Filippo Spriano

Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma.

The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra‐terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti‐tumour activity of the novel bromodomain and extra‐terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma models. BAY 1238097 showed anti‐proliferative activity in a large panel of lymphoma‐derived cell lines, with a median 50% inhibitory concentration between 70 and 208 nmol/l. The compound showed strong anti‐tumour efficacy in vivo as a single agent in two diffuse large B cell lymphoma models. Gene expression profiling showed BAY 1238097 targeted the NFKB/TLR/JAK/STAT signalling pathways, MYC and E2F1‐regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling signatures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC‐inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented in vitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti‐lymphoma preclinical activity in vitro and in vivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK alongside BET bromodomains.

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models. Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors. Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib. Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120–9. ©2017 AACR.

Trabectedin is a novel chemotherapy agent for diffuse large B cell lymphoma

Trabectedin (ET-743, Yondelis ) is a natural product originally derived from Ecteinascidia turbinata, and currently produced semi-synthetically (Larsen et al, 2016). Trabectedin is approved in Europe as a single agent for patients with all soft tissue sarcoma subtypes and, in combination with pegylated liposomal doxorubicin, for patients with relapsed, platinumsensitive ovarian cancer. In the US it is only approved as a single agent for patients with unresectable or metastatic liposarcoma or leiomyosarcoma after anthracycline therapy (Larsen et al, 2016). The drug works by simultaneously interacting with the minor groove of DNA and transcription factors or DNA repair molecules, inducing cell cycle perturbations and cell death (Larsen et al, 2016). Moreover, both trabectedin and its novel derivative lurbinectedin do not only have direct anti-tumour activity, but they also affect the tumour microenvironment, decreasing the number of tumourassociated macrophages (Belgiovine et al, 2017). Trabectedin is active in preclinical models derived from different solid tumours, especially sarcomas (Larsen et al, 2016). Among the latter, Ewing sarcoma exhibits marked sensitivity to trabectedin. Ewing sarcoma is characterized by a balanced chromosomal translocation t(11;22)(q24;q12) resulting in the fusion oncoprotein EWS/FLI1 that acts as an aberrant transcriptional activator with strong transforming capabilities. Trabectedin displaces EWS-FLI1 from the promoters of target genes, thus blocking its transactivating function (Grohar et al, 2011). FLI1 is recurrently deregulated in up to one quarter of diffuse large B cell lymphoma (DLBCL) and its genetic silencing is toxic for cell lines derived from this tumour type (Bonetti et al, 2013). Due to its displacement of EWS-FLI1, we hypothesized that trabectedin would also be able to displace the FLI1 oncoprotein and have anti-tumour activity in DLBCL. We first assessed the anti-tumour activity of trabectedin in DLBCL cell lines derived from the two main DLBCL subtypes, the germinal centre B cell (GCB) type (OCI-LY7, Karpas422 OCI-LY19, and SU-DHL-4), and the activated B celllike (ABC) type (OCI-LY10, and U2932). The cells were exposed to increasing concentrations of the compound for 72 h, as previously described (Boi et al, 2015). All the cell lines were very sensitive to trabectedin with 50% inhibitory concentration (IC50) values in the subnanomolar range at 72 h (Fig 1A, B). These data were comparable to previously reported responses in cell lines derived from sarcomas and other solid tumours (Scotlandi et al, 2002; Grohar et al, 2011). The IC50 values we obtained were also well within the clinically achievable plasma concentrations of the drug (von Mehren et al, 2008). In previous studies, trabectedin mainly induced G2/M cell cycle arrest (Erba et al, 2001). Exposure of DLBCL cells to trabectedin (2 5 nmol/l for 24 h) induced G1/S arrest, a phenotype described in myelomonocytic cells treated with trabectedin but not in sarcoma cells (Mannarino et al, 2018), indicating that the drug might behave differently in different tumour types. The mechanism of action of trabectedin in different tumour types heavily depends on its effect on the regulation of transcripts, due to its interference with RNA polymerase II following DNA binding which frequently occurs at promoter sites of different transcription factors, including the EWSFLI1 fusion protein (Grohar et al, 2011; Larsen et al, 2016). Thus, to determine the effect of trabectedin on the gene expression profile of DLBCL, we exposed one GCB (OCILy7) and one ABC (U2932) DLBCL cell line to 10 nmol/l trabectedin for 8 and 24 h and analysed the RNA as previously described (Bonetti et al, 2013). Trabectedin induced Fig 1. Trabectedin has anti-proliferative activity in DLBCL cell lines. (A) Cells were exposed to trabectedin for 72 h and the 50% inhibitory concentration (IC50) determined. Y-axis, IC50 (nmol/l). X-axis, cell lines. (B) Normalized RNA expression of FLI1 in the tested cell lines as obtained from GSE64820 (Boi et al, 2015). Correspondence

