Discovery and Preclinical Activity of BMS-986351, an Antibody to SIRPα That Enhances Macrophage-mediated Tumor Phagocytosis When Combined with Opsonizing Antibodies.

In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). SIGNIFICANCE: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.

Inactive full-length p53 mutants lacking dominant wild-type p53 inhibition highlight loss of heterozygosity as an important aspect of p53 status in human cancers.

Over 1000 different mutants of the tumor suppressor protein p53 with one amino acid change in the core domain have been reported in human cancers. In mouse knock-in models, two frequent mutants displayed loss of wild-type (wt) p53 function, inhibition of wt p53 and wt p53-independent gain of function. The remaining mutants have been systematically characterized for loss of wt p53 function, but not other phenotypes. We report the concomitant assessment of loss of function and interference with wt p53 using URA3-based p53 yeast and confirmatory mammalian assays. We studied 76 mutants representing 54% of over 15 000 reported missense core domain mutations. The majority showed the expected complete loss of wt p53 function and dominant p53 inhibition. A few infrequent p53 mutants had wt p53-like activity. Remarkably, one-third showed no interference with wt p53 despite loss of wt p53 function at 37 degrees C. Half of this group consisted of temperature-sensitive p53 mutants, but the other half was surprisingly made up of mutants with complete loss of wt p53 function. Our findings illustrate the diverse behavior of p53 mutants and mechanisms of malignant transformation by p53 mutants. The identification of full-length p53 mutants without dominant inhibition of wt p53 highlights the importance of determining the status of the wt p53 allele in human cancers, in particular in the context of clinical studies. In the case of p53 mutants with no or weak dominant p53 inhibition, presence of the wt allele may indicate a good prognosis cancer, whereas loss of heterozygosity may spell an aggressive, therapy-resistant cancer.

Functional census of mutation sequence spaces: the example of p53 cancer rescue mutants.

Many biomedical problems relate to mutant functional properties across a sequence space of interest, e.g., flu, cancer, and HIV. Detailed knowledge of mutant properties and function improves medical treatment and prevention. A functional census of p53 cancer rescue mutants would aid the search for cancer treatments from p53 mutant rescue. We devised a general methodology for conducting a functional census of a mutation sequence space by choosing informative mutants early. The methodology was tested in a double-blind predictive test on the functional rescue property of 71 novel putative p53 cancer rescue mutants iteratively predicted in sets of three (24 iterations). The first double-blind 15-point moving accuracy was 47 percent and the last was 86 percent; r = 0.01 before an epiphanic 16th iteration and r = 0.92 afterward. Useful mutants were chosen early (overall r = 0.80). Code and data are freely available (http://www.igb.uci.edu/research/research.html, corresponding authors: R.H.L. for computation and R.K.B. for biology).

A global suppressor motif for p53 cancer mutants.

The transcription factor and tumor suppressor protein p53 is frequently inactivated in human cancers. In many cases, p53 gene mutations result in high levels of inactive, full-length p53 protein with one amino acid change in the core domain that recognizes p53 DNA-binding sites. The ability to endow function to mutated p53 proteins would dramatically improve cancer therapy, because it would reactivate a central apoptotic pathway. By using genetic strategies and p53 assays in yeast and mammalian cells, we identified a global suppressor motif involving codons 235, 239, and 240. These intragenic suppressor mutations, either alone or in combination, restored function to 16 of 30 of the most common p53 cancer mutants tested. The 235-239-240 suppressor motif establishes that manipulation of a small region of the core domain is sufficient to activate a large number of p53 cancer mutants. Understanding the structural basis of the rescue mechanism will allow the pursuit of small compounds able to achieve a similar stabilization of p53 cancer mutants.

Effect of moderate exercise immediately followed by induced hyperglycemia on gene expression and content of the glucose transporter-4 protein in skeletal muscles of horses.

