Elena Bernasconi

The BET Bromodomain Inhibitor OTX015 Affects Pathogenetic Pathways in Preclinical B-cell Tumor Models and Synergizes with Targeted Drugs.

Purpose: In cancer cells, the epigenome is often deregulated, and inhibition of the bromodomain and extra-terminal (BET) family of bromodomain-containing proteins is a novel epigenetic therapeutic approach. Preliminary results of an ongoing phase I trial have reported promising activity and tolerability with the new BET bromodomain inhibitor OTX015. Experimental Design: We assessed the preclinical activity of OTX015 as single agent and in combination in mature B-cell lymphoma models and performed in vitro and in vivo experiments to identify the mechanism of action and the genetic features associated with sensitivity to the compound. Results: OTX015 showed antiproliferative activity in a large panel of cell lines derived from mature B-cell lymphoid tumors with median IC50 of 240 nmol/L, without significant differences among the different histotypes. In vitro and in vivo experiments showed that OTX015 targeted NFKB/TLR/JAK/STAT signaling pathways, MYC- and E2F1-regulated genes, cell-cycle regulation, and chromatin structure. OTX015 presented in vitro synergism with several anticancer agents, especially with mTOR and BTK inhibitors. Gene expression signatures associated with different degrees of sensitivity to OTX015 were identified. Although OTX015 was mostly cytostatic, the compound induced apoptosis in a genetically defined subgroup of cells, derived from activated B-cell–like diffuse large B-cell lymphoma, bearing wtTP53, mutations in MYD88, and CD79B or CARD11. Conclusions: Together with the data coming from the ongoing phase I study, the in vitro and in vivo data presented here provide the basis for further clinical investigation of OTX015 as single agent and in combination therapies. Clin Cancer Res; 21(7); 1628–38. ©2015 AACR.

Bromodomain inhibitor OTX015 (MK-8628) combined with targeted agents shows strong in vivo antitumor activity in lymphoma

The bromodomain inhibitor OTX015 (MK-8628) has shown anti-lymphoma activity as a single agent in both the preclinical and clinical settings, as well as in vitro synergism with several anticancer agents. Here, we report in vivo data for OTX015 in combination with the histone deacetylase inhibitor vorinostat, the Brutons tyrosine kinase inhibitor ibrutinib, the anti-CD20 monoclonal antibody rituximab, and the mTOR inhibitor everolimus in a diffuse large B cell lymphoma model. The antitumor effect of OTX015-containing combinations in SU-DHL-2 xenografts in mice was much stronger than the activity of the corresponding single agents with almost complete tumor eradication for all four combinations. Pharmacokinetic analyses showed similar OTX015 levels in plasma and tumor samples of approximately 1.5 μM, which is equivalent to the concentration showing strong in vitro activity. For all four combinations, mean terminal levels of the bromodomain inhibitor differed from those in mice exposed to single agent OTX015, indicating a need for thorough pharmacokinetic investigations in phase I combination studies. In conclusion, our results provide a strong rationale to explore OTX015-containing combinations in the clinical lymphoma setting.

Combination of the MEK inhibitor pimasertib with BTK or PI3K-delta inhibitors is active in preclinical models of aggressive lymphomas

BACKGROUND Lymphomas are among the most common human cancers and represent the cause of death for still too many patients. The B-cell receptor with its downstream signaling pathways represents an important therapeutic target for B-cell lymphomas. Here, we evaluated the activity of the MEK1/2 inhibitor pimasertib as single agent and in combination with other targeted drugs in lymphoma preclinical models. MATERIALS AND METHODS Cell lines derived mature B-cell lymphomas were exposed to increasing doses of pimasertib alone. Immunoblotting and gene expression profiling were performed. Combination of pimasertib with idelalisib or ibrutinib was assessed. RESULTS Pimasertib as single agent exerted a dose-dependent antitumor activity across a panel of 23 lymphoma cell lines, although at concentrations higher than reported for solid tumors. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib and the BTK inhibitor ibrutinib in cell lines derived from diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma. The data were confirmed in an in vivo experiment treating DLBCL xenografts with pimasertib and ibrutinib. CONCLUSION The data presented here provide the basis for further investigation of regimens including pimasertib in relapsed and refractory lymphomas.

