowth of Y. enterocolitica, P. fluores- cens, and P. putida deliberately inoculated into WBC-reduced CPD/AS-1 and CP2D/AS-3 RBCs. The results demonstrated the crucial bactericidal role of PEN110 in the INACTINE process.

Background CBL0137 is a novel drug that modulates FAcilitates Chromatin Transcription (FACT), resulting in simultaneous nuclear factor-κB suppression, heat shock factor 1 suppression and p53 activation. CBL0137 has demonstrated antitumor effects in animal models of several adult cancers and neuroblastoma. Procedures CBL0137 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro cell line panel at concentrations ranging from 1.0 nM to 10.0 μM and against the PPTP in vivo solid tumor xenograft and acute lymphocytic leukemia (ALL) panels at 50 mg/kg administered intravenously weekly for 4 weeks. Results The median relative IC50 (rIC50) value for the PPTP cell lines was 0.28 μM (range: 0.13–0.80 μM). There were no significant differences in rIC50 values by histotype. CBL0137 induced significant differences in event-free survival (EFS) distribution compared to control in 10 of 31 (32%) evaluable solid tumor xenografts and in eight of eight (100%) evaluable ALL xenografts. Significance differences in EFS distribution were observed in four of six osteosarcoma lines, three of three rhabdoid tumor lines and two of six rhabdomyosarcoma lines. No objective responses were observed among the solid tumor xenografts. For the ALL panel, one xenograft achieved complete response and four achieved partial response. Conclusions The most consistent in vivo activity for CBL0137 was observed against ALL xenografts, with some solid tumor xenograft lines showing tumor growth delay. It will be important to relate the drug levels in mice at 50 mg/kg to those in humans at the recommended phase 2 dose.

Multiple randomized clinical trials have demonstrated that first line treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) such as afatinib in lung adenocarcinoma patients with EGFR mutations results in better progression free survival, response and quality of life, compared to chemotherapy. Despite excellent initial responses, almost all patients eventually acquire resistance to EGFR-TKI and overall survival for these patients remains dismal. Acquired resistance to EGFR-TKI results from several distinct mechanisms, including second site mutations (such as T790M), Met amplification and NFκB activation. An evolving hypothesis is that most patients achieve an initial incomplete response to EGFR-TKI but have residual disease comprised of cells that have developed an adaptive survival response to EGFR inhibition. Activation of NFkB and acquisition of stem cell like properties are important for this adaptive response. CBL0137 is a curaxin that inhibits NFκB and targets cancer stem cells preferentially by causing chromatin trapping of the FACT (facilitates chromatin transcription) complex. We evaluated the combination of CBL0137 and afatinib in EGFR mutant cell lines and a patient-derived xenograft (PDX) with acquired resistance to EGFR TKI. Drug synergy experiments were done in the EGFR mutant lung adenocarcinoma cell lines H1975 and H1993 with acquired resistance due to T790M and Met amplification, respectively. Cell viability was measured by MTT assay and synergy was quantified by using ChouTalalay combination indices (CI). The PDX was obtained from a patient with an EGFR mutation and progression after treatment with erlotinib with no detectable T790M mutation. To evaluate the efficacy of the afatinib-CBL0137 combination in mice bearing PDX tumors, afatinib (5 mg/kg orally/days 1-5 every week) alone, CBL0137 (50 or 75 mg/kg IV/once a week) alone, a combination of afatinib and CBL0137, or vehicle alone were administered for 52 days and tumor growth was measured every other day. Ten mice were used for each test. In the cell lines, the combination of afatinib and CBL0137 was synergistic with CI less than 1. The combination of afatinib and CBL0137 was well tolerated in the mice. In the treatment groups of vehicle alone, afatinib alone, CBL0137 50 mg/kg alone or 75 mg/kg alone, tumor volumes increased by factors of ∼35, 17.5, 17.5 and 12.5, respectively. There was no significant tumor growth for 3 weeks in mice treated with the combination of afatinib and CBL0137. After 3 weeks of treatment, the tumors started to grow slowly and increased in volume approximately 5 fold by day 52 when the treatment was stopped. We conclude that the combination of CBL0137 and afatinib is synergistic and can overcome acquired resistance to an EGFR TKI in an EGFR mutant lung adenocarcinoma. Phase I oral and IV clinical trials of CBL0137 monotherapy in solid tumors are ongoing, and combination studies with afatinib are being planned. Citation Format: Neelesh Sharma, Josephine Dermawan, Andrei Purmal, Daniel Lindner, Gary Wildey, Afshin Dowlati, Andrei Gudkov, Katerina Gurova, George R. Stark. Targeting FACT complex with CBL0137 to overcome acquired resistance to EGFR-TKI in lung adenocarcinoma. [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 3036.

