Abstract: Introduction: Despite the improvements, still too many patients die for their lymphomas and novel compounds are needed. We present a new small molecule, EG-011 (PCT/EP2018/057678), with in vitro and in vivo anti-cancer activity in lymphoma models. Methods: Lymphoma and solid tumor cell lines were exposed to a large range of concentrations of EG-011 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell viability of twelve acute lymphoblastic leukemia (ALL) primary patient cells from different high-risk subgroups (VNN2+, E2A-HLF, refractory T and IKZF plus) co-culture with marrow-derived MSCs were assayed after 72h of incubation with EG-011 and controls. Apoptosis assay was measured with annexin V by FACS. Xenografts were established s.c. into the left flanks of female NOD-SCID mice; treatment (200 mg/kg, i.p. 5 days per week) started with already established tumors. Combinations were evaluated with Chou-Talalay combination index (CI): synergism ( & lt;0.9), additive (0.9-1.1), antagonism/no benefit ( & gt; 1.1) after 72 hr treatments. Results: EG-011 presented a median IC50 of 2.25 μM in 62 lymphoma cell lines (95% C.I. 1-5μM). A higher activity was observed in a group of 21 cell lines that had a median IC50 of 250 nM (95% C.I. 40-600 nM). Among these there were 11 germinal center B cell (GCB) diffuse large B cell lymphomas (DLBCL) (sensitive n=11/21, resistant n=9/41, P & lt; 0.05), 4 mantle cell lymphoma (MCL) (sensitive n=4/21, resistant n=6/41, P n.s.), 3 marginal zone lymphoma (sensitive n=3/21, resistant n=2/41, P n.s.). EG-011 did not show any anti-proliferative activity in a panel of 25 solid tumor cell lines (IC50s & gt; 10 μM), Among 12 primary ALL samples, 7 were sensitive to EG-011 with IC50 values between 0.3-4.6 µM after 72h, 5 displayed IC50 higher than 20 µM. A dose-dependent increase in cell death (20-55%) was observed in lymphoma cell lines (OCI-LY-19 and REC1) (500 nM and 2 μM; 72h). No cytotoxicity was seen in PBMCs from two healthy donors after treatment at 1 and 10 μM for 24h and 48h. In an in vivo xenograft experiment with the MCL REC-1 cell line, EG-011 delayed tumor growth (Day 6, Day 7, Day 9, P & lt; 0.05) and tumor weight. EG-011-treated tumors were 2.2-fold smaller than controls (P & lt; 0.001). Combinations were tested in DLBCL (OCI-LY-1, OCI-LY-8, TMD8) and MCL (REC1, MINO). EG-011 was synergistic with rituximab, bendamustine, venetoclax, ibrutinib and lenalidomide in all tested cell lines. Conclusion: The selective anti-lymphoma activity, in both in vitro and in vivo models, and the observed in vitro synergisms with FDA approved targeted agents make EG-011 a novel intriguing new drug candidate deserving further preclinical studies. Citation Format: Eugenio Gaudio, Filippo Spriano, Chiara Tarantelli, Matilde Guala, Eugenia Riveiro, Gaetanina Golino, Antonio Lupia, Giosuè Costa, Roberta Rocca, Luciano Cascione, Silvia Jenni, Yi-Chien Tsai, Beat Bornhauser, Stefano Alcaro, Francesco Paduano, Francesco Trapasso, Emanuele Zucca, Anastasios Stathis, Natalina Pazzi, Franco Cavalli, Francesco Bertoni. EG-011 is a novel small molecule with in vitro and in vivo anti-tumor activity against lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4796.