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.

DNA-mediated adjuvant immunotherapy extends survival in two different mouse models of myeloid malignancies.

We have previously shown that a specific promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) DNA vaccine combined with all-trans retinoic acid (ATRA) increases the number of long term survivors with enhanced immune responses in a mouse model of acute promyelocytic leukemia (APL). This study reports the efficacy of a non-specific DNA vaccine, pVAX14Flipper (pVAX14), in both APL and high risk myelodysplastic syndrome (HR-MDS) models. PVAX14 is comprised of novel immunogenic DNA sequences inserted into the pVAX1 therapeutic plasmid. APL mice treated with pVAX14 combined with ATRA had increased survival comparable to that obtained with a specific PML-RARA vaccine. Moreover, the survival advantage correlated with decreased PML-RARA transcript levels and increase in anti-RARA antibody production. In HR-MDS mice, pVAX14 significantly improved survival and reduced biomarkers of leukemic transformation such as phosphorylated mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1. In both preclinical models, pVAX14 vaccine significantly increased interferon gamma (IFNγ) production, memory T-cells (memT), reduced the number of colony forming units (CFU) and increased expression of the adapter molecule signalling to NF-κB, MyD88. These results demonstrate the adjuvant properties of pVAX14 providing thus new approaches to improve clinical outcome in two different models of myeloid malignancies, which may have potential for a broader applicability in other cancers.

The novel atypical retinoid ST5589 down-regulates Aurora Kinase A and has anti-tumour activity in lymphoma pre-clinical models.

Despite the marked improvements in the treatment of lymphomas, there is still a need for new therapeutic agents. Synthetic retinoids represent a class of compounds with anti‐cancer activity. Here, we report the preclinical activity of a new member of this class, the ST1926‐derivative ST5589, in lymphomas. ST5589 presented a dose‐dependent anti‐proliferative activity in almost all of the 25 lymphoma cell lines analysed, with a median 50% inhibitory concentration of 433 nM. Apoptosis was observed in 8/11 cell lines. ST5589 induced changes in the gene expression profiles of the cell lines, including the down‐regulation of Aurora Kinase A (AURKA). Specific gene expression signatures were associated with a higher sensitivity to the compound and combination of ST5589 with carfilzomib revealed the importance of proteasome activity in mediating the anti‐tumour activity of ST5589. In conclusion, we have identified a new mechanism of action of atypical retinoids as anti‐cancer compounds, and the encouraging results obtained with the new ST1926‐derivative ST5589 provide the basis for further developments of the compound.

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.

Abstract B77: The BET bromodomain inhibitor OTX015 (MK-8628) in mantle cell lymphoma (MCL): in vivo activity and identification of novel combinations to overcome adaptive resistance

