Emine Ercikan Abali

NAD+ Kinase as a Therapeutic Target in Cancer

NAD+ kinase (NADK) catalyzes the phosphorylation of nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide phosphate (NADP+) using ATP as the phosphate donor. NADP+ is then reduced to NADPH by dehydrogenases, in particular glucose-6-phosphate dehydrogenase and the malic enzymes. NADPH functions as an important cofactor in a variety of metabolic and biosynthetic pathways. The demand for NADPH is particularly high in proliferating cancer cells, where it acts as a cofactor for the synthesis of nucleotides, proteins, and fatty acids. Moreover, NADPH is essential for the neutralization of the dangerously high levels of reactive oxygen species (ROS) generated by increased metabolic activity. Given its key role in metabolism and regulation of ROS, it is not surprising that several recent studies, including in vitro and in vivo assays of tumor growth and querying of patient samples, have identified NADK as a potential therapeutic target for the treatment of cancer. In this review, we will discuss the experimental evidence justifying further exploration of NADK as a clinically relevant drug target and describe our studies with a lead compound, thionicotinamide, an NADK inhibitor prodrug. Clin Cancer Res; 22(21); 5189–95. ©2016 AACR.

Species-Specific Differences in Translational Regulation of Dihydrofolate Reductase

We have observed that rodent cell lines (mouse, hamster) contain approximately 10 times the levels of dihydrofolate reductase as human cell lines, yet the sensitivity to methotrexate (ED50), the folate antagonist that targets this enzyme, is similar. Our previous studies showed that dihydrofolate reductase protein levels increased after methotrexate exposure, and we proposed that this increase was due to the relief of feedback inhibition of translation as a consequence of methotrexate binding to dihydrofolate reductase. In the current report, we show that unlike what was observed in human cells, dihydrofolate reductase (DHFR) levels do not increase in hamster cells after methotrexate exposure. We provide evidence to show that although there are differences in the putative mRNA structure between hamster and human mRNA in the dihydrofolate reductase binding region previously identified, “hamsterization” of this region in human dihydrofolate reductase mRNA did not change the level of the enzyme or its induction by methotrexate. Further experiments showed that human dihydrofolate reductase is a promiscuous enzyme and that it is the difference between the hamster and human dihydrofolate reductase protein, rather than the DHFR mRNA, that determines the response to methotrexate exposure. We also present evidence to suggest that the translational up-regulation of dihydrofolate reductase by methotrexate in tumor cells is an adaptive mechanism that decreases sensitivity to this drug.

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy.

NAD+ kinase (NADK) is the only known cytosolic enzyme that converts NAD+ to NADP+, which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.

Antitumor and modeling studies of a penetratin-peptide that targets E2F-1 in small cell lung cancer

E2F-1, a key transcription factor necessary for cell growth, DNA repair, and differentiation, is an attractive target for development of anticancer drugs in tumors that are E2F “oncogene addicted”. We identified a peptide isolated from phage clones that bound tightly to the E2F-1 promoter consensus sequence. The peptide was coupled to penetratin to enhance cellular uptake. Modeling of the penetratin-peptide (PEP) binding to the DNA E2F-1 promoter demonstrated favorable interactions that also involved the participation of most of the penetratin sequence. The penetratin-peptide (PEP) demonstrated potent in vitro cytotoxic effects against a range of cancer cell lines, particularly against Burkitt lymphoma cells and small cell lung cancer (SCLC) cells. Further studies in the H-69 SCLC cell line showed that the PEP inhibited transcription of E2F-1 and also several important E2F-regulated enzymes involved in DNA synthesis, namely, thymidylate synthase, thymidine kinase, and ribonucleotide reductase. As the PEP was found to be relatively unstable in serum, it was encapsulated in PEGylated liposomes for in vivo studies. Treatment of mice bearing the human small cell lung carcinoma H-69 with the PEP encapsulated in PEGylated liposomes (PL-PEP) caused tumor regression without significant toxicity. The liposome encapsulated PEP has promise as an antitumor agent, alone or in combination with inhibitors of DNA synthesis.

Over the counter drugs (and dietary supplement) exercise: a team-based introduction to biochemistry for health professional students.

For successful delivery of basic science topics for health‐professional students, it is critical to reduce apprehension and illustrate relevance to clinical settings and everyday life. At the beginning of the Biochemistry course for Physician Assistants, a team‐based assignment was designed to develop an understanding of the mechanism of action, effectiveness, and toxicity of five common over the counter (OTC) drugs and dietary supplements, and place these familiar medicines in a political and historical context. The objectives of this exercise were to stimulate interest in biochemistry; to provide basic information on enzymes and enzyme inhibitors related to these drugs to be expanded upon later in the course; and to encourage active and interactive learning. Teams of five students were formed, and each student was given an information sheet on aspirin, alpha‐galactosidase, orlistat, dextromethorphan, or simvastatin, a low dose statin, which was previously available without prescription at pharmacies in the UK. After each member of the team acquired information on one OTC drug/dietary supplement by reading an assigned information sheet, the team was asked to go through a series of questions, and then submit answers to a quiz as a group. A high rate of success on the quiz, an overwhelmingly positive response on formal course evaluations, and enthusiastic exchanges during class suggested this team‐based session accomplished its goals.