Given an ever-increasing risk of nuclear and radiological emergencies, there is a critical need for development of medical radiation countermeasures (MRCs) that are safe, easily administered, and effective in preventing and/or mitigating the potentially lethal tissue damage caused by acute high-dose radiation exposure. Because the efficacy of MRCs for this indication cannot be ethically tested in humans, development of such drugs is guided by the Food and Drug Administration's Animal Efficacy Rule. According to this rule, human efficacious doses can be projected from experimentally established animal efficacious doses based on the equivalence of the drug's effects on efficacy biomarkers in the respective species. Therefore, identification of efficacy biomarkers is critically important for drug development under the Animal Efficacy Rule. CBLB502 is a truncated derivative of the Salmonella flagellin protein that acts by triggering Toll-like receptor 5 (TLR5) signaling and is currently under development as a MRC. Here, we report identification of two cytokines, granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), as candidate biomarkers of CBLB502's radioprotective/mitigative efficacy. Induction of both G-CSF and IL-6 by CBLB502 1) is strictly TLR5-dependent, 2) occurs in a CBLB502 dose-dependent manner within its efficacious dose range in both nonirradiated and irradiated mammals, including nonhuman primates, and 3) is critically important for the ability of CBLB502 to rescue irradiated animals from death. After evaluation of CBLB502 effects on G-CSF and IL-6 levels in humans, these biomarkers will be useful for accurate prediction of human efficacious CBLB502 doses, a key step in the development of this prospective radiation countermeasure.

Development of safe and effective tumor-preventive treatments for high-risk patient populations and therapies for early-stage cancer remains a critical need in oncology. We have recently discovered compound with anticancer activity, Curaxin-137, which modulates several important signaling pathways involved in even the very early stages of cancer. In tumor cells, Curaxin-137 inhibits NF-κB- and HSF1-dependent transcription (prosurvival pathways) and activates p53 (a proapoptotic pathway) without inducing DNA damage. These effects result from chromatin trapping and inhibition of activity of the FACT (facilitates chromatin transcription) complex by Curaxin-137. FACT has not been previously implicated in cancer, but we found that its subunits are overexpressed in breast cancer. On the basis of this background, we tested whether Curaxin-137 could suppress tumorigenesis in MMTV-neu transgenic mice, which spontaneously develop mammary carcinoma due to steroid receptor–regulated expression of the Her2 proto-oncogene. We found that chronic administration of Curaxin-137 in a preventive regimen to MMTV-neu mice did not cause any detectable changes in normal organs and tissues, yet inhibited tumor onset, delayed tumor progression, and prolonged survival of mice in a dose-dependent manner. Curaxin-137 induced changes in FACT, altered NF-κB localization, and activated p53 in tumor cells as expected from its defined mechanism of action. These results support further investigation of Curaxin-137 as a potential preventive and/or early-stage therapeutic agent for breast cancer. Cancer Prev Res; 5(8); 1025–35. ©2012 AACR.

