Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3.

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics.

Small molecule IAP antagonists - SMAC mimetics (SM) - are being developed as an anticancer therapy. SM therapy was demonstrated not only to sensitize tumor cells to TNFα-mediated cell death but also to exert immunostimulatory properties. Their good safety and tolerability profile, plus promising preclinical data, warrants further investigation into their various effects within the tumor microenvironment. Using in vitro models of human tumor cells and fibroblast spheroids co-cultured with primary immune cells, we investigated the effects of SM on immune cell activation. SM treatment induces the maturation of human PBMC- and patient-derived dendritic cells (DC), and modulates cancer-associated fibroblasts towards an immune interacting phenotype. Finally, SM-induced tumor necroptosis further enhances DC activation, leading also to higher T-cell activation and infiltration into the tumor site. These results highlight the relevance of using heterotypic in vitro models to investigate the effects of targeted therapies on different components of the tumor microenvironment.

Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6.

The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.

Pharmacological Profile of BI 847325, an Orally Bioavailable, ATP-Competitive Inhibitor of MEK and Aurora Kinases.

Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.

Clusterin is protective in pancreatitis through anti-apoptotic and anti-inflammatory properties.

Clusterin is overexpressed in pancreas during the acute phase of pancreatitis. We intended to clarify the role of clusterin expression in stressed exocrine pancreas. We performed in vitro experiments in transfected AR4-2J cells with modified expression levels of clusterin and in vivo studies in clusterin-deficient mice. AR4-2J cells were exposed to agents mimicking cell-stress during pancreatitis (cerulein, hydrogen peroxide, staurosporine or lysophosphatidylcholine). Clusterin-overexpressing AR4-2J cells showed higher viability after cell stress and accordingly reduced rates of apoptosis and lessened caspase-3 activation. Blockage of endogenous clusterin expression reduced viability and enhanced apoptosis. Presence of clusterin reduced NF-kappaB activation and expression of the NF-kappaB target genes TNF-alpha and MOB-1 under cell stress. Clusterin-deficient mice showed a more severe course of acute experimental pancreatitis with enhanced rates of apoptosis and inflammatory cell infiltration. We concluded that clusterin was protective during inflammation of exocrine pancreas because of its anti-apoptotic and anti-inflammatory functions.

Epithelial growth factor receptor status in primary and recurrent ovarian cancer.

Success of epidermal growth factor receptor (EGFR) targeting agents in different cancer types is related to EGFR gene mutations and/or copy number gains. We investigated the EGFR gene status and protein expression by DNA mutational analysis, fluorescence in situ hybridization (FISH), and immunohistochemistry in tumor tissues from 80 patients with primary and corresponding recurrent ovarian serous carcinomas. The patients were classified into six groups with ascending EGFR gene copy numbers. EGFR amplification and high polysomy (FISH+) was present in a significant fraction of the primary (20%) and recurrent (22%) ovarian carcinomas. On mutational analysis, only one tumor with a silent EGFR mutation was observed, and this was the only carcinoma with high-level amplification. EGFR protein immunoexpression was seen in 28% of primary and 33% of recurrent carcinomas and correlated to amplification in the primary tumors (P = 0.003). In recurrent carcinoma, moderate and strong EGFR expression was associated with amplification (P = 0.034). These molecular events potentially have impact on the responsiveness to EGFR targeting agents in ovarian cancer.