Zuzana Zachar

Regulation of white locus expression: The structure of mutant alleles at the white locus of Drosophila melanogaster

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.

On/off regulation of gene expression at the level of splicing

Abstract The past two years have seen the discovery of three independent cases in which expression of a eukaryotic protein gene product is turned on and off by controlling splicing events necessary to produce the corresponding mRNA. Various considerations suggest that such on/off regulation at the level of splicing may be unexpectedly common.

Developmental expression of a regulatory gene is programmed at the level of splicing.

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.

Evidence that a regulatory gene autoregulates splicing of its transcript.

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.

Non-redox-active lipoate derivates disrupt cancer cell mitochondrial metabolism and are potent anticancer agents in vivo

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.

A strategically designed small molecule attacks alpha-ketoglutarate dehydrogenase in tumor cells through a redox process

BackgroundTargeting 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.ResultsCPI-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.ConclusionsOur 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.

Nuclear pre-mRNA metabolism: channels and tracks

We review new evidence suggesting that metazoan nuclear pre-mRNA metabolism occurs in a small subnuclear compartment consisting of a network of channels defined by exclusion from various condensed structures. Nuclear components, including mRNA en route from the gene to the nuclear surface, apparently move through these channels by conventional diffusion.

Safety and tolerability of the first-in-class agent CPI-613 in combination with modified FOLFIRINOX in patients with metastatic pancreatic cancer: a single-centre, open-label, dose-escalation, phase 1 trial

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/m2, leucovorin at 400 mg/m2, irinotecan at 140 mg/m2, and fluorouracil 400 mg/m2 bolus followed by 2400 mg/m2 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/m2 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/m2. 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/m2, 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/m2 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.Background Pancreatic cancer statistics are dismal, with a five-year survival of less than 10%, and over 50% of patients presenting with metastatic disease. Metabolic reprogramming is an emerging hallmark of pancreatic adenocarcinoma, including aerobic glycolysis, oxidative phosphorylation, glutaminolysis, lipogenesis and lipolysis, autophagic status, and anti-oxidative stress. CPI-613 is a novel anti-cancer agent that selectively targets the altered form of mitochondrial energy metabolism in tumor cells, causing changes in mitochondrial enzyme activities and redox status which lead to apoptosis, necrosis and autophagy of tumor cells. Methods This is a phase 1 study to determine the maximum-tolerated dose (MTD) of CPI-613 when used in combination with modified FOLFIRINOX (oxaliplatin at 65 mg/m2 and irinotecan at 140 mg/m2, and fluorouracil 400 mg/m2 bolus and 2400 mg/m2 over 46 h) in combination with CPI-613 in patients with newly diagnosed metastatic pancreatic adenocarcinoma with good bone marrow, liver and kidney function and good performance status (NCT01835041 – closed to recruitment). A two-stage dose-escalation scheme (single patient and traditional 3+3 design) was applied. In the single patient stage, one patient was accrued per dose level. The starting dose of CPI-613 was 500 mg/m2/day; the dose level was then escalated by doubling the previous dose if there was no toxicity greater 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 toxicity attributed as probably or definitely related to CPI-613 was ≥ Grade 2. The dose level for CPI-613 for the first cohort in the traditional dose-escalation stage was the same as used in the last cohort of the single patient dose-escalation stage. Secondary objectives were safety, preliminary efficacy, and tissue collection for future analyses. Response rates, progression-free survival and overall survival data were assessed in the patients treated at the MTD. Findings Twenty patients were enrolled April 22, 2013 – January 8, 2016. The MTD of CPI-613 was 500 mg/m2. The median number of treatment cycles administered at the MTD was 11 (interquartile range, 4–19). Two patients enrolled at a higher dose (1000 mg/m2) both experienced a DLT (dose limiting toxicity). There were 2 unexpected serious adverse events (SAEs), both for the first patient enrolled: 1) possible leaching due to infusion of CPI-613 via non-PVC tubing, and 2) the patient re- accessed her port at home after accidental de-access. Neither incident resulted in a negative clinical outcome. Expected SAEs were: thrombocytopenia, anemia and lymphopenia (all for Patient #2, with anemia and lymphopenia being a DLT); hyperglycemia (Patient #7); hypokalemia, hypoalbuminemia and sepsis (Patient #11); and neutropenia (Patient #20). There was no grade 5 toxicity. For the 18 patients treated at the MTD, the most common Grade 3–4 toxicities were hypokalemia (6/18, 33%), diarrhea (5/18, 28%) and abdominal pain (4/18, 22%). Sensorial neuropathy (17/18, 94%) was managed with dose de-escalation or discontinuation per standard of care. None of the patients experienced grade 4 or 5 neuropathy. No patients died while on active treatment; 11 study participants died, with cause of death as terminal pancreatic cancer. Among the 18 patients treated with the MTD, there were 3 patients with a complete response (CR), 1 with a non-CR/non-progressive disease, 7 with a partial response (PR), 3 with stable disease, and 4 with PD. The partial + complete response rate was 61% (11/18). Interpretation The treatment was well tolerated and all end points were met. The intriguing signal of efficacy will require validation in a phase 2 study. Funding Comprehensive Cancer Center of Wake Forest Baptist Medical Center

Lipoic acid and lipoic acid analogs in cancer metabolism and chemotherapy

The lipoic acid (lipoate) coenzyme is unique in all of mammalian metabolism. It is not only crucial to the function of some of the major enzymes feeding carbon into the tricarboxylic acid cycle, but also generates dynamic regulatory information about the metabolic status of the mitochondrial matrix, ultimately functioning to control these metabolic fluxes. Moreover, these lipoate-sensitive regulatory processes are apparently systematically redesigned in tumor cells and the affected enzymes commonly become especially central to cancer metabolism. Thus, lipoate-sensitive regulatory processes constitute potentially uniquely valuable targets for chemotherapeutic attack. Our goal here is to review the current status of our knowledge relevant to the use of lipoate and lipoate analogs to therapeutically attack malignant disease.

Molecular cloning and genetic analysis of the suppressor-of-white-apricot locus from Drosophila melanogaster

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.