Abstract 154: The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib

Introduction Copanlisib (BAY 80 6946) is a highly selective pan class I PI3K-i with predominant inhibitory activity against PI3Kδ and PI3Kα, in clinical development as single agent and in combination for lymphoma patients. To address single agent antitumor activity in different lymphomas and to understand the molecular basis of resistance mechanisms for rational combination, we performed a screening of copanlisib as single agent and in combination with 15 other anticancer agents in 17 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL). Methods MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) cell lines were exposed to increasing doses of copanlisib alone and in combination with other compounds using the fixed ratio set-up. Tested compounds included approved and experimental inhibitors of key regulatory pathways. Synergy was assessed via Chou-Talalay combination index (CI). Gene expression profiling (GEP) was done using Illumina Human HT12Expression BeadChips and GSEA (FDR Results Copanlisib showed antitumor activity in most cell lines (median IC50=22nM; 95%C.I.: 15-98). The other most active drugs were bortezomib (5nM; 5-7), romidepsin (34nM; 2-94), roniciclib (23nM; 18-29), panobinostat (161nM; 11-1263), MI2 (490nM; 224-1000). The remaining had median IC50s >500nM. Copanlisib-containing combinations often gave synergy/additive effects: copanlisib with venetoclax was beneficial in 16/17; with MI2 in 15; with palbociclib or ibrutinib in 14; with BAY 1125976 or panobinostat in 13; with lenalidomide or BAY 1238097 in 12; with rituximab in 11; with romidepsin in 10; with roniciclib in 9; with bortezomib in 8; with BAY 1143572 in 7; with bendamustine in 6; with ruxolitinib in 2. Combinations with venetoclax and with palbociclib were the most promising, achieving CI values Conclusion Copanlisib was active in MCL, MZL and CLL models. Combinations with BCL2-i venetoclax and CDK4/CDK6-i palbociclib were the most synergistic. Specific GEP features might predict lymphomas that could benefit from these regimens. Citation Format: Eugenio Gaudio, Ivo Kwee, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Thibaud Jourdan, Melanie Berthold, Alberto Arriibas, Anastasion Stathis, Davide Rossi, Ningshu Liu, Martin Lange, Oliver Politz, Emanuele Zucca, Francesco Bertoni. The phosphatidylinositol-3-kinase (PI3K) inhibitor (i) copanlisib is active in preclinical models of B-cell lymphomas as single agent and in combination with conventional and targeted agents including venetoclax and palbociclib [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 154. doi:10.1158/1538-7445.AM2017-154

BET bromodomain inhibitor birabresib in mantle cell lymphoma: in vivo activity and identification of novel combinations to overcome adaptive resistance

Background The outcome of patients affected by mantle cell lymphoma (MCL) has improved in recent years, but there is still a need for novel treatment strategies for these patients. Human cancers, including MCL, present recurrent alterations in genes that encode transcription machinery proteins and of proteins involved in regulating chromatin structure, providing the rationale to pharmacologically target epigenetic proteins. The Bromodomain and Extra Terminal domain (BET) family proteins act as transcriptional regulators of key signalling pathways including those sustaining cell viability. Birabresib (MK-8628/OTX015) has shown antitumour activity in different preclinical models and has been the first BET inhibitor to successfully undergo early clinical trials. Materials and methods The activity of birabresib as a single agent and in combination, as well as its mechanism of action was studied in MCL cell lines. Results Birabresib showed in vitro and in vivo activities, which appeared mediated via downregulation of MYC targets, cell cycle and NFKB pathway genes and were independent of direct downregulation of CCND1. Additionally, the combination of birabresib with other targeted agents (especially pomalidomide, or inhibitors of BTK, mTOR and ATR) was beneficial in MCL cell lines. Conclusion Our data provide the rationale to evaluate birabresib in patients affected by MCL.

Abstract 1894: Identification of novel OTX015-containing combinations for lymphoma treatment

Introduction. Epigenetics represents and intriguing target of therapy for lymphomas. The epigenetic mechanisms are regulated by several players (e.g. HDAC, HAT, BET) and among them the BET proteins became of interest a few years ago with demonstrated preclinical anti-cancer activity with the first compounds and early clinical responses first in class clinical-grade BET inhibitor OTX015 (MK-8628/birabresib). Despite a widespread anti-proliferative activity, the cytotoxicity effect of BET inhibitors is limited, and, here, we aimed to identify drugs that improve OTX015 activity performing a screening with the BET inhibitor with a library 348 compounds in two lymphoma cell lines. Methods. Two cell lines derived from germinal center B cell (GCB) diffuse large B cell lymphoma (DLBCL) (OCI-LY-19 and WSU-DLCL2) were exposed to OTX015 (single dose, 100 nM) in combination with two different doses (20 and 1,000 nM) of 348 compounds. Compounds giving a 1.5-fold decreased proliferation with the combination than with the individual compounds were further evaluated in two additional lymphoma cell lines, exposed (72h) to increasing doses of OTX015 alone and in combination with increasing doses of other selected compounds. Synergy was assessed with Chou-Talalay combination index (CI): synergism ( 1.1). Results. OCI-LY-19 and WSU-DLCL2 were treated with OTX015 (100 nM) and 348 compounds as single agents and in combination. The combinations of OTX015 with a series of compounds achieved improved anti-tumor activity than single agents. Besides HDAC and mTOR inhibitors, in accordance with what previously reported (Boi, Gaudio, Bonetti et al, Clinical Cancer Res 2015), the ABL/SRC inhibitor dasatinib, the AKT1/2/3 inhibitor MK-2206, the JAK2 inhibitor TG101209 and the LRRK2 inhibitor LRRK2-IN appeared interesting combination partners. These screening results were validated in the DLBCL cell lines with targeted experiments, as well as in chronic lymphocytic leukemia (CLL) (OTX015 plus MK-2206) and mantle cell lymphoma (MCL) (REC1) (Dasatinib, LRRK2-IN). Experiments in additional cell lines and assessing the effects on the apoptosis are on-going and will be presented. Conclusion. A chemical screening has identified novel OTX015-containing combinations active in lymphoma cell lines that are worth of further investigations. Work supported by a San Salvatore Foundation grant. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Filippo Spriano, Alberto J. Arribas, Luciano Cascione, Emanuele Zucca, Anastasios Stathis, Francesco Bertoni. Identification of novel OTX015-containing combinations for lymphoma treatment [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 1894.

Abstract 5179: The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma

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 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