A second target of benzamide riboside: dihydrofolate reductase.

Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin’s lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

An online guided e-journal exercise in pre-clerkship years: Oxidative phosphorylation in brown adipose tissue

The rationale for this mandatory, guided online e‐journal exercise is to foster the ability of students to independently read medical and scientific literature in a critical manner and to integrate journal reading with their basic science knowledge. After a lecture on oxidative phosphorylation, students were assigned to read an article on brown adipose tissue published in New England Journal of Medicine and were guided to analyze the article by answering online questions. After two iterations, student surveys about the project, its key pedagogical features, and ways to improve it suggest that the students perceived these exercises as active learning, which is clinically relevant and built on their course material. Furthermore, students agreed that the e‐journal project was useful for learning how to read an article, for reviewing the material learned in class, and for promoting evidence‐based medicine. This online e‐journal exercise models some aspects students will experience as future physicians, where it is essential to keep up with literature and extract relevant information on a tight physicians schedule. This study demonstrated the usefulness of guided e‐journal exercises as a simple effective active teaching tool for preclinical medical students, which can also be used for prehealth undergraduate programs.

Modeling and antitumor studies of a modified L–penetratin peptide targeting E2F in lung cancer and prostate cancer

E2F1-3a overexpression due to amplification or to mutation or loss of the retinoblastoma gene, induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more of these activating E2Fs is a recognized target in cancer therapeutics. In previous studies we identified by phage display, a novel 7-mer peptide (PEP) that bound tightly to an immobilized consensus E2F1 promoter sequence, and when conjugated to penetratin to increase its uptake into cells, was cytotoxic to several malignant cell lines and human prostate and small cell lung cancer xenografts. Based on molecular simulation studies that showed that the D-Arg penetratin peptide (D-Arg PEP) secondary structure is more stable than the L-Arg PEP, the L-Arg in the peptide was substituted with D-Arg. In vitro studies confirmed that it was more stable than the L- form and was more cytotoxic as compared to the L-Arg PEP when tested against the human castrate resistant cell line, DU145 and the human lung cancer H196 cell line. When encapsulated in PEGylated liposomes, the D-Arg-PEP potently inhibited growth of the DU145 xenograft in mice. Our findings validate D- Arg PEP, an inhibitor of E2F1and 3a transcription, as an improved second generation drug candidate for targeted molecular therapy of cancers with elevated levels of activated E2F(s).

Abstract 1237: Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer

BACKGROUND: Mutation or inactivation of the retinoblastoma protein is frequently involved in prostate cancer tumorigenesis resulting in overexpression/deregulation of E2F activity. E2F1-3a overexpression induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) bound tightly to an immobilized consensus E2F1 promoter sequence, was cytotoxic at low micro molar concentrations to many malignant cell lines and as the PEP was unstable in serum, the PEP was encapsulated in PEGylated liposomes and treatment of tumor xenografts of small cell lung cancer H-69 and DU145 tumors propagated in mice caused tumor regression. OBJECTIVE: To determine the antitumor activity and stability of two different modified penetratin peptides: D-Arg PEP (substituting L-Arginine with D-Arginine in the peptide sequence) and N-acetylated as well as C-methylated PEP analog. METHODS: DU145 (prostate cancer) and H196 (small cell lung cancer) cells were used. To compare the efficacy of the peptides, we tested the IC50s of peptides at different time points using the MTS assay. Drug combination experiment results were analyzed using the combination index (CI) method. Peptide conformational studies were carried out using the Amber 12 suite of biomolecular simulation programs. RESULTS: Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L-Arg peptide structure in water. D-Arg PEP was more potent compared to L-Arg PEP, and it was also found to be more resistant to degradation by serum proteases than the L-form. The other modified form, N-acetylated, C-methylated PEP was marginally more effective than the unmodified PEP. Drug combination studies showed that the D-Arg PEP in combination with docetaxel, caused synergistic cytotoxicity against DU 145 cells. Our findings validate D-Arg peptide, an inhibitor of E2F1and 3a transcription, as a drug candidate for targeted molecular therapy of prostate cancers with elevated levels of activated E2F’s. Studies in progress are evaluating the combination of the PEGylated liposome encapsulated D-Arg PEP in combination with docetaxel against DU145 xenografts and against primary prostate cancer cells. Supported in part by a grant from the Lung Cancer Research Foundation. Citation Format: Tazeem Shaik, Nitu Bansal, Nadine Johnson Farley, John Kerrigan, Olga Garbuzenko, Tamara Minko, Emine Abali, Zoltan Szekely, Kathleen Scotto, Debabrata Banerjee, Joseph Bertino. Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer. [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 1237. doi:10.1158/1538-7445.AM2015-1237