Abstract 5179: The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma
Authors: Spriano Filippo , Tarantelli Chiara , Gaudio Eugenio , Chung Elaine YL , Arribas Alberto J. , Cascione Luciano , Napoli Sara , Kwee Ivo , Rinaldi Andrea , Rossi Davide , Zucca Emanuele , Stathis Anastasios , Jessen Katti , Lannutti Brian , Toretsky Jeffrey , Bertoni Francesco 
Published: July 2017

Abstract
Background. ETS transcription factors, such as FLI1 and SPIB, are recurrently deregulated in human lymphomas (Bonetti et al, Blood 2013; Lenz et al, PNAS 2008). The small molecule YK-4-279 inhibits binding of EWS1-FLI1 fusion protein to RHA resulting in growth arrest and apoptosis in Ewing sarcoma cells (Erkizan et al, Nat Med 2009) and we previously showed that YK-4-279 has in vitro anti-lymphoma activity (Chung et al, AACR 2015). TK-216 is a YK-4-279 clinical derivative that is in phase 1 for patients with relapsed or refractory Ewing sarcoma (NCT02657005). Here, we present extensive preclinical results obtained with TK-216 in lymphoma models.
Methods. 56 cell lines [27 diffuse large B cell lymphoma (DLBCL); 10 mantle cell lymphoma; 6 marginal zone lymphoma; 5 anaplastic large T-cell lymphoma; 8 others] were exposed to TK-216 increasing doses for 72h using a Tecan D300e Digital Dispenser and 384well plates; cell proliferation was measured with MTT. In vivo studies were performed in NOD-SCID mice and treatments started with approximately sc 60mm3 tumor volumes.
Results. TK-216 displayed high activity: median IC50 was 449 nM (95%CI: 367-506). Sensitivity was not affected by the lymphoma cell of origin [B vs T; activated B cell type (ABC) vs germinal center type DLBCL] or MYC and TP53 status. There was a non-statistically significant trend for lower sensitivity in cell lines bearing BCL2 chromosomal translocation (P=0.07, DLBCL only; P=0.06, all cell lines). Anti-tumor activity was mainly cytotoxic as confirmed by performing cell cycle analysis and Annexin V staining in 6 DLBCL cell lines (TMD8, U2932, HBL1, OCI-LY-18, WSU-DLCL2, DOHH2 for 24, 48, 72h), in which a time-dependent apoptosis was preceded by G2/M arrest.
Antitumor activity was confirmed in DLBCL TMD8 xenografts. Compared with control group (n=10), mice treated with TK-216 (100 mg/Kg, BID; n=9) clearly presented a reduction in tumor growth, already evident at day 3 and becoming much stronger with time (D3, D5, D8, D11: P&lt;0.01; D13, P not available since control group had to be stopped due to tumor volume) and a 4 times reduction in tumor volume at D11 (P&lt;0.01).
TK-216 was tested in combination with other targeted agents in DLBCL cell lines. A benefit was observed with the combination of TK-216 with the immunomodulator lenalidomide (synergism in 2/2 ABC DLBCL), with the BET inhibitor OTX015 (MK-8628) (synergism in 2/4 cells and additive effect in 1/4), the anti-CD20 monoclonal antibody rituximab (synergism in 2/3 cells) and the BCL2 inhibitor venetoclax (synergism in 3/4 cells). The latter synergism could be linked to the previously mentioned negative trend between TK-216 IC50 values and the presence of BCL2 translocation.
Conclusions. The novel small molecule TK-216 presented strong preclinical anti-lymphoma activity, which provides evidence for further preclinical and clinical development as single agent and in combination.
Citation Format: Filippo Spriano, Chiara Tarantelli, Eugenio Gaudio, Elaine YL Chung, Alberto J. Arribas, Luciano Cascione, Sara Napoli, Ivo Kwee, Andrea Rinaldi, Davide Rossi, Emanuele Zucca, Anastasios Stathis, Katti Jessen, Brian Lannutti, Jeffrey Toretsky, Francesco Bertoni. The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5179. doi:10.1158/1538-7445.AM2017-5179