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Summary Background Pancreatic cancer statistics are dismal, with a 5-year survival of less than 10%, and more than 50% of patients presenting with metastatic disease. Metabolic reprogramming is an emerging hallmark of pancreatic adenocarcinoma. CPI-613 is a novel anticancer agent that selectively targets the altered form of mitochondrial energy metabolism in tumour cells, causing changes in mitochondrial enzyme activities and redox status that lead to apoptosis, necrosis, and autophagy of tumour cells. We aimed to establish the maximum tolerated dose of CPI-613 when used in combination with modified FOLFIRINOX chemotherapy (comprising oxaliplatin, leucovorin, irinotecan, and fluorouracil) in patients with metastatic pancreatic cancer. Methods In this single-centre, open-label, dose-escalation phase 1 trial, we recruited adult patients (aged ≥18 years) with newly diagnosed metastatic pancreatic adenocarcinoma from the Comprehensive Cancer Center of Wake Forest Baptist Medical Center (Winston-Salem, NC, USA). Patients had good bone marrow, liver and kidney function, and good performance status (Eastern Cooperative Oncology Group [ECOG] performance status 0–1). We studied CPI-613 in combination with modified FOLFIRINOX (oxaliplatin at 65 mg/m 2 , leucovorin at 400 mg/m 2 , irinotecan at 140 mg/m 2 , and fluorouracil 400 mg/m 2 bolus followed by 2400 mg/m 2 over 46 h). We applied a two-stage dose-escalation scheme (single patient and traditional 3+3 design). In the single-patient stage, one patient was accrued per dose level. The starting dose of CPI-613 was 500 mg/m 2 per day; the dose level was then escalated by doubling the previous dose if there were no adverse events worse than grade 2 within 4 weeks attributed as probably or definitely related to CPI-613. The traditional 3+3 dose-escalation stage was triggered if toxic effects attributed as probably or definitely related to CPI-613 were grade 2 or worse. The dose level for CPI-613 for the first cohort in the traditional dose-escalation stage was the same as that used in the last cohort of the single-patient dose-escalation stage. The primary objective was to establish the maximum tolerated dose of CPI-613 (as assessed by dose-limiting toxicities). This trial is registered with ClinicalTrials.gov, number NCT01835041, and is closed to recruitment. Findings Between April 22, 2013, and Jan 8, 2016, we enrolled 20 patients. The maximum tolerated dose of CPI-613 was 500 mg/m 2 . The median number of treatment cycles given at the maximum tolerated dose was 11 (IQR 4–19). Median follow-up of the 18 patients treated at the maximum tolerated dose was 378 days (IQR 250–602). Two patients enrolled at a higher dose of 1000 mg/m 2 , and both had a dose-limiting toxicity. Two unexpected serious adverse events occurred, both for the first patient enrolled. Expected serious adverse events were: thrombocytopenia, anaemia, and lymphopenia (all for patient number 2; anaemia and lymphopenia were dose-limiting toxicities); hyperglycaemia (in patient number 7); hypokalaemia, hypoalbuminaemia, and sepsis (patient number 11); and neutropenia (patient number 20). No deaths due to adverse events were reported. For the 18 patients given the maximum tolerated dose, the most common grade 3–4 non-haematological adverse events were hyperglycaemia (ten [55%] patients), hypokalaemia (six [33%]), peripheral sensory neuropathy (five [28%]), diarrhoea (five [28%]), and abdominal pain (four [22%]). The most common grade 3–4 haematological adverse events were neutropenia (five [28%] of 18 patients), lymphopenia (five [28%]), anaemia (four [22%], and thrombocytopenia in three [17%]). Sensory neuropathy (all grade 1–3) was recorded in 17 (94%) of the 18 patients and was managed with dose de-escalation or discontinuation per standard of care. No patients died while on active treatment; 11 study participants died, with cause of death as terminal pancreatic cancer. Of the 18 patients given the maximum tolerated dose, 11 (61%) achieved an objective (complete or partial) response. Interpretation A maximum tolerated dose of CPI-613 was established at 500 mg/m 2 when used in combination with modified FOLFIRINOX in patients with metastatic pancreatic cancer. The findings of clinical activity will require validation in a phase 2 trial. Funding Comprehensive Cancer Center of Wake Forest Baptist Medical Center.