Uracil DNA N-glycosylase (UDG) has been used as a model enzyme to test a novel universal approach to discriminate between two possible enzymatic mechanisms of specific site location in DNA, processive (DNA-scanning mechanism) and distributive (random diffusion-mediated mechanism). Two double-stranded concatemeric polynucleotides of defined length (440-480 nucleotides) containing deoxyuridine at either every 10th or 20th nucleotide in the DNA chain were prepared by the ligation of self-complementary 10- or 20-mer oligodeoxyribonucleotides. Incubation of these polynucleotides with Escherichia coli UDG, followed by thermal breakage of the abasic sites, formed fragments that were multiples of either the 10- or the 20-mer. Since the processive and distributive mechanisms of uracil removal by UDG would be very different, the fragment distribution, generated at each time interval during the UDG reaction, should be unique. To show this, we developed a computer model illustrating both possible mechanisms of UDG functioning. The distribution of DNA fragments experimentally generated during the time course of the UDG reaction was compared with the results of the computer programs that modeled the distributive and processive mechanisms. The data indicated that uracil removal, catalyzed by UDG, is consistent with a distributive model.

5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.

Oligonucleotides containing a unique alpha-deoxyadenosine or tetrahydrofuran (a model abasic site) were synthesized using phosphoramidite chemistry. Repair enzymes from Escherichia coli, including endonucleases III, IV, and VIII, exonuclease III, formamidopyrimidine N-glycosylase, and deoxyinosine 3'-endonuclease, as well as UV dimer N-glycosylases from T4 (den V) and Micrococcus luteus, were examined for their ability to recognize alpha-deoxyadenosine and tetrahydrofuran. In agreement with prior studies, a tetrahydrofuran-containing oligonucleotide was a substrate for endonuclease IV and exonuclease III, but not for the other repair enzymes. However, an oligonucleotide containing alpha-deoxyadenine was a substrate only for endonuclease IV. Competitive inhibition studies with both substrates confirmed that the activity recognizing alpha-deoxyadenine was endonuclease IV and not a possible contaminant in the endonuclease IV preparation. Using E. coli extracts, the activity that recognized alpha-deoxyadenine was dependent on nfo, the structural gene of endonuclease IV, further substantiating that endonuclease IV is the enzyme that recognized alpha-deoxyadenine. Kinetic measurements indicated that alpha-deoxyadenosine was as good a substrate for endonuclease IV as tetrahydrofuran; the Km and Vmax values for both substrates were similar. Using substrates that were labeled at either the 3'- or 5'-terminus, endonuclease IV was shown to hydrolyze the phosphodiester bond 5' to either alpha-deoxyadenosine or tetrahydrofuran, leaving the lesion, alpha-deoxyadenosine or tetrahydrofuran, on the 5'-terminus of the nicked site. The ability of endonuclease IV to recognize alpha-deoxyadenosine suggests that endonuclease IV is able to recognize a new class of DNA base lesions that is not recognized by other DNA N-glycosylases and AP endonucleases.

Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU). In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized. Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free). However, 5-OHdUTP can only be incorporated into DNA in place of dTTP. To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used. Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed. The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent. In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU. However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC. In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU. These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions.

Recent studies with 8-oxodeoxyguanosine triphosphate (8-oxodGTP) have suggested that incorporation of oxidized nucleotides from the precursor pool into DNA may have deleterious effects. Here we show that 5-hydroxydeoxycytosine triphosphate (5-OHdCTP) and 5-hydroxydeoxyuridine triphosphate (5-OHdUTP) are more efficient substrates than 8-oxodGTP for Escherichia coli DNA polymerase I Klenow fragment lacking proofreading activity, while 8-oxodeoxyadenosine triphosphate (8-oxodGTP, 5-OHdCTP can mispair with dA in DNA but with lower efficiency. Since the 5-hydroxypyrimidines are present in normal and oxidized cellular DNA in amounts similar to the 8-oxopurines, these data suggest that enzymatic mechanisms might exist for removing them from the DNA precursor pools.

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.