OBJECTIVE: To determine the effect of a single bout of exercise and increased substrate availability after exercise on gene expression and content of the glucose transporter-4 (GLUT-4) protein in equine skeletal muscle. ANIMALS: 6 healthy adult Thoroughbreds. PROCEDURES: The study was designed in a balanced, randomized, 3-way crossover fashion. During 2 trials, horses were exercised at 45% of their maximal rate of oxygen consumption for 60 minutes after which 1 group received water (10 mL/kg), and the other group received glucose (2 g/kg, 20% solution) by nasogastric intubation. During 1 trial, horses stood on the treadmill (sham exercise) and then received water (10 mL/kg) by nasogastric intubation. Muscle glycogen concentration and muscle GLUT-4 protein and mRNA content were determined before exercise and at 5 minutes and 4, 8, and 24 hours after exercise. RESULTS: Although exercise resulted in a 30% reduction in muscle glycogen concentration, no significant difference was detected in muscle GLUT-4 protein or mRNA content before and after exercise. Glycogen replenishment was similar in both exercised groups and was not complete at 24 hours after exercise. Horses that received glucose had significantly higher plasma glucose and insulin concentrations for 3 hours after exercise, but no effect of hyperglycemia was detected on muscle GLUT-4 protein or mRNA content. CONCLUSION: Under the conditions of this study, neither exercise nor the combination of exercise followed by hyperglycemia induced translation or transcription of the GLUT-4 protein in horses.

An AU-rich element in the 3' untranslated region of the C/EBP delta mRNA is important for protein binding during G0 growth arrest.

Posttranscriptional regulation at the level of mRNA stability is becoming increasingly recognized as an important mechanism to control the levels of mRNAs that encode key cell fate determining proteins. Previous work from our laboratory demonstrated that C/EBPdelta is a highly unstable mRNA in G(0) growth arrested mammary epithelial cells. In this report we investigated trans-acting factor binding to the C/EBPdelta 3'-UTR and identified a cis-acting element important for this interaction. RNA electromobility shift assays (REMSAs) demonstrate that the C/EBPdelta mRNA 3'-UTR binds trans-acting factor(s) present in G(0) growth arrested mammary epithelial cell lysates. This binding was not detected in the presence of lysates from growing cells. UV-binding analysis detected a RNA/protein complex of approximately 35kDa following incubation of the full-length C/EBPdelta 3'UTR with lysates from G(0) growth arrested mammary epithelial cells. Competition assays indicate that a specific AU-rich region (U1) is necessary for trans-acting factor binding to the C/EBPdelta 3'-UTR. These studies have identified an AU-rich element located within the C/EBPdelta 3'-UTR that interacts with a putative G(0) growth arrest-specific trans-acting factor(s), which may regulate C/EBPdelta mRNA decay.

Posttranscriptional and posttranslational regulation of C/EBP delta in G0 growth-arrested mammary epithelial cells.

Previous work from our laboratory demonstrated that CCAAT/enhancer-binding protein delta (C/EBP delta) functions in the initiation and maintenance of G(0) growth arrest in mouse mammary epithelial cells (MECs). In this report, we investigated the posttranscriptional and posttranslational regulation of C/EBP delta in G(0) growth-arrested mouse MECs. The results of transcriptional inhibitor studies demonstrated that the C/EBP delta mRNA exhibits a relatively short half-life in G(0) growth-arrested mouse MECs (t(1/2) approximately 35 min). In contrast, C/EBP delta mRNA has a longer half-life in G(0) growth-arrested mouse fibroblast cells (t(1/2) >100 min). Oligo/RNase H cleavage analysis and rapid amplification of cDNA ends-poly(A) test both confirmed the short C/EBP delta mRNA half-life observed in MECs and demonstrated that the C/EBP delta mRNA poly(A) tail is relatively short (approximately 100 nucleotides). In addition, the poly(A) tail length was not shortened during C/EBP delta mRNA degradation, which suggested a deadenylation-independent pathway. The C/EBP delta protein also exhibited a relatively short half-life in G(0) growth-arrested mouse MECs (t(1/2) approximately 120 min). The C/EBP delta protein was degraded in a ubiquitin-dependent manner, primarily in the nucleus, during G(0) growth arrest. In conclusion, these studies indicated that the C/EBP delta mRNA and protein content are under tight regulation in G(0) growth-arrested mouse MECs, despite the general concept that G(0) growth arrest is associated with a decrease in cellular activity.