Evolutionary psychology has made enormous progress in understanding how individual and kin selection shape our sexual and family behaviors. In striking contrast, our understanding of the evolution of our uniquely massive scale of social cooperation (kinship-independent; subjectively, the “public” sphere) has been seriously incomplete. We briefly critique theories of human social evolution to identify specific limitations. We then review and expand a specific theory of the evolution of the uniquely human public domain. This theory is coherent and well-supported empirically. Moreover, this theory has the broad predictive fecundity not displayed by earlier, less complete theories. For example, we can predict/account for both individual human novelties (speech, cognitive virtuosity, etc.) and the salient features of the human historical record through the present. We argue that our discipline can now catalyze the long-sought unification of the social and natural sciences. Further, this new theoretical power allows us to understand and address diverse elements of contemporary human welfare with substantially improved clarity. We argue that evolutionary psychology is now robustly positioned to contribute to formulation of potent local and global public policies that can build and sustain a very substantially improved human future. We explore specific examples of such policy implications.

We report the analysis of CPI-613, the first member of a large set of analogs of lipoic acid (lipoate) we have investigated as potential anticancer agents. CPI-613 strongly disrupts mitochondrial metabolism, with selectivity for tumor cells in culture. This mitochondrial disruption includes activation of the well-characterized, lipoate-responsive regulatory phosphorylation of the E1α pyruvate dehydrogenase (PDH) subunit. This phosphorylation inactivates flux of glycolysis-derived carbon through this enzyme complex and implicates the PDH regulatory kinases (PDKs) as a possible drug target. Supporting this hypothesis, RNAi knockdown of the PDK protein levels substantially attenuates CPI-613 cancer cell killing. In both cell culture and in vivo tumor environments, the observed strong mitochondrial metabolic disruption is expected to significantly compromise cell survival. Consistent with this prediction, CPI-613 disruption of tumor mitochondrial metabolism is followed by efficient commitment to cell death by multiple, apparently redundant pathways, including apoptosis, in all tested cancer cell lines. Further, CPI-613 shows strong antitumor activity in vivo against human non-small cell lung and pancreatic cancers in xenograft models with low side-effect toxicity.

CPI-613 is a novel inhibitor of mitochondrial metabolism that has recently shown promise in early stage clinical trials for pancreatic cancer, lymphoma, and leukemia. It is a lipoate analogue that selectively inhibits pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). Since these two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle, CPI-613 significantly inhibits mitochondrial metabolism. Importantly, it has demonstrated a favorable safety profile in clinical trials, presumably due to its documented selectivity to cancer metabolism. Early phase clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. For example, in a recently published Phase I clinical trial in 18 patients with Stage 4 pancreatic cancer in combination with a modified FOLFIRINOX regiment, three (17%) had a complete response, eight (44%) had a partial response, three (17%) had stable disease, and four (22%) had progressive disease. Since altered cancer metabolism is a hallmark of cancer in general, we hypothesized that CPI-613 should have efficacy in other cancers, including prostate cancer, which is the second most common malignancy in men that affects over 160,000 men in the U.S annually. To test our hypothesis, we treated prostate cancer cell lines with CPI-613 in vitro and in vivo in a preclinical murine flank model. We found that CPI-613 was highly effective at killing prostate cancer cells in vitro, which was similar to its previously published potency in pancreatic cancer cells. Furthermore, we found in vitro therapeutic synergy with docotaxel, a standard of care treatment of advanced prostate cancer. We therefore tested CPI-613 in a preclinical Pc-3 flank model of androgen-resistant prostate cancer. Groups of mice (n=8) were treated 2-3 times per a week for 5 weeks with 2.5mg/kg of CPI-613, 10mg/kg of CPI-613 and control saline after tumors reached approximately 40mm3. CPI-613 was formulated in the same manner as it was for clinical trials. Both groups of mice treated with CPI-613 demonstrated a significant decrease in tumor growth without any toxicity. There was no observed difference between doses, which may be due to having crossed the threshold of drug uptake and concentration required in the mitochondria for a response. Furthermore, there was a significant increase in survival between treated groups and the control group, again without a dose effect. These results for the first time indicate biological activity of CPI-613 both in vitro and in vivo against prostate cancer and therefore warrants further investigation in representative preclinical prostate cancer models and in combination with standard-of-care therapies. Citation Format: Kiran K. Solingapuram Sai, Anirudh Sattiraju, Zuzana Zachar, Michael S. Dahan, Robert Shorr, Timothy S. Pardee, Paul M. Bingham, Akiva Mintz. Initial preclinical evaluation of a novel inhibitor of mitochondrial metabolism against prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4861.