In prostate cancer (PCa) patients, initial responsiveness to androgen deprivation therapy is frequently followed by relapse due to development of treatment-resistant androgen-independent PCa. This is typically associated with acquisition of mutations in AR that allow activity as a transcription factor in the absence of ligand, indicating that androgen-independent PCa remains dependent on AR function. Our strategy to effectively target AR in androgen-independent PCa involved using a cell-based readout to isolate small molecules that inhibit AR transactivation function through mechanisms other than modulation of ligand binding. A number of the identified inhibitors were toxic to AR-expressing PCa cells regardless of their androgen dependence. Among these, some only suppressed PCa cell growth (ARTIS), while others induced cell death (ARTIK). ARTIK, but not ARTIS, compounds caused disappearance of AR protein from treated cells. siRNA against AR behaved like ARTIK compounds, while a dominant negative AR mutant that prevents AR-mediated transactivation but does not eliminate the protein showed only a growth suppressive effect. These observations reveal a transcription-independent function of AR that is essential for PCa cell viability and, therefore, is an ideal target for anti-PCa treatment. Indeed, several of the identified AR inhibitors demonstrated in vivo efficacy in mouse models of PCa and are candidates for pharmacologic optimization.

Neuroblastoma is a childhood cancer of the sympathetic nervous system. Approximately 20% of patients present with an aggressive metastatic disease characterized by amplification of the MYCN locus. However, an additional 30% of patients suffer from an equally aggressive disease, yet there are no unified genetic markers available. To identify potential biomarkers of poor prognosis in neuroblastoma, we conducted unsupervised hierarchical clustering on 649 primary neuroblastoma tumors based on a 51 gene MYC core target signature. This demonstrated strong activation within the MYCN amplified cohort as expected, but surprisingly there was a large subset of MYCN non-amplified patients that exhibited high MYC signaling. High expression of this MYC signature proved to be strongly predictive of poor overall survival, in both the full cohort (Hazard ratio (HR): 47.6, p= 2.7x10-35, n=649) and in MYCN non-amplified patients (HR=32.8, p=1.3x10-13, n=554), suggesting that neuroblastoma can be driven by MYC signaling in the absence of MYCN amplification. To evaluate potential therapeutic targets within this MYC signature, we conducted cox regression analysis on the 51 individual members and identified that SPT16 was a particularly potent predictor of poor outcome in these 649 neuroblastoma patients (HR: 7.2, p=2.7x10-17). SPT16 is a subunit of the FACT histone chaperone complex, which together with its heterodimer partner SSRP1, has previously been shown to function in dynamic regulation of chromatin to drive transcription, DNA replication and DNA repair. Similar to SPT16, high SSRP1 expression was a strong predictor of poor prognosis in 649 neuroblastoma patients (HR: 12.5, p=5.812x10-26) implicating FACT as a mediator of MYC driven neuroblastoma. Next we investigated the biological relationship between FACT and MYCN in neuroblastoma. siRNA experiments showed that FACT and MYCN participate in a positive feedback transcriptional loop that is essential for maintaining mutual high expression. Immunohistochemistry in sympathetic ganglia of pre-tumor TH-MYCN mouse model of neuroblastoma identified that FACT was highly expressed at tumor initiation and chemical inhibition of FACT by CBL0137 markedly reduced tumor initiation and subsequent tumorigenesis in vivo. Next we tested CBL0137 against established neuroblastoma in TH-MYCN mice. CBL0137 was highly cytotoxic to tumors either as a single agent or by synergistically promoting clinically used chemotherapeutics. This was accompanied by marked MYCN depletion and prolonged tumor regression in a majority of recipient mice. Pulsed-field electrophoresis showed that CBL0137 impaired FACT dependent DNA damage repair in the presence of genotoxic chemotherapy, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells. Together our data define FACT as a mediator of neuroblastoma tumorigenesis and MYCN-related treatment target. Given the current poor survival rates of children with high-risk neuroblastoma, our findings provide support for the clinical development of CBL0137 as a novel treatment approach for this refractory malignancy. Citation Format: Daniel R. Carter, Jayne Murray, Belamy B. Cheung, Heyam Kalla, Laura Gamble, Joanna Tsang, Selina Sutton, Jessica Koach, Sarah Syed, Andrew Gifford, Natalia Issaeva, Asel Biktasova, Bernard Atmadibrata, Yuting Sun, Nicolas Sokolowski, Dora Ling, Patrick Y. Kim, Hannah Webber, Ashleigh Clark, Michelle Ruhle, Bing Liu, André Oberthuer, Matthias Fischer, Jennifer Byrne, Federica Saletta, Andrei Purmal, David Ziegler, Tao Liu, Katerina V. Gurova, Andrei V. Gudkov, Murray D. Norris, Michelle Haber, Glenn M. Marshall. MYCN and is a therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR09.