Abstract: Copanlisib is a pan–class I phosphoinositide 3-kinase (PI3K) inhibitor with preferred activity toward PI3Kα and PI3Kδ. Despite the clear overall clinical benefit, the number of patients achieving complete remissions with the single agent is relatively low, a problem shared by the vast majority of targeted agents. Here, we searched for novel copanlisib-based combinations. Copanlisib was tested as a single agent, in combination with an additional 17 drugs in 26 cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and T-cell lymphomas. In vivo experiments, transcriptome analyses, and immunoblotting experiments were also performed. Copanlisib as a single agent showed in vitro dose-dependent antitumor activity in the vast majority of the models. Combination screening identified several compounds that synergized with copanlisib. The strongest combination was with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax. The benefit of the combination over single agents was also validated in an MZL xenograft model and in MCL primary cells, and was due to increased induction of apoptosis, an effect likely sustained by the reduction of the antiapoptotic proteins myeloid cell leukemia 1 (MCL1) and BCL-XL, observed in MCL and MZL cell lines, respectively. These data supported the rationale for the design of the Swiss Group for Clinical Cancer Research (SAKK) 66/18 phase 1 study currently exploring the combination of copanlisib and venetoclax in relapsed/refractory lymphomas.

Abstract: Introduction. CD22 is a cell surface marker expressed by the vast majority of normal and neoplastic B-cells. ADCT-602 is an antibody drug conjugate (ADC) composed of Emab-C220, an engineered version of the anti-CD22 humanized IgG1 antibody epratuzumab, site-specifically conjugated to SG3249, which includes the DNA minor groove crosslinking pyrrolobenzodiazepine (PBD) dimer SG3199 linked to the antibody via a protease-cleavable linker (Zammarchi et al, ASH 2016). ADCT-602 is currently being tested in a phase I/II clinical trial (NCT03698552) in recurrent or refractory B-cell acute lymphoblastic leukemia (B-ALL) patients. Here, we assessed its in vitro anti-lymphoma activity, also exploring for potential biomarkers and mechanisms of resistance. Methods. Fifty-seven human lymphoma cell lines were exposed to ADCT-602, an isotype-control ADC and the PBD dimer SG3199 as single agents for 96h, followed by MTT proliferation assay and IC50 calculation. Quantum Simply Cellular (QSC) microspheres were used for the quantitative determination of cellular CD22 antigen expression (Bangs Laboratories. Inc). Differences in IC50 values among lymphoma subtypes were calculated using the Wilcoxon rank-sum test. Statistical significance was defined by P values of 0.05 or less. Sensitivity analysis to ADCT-602 was performed by integrating different omics data, such as Illumina HT-12 microarray data (GSE94669), HTG EdgeSeq Oncology Biomarker Panel data (GSE103934) and DNA copy number variations. Results. The median IC50 for ADCT-602 was 200 pM (95%C.I, 90-400 pM) in 48 B-cell lymphoma lines (including three Hodgkin lymphoma cell lines), and 1850 pM in nine T-cell lymphoma lines (95%C.I, 700-15000 pM). ADCT-602 was more active in B- than in T-cell lymphomas, as expected based on the pattern of CD22 expression (P & lt; 0.005). Focusing on B-cell lymphomas, ADCT-602 in vitro activity was not correlated with its target expression measured both at the cell surface protein level (absolute quantitation, n=48, r=0.06 P=ns) and at the RNA level (Illumina HT-12 arrays, n=42, r=0.28, P=ns; HTG EdgeSeq Oncology Biomarker Panel, n=36, r=0.24, P=ns). In vitro activity was stronger in marginal zone lymphoma (MZL) cell lines than other B-cell lymphoma models (median IC50s 62.5 vs 312.5 pM; P = 0.03) as well as in diffuse large B-cell lymphoma (DLBCL) cell lines with BCL2 and MYC translocations (DHT DLBCL) versus DLBCL with none or a single translocation (median IC50s 25 vs 400 pM, P = 0.03). No associations were seen with TP53 status or other histology (mantle cell lymphoma, DLBCL, DLBCL cell of origin). We then exploited the gene expression profiling data using the Illumina HT-12 microarray