CPI-613 is a lipoate analogue that has been shown to inhibit the pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (KGDH) complexes selectively in tumor cells (reviewed in Exp.Rev.Clin.Pharma. 7, 837). These two enzymes control the vast majority of carbon flow into the tricarboxylic acid (TCA) cycle and play a central role in mitochondrial metabolism. PDH converts pyruvate into acetyl-CoA, which in turn can enter the TCA cycle for cellular respiration. Since pyruvate is the final product of glycolysis, PDH serves to link glycolysis to the TCA cycle. Early clinical trials with CPI-613 have demonstrated very promising clinical responses in pancreatic cancer, leukemia, and lymphoma when used in combination with standard chemotherapy. Moreover, apparently homeostatic responses of tumor cells to CPI-613 inhibition of mitochondrial metabolism include compensatory upregulation of glucose uptake. Thus, we hypothesized that we can use 18F-Fludeoxyglucose (FDG)-PET/CT, a translational imaging approach that measures cellular glucose uptake, as a biomarker for CPI-613 cellular delivery and its targeting of mitochondrial metabolism. Therefore, in this work we evaluated the in vitro and in vivo glucose uptake in cancer cells and tumor xenografts after treatment with CPI-613. To measure glucose uptake in vitro we pulsed BxPc3 pancreatic cancer cells with 3H 2-deoxyglucose for 15 or 60 minutes following 2 hours of treatment with CPI-613. We observed a significant upregulation (~100% increase) of cellular 2-deoxyglucose uptake, consistent with a compensatory increase in glucose uptake as a result of successful targeting of mitochondrial metabolism. We therefore examined whether this upregulation occurs in vivo using FDG-PET/CT. Mice bearing BxPc3 flank tumors were treated with 50mg/kg of CPI-613 and underwent FDG-PET/CT scans 4 hours and 24 hours post-CPI-613 treatment. Similar to the in vitro response, tumors treated with CPI-613 exhibited a 75% increase in 18F-FDG uptake compared to untreated controls at 4 hours post therapy. In the 24 hour post-therapy scans, tumor 18F-FDG was significantly decreased, indicating tumor cell killing, which corresponds to previously published data that demonstrated efficacy of CPI-613 against BxPc3 tumor xenografts. These results indicate the potential for using the initial FDG flare seen on PET imaging as a biomarker to detect mitochondrial targeting by CPI-613 immediately after CPI-613 treatment, a strategy that may be used, after further clinical validation, to stratify responders to this novel mitochondrial inhibitor. Citation Format: Kiran Solingapuram Sai, Zuzana Zachar, Frankis Almaguel, Shawn D. Stuart, Michael S. Dahan, Moises Guardado, Stephanie Rideout, Minghui Wang, Anirudh Sattiraju, Paul M. Bingham, Boris Pasche, Akiva Mintz. FDG-PET imaging as a potential biomarker of mitochondrial targeting by CPI-613, a novel inhibitor of mitochondrial metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2867. doi:10.1158/1538-7445.AM2017-2867

