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Background: Triple negative breast cancer (TNBC) has a relative paucity of effective targeted therapies compared to other molecular subtypes, and with poor prognosis. Thus, new targeted therapeutics are needed. ONC201 is a first-in-class small molecule that induces caspase-mediated apoptosis in cancers. It recently showed efficacy in pediatric glioma, harboring the potential to be translated in advanced breast cancer. Here we evaluated the efficacy of ONC201 in TNBC cell lines, investigated proteomic/genomic markers that predict efficacy, and performed a synthetic lethal RNAi screening to identify rational combinational targets. Method: The CellTiter-Blue (CTB) cell viability or sulforhodamine B assay was used to measure the range of half-maximal inhibitory concentrations (IC50) of ONC201 in TNBC cell lines. 2D/3D high-throughput RNAi kinome screening was performed using ONC201-sensitive CAL51, resistant HCC70 cells. The top genes noted to augment ONC201 sensitivity by siRNA were analyzed using both protein-protein interactome analysis (STRING V.11) and Ingenuity Pathway Analysis (IPA) to identify contributing canonical pathways. Combinational anti-proliferative activity of ONC201 and target inhibitors were evaluated by CTB cell viability assay and quantified by CalcuSyn software. Identified potential partners were subsequently tested using 3D ex vivo spheroid assay, tumor xenograft animal model to measure tumor growth inhibition (TGI). Five NOD/SCID mice were treated per each group. Mice were treated with 50mg/kg twice daily dosing of ONC201, and/or 25mg /kg of palbociclib after tumor grew to 100mm3. Reverse phase protein array (RPPA) of both ONC201 treated and non-treated TNBC cell lysates was performed to identify pathways affecting the sensitivity to ONC201 by comparing the effect of protein level changes (individual and set) using 3-way ANOVA, R program. Results: Twenty TNBC cell lines exhibited varying IC50 values (2 μM to 40 μM). No correlation between Vanderbilt subtypes and IC50 was observed. RNAi kinome screening identified 65 overlapping target kinases. Pathway analyses revealed PI3K/Akt, MEK/ERK, CDK 2/4/6 as potential combination therapeutic partners. In the in vitro study, the MEK inhibitor trametinib and CDK 4/6 inhibitor palbociclib consistently showed synergistic TGI with ONC201 (combination index values [CI] = 0.1 - 0.7). AKT inhibitor MK-2206 showed moderate combinational TGI (CI = 0.5 - 0.9). PI3K inhibitors PF04691052, buparlisib, and dactolisib showed cell type-specific combinational TGI. RPPA analysis did not reveal direct apoptosis-regulators mediators of sensitivity to ONC201, yet identified 7 potential predictive markers (fibronectin, pHER2, PAR, PLK1, pRb, SOD2, and EMA) of resistance/sensitivity. It also provided the rationale to study CDK 4/6 inhibition as a combination partner. Ex vivo study of ONC201/palbociclib (CDK 4/6 inhibitor) treatment inhibited of the colony formation in CAL51 cells. Interestingly, ONC201/palbociclib did not induce synergy in HCC70 cells, while the ONC-201/trametinib pair was synergistic in both cell lines. CAL51 tumor xenograft in vivo study confirmed the synergy of ONC201/palbociclib. Conclusion: MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib demonstrated synergy with ONC201 yet palbociclib only in ONC201 sensitive CAL51. Detailed mechanisms of synergy are being investigated that can further guide clinical translation. Citation Format: Bora Lim, Christine Peterson, Elin Cho, Alexander Davis, Troy Pearson, Huey Liu, Minha Hwang, Debu Tripathy, Naoto T Ueno, Jangsoon Lee. ONC201 synergistically induces cell death in triple negative breast cancer with CDK 4/6 and MEK inhibition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-14.

