Andrei A. Purmal

Oxidative DNA Lesions as Blocks to in Vitro Transcription by Phage T7 RNA Polymerase

In recent years, a link between the transcriptional state of damaged DNA and the rate at which it is repaired has been demonstrated in both prokaryotes and eukaryotes. DNA containing bulky adducts, cross-links, and UV damage processed by nucleotide excision repair is repaired at a higher rate when it is actively transcribed. For these damages, evidence exists that an RNA polymerase molecule, stalled opposite a lesion, works as a signal to initiate repair, thus linking the two processes. However, no conclusive demonstration exists between base excision repair processing and transcription. Accordingly, we have examined the ability of several oxidative DNA lesions to block in vitro transcription by phage T7 RNA polymerase. Previous and ongoing work in this laboratory suggests that the effect that these lesions have on DNA polymerases is greatly influenced by the sequence context in which they are found. Future work will examine if sequence context regulates the role of these lesions as blocks to transcription.

A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E. coli and T4

Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.

The Toll-Like Receptor 5 Agonist Entolimod Mitigates Lethal Acute Radiation Syndrome in Non-Human Primates

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<0.01) absolute survival advantage of 40–60% compared to vehicle treatment. Similar magnitude of survival improvement was also achieved with drug delivered 48 hours after irradiation. Improved survival was accompanied by predominantly significant (P<0.05) effects of entolimod administration on accelerated morphological recovery of hematopoietic and immune system organs, decreased severity and duration of thrombocytopenia, anemia and neutropenia, and increased clonogenic potential of the bone marrow compared to control irradiated animals. Entolimod treatment also led to reduced apoptosis and accelerated crypt regeneration in the gastrointestinal tract. Together, these data indicate that entolimod is a highly promising potential life-saving treatment for victims of radiation disasters.

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

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

Small-Molecule Xenomycins Inhibit All Stages of the Plasmodium Life Cycle

ABSTRACT 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.

Processing and Consequences of Oxidative DNA Base Lesions

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

Genotoxicity of two new carbazole derivatives with antifungal activity

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.

Abstract 3036: Targeting FACT complex with CBL0137 to overcome acquired resistance to EGFR-TKI in lung adenocarcinoma

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.

Preclinical Validation of a Single-Treatment Infusion Modality That Can Eradicate Extremity Melanomas

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.

Abstract 1611: 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

Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children9s 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