Background: Bromodomain and Extra-Terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidences sustain BET targeting as a promising novel approach for cancer treatment. BET degraders are chimeric compounds merging a BET inhibitor, which allows the binding to BET bromodomains, linked to an additional small molecule, which mediates the binding to an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These compounds are based on the concept of the PROTAC (Proteolysis targeting Chimera) (Sakamoto et al, PNAS 2001). MZ1 is a potent BRD4 degrader that exploits JQ1 for BET bromodomain binding and the von Hippel Lindau-containing complex for protein degradation (Zengerle et al, ACS Chem Biol 2015; Gadd et al., Nat Chem Biol 2017). MZ1 showed potent antiproliferative activity in AML cell lines (Chan et al. J Med Chem 2017). Here, we present data on the antitumor activity of MZ1 in diffuse large B cell lymphoma of the activated B cell like (ABC DLBCL). Materials and Methods: Established lymphoma cell lines were exposed for 72 hours to increasing doses of the compounds. Cell proliferation was evaluated by using MTT assay. FACS analysis was performed to measure apoptotic activation (Annexin V, threshold of 1.5 fold) after 72 hours of treatment. Results: The antitumor activity of the BET degrader MZ1, of its negative control epimer cisMZ1 and, as comparison, of the BET inhibitor OTX015/MK-8628 was assessed in seven cell lines derived from diffuse large B cell lymphoma of the activated B cell like (ABC DLBCL). MZ1 was very active with a median IC50 of 50nM (range, 5-150 nM). No activity was seen with cisMZ1. BET degrader was more active in vitro than the BET inhibitor, which presented a median IC50 of 125 nM (range, 80-400 nM) (P=0.024). Importantly, MZ1 (500nM, 72 h) induced apoptosis in all the seven ABC DLBCL cell lines, while, in accordance with previous data (Boi et al, Clin Cancer Res 2015), OTX015 (500nM, 72 h) was cytotoxic only in 2/7 cell lines (P=0.021). No apoptosis was observed with cisMZ1 (500nM, 72 h). Conclusion: The BRD4 degrader MZ1 had a strong cytotoxic activity in all the ABC-DLBCL cell lines we tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations. Citation Format: Chiara Tarantelli, Hillarie Ekeh, Carmelo Moscatello, Eugenio Gaudio, Andrea Testa, Emanuele Zucca, Anastasios Stathis, Alessio Ciulli, Francesco Bertoni. The BRD4 degrader MZ1 exhibits potent antitumoral activity in diffuse large B cell lymphoma of the activated B cell-like type [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A179.

Aim: Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that play a fundamental role in transcription regulation. Preclinical and early clinical evidence sustain BET targeting as an anti-cancer approach. BET degraders are chimeric compounds comprising of a BET inhibitor, which allows the binding to BET bromodomains, linked to a small molecule, binder for an E3 ubiquitin ligase complex, triggering BET proteins degradation via the proteasome. These degraders, called proteolysis-targeting chimeras (PROTACs), can exhibit greater target specificity compared to BET inhibitors and overcome some of their limitations, such as the upregulation of the BET proteins themselves. Here are presented data on the anti-tumor activity and the mechanism of action of the BET degrader MZ1 in diffuse large B cell lymphoma (DLBCL) of the activated B-cell like (ABC, ABC DLBCL), using a BET inhibitor as a comparison. Methods: Established lymphoma cell lines were exposed for 72 h to increasing doses of the compounds. Cell proliferation was evaluated by using an 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assay. Fluorescent-Activated Cell Sorter (FACS) analysis was performed to measure apoptotic activation and RNA sequencing (RNA-Seq) to study the transcriptional changes induced by the compounds. Results: MZ1, and not its negative control epimer cisMZ1, was very active with a median half maximal inhibitory concentration (IC50) of 49 nmol/L. MZ1 was more in vitro active than the BET inhibitor birabresib (OTX015). Importantly, MZ1 induced cell death in all the ABC DLBCL cell lines, while the BET inhibitor was cytotoxic only in a fraction of them. BET degrader and inhibitor shared partially similar changes at transcriptome level but the MZ1 effect was stronger and overlapped with that caused cyclin-dependent kinase 9 (CDK9) inhibition. Conclusions: The BET degrader MZ1 had strong cytotoxic activity in all the ABC DLBCL cell lines that were tested, and, at least in vitro, it elicited more profound effects than BET inhibitors, and encourages further investigations.