Since Darwin we have been in possession of two superficially dissonant facts. On one hand, humans are merely one of millions of animal species, all products of common ancestry. On the other, humans enjoy a level of ecological dominance that is spectacularly, qualitatively greater than that of any other animal that ever lived, including our closest relatives. Moreover, this unique ascendancy results from a complex suite of attributes that are each individually also unprecedented, including cognitive virtuosity, complex language, and an expanded ethical sense. Collectively, these facts constitute the human uniqueness problem. In spite of its importance, the superficial complexity of this problem has frustrated attempts to resolve it. Though a vast body of earlier work produced important isolated insights, no earlier theory has proven complete or convincing. I briefly review here a new resolution of the human uniqueness problem.1 This new hypothesis appears to be the necessary theory-of-everything. It ostensibly accounts parsimoniously for every major nonstochastic feature of the human story from the origin of Homo approximately 2.0 to 2.5 million years ago through the present instant. I use secondary, review literature where possible here to improve interdisciplinary accessibility. As well, I apologize to the many investigators whose important work could not be directly referenced because of length constraints.

The extensive, persistent ecological dominance of humans is unprecedented. We display a highly derived social adaptation involving uniquely extensive cooperation among nonclose kin. Further, humans possess adaptive capabilities, including language, high cognitive function, and technological virtuosity not previously seen on this planet. Moreover, this suite of properties emerged and was refined very rapidly on a geological time scale. These diverse features of humans present what is referred to as the "human uniqueness problem." A theoretical interpretation of these phenomena is one of the largest remaining challenges to the scientific enterprise. While many interpretations have been proposed-several containing important individual insights-none has yet proven robust or complete. A straightforward resolution of the human uniqueness problem is proposed. It is argued that coalitional enforcement is necessary and sufficient to allow extensive nonkin cooperation, leading to all major elements of human uniquen...

© 2012 Bingham and Zachar, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Pyruvate Dehydrogenase Complex in Cancer: Implications for the Transformed State and Cancer Chemotherapy

The Drosophila suppressor-of-white-apricot [su(wa)] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(wa) expression is autoregulated at the level of pre-mRNA splicing is reported. su(wa) protein represses accumulation of the fully spliced su(wa) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(wa) pre-mRNA. Second, the fully spliced su(wa) mRNA is sufficient for all known su(wa) genetic functions indicating that it encodes the sole su(wa) protein. Third, the incompletely spliced su(wa) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(wa) expression during oogenesis allows maternal deposition exclusively of fully spliced su(wa) mRNA. Fifth, su(wa) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(wa) expression.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.

We report studies using an enhanced experimental system to investigate organization of nuclear pre-mRNA metabolism. It is based on the powerful genetic system and polytene nuclei of Drosophila. We observe (at steady state) movement of a specific pre-mRNA between its gene and the nuclear surface. This movement is isotropic, at rates consistent with diffusion and is restricted to a small nuclear subcompartment defined by exclusion from chromosome axes and the nucleolus. Bulk polyadenylated nuclear pre-mRNA precisely localizes in this same subcompartment indicating that most or all pre-mRNAs use the same route of intranuclear movement. In addition to association with nascent transcripts, snRNPs are coconcentrated with pre-mRNA in this subcompartment. In contrast to constitutive splices, at least one regulated splice occurs slowly and may undergo execution remotely from the site of pre-mRNA synthesis. Details of our results suggest that retention of incompletely spliced pre-mRNA is a function of the nuclear surface. We propose a simple model--based on channeled diffusion--for organization of intranuclear transport and metabolism of pre-mRNAs in polytene nuclei. We argue that this model can be generalized to all metazoan nuclei.

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.