gene expression technology. Within all the B-cell lymphoma cell lines (sensitive, n= 25; resistant, n= 23) we identified 1.207 genes down-regulated (FC-) and 1,104 genes up-regulated (FC+) in resistant cell lines. To delineate the pathways associated with the different degrees of sensitivity to ADCT-602, we performed a gene set enrichment analysis (GSEA; GSEA hallmarks and c2.common pathways) on the pre-ranked limma data. Transcripts up-regulated in resistant cell lines were enriched of genes coding for proteins involved in respiratory electron transport, oxidative phosphorylation and proteasome. Conversely, transcripts up-regulated in the sensitive cell lines were enriched of genes coding for proteins involved in inflammation, chemokine signaling, p53 response, IL2/STAT5 signaling, hypoxia, TGF-beta and interferon response. Similar gene signatures were picked up using the HTG platform, which can be applied to formalin-fixed paraffin-embedded clinical specimens, despite the smaller number of investigated genes. Conclusion. ADCT-602 showed in vitro anti-tumor activity across a large panel of B-cell lymphoma models of various histology. Expression signatures and other features (MZL or DHT DLBCL histology), but not the expression levels of its target, were associated with different sensitivity to the ADC. Our data supports the clinical evaluation of ADCT-602 in patients with B-cell lymphoma in addition to B-ALL. Disclosures Zucca: Kite: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; Celltrion Healthcare: Membership on an entity's Board of Directors or advisory committees. Stathis:PharmaMar: Other: Travel Grant; Member of the steering committee of the trial of this abstract: Other; Loxo: Honoraria, Other, Research Funding; Cellestia: Research Funding; Roche: Other, Research Funding; Novartis: Other, Research Funding; Bayer: Other, Research Funding; Merck: Other, Research Funding; Pfizer: Other, Research Funding; MEI Pharma: Other, Research Funding; ADC Therapeutcis: Other, Research Funding; Abbvie: Other: Travel Grant. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bertoni:ADC-Therapeutics: Research Funding; Bayer AG: Research Funding; Helsinn: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Nordic Nanovector ASA: Research Funding; Astra Zeneca: Other: travel grant; Amgen: Other: travel grant.

Abstract: Background: Lymphoma cells have frequent deregulation of their epigenome. The Bromodomain (BRD) and Extra-Terminal domain (BET) proteins are key regulators of the transcription process (Stathis & Bertoni, Cancer Discovery 2018). The acetyltransferases cyclic AMP response element binding protein (CREB)-binding protein (CBP) and the E1A interacting protein of 300 kDa (EP300 or P300) are highly homologous BRD-containing transcriptional co-activators and their genes are often mutated in diffuse large B cell lymphoma (DLBCL) (Pasqualucci et al, Nature 2011; Morin et al, Nature 2011). Targeting the individual classes of proteins is a new therapeutic approach, as shown especially by BET inhibitors with both preclinical and early clinical anti-lymphoma activity (Stathis & Bertoni, Cancer Discovery 2018). NEO2734 (Epigene Therapeutics Inc) is a novel oral dual inhibitor of BET and CREBBP/EP300 proteins with pre-clinical activity in a spectrum of solid tumors (Giles et al, ESMO 2018). Here, we present the first data exploring its anti-tumor activity in DLBCL models. Methods: Lymphoma cell lines were exposed to increasing doses of compounds for 72h. Cell proliferation was measured by using the MTT assay. Results: Twenty-seven DLBCL cell lines were exposed to NEO2734. The compound showed anti-tumor activity with a median IC50 of 157 nM (95% C.I., 135-214). Cell lines derived from activated B-cell-like DLBCL (ABC DLBCL) (n.=7) were more sensitive than the ones derived from germinal center B-cell (GCB) DLBCL (n.=20) (P = 0.04). No difference were observed based on MYC gene status (translocation: yes, n=8; no, n.=13), BCL2 gene status (translocation: yes, n=12; no, n.=6), TP53 gene status (inactive: yes, n=14; no, n.=6), double hit MYC/BCL2 (yes, n.=6; no, n.=14), CREBBP gene status (mutated, n.=10; wild type, n.