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Background: Tyrosine kinase (TK) inhibitors represent the class of targeted antitumoral drugs that has been so far more successful. Here, we present two novel TK inhibitors, UJ26 and UJ30, with antitumor activity in preclinical models of solid tumors and hematologic cancers. Materials and Methods: Biochemical kinase profiling was performed (DiscoverX, CA, USA). Cell lines included in the NCI60 panel and lymphoma cell lines were exposed to increasing doses of UJ26, UJ30, and, as control, to the SRC inhibitor dasatinib for 72 hours. Cell proliferation was evaluated by using MTT assay. In vitro synergy was assessed with the Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). DLBCL RI-1 cells (15x106) were s.c. inoculated in NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice. Tumor size was measured on regular basis and drug treatments with UJ26 and UJ30 both given i.p, at the dose of 100mg/kg started when tumors reached 5 mm in diameter (100 mm3). Results: The two novel kinase inhibitors UJ26 and UJ30 presented broad specificity comparable to that of dasatinib, able to inhibit clinically relevant kinases ABL1, EGFR, FLT3, KIT, and MET. UJ26 and UJ30 presented antitumor activity in the NCI60 cell line panel against models of hematologic malignancies and solid tumors, in particular in the K562 leukemia and KM12 colon cancer cell lines (GI50 < 50 nM). Antiproliferative activity of UJ26 and UJ30 was then assessed in 8 B-cell lymphoma cell lines derived from mantle cell lymphoma (MCL) (Jeko1, MAVER1), chronic lymphocytic leukemia (MEC-1, PCL-12), and diffuse large B cell lymphoma (DLBCL) of the activated B cell like (ABC) (RI-1, SU-DHL-2) or of the germinal center B cell (GCB) (VAL, SU-DHL-4). Compounds were active with median IC50s of 600nM (95%C.I. 300-1065nM) and 250nM (95%C.I. 100-1049nM), respectively. Their antitumor activity appeared more potent than dasatinib (median IC50=1325nM, 95%C.I. 100-16625nM), in particular in 3 of the cell lines (MAVER1, SU-DHL-2 and MEC1). UJ26 and UJ30 were evaluated in combination with the BET inhibitor OTX015, exposing the four most sensitive lymphoma cell lines to increasing concentrations of the single compound or of both compounds for 72h. Both the UJ26/OTX015 combination, as well as the UJ30/OTX015 combination, were synergistic in all the cell lines. In vivo experiments showed that UJ26 was very toxic already after the first dose with swelling of the injection area. UJ26-treated mice started to recover after 2 weeks and were not treated any more, and there was no antitumor effect. UJ30 given once every 5 days reduced tumor volume compared to control group (D5, D11, D15; P < 0.05) and was well tolerated. UJ30-treated xenografts were three times smaller than both control and UJ26 groups at the end of the experiment (D15, UJ30 median tumor volume=867, 95%C.I. 600-1267.5). Body weight loss was observed after treatment with compound UJ26 whereas UJ30 showed similar weight growth as in the control group. Experiment was terminated after 2 weeks for vehicle- and UJ26-treated groups (D15) while UJ30-treated mice were sacrificed at day 20. Conclusions: The novel TK inhibitors UJ26 and UJ30 showed preclinical antitumor activity and in particular UJ30 is worth further investigations. CN, AC, FB: co-senior authors. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Andrea Unzue, Pawel Sledz, Hillarie Ekeh, Elena Bernasconi, Filippo Spriano, Cristina Nevado, Amedeo Caflisch, Francesco Bertoni. The novel tyrosine kinase inhibitors UJ26 and UJ30 are active in solid tumor and hematologic cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B182.