Iron uptake in Uslilago maydis is mediated by production of extracellular hydroxamate siderophores. L-Ornitne N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the frost committed step of ferrichrome and ferrichrome A biosynthesis in U. maydis. We have characterized sidi, a gene coding for this enzyme, by complementation in trans, gene disruption, and DNA sequence analysis. A comparison of genomic DNA and cDNA sequences has shown that the gene is interrupted by three introns. The putative amino acid sequence revealed similarity with Escheichia coli lysine N6-hydroxylase, which catalyzes the hydroxylation of lysine, the first step in biosynthesis of aerobactin. Two transcription initiation points have been determined, both by PCR amplification of the 5' end of the mRNA and by primer extension. A 2.3-kb transcript which accumulates in cells grown under low iron conditions was detected by Northern hybridization. A less abundant 2.7-kb transcript was observed in cells grown in iron-containing medium. By contrast, constitutive accumulation ofthe 2.3-kb transcript was observed in a mutant carrying a disruption of urbsI, a gene involved in regulation of siderophore biosynthesis. Analysis of the pathogenicity of mutants carrying a null allele of sidl suggests that the biosynthetic pathway of siderophores does not play an essential role in the infection of maize by U. maydis.

The sid1 and urbs1 genes encode L-ornithine N5-oxygenase and a GATA family transcription regulator, respectively, for siderophore biosynthesis in Ustilago maydis. The basic promoter and iron-regulatory sequences of the U. maydis sid1 gene were defined by fusing restriction and Bal31 nuclease-generated deletion fragments of the promoter region with the Escherichia coli beta-glucuronidase (GUS) reporter gene. Sequences required for basal expression of sid1 mapped within 1043 bp upstream of the translation start site and include the first untranslated exon and first intron. Sequences needed for iron-regulated expression of sid1 were localized to a 306 bp region mapping 2.3 and 2.6 kb upstream of the ATG. The 306 bp region contains two G/TGATAA sequences, consensus DNA binding sites of GATA family transcription factors. Deletion or site-directed mutation of either or both GATA sequences resulted in deregulated expression of sid1. In vitro DNA binding studies showed that Urbs1 binds to the 3'-GATA site in the 306 bp iron-responsive region. However, deletion of 1.1 kb between the distal GATA sites and the basal promoter region led to deregulated expression of GUS, indicating that these GATA sequences are by themselves insufficient to regulate sid1. In vitro DNA binding and in vivo reporter gene analysis revealed that siderophores are not co-repressors of Urbs1.

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TPS3113 Background: RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of RNAi and antisense is to harness the activity of naturally occurring enzymes that degrade RNA. Variants of human ribonucleases (RNase) have been generated with diminished binding to their natural inhibitor inside cells, which allows the new proteins to kill cancer cells. QBI-139, a variant that retains 95% sequence identity to a naturally occurring RNase, has demonstrated efficacy as both a single agent and in combination against multiple tumor types in in vivo models of human cancer. A first in human phase I trial was designed and initiated for QBI-139. Methods: Since QBI-139 showed efficacy against multiple cancers in model systems, a first in human phase I trial was designed for patients with advanced solid tumors (NCT00818831). Patients receive QBI-139 by intravenous infusion once weekly for a cycle of three weeks. In the absence of disease progression or unacceptable toxicity, treatment can continue on the twenty one day cycle. The trial is a standard 3+3 design with cohorts of three to six patients receiving escalating doses of QBI-139 until the maximum tolerated dose (MTD) and/or recommended phase II dose is determined. The inclusion/exclusion criteria are typical for the patient population. Forty three patients have been treated to date (January 2012) without identification of dose limiting toxicity. The starting dose in the clinical trial was 3 mg/m2 while the most recently completed cohort was treated with a dose of 50.4 mg/m2. Dose escalation is ongoing. The primary outcome is to evaluate the toxicity and tolerability of QBI-139 in patients with advanced refractory solid tumors. This information will allow for identification of the maximum tolerated dose. Clinical exposure levels are being monitored by measuring the pharmacokinetics of QBI-139. Tumor response to QBI-139 will be measured using RECIST criteria. Since QBI-139 demonstrated broad efficacy in model systems, another outcome of the phase I trial may be identification of the indications for the next stage of clinical development.

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RNA has been recognized as a drug target for cancer therapy, as evidenced by the ongoing clinical trials of RNAi and antisense therapies. An alternative approach that circumvents the delivery and stability issues of those drugs is to harness the activity of naturally occurring RNA degradation enzymes. Human ribonuclease (RNase) variants have been generated with diminished binding to their natural inhibitor inside cells, which allows the new proteins to kill cancer cells. One of these RNase variants, referred to as QBI-139, demonstrated efficacy against multiple tumor types in xenograft models of human cancer and was selected for an ongoing first in human Phase I clinical trial. Non-small cell lung and ovarian cancer are two of the indications where QBI-139 has shown significant efficacy as a single agent in xenograft models. In planning for the next stage of clinical trials, the impact of combinations of QBI-139 plus standard of care agents in these diseases was determined both in vitro and in vivo. QBI-139 showed additive and synergistic activity in combination with cisplatin and docetaxel, which are standard of care for non-small cell lung and ovarian cancer, respectively. Fixed ratios of QBI-139 and the combination drug based on their EC50 values were tested. The Combination Index (CI) was determined using median effect analysis. Synergy was demonstrated with QBI-139 in combination with cisplatin against non-small cell lung cancer (A549 and H1975). An additive effect of QBI-139 and cisplatin was seen in ovarian (Ovcar-3) cancer. In ovarian cancer (SK-OV-3), a combination of docetaxel and QBI-139 was synergistic with QBI-139. The benefit of the combinations were repeated in xenograft models of both cancers and were signifcat relative to each agent alone. Non-small cell lung and ovarian cancer represent critical unmet needs and new, differentiated treatment approaches such as QBI-139 combination therapy are critical. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1838. doi:1538-7445.AM2012-1838