Andressa Ardiani

Bacterial Cytosine Deaminase Mutants Created by Molecular Engineering Show Improved 5-Fluorocytosine–Mediated Cell Killing In vitro and In vivo

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.

Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo

Herpes simplex virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We described earlier two strategies to overcome this limitation: (1) generation of HSVTK mutants with improved GCV activation potential and (2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants, mutant 30 and SR39, were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, shows significant reduction in IC50 values of approximately 12 500-fold, 100-fold, and 125-fold compared with HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses show that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1 mg kg−1) that has no effect on wild-type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2500-fold or 25-fold compared with HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC50 values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 seems to elicit stronger bystander killing effects in which 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1 mg kg−1 GCV. However, at a lower dose of GCV (0.1 mg kg−1), MGMK/SR39 seems to have slightly greater tumor growth inhibition properties compared with SR39 (P⩽0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1 mg kg−1 GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.

Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.

Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications

Bacterial- and yeast- encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD1525) or increased thermostability (yCDdouble, yCDtriple) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC50 values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD1525 displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD1525 appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications.

A poxviral-based cancer vaccine the transcription factor twist inhibits primary tumor growth and metastases in a model of metastatic breast cancer and improves survival in a spontaneous prostate cancer model

Several transcription factors play a role in the alteration of gene expression that occurs during cancer metastasis. Twist expression has been shown to be associated with the hallmarks of the metastatic process, as well as poor prognosis and drug resistance in many tumor types. However, primarily due to their location within the cell and the lack of a hydrophobic groove required for drug attachment, transcription factors such as Twist are difficult to target with conventional therapies. An alternative therapeutic strategy is a vaccine comprised of a Modified vaccinia Ankara (MVA), incorporating the Twist transgene and a TRIad of COstimulatory Molecules (B7-1, ICAM-1, LFA-3; TRICOM). Here we characterize an MVA-TWIST/TRICOM vaccine that induced both CD4+ and CD8+ Twist-specific T-cell responses in vivo. In addition, administration of this vaccine reduced both the primary tumor growth and metastasis in the 4T1 model of metastatic breast cancer. In the TRAMP transgenic model of spontaneous prostate cancer, MVA-TWIST/TRICOM alone significantly improved survival, and when combined with the androgen receptor antagonist enzalutamide, the vaccine further improved survival. These studies thus provide a rationale for the use of active immunotherapy targeting transcription factors involved in the metastatic process and for the combination of cancer vaccines with androgen deprivation.

Androgen deprivation therapy sensitizes triple negative breast cancer cells to immune-mediated lysis through androgen receptor independent modulation of osteoprotegerin

Among breast cancer types, triple-negative breast cancer (TNBC) has the fewest treatment options and the lowest 5-year survival rate. Androgen receptor (AR) inhibition has displayed efficacy against breast cancer preclinically and is currently being examined clinically in AR positive TNBC patients. Androgen deprivation has been shown to induce immunogenic modulation; the alteration of tumor cell phenotype resulting in increased sensitivity to immune-mediated killing. We evaluated the ability of AR inhibition to reduce the growth and improve the immune-mediated killing of breast cancer cells with differing expression of the estrogen receptor and AR. While AR expression was required for the growth inhibitory effects of enzalutamide on breast cancer cells, both enzalutamide and abiraterone improved the sensitivity of breast cancer cells to immune-mediated lysis independent of detectable AR expression. This increase in sensitivity was linked to an increase in cell surface tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor expression as well as a significant reduction in the expression of osteoprotegerin (OPG). The reduction in OPG was further examined and found to be critical for the increase in sensitivity of AR- TNBC cells to immune-mediated killing. The data presented herein further support the use of AR inhibition therapy in the AR+ TNBC setting. These data, however, also support the consideration of AR inhibition therapy for the treatment of AR- TNBC, especially in combination with cancer immunotherapy, providing a potential novel therapeutic option for select patients.