Triple-negative breast cancer (TNBC) is a heterogeneous group of estrogen, progesterone, and HER2-negative breast cancers with poor clinical outcomes. The imipridone ONC201 is a G-protein-coupled dopamine receptor D2 modulator and an allosteric agonist of the mitochondrial protease caseinolytic protease P(ClpP), which induces apoptosis. Here, we aimed to develop a novel ONC201-based combination therapy targeting TNBC. We performed a reverse-phase protein array analysis of ONC201-treated/-untreated and -sensitive/-resistant cell lines to identify potential predictive biomarkers. A principal component analysis using measured protein expression levels, the apoptosis score (AS), and heatmaps of all the measured protein and AS-related protein expression levels did not show a clear correlation between the expression levels of a specific protein and ONC201 efficacy. Three-dimensional RNA interference kinome-wide library screening revealed the MAPK and PI3K/Akt pathways as potential synergistic therapeutic partners. The combination with the MEK inhibitor trametinib successfully inhibited the growth of both ONC201-sensitive/-resistant TNBC cell lines. The baseline ClpP level correlated with the efficacy of single-agent ONC201. Single and combination therapy increased caspase 3/7 activity. The predictive biomarkers and a detailed mechanism of synergy beyond an induction of caspase activation should be tested for translation into future studies.

International audience; Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is an autoimmune disorder which affects small-and, to a lesser degree, medium-sized vessels. ANCA-associated vasculitis encompasses three disease phenotypes: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). This classification is largely based on clinical presentations and has several limitations. Recent research provided evidence that genetic background, risk of relapse, prognosis, and co-morbidities are more closely related to the ANCA serotype, proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA, compared to the disease phenotypes GPA or MPA. This finding has been extended to the investigation of biomarkers predicting disease activity, which again more closely relate to the ANCA serotype. Discoveries related to the immunopathogenesis translated into clinical practice as targeted therapies are on the rise. This review will summarize the current understanding of the immunopathogenesis of ANCA-associated vasculitis and the interplay between ANCA serotype and proposed disease biomarkers and illustrate how the extending knowledge of the immunopathogenesis will likely translate into development of a personalized medicine approach in the management of ANCA-associated vasculitis.

Background: Aberrant expression of members of the ErbB family of receptor tyrosine kinases and its downstream pathways is known to play pivotal roles in breast cancer malignancy. HER2-positive breast cancer has an aggressive nature that is driven by HER2 gene amplification. Inflammatory breast cancer is a rare but the most aggressive clinical subtype of breast cancer. Triple-negative breast cancer is an aggressive receptor subtype of breast cancer. More than 30% of cases of each of these groups express EGFR. Emerging therapeutic resistance continues to be a challenge in recurrent and/or metastatic disease. Neratinib is a potent irreversible EGFR, HER2, and HER4 tyrosine kinase inhibitor that blocks signal transduction generated via these receptors. In this study, we sought to identify the optimal targets in different subtypes of breast cancer and synergistic partners to maximize the antitumor effect of neratinib against breast cancer cells in vitro and in vivo. Methods: Expression of EGFR and HER2 was assessed via Western blot in 31 breast cancer cell lines, including HER2-targeted drug-resistant cell lines (14 triple-negative breast cancer and 10 HER2-positive). The antiproliferative effects of neratinib were measured using cell proliferation and anchorage-independent colony-formation (soft agar) assays. A reverse-phase protein array was used to profile and validate the signaling networks induced by neratinib-based treatment. To identify the synergistic targets, RNA interference screening of a 779-kinome library was performed under multicellular tumor spheroid culture conditions. Results: In vitro proliferation data demonstrated that neratinib had a nanomolar range of half-maximal inhibitory concentrations in most of the tested cell lines, including T-DM1-resistant HER-2 breast cancer cell lines. Neratinib inhibited colony formation in a dose-dependent manner (P = 0.0001). Reverse-phase protein array data revealed that the efficacy of neratinib correlated with phosphorylated EGFR (R2 = 0.6055) or phosphorylated HER2 (R2 = 0.6734) expression level. Statistical analysis identified the 25 most relevant targets in the RNA interference screening, which we will validate for combinational antitumor effects with neratinib in in vitro and in vivo studies. Conclusion: We determined the efficacy of neratinib and identified target molecules to enhance neratinib efficacy in different breast cancer subtype cell lines with ErbB alterations. Our data inform new combined treatment strategies with neratinib for breast cancer that will lead to innovative designs using optimized neratinib combinations in clinical trials. Citation Format: Bora Lim, Jangsoon Lee, Troy Pearson, Liu Huey, Minha Hwang, Kuicheon Choi, Francesca Avogadri-Connors, Richard E. Cutler, Debu Tripathy, Naoto T. Ueno. Identification of molecular therapeutic targets that enhance the antitumor activity of neratinib in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 825.