OTX015 (MK-8628) has shown anti-lymphoma activity in both the preclinical and clinical settings (Boi et al, CCR 2015; Stathis et al, EORT-NCI-AACR 2014). Now, we report in vivo and in vitro data on OTX015 in MCL, including combination regimens. Methods. NOD-Scid (NOD.CB17- Prkdcscid /NCrHsd) mice were subcutaneously inoculated with 15 x10 6 MCL REC1 cells. Tumor size was measured twice weekly using digital calipers. Tumor volumes were calculated using the equation V = [length x width 2 ]/2, in which the width and the length are the shortest and the longest diameters of each tumor, respectively. Gene expression profiling (GEP) was performed with Illumina HumanHT12 Expression BeadChips. Combinations were evaluated using the Chou-Talalay combination index (CI): 1.2, no benefit. Results. Since we had previously reported in vitro activity of OTX015 as single agent in MCL cell lines (Boi et al, CCR 2015), we first confirmed its antitumor activity in an in vivo model. Mice were treated with OTX015 (50 mg/kg once daily; QDx7/w x5w) started 3 days after the graft. OTX015-treated mice did not show any weight loss. OTX015-treated REC1 xenografts presented a 2-fold decrease in volume compared with the control group (p 10000 nM) exposed to DMSO or OTX015 (500 nM) for 2, 4, 8 or 12 hr. Despite the difference in the IC50 values between the three OTX015 sensitive cell lines and Granta-519, GEP changes were similar, and support the previously reported synergisms of OTX015 with ibrutinib and lenalidomide (Bernasconi et al, ASH 2014). Downregulated transcripts were enriched for MYC targets, genes involved in interferon response, in NFKB signaling, in MYD88 signaling, in B cell activation, genes more expressed in MCL than in CLL or DLBCL. TNFRSF17 , TNFRSF13B , TLR10 , CD72 , VPREB3 , PLEK , PTPN6 were among the most down-regulated transcripts, while the most upregulated ones comprised genes coding different histones of the clusters 1-2, PPP1R13B, CDKN2D (p19), IRF7, FGFR3, HES6 . The upregulation of few transcripts, including HES and FGFR3 , more pronounced in the resistant cell line, were suggestive of adaptive resistance mechanisms. We thus evaluated the combination of OTX015 with inhibitors of FGFR (PD173074) or MEK (pimasertib), which might counteract FGFR3 upregulation. Both combinations increased the antiproliferative effect of OTX015 in the sensitive cell line Jeko1 (CI values of 0.83 and 0.37, respectively). The combination with pimasertib was able to increase the sensitivity to OTX015 also in the resistant cell line (CI = 0.18), although it was not able to completely reverse its primary resistance. Conclusions. OTX015 showed in vivo activity as a single agent in MCL. GEP identified new combinations with MEK and FGFR inhibitors with the potential to overcome adaptive resistance. Citation Format: Eugenio Gaudio, Elena Bernasconi, Chiara Tarantelli, Luciano Cascione, Ivo Kwee, Andrea Rinaldi, Maurilio Ponzoni, Maria E. Riveiro, Emanuele Zucca, Francesco Bertoni. The BET bromodomain inhibitor OTX015 (MK-8628) in mantle cell lymphoma (MCL): in vivo activity and identification of novel combinations to overcome adaptive resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B77.

Abstract 2676: The MEK-inhibitor pimasertib is synergistic with PI3K-delta and BTK inhibitors in lymphoma models

Pimasertib (AS-703026) is a potent and highly selective ATP noncompetitive MEK1/2 inhibitor that has shown anti-tumor activity in different pre-clinical models. We have previously reported pimasertib activity as single agent in lymphoma models and preliminary combinations results (Gaudio et al, AACR 2014). Here, we report detailed data on combinations of pimasertib with the PI3K-delta inhibitor idelalisib and with the BTK-inhibitor ibrutinib. Methods. Cell lines derived from activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL) (OCI-Ly10, TMD8), germinal center B-cell like (GCB) DLBCL (DOHH2, RCK8) and from mantle cell lymphoma (REC1, JEKO1) were exposed to increasing doses of pimasertib alone and in combination with idelalisib and ibrutinib. Synergy was assessed by Chou-Talalay combination index (CI). ERK phosphorylation level and PARP cleavage were detected by western blotting in cell treated with single agents or combination of pimasertib with idelalisib or ibrutinib. NOD-Scid (NOD.CB17-Prkdcscid/NCrHsd) mice were subcutaneously inoculated with OCI-Ly-10 (10 x106) DLBCL cell line. Mice developed palpable tumors (100 mm3) and were randomized to receive pimasertib, orally once per day (30 mg/kg), ibrutinib (5 mg/Kg), the combination of the two, or control vehicle alone. Tumor size was measured two times per week using a digital caliper [tumor volume (mm3) = (I x W x W)/2]. Results. Strong synergism was observed with pimasertib combined with the PI3K inhibitor idelalisib in the ABC-DLBCL OCI-Ly10 (median CI = 0.026) and TMD8 (CI = 0.25), whereas synergistic/additive effects were detected in GCB-DLBCL (DOHH2, RCK8) and MCL (REC1, JEKO1). Synergism was observed with the BTK-inhibitor ibrutinib in all the cell lines: OCI-Ly10 (CI = 0.32), TMD8 (CI = 0.63), DOHH2 (CI = 0.66), RCK8 (CI = 0.87), REC1 and JEKO1 (CI = 0.2). Thirty minutes of exposure time with pimasertib were sufficient to knock-down phospho-ERK1/2 proteins in the mentioned ABC-DLBCL and MCL cell lines stimulated with anti-IgM (20μg/mL) for 20 minutes. Stronger down-regulation of phospho-ERK1/2 was seen in ABC-DLBCL and MCL cell lines treated with combination of pimasertib with idelalisib or pimasertib with ibrutinib rather than single agent treatment conditions. Notably, apoptosis and PARP cleavage was observed in cell lines treated with pimasertib in combination with ibrutinib or idelalisib. OCI-Ly10 xenograft tumors that received a combination of pimasertib (30mg/Kg) and ibrutinib (5mg/Kg) for 14 days, showed a five-fold reduction of both tumor volume and weight as compared to the control and single compound groups. Conclusions. Pimasertib-containing combinations with PI3K-delta and BTK inhibitors show very promising activity in preclinical models of mature lymphomas. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Chiara Barassi, Elena Bernasconi, Ivo Kwee, Luciano Cascione, Maurilio Ponzoni, Andrea Rinaldi, Anastasios Stathis, Samantha Goodstal, Emanuele Zucca, Francesco Bertoni. The MEK-inhibitor pimasertib is synergistic with PI3K-delta and BTK inhibitors in lymphoma models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2676. doi:10.1158/1538-7445.AM2015-2676