There are currently no approved medical radiation countermeasures (MRC) to reduce the lethality of high-dose total body ionizing irradiation expected in nuclear emergencies. An ideal MRC would be effective even when administered well after radiation exposure and would counteract the effects of irradiation on the hematopoietic system and gastrointestinal tract that contribute to its lethality. Entolimod is a Toll-like receptor 5 agonist with demonstrated radioprotective/mitigative activity in rodents and radioprotective activity in non-human primates. Here, we report data from several exploratory studies conducted in lethally irradiated non-human primates (rhesus macaques) treated with a single intramuscular injection of entolimod (in the absence of intensive individualized supportive care) administered in a mitigative regimen, 1-48 hours after irradiation. Following exposure to LD50-70/40 of radiation, injection of efficacious doses of entolimod administered as late as 25 hours thereafter reduced the risk of mortality 2-3-fold, providing a statistically significant (P

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

3063 Background: Entolimod is a specific, TLR5 agonist derived from salmonella flagellin, with immunotherapeutic effects in preclinical cancer models. Entolimod activates innate and adaptive immune responses and mobilizes immunocytes to organs such as liver and bladder, with functional TLR5. A phase 1 trial was undertaken to evaluate entolimod’s safety, tolerability, pharmacokinetics, immunoactivity, and preliminary antitumor activity. Methods: Pts with advanced cancers and ECOG PS of 0-1 were treated at 5 subcutaneous doses (5, 10, 15, 20, 30, 40 µg/day) over a 2-week period. PK/PD samples were obtained pre, and 2, 4, 6, 8, 24 hrs post-dose. Immune function samples were obtained pre and 24 hrs post-dose on days1, 8, 11 and 22. Results: 26 pts (38.4% colorectal cancer [CRC] and 19% NSCLC) were enrolled. Common toxicities were transient hypotension (9 grade 1/4, 7 grade 2/4), hyperglycemia (8 grade 1/4, 6 grade 2/4), and fever (10 grade 1/4, 3 grade 2/4). Three DLTs were observed at 40 µg/day. One pt devel...

Development and characterization of methods for preventing transfusion-associated GVHD have utilized in vitro studies with human WBCs and in vivo studies in animal models. The limitation of these assays is that the in vivo GVHD response of treated human WBCs has not been tested directly.PBMNCs isolated from nonleukoreduced RBC units exposed to gamma irradiation, treated with PEN110 or PBS, were tested for their ability to induce xenogeneic GVHD when injected into severe combined immunodeficient (SCID) mice.These studies showed that the SCID mice injected with PBS-treated PBMNCs developed serum levels of human immunoglobulin that were followed by weight loss and display of ruffled fur characteristic of xenogeneic GVHD in these mice. In contrast, SCID mice injected with PEN110-treated or gamma-irradiated PBMNCs did not exhibit any of these responses.In these studies PEN110 treatment and gamma irradiation were equally effective at preventing in vivo GVHD responses when the treated cells were injected into SCID recipients. These results are consistent with previous results obtained when these two treatment methods were compared with in vitro studies with PBMNCs and in vivo studies in mouse models.

