No abstract

Human SIRT1 is an enzyme that deacetylates the p53 tumor suppressor protein and has been suggested to modulate p53-dependent functions including DNA damage-induced cell death. In this report, we used EX-527, a novel, potent, and specific small-molecule inhibitor of SIRT1 catalytic activity to examine the role of SIRT1 in p53 acetylation and cell survival after DNA damage. Treatment with EX-527 dramatically increased acetylation at lysine 382 of p53 after different types of DNA damage in primary human mammary epithelial cells and several cell lines. Significantly, inhibition of SIRT1 catalytic activity by EX-527 had no effect on cell growth, viability, or p53-controlled gene expression in cells treated with etoposide. Acetyl-p53 was also increased by the histone deacetylase (HDAC) class I/II inhibitor trichostatin A (TSA). EX-527 and TSA acted synergistically to increase acetyl-p53 levels, confirming that p53 acetylation is regulated by both SIRT1 and HDACs. While TSA alone reduced cell survival after DNA damage, the combination of EX-527 and TSA had no further effect on cell viability and growth. These results show that, although SIRT1 deacetylates p53, this does not play a role in cell survival following DNA damage in certain cell lines and primary human mammary epithelial cells. SIRT1 is an enzyme that catalyzes the deacetylation of acetyl-lysine residues by a mechanism in which NADϩ is cleaved and a unique product, O-acetyl ADP-ribose, is generated (4,19,21). In addition, the reaction results in the release of nicotinamide, which acts as an end product inhibitor (3). SIRT1 is distinct from the class I and II histone deacetylase (HDAC) enzymes that remove acetyl groups without hydrolysis of NAD ϩ (13). Indeed, SIRT1 catalytic activity is not affected by the class I and II HDAC inhibitor trichostatin A (TSA) (19).SIRT1 plays a role in a wide variety of processes including stress resistance, metabolism, differentiation, and aging (4). Overexpression of SIRT1 orthologs in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster leads to an increased life span of these organisms (23,43,49), and this has excited speculation that SIRT1 might also regulate life span in mammals (5). SIRT1 binds to and regulates the activity of several transcription factors, including FOXO1, FOXO3a, and FOXO4 (8,16,37,38,51), HES-1 and HEY-2 (48), MyoD (15), CTIP2 (46), PPAR␥ (40), NF-B (55), and PGC1␣ (42). SIRT1 has also been shown to interact with and deacetylate the p53 tumor suppressor protein (25,30,53). p53 is a key transcriptional regulator of genes involved in cell cycle progression, apoptosis, and DNA repair. Indeed, many human tumors have inactivated p53 protein (54). p53 becomes acetylated after DNA damage, and the acetylated form has been reported to have increased transcriptional activity, promote coactivator recruitment, and enhance site-specific DNA binding (2). Acetylation of p53 is also believed to increase p53 stability by preventing ubiquitination of key lysine residues and subsequent proteasom...

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.

This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.

The receptor mechanisms mediating the retrograde axonal transport of the neurotrophins have been investigated in adult rats. We show that transport of the TrkB ligands NT-4 and BDNF to peripheral neurons is dependent on the low affinity neurotrophin receptor (LNR). Pharmacological manipulation of LNR in vivo using either an anti-LNR antibody or a soluble recombinant LNR extracellular domain completely blocked retrograde transport of NT-4 and BDNF to sensory neurons, while having minimal effects on the transport of NGF in either sensory or sympathetic neurons. Furthermore, in mice with a null mutation of LNR, the transport of NT-4 and BDNF, but not NGF, was dramatically reduced. These observations demonstrate a selective role for LNR in retrograde transport of the various neurotrophins from distinct target regions in vivo.

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G 1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-⌬11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-⌬11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein.Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-⌬11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-⌬11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-⌬11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-⌬11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-⌬11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.Germ line mutations in BRCA1 predispose women to earlyonset breast and ovarian cancers (18,38). The BRCA1 gene is composed of 23 exons that encode a 1,863-amino-acid fulllength protein, over half of which is encoded by an unusually large exon, exon 11, which is 3.4 kb in length. In addition to the full-length BRCA1 protein, p220 BRCA1 , human cells contain alternatively spliced variants referred to as BRCA1-⌬11 (referred to here as p97 BRCA1 ) and BRCA1-⌬11b (referred to here as p110 BRCA1 ), which lack all and most of exon 11, respectively (54, 58). These isoforms arise from in-frame splicing events and retain the highly conserved amino-terminal RING finger and carboxyl-terminal BRCT domains found in fulllength BRCA1 but lack the nuclear localization signals previously identified in exon 11 (11,54,58). The abundant expression of p97 BRCA1 and p110 BRCA1 has been demonstrated in a variety of adult tissues, including the human mammary gland, in which transcripts encoding p110 BRCA1 are expressed at levels comparable to those encoding p220 BRCA1 (33,54,58). The observation that human BRCA1 is phosphorylated in response to UV light, ionizing radiation, and other agents that damage DNA, and the identification of BRCA1-interacting proteins such as RAD51 and RAD50-Mre11-p95 complexes that colocalize with BRCA1 following DNA damage have suggested a role for BRCA1 in DNA r...