We will describe recent progress in understanding the role of cancer metabolic remodeling in determining the response of tumors and tumor cells to a novel, first-in-class clinical agent class selectively targeting cancer cell mitochondrial metabolism. The reconfiguration of mitochondrial metabolism in cancer in general, and the tricarboxylic acid (TCA) cycle in particular, presents a rich source of potential therapeutic targets. Pyruvate dehydrogenase (PDH) and alpha ketoglutarate dehydrogenase (KGDH) are two gatekeeper enzymes regulating carbon flux into the mitochondrial TCA cycle. A key component of the altered regulatory processes in cancer is lipoic acid (lipoate), which not only functions as a cofactor for both PDH and KGDH, but also as source of regulatory information. Moreover, the lipoate-sensitive regulatory components of PDH and KGDH are substantially reconfigured in tumor cells, providing cancer-specific targets that can be successfully attacked with properly designed lipoate analogs. We developed and characterized CPI-613, a lipoic acid analog, which selectively targets mitochondrial metabolism in tumor cells by mobilizing lipoate-sensitive, cancer-specific regulation of PDH and KGDH, shutting down the two enzymes and thereby choking carbon flow through the TCA cycle. This attack on mitochondrial metabolism redundantly activates multiple cell death pathways (Zachar, et al., 2011, J Mol Med 89, 1137; Stuart, et al., 2014, Cancer&Metab 2, 4). In vitro, CPI-613 is uniformly effective in killing tumor cell lines (more than 50 tumor lines tested to date). Moreover, these drugs have produced compelling clinical responses (including complete remissions) in some, but not all patients with advanced, refractory, heavily pre-treated cancers in initial Phase I/II clinical trials (Pardee et al., 2014, Clin Cancer Res 20:5255). In an effort to explore the mechanisms of CPI-613 activity and characterize the reasons for differential patient responses, we have shown that the kinetics of the cancer cell death response to CPI-613 is profoundly affected by the carbon sources available to the cell (Zachar, et al., 2011, J Mol Med 89, 1137). For example, in media where pyruvate and glutamine (capable of producing net ATP synthesis only in the mitochondrial compartment) are the major carbon sources, death is rapid, completed within 4 hours (op cit). In contrast, when glucose is substituted for pyruvate, the cell death programs take up to 48 hours to complete. We have analyzed the mechanistic basis of this and related observations, including steady state and flux metabolomics, pharmacological inhibitors of non-TCA metabolic pathways, and manipulation of media composition to systematically characterize these dramatic effects of nutrient availability on cancer cell drug response. Our results demonstrate that CPI-613 induced inactivation of tumor cell KGDH is modulated by the potency of mitochondrial redox detoxification (Stuart, et al., 2014, Cancer&Metab 2, 4). Given that various metabolic pathways (for example, glycolysis) can ultimately contribute to mitochondrial ROS detoxification potential, it appears that substrate availability powerfully influences the cellular response to CPI-613. These insights open a number of fundamentally new approaches to increasing the fraction of patients who respond strongly to CPI-613 in the clinic. We will also report pre-clinical studies targeted at allowing planned future clinical trials to exploit these new insights. Citation Format: Shawn D. Stuart, Moises Guardado, Michael Dahan, Paul M. Bingham, Zuzana Zachar. Tumor metabolic remodeling can modulate anticancer drug response: CPI-613 attack on tumor cell mitochondrial metabolism is mediated by metabolite availability. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A61.

Discovery that a process might be ancient and phylogenetically widely distributed usually leads to dramatic increases in insight as diverse, idiosyncratic sources of experimental power are simultaneously brought to bear. Addition of the Drosophila genetic system to the power of plant and fungal genetics can be expected to produce substantive new understanding of cosuppression in the next several years. Moreover, it will be of considerable interest and potential technical value to establish whether cosuppression occurs in other animal lineages, including vertebrates, as the newly expanded body of evidence clearly predicts. In this context it will be of great interest to see further analysis of the recently reported transgene silencing of a normally imprinted mammalian gene to determine if this represents cosuppression or a more specialized mechanism associated with imprinting (Hatada et al. 1997xHatada, I., Nabetani, A., Arai, Y., Ohishi, S., Suzuki, M., Miyabara, S., Nishimune, Y., and Mukai, T. J. Biol. Chem. 1997; 272: 9120–9122Crossref | PubMed | Scopus (25)See all ReferencesHatada et al. 1997).