Mycoplasmas have been associated with multiple acute and chronic diseases. Mycoplasma genome is found in the blood of 10 to 15 percent of subjectively healthy individuals. If blood borne and viable in donated blood, mycoplasmas could potentially be transfusion transmissible. The INACTINE PEN110 technology is a pathogen reduction process that is in Phase 3 clinical studies. The present study investigated the ability of this process to eradicate mycoplasmas in human blood.Identical whole blood or RBC units inoculated with Mycoplasma arthritidis or M. pneumoniae were incubated with PEN110 (inactivating agent) for 24 hours at 23 degrees C. Sham controls were treated with buffer under the same conditions. 4 degrees C controls were put on storage immediately after the spike.No viable microorganisms were detected in PEN110-treated units after 24 hours of incubation. Sham controls showed no changes to mycoplasma titers during the incubation. In 4 degrees C controls, minor decrease of mycoplasma titers was observed during the storage.The INACTINE process inactivates more than 107 mycoplasma CFU per mL in whole blood and RBCs. This study is the first demonstration of susceptibility of mycoplasmas to pathogen reduction. The data provide further support for the ability of INACTINE technology to address microbial safety issues that are not well characterized.

BACKGROUND : The presence of WBCs in blood components is the primary factor influencing the immunologic consequences of transfusion, such as GVHD and alloimmunization. Depletion or inactivation of WBCs can reduce the deleterious responses. Because treatment with PEN110 (Inactine, V. I. Technologies), an ethyleneimine derivative that disrupts nucleic acid replica- tion, was shown to inactivate in vitro human PBMNC function, the ability of PEN110-treated cells to trigger GVHD or alloantibodies was studied with in vivo murine models. STUDY DESIGN AND METHODS : In vitro assays were employed to confirm that PEN110 treatment inactivated murine splenocyte function as effectively as for human PBMNCs. In vivo experiments in mice examined the ability of PEN110-treated cells to induce GVHD responses in a parent into F 1 hybrid GVHD model, to induce alloantibodies, to stimulate MHC-restricted cytolytic T lymphocyte responses, and to persist after injection. RESULTS : PEN110-treated murine splenocytes did not respond or induce responses in any in vitro or in vivo assay. The PEN110-treated cells were eliminated from blood and secondary lymphoid organs much more rapidly than were untreated cells. CONCLUSION : PEN110 treatment prevents the development of GVHD and alloantibody production following WBC transfusion in a murine model system, supporting the continued development of PEN110 treatment of cellular blood components as an alternative to gamma irradiation for the prevention of GVHD.

BACKGROUND : The presence of WBCs in blood components is the major factor influencing the immunologic consequences of transfusion. Attempts to ameliorate these responses have used WBC reduction or inactivation by ionizing radiation. PEN110 (Inactine, V. I. Technologies) is a chemical that inhibits the replication of infectious pathogens by modifying their nucleic acids. These experiments compared effects of PEN110 treatment or gamma irradiation on WBC function. STUDY DESIGN AND METHODS : Aliquots of non-WBC-reduced RBC units were treated with PEN110 or gamma irradiation with appropriate controls, and PBMNCs from these units were tested with in vitro assays. The assays included immunophenotyping, activating T cells with phorbol ester, proliferation of cells in response to mitogens or allogeneic cells, and determining the ability of cells to stimulate proliferative responses and to produce IL-8. DNA fragmentation following PEN110 treatment was examined by PCR and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays. RESULTS : Treatment of non-WBC-reduced RBC units with PEN110 functionally inactivated WBC in all in vitro assays used. In contrast, while gamma irradiation inhibited proliferation of the WBCs, it did not or only partially inhibited the ability of WBC to function in the other assays. PEN110, but not gamma irradiation, rapidly induced fragmentation of cellular DNA. CONCLUSION : Because PEN110 was as effective as gamma irradiation at inhibiting WBC proliferation, PEN110 treatment could potentially be used to prevent the development of GVHD following transfusion.

