Anti-NaPi2b antibody-drug conjugate lifastuzumab vedotin (DNIB0600A) compared with pegylated liposomal doxorubicin in patients with platinum-resistant ovarian cancer in a randomized, open-label, phase II study.

Background: Lifastuzumab vedotin (LIFA) is a humanized anti-NaPi2b monoclonal antibody conjugated to a potent antimitotic agent, monomethyl auristatin E, which inhibits cell division by blocking the polymerization of tubulin. This study is the first to compare an antibody-drug conjugate (ADC) to standard-of-care in ovarian cancer (OC) patients. Patients and methods: Platinum-resistant OC patients were randomized to receive LIFA [2.4 mg/kg, intravenously, every 3 weeks (Q3W)] or pegylated liposomal doxorubicin (PLD) (40 mg/m2, intravenously, Q4W). NaPi2b expression and serum CA-125 and HE4 levels were assessed. The primary end point was progression-free survival (PFS) in intent-to-treat (ITT) and NaPi2b-high patients. Results: Ninety-five patients were randomized (47 LIFA; 48 PLD). The stratified PFS hazard ratio was 0.78 [95% confidence interval (95% CI), 0.46-1.31; P = 0.34] with a median PFS of 5.3 versus 3.1 months (LIFA versus PLD arm, respectively) in the ITT population, and 0.71 (95% CI, 0.40-1.26; P = 0.24) with a median PFS of 5.3 months versus 3.4 months (LIFA versus PLD arm, respectively) in NaPi2b-high patients. The objective response rate was 34% (95% CI, 22% to 49%, LIFA) versus 15% (95% CI, 7% to 28%, PLD) in the ITT population (P = 0.03), and 36% (95% CI, 22% to 52%, LIFA) versus 14% (95% CI, 6% to 27%, PLD) in NaPi2b-high patients (P = 0.02). Toxicities included grade ≥3 adverse events (AEs) (46% LIFA; 51% PLD), serious AEs (30% both arms), and AEs leading to discontinuation of drug (9% LIFA; 8% PLD). Five (11%) LIFA versus 2 (4%) PLD patients had grade ≥2 neuropathy. Conclusion: LIFA Q3W was well tolerated and improved objective response rate with a modest, nonstatistically significant improvement of PFS compared with PLD in platinum-resistant OC. While the response rate for the monomethyl auristatin E-containing ADC was promising, response durations were relatively short, thereby highlighting the importance of evaluating both response rates and duration of response when evaluating ADCs in OC. Clinical trials.gov: NCT01991210.

Autonomously replicating macronuclear DNA pieces are the physical basis of genetic coassortment groups in Tetrahymena thermophila.

The macronucleus of the ciliate Tetrahymena thermophila contains a fragmented somatic genome consisting of several hundred identifiable chromosome pieces. These pieces are generated by site-specific fragmentation of the germline chromosomes and most of them are represented at an average of 45 copies per macronucleus. In the course of successive divisions of an initially heterozygous macronucleus, the random distribution of alleles of loci carried on these copies eventually generates macronuclei that are pure for one allele or the other. This phenomenon is called phenotypic assortment. We have previously reported the existence of loci that assort together (coassort) and hypothesized that these loci reside on the same macronuclear piece. The work reported here provides new, rigorous genetic support for the hypothesis that macronuclear autonomously replicating chromosome pieces are the physical basis of coassortment groups. Thus, coassortment allows the mapping of the somatic genome by purely genetic means. The data also strongly suggest that the random distribution of alleles in the Tetrahymena macronucleus is due to the random distribution of the MAC chromosome pieces that carry them.

Pronuclear fusion failure: an alternate conjugational pathway in Tetrahymena thermophila, induced by vinblastine.

Vinblastine is shown to induce pronuclear fusion failure in conjugating Tetrahymena thermophila. In this alternate conjugational pathway gametic pronuclei are exchanged between conjugants but do not fuse. Each pronucleus undergoes one mitotic division to produce a new macro- and micronucleus. Genetic consequences of pronuclear fusion failure include the following: (1) the progeny are whole genome homozygotes with nuclei derived from single meiotic products, and (2) half of the progeny are heterokaryons with micro- and macronuclei of different genetic origins. These facts make this process extremely useful in strain construction and mutant isolation. The induction of pronuclear fusion failure by vinblastine suggests that microtubules play an essential role in pronuclear fusion.

Three binding sites for AraC protein are required for autoregulation of araC in Escherichia coli.