The targeting of tumor cell metabolism has emerged as an attractive candidate for chemotherapeutics. In particular, tumor cell mitochondria are dramatically reprogrammed to generate large amounts of biosynthetic intermediates to fuel cell division. We previously described a novel class of non-redox active lipoic acid analogs (“thioctoids”) which effect dramatic inactivation of the mitochondrial enzyme complex pyruvate dehydrogenase (PDH) (PMID:21769686). We have recently completed a comprehensive metabolomic study indicating that thioctoids down-regulate multiple mitochondria-associated enzymes. Each of these target enzymes are key control points for entry of carbon into the TCA cycle, including the alpha-ketoglutarate dehydrogenase complex and the branch-chain dehydrogenase/amino trasferase complex. In addition we will present data that reactive oxygen species (ROS) play a direct regulatory role in these inactivations and are a major contributor to the resultant cell cycle arrest and multiple cell death pathways which follow. Taken together, our results indicate that thioctoids are potent inhibitors of tumor cell mitochondrial energy metabolism, owing, apparently, to the central regulatory role of lipoic acid in mitochondrial lipoamide dehydrogenase-containing enzyme complexes. We hypothesize that thioctoids are ‘misread’ by these complexes resulting in increased levels of ROS leading to the further shutdown of redox-sensitive metabolic enzymes and ultimately cell death. As a consequence of these novel properties, thioctoids attack tumor-specific metabolism far more broadly and pervasively than other agents directed at individual metabolic steps or activities. Thioctoids are exceptionally well tolerated in animal studies and as a result may have high clinical potential. They are currently in early stage human clinical trials. 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 1122. doi:1538-7445.AM2012-1122

The copia insertion responsible for the wamutation is 3' to the white promotor and in the same transcriptional orientation as white. First, we have analyzed the effects of the wa copia insertion on levels of polyadenylated white transcripts and find large, developmentally programmed effects. Second, we have isolated and sequenced an LTR-excision event involving the copia insertion at wa. This represents the first documented case of an LTR-excision event in Drosophila. This single copia LTR has developmentally programmed effects on white transcript levels qualitatively similar to the intact copia element. Third, we have characterized the structures of white transcripts from wa. We find polyadenylated white transcripts apparently having 3' termini in or near the 3' LTR of the wa copia insertion, as has been reported in limited studies of wa transcription in adults by others. These earlier studies also revealed wa transcripts apparently corresponding to polyadenylated terminus formation in the 5' LTR of the copia transposon; however, our more detailed studies reveal that these transcripts probably have other origins and that little, if any, polyadenylated terminus formation for white transcripts occurs in the 5' LTR of the wa copia insertion. Moreover, we find no polyadenylated terminus formation for white transcripts occurring in the single LTR of the wa LTR-excision product. Fourth, we find that each of three mutant alleles at su(wa) produces elevated levels of several classes of RNAs apparently corresponding to transcriptional readthrough of the wa copia transposon. Elevated levels of one presumptive readthrough transcript were observed previously in one su(wa)1 mutant strain. Fifth, we have confirmed the existence of a transcript initiated in the 3' LTR of the wa copia insertion and find the levels of this transcript to be strongly influenced by developmental stage and genetic background. Lastly, we have analyzed white transcripts produced by the whd 81b11 allele, which carries an insertion of copia in the opposite transcriptional orientation and in a different position than the wa copia insertion. In contrast to the wa copia insertion allele, the whd 81b11 allele produces polyadenylated white transcript levels very similar to the w + case at the stages examined. Moreover, the whd 81b11 copia element apparently produced polyadenylated terminus formation in white transcripts and we observe no effect of the allelic state of su(wa) on apparent readthrough of this stop site. We discuss some possible implications of our results for the properties of retrotransposons as developmentally complex insertional mutagens and for the functional organization of the copia transcription unit.

