A phase I dose-escalation and dose-expansion study of brontictuzumab in subjects with selected solid tumors.

Background: Brontictuzumab is a monoclonal antibody that targets Notch1 and inhibits pathway activation. The purpose of this first-in-human study was to determine the maximum tolerated dose (MTD), safety, pharmacokinetics, immunogenicity and preliminary efficacy of brontictuzumab in patients with solid tumors. Patients and methods: Subjects with selected refractory solid tumors were eligible. Brontictuzumab was administered intravenously at various dose levels and schedule during dose escalation, and at 1.5 mg/kg every 3 weeks (Q3W) during expansion. Evidence of Notch1 pathway activation as determined by an immunohistochemistry assay was required for entry in the expansion cohort. Adverse events were graded according to the NCI-CTCAE v 4.03. Efficacy was assessed by RECIST 1.1. Results: Forty-eight subjects enrolled (33 in dose escalation and 15 in the expansion phase). The MTD was 1.5 mg/kg Q3W. Dose-limiting toxicities were grade 3 diarrhea in two subjects and grade 3 fatigue in one subject. The most common drug-related adverse events of any grade were diarrhea (71%), fatigue (44%), nausea (40%), vomiting (21%), and AST increase (21%). Brontictuzumab exhibited nonlinear pharmacokinetics with dose-dependent terminal half-life ranging 1-4 days. Clinical benefit was seen in 6 of 36 (17%) assessable subjects: 2 had unconfirmed partial response (PR) and 4 subjects had prolonged (≥ 6 months) disease stabilization (SD). Both PRs and three prolonged SD occurred in adenoid cystic carcinoma (ACC) subjects with evidence of Notch1 pathway activation. Pharmacodynamic effects of brontictuzumab were seen in patients' blood and tumor. Conclusion: Brontictuzumab was well tolerated at the MTD. The main toxicity was diarrhea, an on-target effect of Notch1 inhibition. An efficacy signal was noted in subjects with ACC and Notch1 pathway activation. ClinicalTrials.gov identifier: NCT01778439.

Inhibition of p38alpha MAPK enhances proteasome inhibitor-induced apoptosis of myeloma cells by modulating Hsp27, Bcl-X(L), Mcl-1 and p53 levels in vitro and inhibits tumor growth in vivo.

Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.

Altered patterns of gene expression in response to myocardial infarction.

The use of cDNA microarrays has made it possible to simultaneously analyze gene expression for thousands of genes. Microarray technology was used to evaluate the expression of >4000 genes in a rat model of myocardial infarction. More than 200 genes were identified that showed differential expression in response to myocardial infarction. Gene expression changes were monitored from 2 to 16 weeks after infarction in 2 regions of the heart, the left ventricle free wall and interventricular septum. A novel clustering program was used to identify patterns of expression within this large set of data. Unique patterns were revealed within the transcriptional responses that illuminate changes in biological processes associated with myocardial infarction.

Preferential activation of Fgf8 by proviral insertion in mammary tumors of Wnt1 transgenic mice.

Mouse mammary tumor virus (MMTV) is an insertional mutagen that has been demonstrated to transcriptionally activate flanking cellular proto-oncogenes. Previously we have used MMTV infection to accelerate mammary tumorigenesis in Wnt1 transgenic mice in order to identify genes that cooperate with the Wnt1 oncogene. Initial investigations into the resulting tumor collection, screened primarily by Southern analysis, showed that three fibroblast growth factor genes, Fgf8, Fgf3 and Fgf4, sustain activating insertion mutations in 10%, 42% and 6% of the tumors, respectively. Here, in an examination of the tumors from MMTV-infected Wnt1 transgenic mice that emphasizes Northern analysis, we report transcriptional activation of Fgf8 in 30 additional tumors (increasing the percentage of activations to 50%), while no significant changes in the activation frequency of Fgf3 or Fgf4 were found. To determine the frequency of insertional activation in normal mice, we examined tumors from MMTV-infected nontransgenic littermates of the Wnt1 transgenics and from MMTV-infected BALB/c mice. Fgf8, Fgf3 and Fgf4 were found to be activated in 11%, 80% and 5%, respectively, of the tumors in the combined nontransgenic groups. Thus, there appears to be an increased predisposition for Fgf8 activations in Wnt1 transgenic mice versus normal mice, suggesting that cells expressing Wnt1 are especially sensitized to stimulation by FGF8 compared with FGF3 or FGF4. In contrast, the activation frequency of Fgf3 in tumors from MMTV-infected Wnt1 transgenic mice was approximately one-half that of normal mice. Our results show that this in vivo model of multistep tumorigenesis reveals significant differences in the activation rates of Fgf3 and Fgf8 depending upon the status of Wnt1 expression in the mammary gland. The differential activation of these Fgfs may relate to differences in their signaling pathways.