=16), or EP300 gene status (mutated, n.=5; wild type, n.=20). As comparison, all the cell lines were also exposed to a BET inhibitor (birabresib, OTX015) (Boi et al, Clinical Cancer Res 2015) and to a CREBBP/EP300 inhibitor (CBP30) (Hammitzsch et al, PNAS 2015). The median IC50 values of the two molecules were 237 nM (95% C.I., 171-344) and 5.5 μM (95% C.I., 4.2-8.3 μM), respectively. The data obtained for birabresib were in agreement with what we had previously reported (Boi et al, Clinical Cancer Res 2015). The three compounds presented a similar pattern of anti-proliferative activity across all the cell lines (NEO2734 and birabresib: R2 =0.84, P 〈 0.001; NEO2734 and CBP30, R2 = 0.73, P 〈 0.001; birabresib and CBP30, R2 = 0.73, P 〈 0.001) but with different degrees of IC50. Both NEO2734 and birabresib were more potent than CBP30 (P 〈 0.0001). The novel dual inhibitor was more potent than birabresib (P=0.0182) and the difference was even bigger considering the compounds' IC90 (P = 0.0025): median values were 1.1 μM (95% C.I., 735 nM - 2.7 μM) and 20 μM (95% C.I., 2 - 30 μM) for the dual inhibitor and for the BET inhibitor, respectively. Conclusions: The novel dual BET and CREBBP/EP300 inhibitor NEO2734 showed strong in vitro anti-tumor activity across a large panel of DLBCL cell lines and it appeared more potent than single BET or CREBBP/EP300 inhibitors. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Stathis:Oncology Therapeutic Development: Research Funding. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership.

Abstract: Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P 〈 0.005) and RIVA (D14, D16, D19, D21; P 〈 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P 〈 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P 〈 0.001) and PQR309 (D7, D9, D11; P 〈 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

Abstract: Background. Currently, 3 histone deacetylase inhibitors (HDACi) are clinically approved for the treatment of T-cell lymphomas: vorinostat, romidepsin and belinostat. Pracinostat (SB939) is a class I, II and IV HDACi (Novotny-Diermayr et al, Mol Cancer Ther 2010), in phase 3 in combination with azacitidine for acute myeloid leukemia patients who are unfit to receive intensive remission induction chemotherapy (NCT03151408). Here, we tested it on a large panel of lymphoma cell lines alone or in combination with a BCL2 inhibitor, venetoclax and with 5-azacitidine. Methods. 60 cell lines derived from T-cell lymphoma (10), diffuse large B cell lymphoma (DLBCL, 25), mantle cell lymphoma (10), marginal zone lymphoma (5), Hodgkin lymphoma (4), chronic lymphocytic leukemia (2), primary mediastinal B-cell lymphoma (1), murine (2) and canine (1) lymphomas were exposed to increasing doses of pracinostat and vorinostat alone or to pracinostat in combination with venetoclax or 5-azacitidine for 72h. Cell proliferation was assessed using the MTT assay. Chou-Talalay index was used to determine synergism. For apoptosis and cell cycle analyses, cells were treated with 250 nM pracinostat for 72h then stained with Annexin V and 7-AAD (apoptosis) or fixed then stained with 7-AAD (cell cycle) before flow cytometry. Results. All lymphoma subtypes responded well to pracinostat (median IC50 250 nM; 95%C.I., 171-324 nM). Pracinostat compared favorably with vorinostat (306 nM; 95%C.I., 241-363 nM), and the anti-proliferative activity of the two compounds was correlated (R=0.8, P & lt;0.0001). Comparison of consensus cluster classification DLBCL subgroups showed that BCR DLBCL were more sensitive to HDACi than OxPhos DLBCL (pracinostat median IC50 BCR: 171 nM, OxPhos: 865 nM, P 0.001; vorinostat median IC50 BCR: 246 nM, OxPhos: 829 nM, P 0.01. No differences were observed between the DLBCL subtypes based on the cell of origin. Pracinostat mostly provoked cell cycle arrest in the S and G2/M phases with S-phase arrest associated with OxPhos and G2/M arrest with BCR DLBCL. Pracinostat caused pronounced apoptosis in 2/8 DLBCL cell lines. Functional annotation analysis to compare DLBCL with IC50 values & lt; 200 nM (n=12) and DLBCL with IC50 & gt; 400 nM (n=7) revealed an enrichment of mitochondrial metabolic pathways, DNA repair and cell