BACKGROUND: Despite extensive reductions in risk, donor selection and testing cannot eliminate pathogen transmission. The objective of this study was to evaluate clinically the viability of pathogen-inactivated RBCs. STUDY DESIGN AND METHODS: Twelve healthy subjects each donated two units of blood that were handled as additive system control units or were inactivated using the PEN110 process (INACTINE, V.I. Technologies) in randomized order. PEN110, which inactivates pathogens by reacting with nucleic acid bases, was incubated with RBCs for 6 hours, followed by washing and quenching with sodium thiosulfate. Radiolabeled RBC recovery and survival determinations were undertaken after 28 days of storage; biochemical and hematologic variables were assessed over 42 days. RESULTS: After 28 days, treated units had a 24-hour double-label (51Cr/99mTc) recovery (85.0 ± 5.0%) that was indistinguishable from control units (85.9 ± 2.7%); the times required for reduction of radioactivity of labeled cells to half the Day 1 activity (T50) were similar for both groups (treated, 31.9 ± 8.2 days; control, 32.9 ± 3.3 days). No deleterious effects of PEN110 treatment were found on RBC antigens tested. Treatment reduced 2,3-DPG levels by half and slightly lowered pH levels. Throughout the 42-day storage period, treated RBCs had a lower level of lactate production and a trend toward lower glucose consumption. Hemolysis remained below 1 percent; supernatant potassium and ATP levels were lower than those seen in control RBCs. CONCLUSION: PEN110-treated RBCs stored for 28 days meet all critical requirements for therapeutically useful units.

BACKGROUND: A pathogen-inactivation process for RBC concentrates is being developed by using PEN110 chemistry (INACTINE, V.I. Technologies). The objective of this study was to characterize the quality of RBCs prepared by using the PEN110 process and to measure the virucidal effect achieved against two viruses. STUDY DESIGN AND METHODS: Virology and RBC studies were conducted with standard RBC units treated with 0.1-percent (vol/vol) PEN110 at 22°C for 6 hours. The quality of PEN110-treated human RBCs was assessed with biochemical and phenotypic variables. The in vivo viability of PEN110-treated RBCs in baboons was studied with the double-label 51Cr/125I method. RESULTS: Decreases in infectious titer by inactivation of greater than a 5 log 50-percent tissue culture infectious doses per mL of bovine viral diarrhea virus (an enveloped RNA virus) and porcine parvovirus (a nonenveloped DNA virus) was observed. RBC hemolysis was less than 1 percent after 42 days of storage, and no changes in RBC antigens were observed. The in vivo viability of PEN110-treated baboon RBCs was unchanged from control. CONCLUSION: The preparation of RBCs by using the PEN110 process achieved a significant viral reduction of two diverse viruses without causing adverse effects to the RBCs. The process appears to be a promising approach, thus justifying further study.

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

DNA base lesions produced by free radicals are common products of normal oxidative metabolism. These lesions are removed by base excision repair processing, the first step of which is recognition of the base lesion by a DNA-glycosylase. In general, the oxidative DNA base lesions are recognized by either a pyrimidine-specific or a purine-specific DNA-glycosylase. In this chapter, we describe the biochemical and biological properties of the newest of these activities the pyrimidine-specific oxidative DNA glycosylase, endonuclease VIII (nei) of Escherichia coli. We also describe an in vitro reconstitution of the base excision repair pathway using model DNAs that contain closely opposed lesions similar to multiply damaged sites produced by ionizing radiation. Here we show that when closely opposed lesions are more than three nucleotides apart, processing by base excision repair can lead to a potentially lethal double strand break. Finally, we describe the interaction between two ring saturation products of pyrimidines, one derived from cytosine, uracil glycol, the other from thymine, thymine glycol, with a model DNA polymerase, DNA polymerase I of E. coli. Both incorporation of the modified nucleoside triphosphate and translesion synthesis past the lesion in the DNA template, are considered