Regulatory regions of the homeotic gene proboscipedia are sensitive to chromosomal pairing.

We have identified regulatory regions of the homeotic gene proboscipedia that are capable of repressing a linked white minigene in a manner that is sensitive to chromosomal pairing. Normally, the eye color of transformants containing white in a P-element vector is affected by the number of copies of the transgene; homozygous flies have darker eyes than heterozygotes. However, we found that flies homozygous for select pb DNA-containing transgenes had lighter eyes than heterozygotes. Several pb DNA fragments are capable of causing this pairing sensitive (PS) negative regulation of white. Two fragments in the upstream DNA of pb, 0.58 and 0.98 kb, are PS; additionally, two PS sites are located in the second intron, including a 0.5-kb region and 49-bp sequence. This phenotype is not observed when two PS sites are located at different chromosomal insertion sites (in trans-heterozygous transgenic animals), indicating that the pb-DNA-mediated repression of white is dependent on the pairing or proximity of the PS regions. The observed phenomenon is similar to transvection in which certain alleles of a gene can complement each other, but only when homologous chromosomes are paired. Interestingly, the intronic PS regions contain positive regulatory sequences for pb, whereas the upstream PS sites contain pb negative regulatory elements.

Molecular control of the induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster larvae.

The activity of alcohol dehydrogenase (ADH:EC 1.1.1.1), the initial enzyme in the major pathway for ethanol degradation, is induced in Drosophila melanogaster larvae by low concentrations of dietary ethanol. Two lines of evidence indicate that the metabolic products of the ADH pathway for ethanol degradation are not directly involved in the induction of Adh. First, the accumulation of the proximal transcript in Adhn2 larvae was increased when the intracellular level of ethanol was elevated. In addition, the ADH activity, the proximal Adh mRNA, and the intracellular concentration of ethanol were elevated coordinately in wild-type larvae fed hexadeuterated-ethanol, which is metabolized more slowly than normal ethanol. An examination of P element transformant lines with specific deletions in the 5' regulatory DNA of the Adh gene showed that a DNA sequence between +527 and +604 of the distal transcript start site is essential for the induction of the Adh gene [corrected]. The DNA sequence between -660 and about -5000 of the distal transcript start site was important for the down-regulation of the induction response.

A functional analysis of 5', intronic and promoter regions of the homeotic gene proboscipedia in Drosophila melanogaster.

In Drosophila, the homeotic gene proboscipedia (pb) is required for the formation of the adult mouthparts. To determine the functional significance of putative pb regulatory DNA, we have performed an in vivo analysis of sequences upstream of and within pb using a series of minigenes. Additionally, we have initiated a dissection of pb's promoter and enhancer elements using lacZ reporter gene constructs. Our results establish that a conserved region located in the second intron is essential for proper formation of the adult mouthparts. A 0.5 kb fragment from this region was shown to direct lacZ expression in a pb pattern in both embryos and third instar labial discs when combined with a 600 bp pb basal promoter sequence. A 32 bp element contained within the 0.5 kb region functions as a labial disc enhancers for pb. Surprisingly, the conserved second intron pb enhancers do not function properly with a heterologous hsp70 promoter, suggesting that promoter-specific interactions occur at the pb locus. We also found redundant and cryptic enhancers in the large introns of pb that are not required for pb function. Finally, we demonstrate that the pb transcription unit does not require sequences upstream of -98 bp to provide pb function in the labial discs. Rather, pb's upstream DNA appears to contain negative regulatory DNA required for silencing PB accumulation in inappropriate domains of third instar imaginal discs. Thus, we have defined many of pb's cis-controlling sequences to an experimentally manageable size, thereby making this an attractive system for the discovery of transacting proteins and, consequently, for elucidating the mechanisms of homeotic gene regulation.