The white locus of Drosophila melanogaster is a genetically well-characterized locus, mutations in which alter the degree of pattern of pigmentation of the eyes. Using a previously cloned DNA segment containing a portion of the white locus of a mutant allele, we have cloned and characterized the DNA of a 48-kilobase chromosomal region of the Canton S wild-type strain. We have mapped the positions, relative to restriction endonuclease cleavage sites, of several previously characterized chromosomal rearrangement breakpoints that bracket the while locus. These results define a segment of 14 kilobase that contains all of the white locus sequences necessary for the production of a wild-type eye color phenotype. By conventional criteria, no repetitive sequences are present within this 14-kilobase segment; however, we have identified an extremely weak DNA sequence homology between a portion of this segment and a chromosomal region in the vicinity of the zeste locus.

A new mutant allele (wDZL)at the white locus of Drosophila melanogaster is dominant to the wild-type allele, but apparently only when the two alleles are synapsed. When chromosomal rearrangements prevent somatic pairing between the two white alleles, wDZL is rendered recessive to wild type. This observation suggests that the dominance of wDZL is sensitive to a synapsis (transvection) effect. On the basis of this and other properties, it is proposed that wDZL causes the repression of transcription of a synapsed w+ allele, but not of a w+ allele elsewhere in the same nucleus. One model to account for this supposes that wDzL produces a repressor of white-locus transcription. This repressor is presumed to be so unstable that other white genes, removed from wDZL but in the same nucleus, are not detectably repressed. These properties may be simply understood if it is assumed that the repressor produced by the wDZL allele is an RNA molecule.

We report sequence and transcript structures for a 6191-base chromosomal segment containing the presumptive regulatory gene from Drosophila, suppressor-of-white-apricot [su(wa)]. Our results indicate that su(wa) expression is controlled by regulating occurrence of specific splices. Seven introns are removed from the su(wa) primary transcript during precellular blastoderm development. The sequence of this mature RNA indicates that it is a conventional messenger RNA. In contrast, after cellular blastoderm the first two of these introns cease to be efficiently removed. The mature RNAs resulting from this failure to remove the first two introns have structures quite unexpected of mRNAs. We propose that postcellular blastoderm su(wa) expression is repressed by preventing splices necessary to produce a functional mRNA. Implications and mechanisms are discussed.

We report genetic and molecular analysis of the suppressor-of-white-apricot [su(wa)] locus, one of several retrotransposon insertion allele-specific suppressor loci in Drosophila melanogaster. First, we isolated and characterized eight new mutations allelic to the original su(wa)1 mutation. These studies demonstrated that su(wa) mutations allelic to su(wa)1 affected a conventional D. melanogaster complementation group. Second, we cloned the chromosomal region containing the su(wa) complementation group by P element transposon tagging. The ca. 14-kilobase region surrounding the su(wa) complementation group contained five distinct transcription units, each with a different developmentally programmed pattern of expression. Third, we used a modified procedure for P-mediated gene transfer to identify the transcription unit corresponding to su(wa) by gene transfer. Fourth, we found that the presumptive su(wa) transcription unit produced a family of transcripts (ranging from ca. 3.5 to ca. 5.2 kilobases) in all developmental stages, tissue fractions, and cell lines we examined, suggesting that the gene is universally expressed.

We report results indicating that the temperature-sensitive white-blood (wbl) mutation has a novel molecular basis, involving the formation of RNA transcripts of the affected gene rather than the behavior of the polypeptide product. First, we show that the temperature-sensitive mutant phenotype of wbl correlates with (and presumably results from) a temperature-dependent effect on levels of the 2.6-kb polyadenylated white transcript. Second, DNA sequence analysis and other studies show that wbl is associated with the insertion into the second white intron of a previously uncharacterized retrotransposon (designated the blood transposon). We discuss the potential origins of the novel temperature-sensitive molecular phenotype of wbl and the prospects for exploiting wbl to engineer temperature-sensitive mutations in other genes.