Abstract 2652: Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas

PQR309 is a novel oral PI3K/mTOR inhibitor, being now evaluated as single agent in a phase I study for solid tumors patients (NCT01940133). Here, we present the activity of the compound in lymphoma pre-clinical models, also integrating response data with genomic features. Methods. IC50s were calculated with the MTT assay at 72h on 40 cell lines [27 diffuse large B-cell lymphoma (DLBCL), 10 mantle cell lymphoma (MCL), 3 splenic marginal zone lymphoma (SMZL)] treated with increasing doses of PQR309, a second dual PI3K/mTOR inhibitor (GDC0980) and the PI3Kdelta inhibitor Idelalisib. Gene expression profiling (GEP) was performed with Illumina HumanHT-12 Expression BeadChips. Results. PQR309 had potent anti-proliferative activity in most of the cell lines. Median IC50 were 166 nM in DLBCL (95%CI 128-343 nM), 235 in MCL (155-381), 214 in SMZL (188-304). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R = 0.9). Idelalisib appeared significantly less active, with a pattern of sensitive cell lines less correlated with PQR309 (R = 0.6) or GDC0980 (R = 0.6). Apoptosis after PQR309 (500 nM, 72h) was limited to 1/7 of cell lines. GEP was performed on 8 DLBCL cell lines (4 GCB, 4 ABC) after treatment with DMSO or PQR309 (1 mcM) for 4, 8 and 12h. PQR309 affected, in a time-dependent manner, relevant biologic pathways in both subtypes. Down-regulated transcripts were enriched of MYC targets, genes involved in NFKB/MYD88/BCR/IFN signaling, apoptosis, DNA damage and proteasome. Transcripts up-regulated were enriched of genes involved in cell cycle and senescence, up-regulated after MYD88 silencing, down-regulated by PI3K, involved in packaging of telomere, in autophagosome, up-regulated by inhibitors of HDAC, BET Bromodomain and JAK2. CXCR4, PIM1, PIM2, YPEL5 (up-regulated), LYAR, CCDC86, DDX21, HSPA8, STIP1, and PAK1IP1 (down-regulated) were among the genes changing after PQR309 treatment in more than one time-point or DLBCL cell-type. To identify markers of resistance we looked for transcripts differently regulated between cell lines with higher or lower sensitivity to PQR309 and we also compared baseline GEP between very sensitive (IC50 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in BCR pathway/signaling, kinases regulation, and immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 3/3 DLBCL and 3/3 MCL cells. Conclusions. PQR309 showed strong anti-proliferative activity in lymphomas and GEP identified affected biologic pathways and features possibly associated with response to the molecule. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Ivo Kwee, Andrea Rinaldi, Elena Bernasconi, Luciano Cascione, Petra Hillmann, Anastasios Stathis, Laura Carrassa, Massimo Broggini, Georg Stussi, Doriano Fabbro, Florent Beaufils, Anna Melone, Thomas Bohnacker, Matthias P. Wymann, Andreas Wicki, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2652. doi:10.1158/1538-7445.AM2015-2652