Mutations at serine 37 in mouse guanylate kinase confer resistance to 6-thioguanine.

Guanylate kinase (GMK) is an essential nucleoside monophosphate kinase that catalyzes the phosphorylation of guanine-monophosphate (GMP) and dGMP to yield GDP and dGDP, respectively, important precursors for nucleotide synthesis. GMK is also responsible for the activation of 6-thioguanine (6-TG), a drug widely used as chemotherapeutic agent to treat leukemia. Several mechanisms of resistance to 6-TG have been reported but a subset of drug resistant cells cannot be explained by these mechanisms. We propose that mutations in GMK could result in drug resistance. Because cells require the presence of a functional GMK for viability, mutations that arise that lead to 6-TG resistance must retain activity toward GMP. We report three amino acid substitutions at serine 37 (S37) in mouse GMK that display activity toward GMP by conferring genetic complementation to a conditional GMK-deficient Escherichia coli and in enzyme assays. When 6-TG is included in complementation studies, cells expressing wild-type GMK are sensitive whereas all S37 mutants examined are able to effectively discriminate against 6-TG and display a drug resistance phenotype. Activity of the three S37 mutant enzymes toward clinically relevant concentrations of 6-TGMP is undetectable. Mutations in GMK, therefore, represent a previously undescribed mechanism for 6-TG resistance.

Abstract 1341: Endocrine deprivation therapy increases the sensitivity of breast cancer cells to T cell-mediated lysis independently of estrogen receptor or androgen receptor status

Estrogen deprivation therapy has been used as the first line adjuvant hormonal therapy for breast cancer for over 20 years. Tamoxifen, the first drug discovered to inhibit estrogen receptor signaling, is used to treat premenopausal women with estrogen receptor positive tumors. Although tamoxifen can be therapeutic in most women with estrogen receptor positive tumors, some women do not respond and others eventually develop resistance. In addition, tamoxifen has minimal effect on the growth of estrogen receptor negative tumors, including triple negative breast cancer, which has the poorest prognosis. Furthermore, prolonged administration of estrogen deprivation therapy can increase a patient9s risk of developing secondary uterine/endometrial cancer, stroke and pulmonary embolism. As breast tumors develop resistant to estrogen deprivation therapy, in some cases, they can switch from estrogen to androgen-dependent growth. Recent studies have shown that enzalutamide, an androgen receptor signaling inhibitor, can reduce the growth of breast tumor cells regardless of their estrogen receptor status. We sought to determine if tamoxifen or enzalutamide could increase the sensitivity of breast tumor cells to T cell-mediated lysis, which would improve their therapeutic value by increasing the number of patients that could derive benefit from these therapies and possibly reducing the duration of treatment required for clinical benefit. We have observed that endocrine deprivation therapy, with either tamoxifen or enzalutamide, was able to increase the sensitivity of breast cancer cells to T cell-mediated killing and that this increased sensitivity occurs independently of estrogen or androgen receptor status. We have also been able to show that tamoxifen or enzalutamide increased the T cell-mediated killing of triple negative breast cancer cell lines, which are normally refractory to most standard treatments. Our hypothesis is that endocrine deprivation therapy has altered the expression of pro-apoptotic and anti-apoptotic genes leading to this increase in immune-mediated lysis. These data suggest that tamoxifen and enzalutamide may be useful in treating both estrogen/androgen receptor positive and estrogen/androgen receptor negative breast cancer, including triple negative breast cancer, when combined with immunotherapy. By combining endocrine deprivation therapy with cancer immunotherapy additional patients who may not have benefitted from the therapy alone may be able to achieve clinical benefit due to its synergistic activity with cancer immunotherapy. Citation Format: Anna R. Kwilas, Andressa Ardiani, Sofia R. Gameiro, James W. Hodge. Endocrine deprivation therapy increases the sensitivity of breast cancer cells to T cell-mediated lysis independently of estrogen receptor or androgen receptor status. [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 1341. doi:10.1158/1538-7445.AM2015-1341