cycle regulation in DLBCL with IC50 & gt;400 nM. Combination with venetoclax was beneficial in 3/3 cell lines, while the addition of 5-azacitidine benefited 2 out of 3 cell lines. Conclusions. Pracinostat robustly inhibits the proliferation of lymphoma cells with a similar IC50 range to vorinostat. Its anti-proliferative activity can be cytotoxic or cytostatic. Combination of pracinostat with other compounds further inhibits lymphoma cell proliferation. In DLBCL, the OxPhos phenotype and an enrichment of mitochondrial metabolism, DNA repair and cell cycle pathways are associated with a poorer response to pracinostat. Citation Format: Afua A. Mensah, Filippo Spriano, Eugenio Gaudio, Chiara Tarantelli, Luciano Cascione, Luca Aresu, Emanuela Lovati, Emanuele Zucca, Anastasios Stathis, Claudio Pietra, Francesco Bertoni. The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent [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 799.

Abstract: 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 & lt;0.25). 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 & gt;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 & lt;0.5 in 7 and 6 cell lines, respectively. GEP before treatment identified genesets associated with different sensitivity to these 2 combinations. High expression of genes involved in IFN signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of cell cycle genes were associated with synergism to copanlisib/venetoclax. Largely the opposite was observed for the palbociclib combination, more active with high expression of E2F/MYC targets and cell cycle genes and low expression of genes involved in IFN PI3K/AKT/mTOR and IL6/JAK/STAT signaling. 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

Abstract: Background: Pracinostat (SB939) is a class I, II and IV histone deacetylase inhibitor (HDACi), in phase 3 in combination with azacitidine for acute myeloid leukemia (NCT03151408). We reported a strong preclinical activity as a single agent in lymphomas (Mensah et al, AACR 2018). Interestingly, anti-tumor activity differed between metabolically-defined subtypes of diffuse large B-cell lymphoma (DLBCL): lower in OxPhos than in B cell receptor (BCR) cell lines. Thus, we now report RNA-Seq on OxPhos and BCR DLBCL cell lines treated with pracinostat. We also present results of a combination screening with 9 anti-lymphoma drugs for future clinical development of this HDACi. Methods: For RNA-Seq, OxPhos (Toledo, Pfeiffer, WSU-DLCL2) and BCR (SU-DHL-4, SU-DHL-6, OCI-LY-1) cell lines were treated with pracinostat or DMSO for 6 hours (h) or 14 days (d). Absolute fold change & gt; 2 with adjusted P & lt;0.01 were used as thresholds. Combinations (72 h) with 5-azacytidine, ibrutinib, lenalidomide, bendamustine, everolimus, rituximab, idelalisib, bortezomib and copanlisib were evaluated on germinal center B-cell (GCB: SU-DHL-6, VAL) and activated B cell-like (ABC: OCI-LY-10, TMD8) DLBCL using the Chou-Talalay combination index (CI). Results: At transcriptome level, pracinostat determined a time-dependent modulation of proliferation and cell cycle genes with differences between OxPhos and BCR cell lines. The more sensitive BCR showed a higher number of modulated genes at both timepoints than the less sensitive OxPhos DLBCL. Moreover, while the upregulated transcripts increased with time in both metabolic clusters [doubled in BCR (1201 vs 591) and almost tripled in OxPhos (330 vs 120)], the opposite was true for downregulated genes in BCR (358 vs 600) but not in less sensitive OxPhos (186 vs 187). Combinations of pracinostat with ibrutinib [0.37 (0.01 - 0.73)] or lenalidomide [0.29 (0.11 - 0.46)] were synergistic in ABC. Idelalisib [0.67 (0.46 - 0.92)] and everolimus [0.78 (0.57 - 1.11)] were both effective in combination with pracinostat in all ABC and 1 GCB, while rituximab was synergistic in 1 ABC and all GCB [0.61 (0.32 - & gt;1.1)]. Copanlisib [0.87 (0.45 - 0.94)] and 5-azacytidine [0.96 (0.64 - 1.14)] were additive with pracinostat in 3/4 cell lines and were synergistic in the remaining. Combination with bortezomib was beneficial in 3 cell lines [0.94 (0.5 - 1.53)] and bendamustine combined effectively with pracinostat