We have identified and sequenced the portion of the white locus affected by an idiosyncratic set of white mutant alleles (the wsp alleles). The affected white locus portion (wsp region) extends from ca. 590 base pairs (bp) to ca. 1270 bp 5' to the apparent start site for the major white transcription unit. Based on the properties of these mutant alleles, we infer the existence of two distinct cis-acting regulatory elements in the wsp region and a third element mapping 3' to this region (3' to position ca. -670). Our analysis allows us to define the apparent position of one of the two wsp region elements with substantial precision. Examination of the DNA sequences in this region suggests that it is functionally similar to the enhancers identified in vertebrates. This same element participates in synapsis-dependent genetic interactions, suggesting a largely unexpected relationship between enhancer-like, cis-acting genetic elements and the genetic elements responsible for the synapsis-dependent genetic interactions in trans revealed by the existence of transvection effects. Our results further suggest that a presumptive regulatory locus (suppressor-of-white-spotted) regulates white transcription in adult tissues and is not involved in regulating white expression in larvae. We discuss the regulation of white expression in light of our studies. We also demonstrate unusual structures for an X-ray-induced deletion and a spontaneous deletion.

G-C leads to A-T transitions are induced by heat, and arise from the deamination of cytosine (5-hydroxymethylcytosine in the case of bacteriophage T4) generating uracil. The reaction is proton-catalyzed, and is also characteristic of acid mutagenesis. Mutation rates and activation energies of mutation are site-specific, and are presumably influenced by neighboring bases. Rates of heat-induced mutation in bacteriophage T4 under conditions of temperature, pH, and ionic strength similar to those prevailing in higher eukaryotic cells suggest that heat mutagenesis may present a serious challenge to organisms with large genomes, and may comprise an important determinant of the rates of spontaneous mutation.

Expression of the presumptive regulatory gene, suppressor-of-white-apricot [su(wa)], is controlled at the level of splicing. Results reported here indicate that this control represents autorepression of su(wa) expression. Specifically, reverse genetic studies demonstrate that the 3.5 kb mature su(wa) RNA (produced by removal of seven introns) is a message essential for su(wa)+ function and indicate that the abundant 4.4 kb and 5.2 kb mature su(wa) RNAs (resulting when the first or first and second of the seven introns are not removed) are, unexpectedly, byproducts of repression of production of the functional 3.5 kb RNA. Moreover, several experiments indicate that this repression of splices necessary to produce the 3.5 kb RNA is dependent on the translation product of the 3.5 kb RNA itself. We propose that this regulatory gene autoregulates its expression by controlling splicing of its primary transcript.

Background Targeting cancer cell metabolism is recognized as a promising arena for development of cancer chemotherapeutics. Moreover, redox metabolism is also systematically altered in tumor cells. Indeed, there is growing reason to believe that tumor-specific alteration of redox control of metabolism will be central to understanding and attacking malignancy. We report here that lipoate analog CPI-613 attacks a gate-keeping, lipoate-using metabolic enzyme, alpha-ketoglutarate dehydrogenase (KGDH), by a redox mechanism selectively in tumors cells. Results CPI-613 inhibited KGDH function strongly and rapidly, selectively in tumor cells. Moreover, CPI-613 induced a correspondingly rapid, powerful redox signal in tumor cell mitochondria. This signal was associated with redox modification of KGDH (including extensive enzyme glutathionylation and redox blockage of enzyme lipoate sulfhydryls), correlating with KGDH inactivation. The source of this tumor-specific mitochondrial redox modulatory signal was not electron transport complexes (I or III), but was largely or entirely the E3 (dihydrolipoamide dehydrogenase) component of dehydrogenases, including KGDH. Finally, we demonstrated that KGDH activity was redox regulated (in tumor cells), as expected if a tumor-specific redox process (auto)regulates KGDH. Conclusions Our data demonstrate that lipoate analog CPI-613 attacks redox control of KGDH activity in tumor cells, perhaps by modulation of an existing lipoate-sensitive allosteric process normally governing tumor cell KGDH activity. Together with its previously reported, mechanistically distinct (non-redox) effects on the other major, lipoate-using mitochondrial metabolic enzyme, pyruvate dehydrogenase, CPI-613’s KGDH effects indicate that this agent simultaneously attacks multiple central, essential components of tumor cell metabolic regulation.