Three binding sites for AraC protein were shown to be required for the autoregulation of araC: araI1, araO1, and araO2. Selective inactivation of AraC-binding sites on the DNA demonstrated that araO1 and araO2 are required in vivo to produce repression of araC in the presence of arabinose, whereas araI1 and araO2 are required in its absence. We found that the low-affinity site araO2 is essential for araC autoregulation; araO1 and araI1 provide high-affinity AraC-binding sites, which allow cooperative binding at araO2. Profound effects on the araBAD promoter and the araC promoter are produced by ligand-induced changes in AraC occupancy of functional sites on the DNA. We suggest that AraC exerts its multiplicity of controls through two alternative states of cooperative interactions with DNA and we illustrate this with a model. This model presents our interpretations of activation and repression of the araBAD operon and the autoregulation of the araC gene.

Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter.

The state of Escherichia coli araI DNA occupancy by AraC protein has been found to change from a two-turn to a four-turn occupancy upon the addition of the inducer arabinose. The araI site is separable into two contiguous regions, araI1 and araI2. araI1 binds both ligand-bound and ligand-free AraC protein, whereas araI2 binds AraC protein in the presence of arabinose only. A mutation in araI and a known mutation in araC led to the loss of araI2 binding, while binding to araI1 was unaffected. Both mutants failed to activate the promoter of the araBAD operon. We propose that araI2 occupancy by AraC protein leads to RNA polymerase recognition of the araBAD promoter and that araI1 acts as a switch mechanism allowing both the repressor and the activator forms of AraC protein to regulate the araBAD promoter.

Repression and catabolite gene activation in the araBAD operon.

Catabolite gene activation of the araBAD operon was examined by using catabolite gene activator protein (CAP) site deletion mutants. A high-affinity CAP-binding site between the divergently orientated araBAD and araC operons has been previously identified by DNase I footprinting techniques. Subsequent experiments disagreed as to whether this site is directly involved in stimulating araBAD expression. In this paper, we present data showing that deletions generated by in vitro mutagenesis of the CAP site led to a five- to sixfold reduction in single-copy araBAD promoter activity in vivo. We concluded that catabolite gene activation of araBAD involves this CAP site. The hypothesis that CAP stimulates the araBAD promoter primarily by relieving repression was then tested. The upstream operator araO2 was required for repression, but we observed that the magnitude of CAP stimulation was unaffected by the presence or absence of araO2. We concluded that CAP plays no role in relieving repression. Other experiments showed that when CAP binds it induces a bend in the ara DNA; similar bending has been reported upon CAP binding to lac DNA. This conformational change in the DNA may be essential to the mechanism of CAP activation.

A microtubule meshwork associated with gametic pronucleus transfer across a cell-cell junction.

In conjugating Tetrahymena, a cellular assembly composed of a microtubule meshwork appears to be required for the transfer of gametic pronuclei across the junction that separates the conjugating cells. This assembly is suggestive of a gametogenic cell division in ancient predecessors of ciliates, with Tetrahymena retaining only the associated nuclear division and export.

Cytogamy: An Inducible, Alternate Pathway of Conjugation in TETRAHYMENA THERMOPHILA.

We report the occurrence of cytogamy in Tetrahymena thermophila. By analogy to Paramecium, cytogamy generates exconjugant clones that derive their entire genetic information from a single meiotic product of their cytoplasmic parent. Thus, "instant" whole-genome homozygotes are created. Cytogamy has been induced in every strain of T. thermophila tested, and most of the excytogamous progeny have exhibited high fertility. The high frequency with which cytogamy can be induced by hyperosmotic shock, coupled with the foregoing genetic properties, make this process a practical (and already proven) method for the isolation of recessive mutants in T. thermophila. We also report that the cytogamy-inducing treatment induces other rare abnormalities of genetic transmission, which have not yet been characterized.

Osmotic shock prevents nuclear exchange and produces whole-genome homozygotes in conjugating Tetrahymena.

Exposure of conjugating Tetrahymena to a hyperosmotic shock blocks the exchange of gametic nuclei and produces self-fertilized exconjugants that are homozygous for their whole genome. Cells are sensitive to this induction during a brief period after meiosis. The high efficiency of the treatment and the fertility of the progeny make this a useful method for the isolation of induced recessive mutations and enhances the value of Tetrahymena as an animal-cell model system in which genetic dissection is practical. The sharp peak of sensitivity is useful in the study of those cellular mechanisms responsible for the independent handling of several functionally distinct nuclei during conjugation.