in 2 of the 4 cell lines [1.1 (0.89 - 1.3)]. Conclusions: Modulation of the DLBCL transcriptome by pracinostat occurs already at 6 h, suggestive of a rapid effect of pracinostat on chromatin architecture, but it differs between OxPhos and BCR DLBCL. In terms of combinations, pracinostat addition to other targeted agents was largely beneficial. Citation Format: Afua A. Mensah, Filippo Spriano, Eugenio Gaudio, Chiara Chiara Tarantelli, Luciano Cascione, Andrea Rinaldi, Emanuela Lovati, Emanuele Zucca, Anastasios Stathis, Claudio Pietra, Francesco Bertoni. The histone deacetylase inhibitor pracinostat modulates the transcriptome of diffuse large B-cell lymphoma cells and is active in combination with several targeted agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4735.

Abstract: Background: NOTCH pathway is recurrently activated in human cancers, including lymphomas, and overactivation of the pathway has been correlated to a worse prognosis in patients with solid tumors, leukemias, and lymphomas, making it an attractive target for treatment (Rossi et al, JEM 2012; Rossi et al, Blood 2012; Kridel et al, Blood 2012). However, so far, pharmacologic approaches to inhibit its activity are limited to the use of nonspecific gamma secretase inhibitors and blocking antibodies with limited application. None of these therapeutic agents have been approved yet for cancer treatment. CB-103 is a novel first-in-class pan-NOTCH inhibitor that blocks signaling by directly and specifically targeting the NOTCH transcription complex, and it has shown preclinical activity in a variety of solid tumor and leukemia models (Lehal et al, AACR 2017). Here, we present the very first results with CB-103 in a large panel of B and T cell lymphoma cell lines. Methods: 58 human lymphoma cell lines [27 diffuse large B cell lymphomas (DLBCL); 9 mantle cell lymphomas; 6 marginal zone lymphomas; 9 T-cell lymphomas; 7 others], 2 murine and 1 canine, were exposed to increasing doses of CB-103 for 72h. Cell proliferation was measured by using the MTT assay. Transcriptome analysis was performed using the Illumina HumanHT 12 Expression BeadChips and with the HTG EdgeSeq Oncology Biomarker Panel, a targeted RNA-Seq investigating over 2500 genes, followed by a limma moderate t-test, and GSEA. Results: CB-103 presented a median IC50 above 20 μM across the whole panel of 61 lymphoma cell lines (range from 400 nM to & gt; 20 μM), without significant differences among lymphoma subtypes. Importantly, 13/58 cell lines had an IC50 & lt;10 μM and were defined as sensitive. These cell lines were derived from ABC DLBCL (3/7, 43%), GCB DLBCL (5/20, 25%), MCL (3/9, 33%), marginal zone lymphoma (1/6, 17%), and canine DLBCL (1/1, 100%). The sensitive cell lines, when compared with those presenting IC50 & gt;10 μM, presented a gene expression signature significantly enriched with genes involved in the epithelial-mesenchymal transition (FDR 0.0014), a Notch-related process. Indeed, the transcripts higher in the sensitive cell lines included, among others, DLL1 (P 0.010; Log Fold Change 2.39), BAMBI (P 0.046; Log Fold Change 0.92), MCM7 (P 0.015; Log Fold Change 1), and ULK1 (P 0.045; Log Fold Change 2.39), all connected to NOTCH pathway. Finally, 10 of the CB-103 sensitive human cell lines were also exposed to another NOTCH inhibitor, the gamma secretase inhibitor LY-3039187, which resulted active only in one MCL cell line, while it had IC50 & gt;20 μM in the other cell lines tested. Conclusions: CB-103 presented in vitro antilymphoma activity in a restricted number of B cell lymphoma cell lines, which were characterized by a NOTCH-related gene expression signature. Further studies with the compound are ongoing and a phase I is planned to start in Q3 2017 (EUDRACT Number 2017-001491-35). Citation Format: Filippo Spriano, Chiara Tarantelli, Alberto Arribas, Eugenio Gaudio, Luciano Cascione, Luca Aresu, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Maximilien Murone, Dirk Weber, Rajwinder Lehal, Freddy Radtke, Francesco Bertoni. Targeting lymphomas with the novel first-in-class pan-NOTCH transcription inhibitor CB-103 [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 B061.

Abstract: Background: PI3Kδ is expressed in B cells and has a central role in the B-cell receptor signaling. Copanlisib is a highly selective PI3Kδ and PI3Kα inhibitor, and it is currently under clinical development in indolent lymphomas including marginal zone lymphoma (MZL). Copanlisib is Food and Drug Administration (FDA) approved for the treatment of patients with relapsed or refractory follicular lymphoma. Nevertheless, a subset of patients can eventually relapse due to acquired resistance. A better understanding of resistance mechanisms could help to design improved therapies; hence, we generated MZL cell lines resistant to copanlisib. Materials and Methods: Cells were kept on copanlisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 3 weeks of drug-free culture. Multidrug resistance phenotype was ruled out by confirming sensitivity to vincristine. Cells underwent transcriptome profiling (RNA-Seq) and immunophenotypic analysis. Results: The RES models were obtained from VL51 cell line with over 50-fold times higher IC50s than PAR counterparts. Of note, the copanlisib-resistant lines showed decreased sensitivity to other PI3K inhibitors such as duvelisib (50-fold) and idelalisib (5-fold) and to the BTK inhibitor ibrutinib (15-fold), suggesting that the mechanism observed here might drive resistance to other downstream B-cell receptor inhibitors. Gene expression profiles of RES showed the upregulation of cytokine signaling (IL1A, IL1B, CXCR4), NFkB (LTA, TNF), MAPK (RASGRP4, RASGRP2), and JAK-STAT (STAT3, JAK3) signaling pathways and negative regulators of apoptosis (CD44, JUN). Conversely, repressed genes in RES were involved in cell adhesion (ITGA4, ITGB1), antigen presentation (HLAs), and IFN response (PARP12, GBP6). Consistent with the overexpression of antiapoptotic signaling genes, RES cells exhibited also resistance to the BCL2-inhibitor venetoclax, either as a single as in combination with copanlisib. Flow cytometry confirmed the CXCR4 upregulation and the downregulation of CD49d (ITGA4), paired with reduced CD20 and CD81 surface expression. In accordance, addition of a CXCR4 inhibitor overcame resistance to copanlisib. Conclusions: We created a model of secondary resistance to the PI3K inhibitor copanlisib, derived from an MZL cell line. This model will help in clarifying mechanisms of resistance to the drug and to evaluate alternative therapeutic approaches. Indeed, we already identified novel potential targets, such as IL1 and CXCR4, that might be exploited in overcoming resistance to copanlisib and are worthy of further investigation. Citation Format: Alberto J. Arribas, Sara Napoli, Luciano Cascione, Eugenio Gaudio, Roberta Bordone-Pittau, Marilia Barreca, Giulio Sartori, Chiara Tarantelli, Filippo Spriano, Andrea Rinaldi, Anastasios Stathis, Georg Stussi, Davide Rossi, Emanuele Zucca, Francesco Bertoni. Mechanisms of resistance to the PI3K inhibitor copanlisib in marginal zone lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-46.