Pascale Lejeune

Preclinical Antitumor Activity of Cabazitaxel, a Semisynthetic Taxane Active in Taxane-Resistant Tumors

Purpose: Taxanes are important chemotherapeutic agents with proven efficacy in human cancers, but their use is limited by resistance development. We report here the preclinical characteristics of cabazitaxel (XRP6258), a semisynthetic taxane developed to overcome taxane resistance. Experimental Design: Cabazitaxel effects on purified tubulin and on taxane-sensitive or chemotherapy-resistant tumor cells were evaluated in vitro. Antitumor activity and pharmacokinetics of intravenously administered cabazitaxel were assessed in tumor-bearing mice. Results: In vitro, cabazitaxel stabilized microtubules as effectively as docetaxel but was 10-fold more potent than docetaxel in chemotherapy-resistant tumor cells (IC50 ranges: cabazitaxel, 0.013–0.414 μmol/L; docetaxel, 0.17–4.01 μmol/L). The active concentrations of cabazitaxel in these cell lines were achieved easily and maintained for up to 96 hours in the tumors of mice bearing MA16/C tumors treated with cabazitaxel at 40 mg/kg. Cabazitaxel exhibited antitumor efficacy in a broad spectrum of murine and human tumors (melanoma B16, colon C51, C38, HCT 116, and HT-29, mammary MA17/A and MA16/C, pancreas P03 and MIA PaCa-2, prostate DU 145, lung A549 and NCI-H460, gastric N87, head and neck SR475, and kidney Caki-1). Of particular note, cabazitaxel was active in tumors poorly sensitive or innately resistant to docetaxel (Lewis lung, pancreas P02, colon HCT-8, gastric GXF-209, mammary UISO BCA-1) or with acquired docetaxel resistance (melanoma B16/TXT). Conclusions: Cabazitaxel is as active as docetaxel in docetaxel-sensitive tumor models but is more potent than docetaxel in tumor models with innate or acquired resistance to taxanes and other chemotherapies. These studies were the basis for subsequent clinical evaluation. Clin Cancer Res; 19(11); 2973–83. ©2013 AACR.

SAR650984, A Novel Humanized CD38-Targeting Antibody, Demonstrates Potent Antitumor Activity in Models of Multiple Myeloma and Other CD38+ Hematologic Malignancies

Purpose: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. Experimental Design: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. Results: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. Conclusion: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies. Clin Cancer Res; 20(17); 4574–83. ©2014 AACR.

Discovery and optimization of new benzimidazole- and benzoxazole-pyrimidone selective PI3Kβ inhibitors for the treatment of phosphatase and TENsin homologue (PTEN)-deficient cancers.

Most of the phosphoinositide-3 kinase (PI3K) kinase inhibitors currently in clinical trials for cancer treatment exhibit pan PI3K isoform profiles. Single PI3K isoforms differentially control tumorigenesis, and PI3Kβ has emerged as the isoform involved in the tumorigenicity of PTEN-deficient tumors. Herein we describe the discovery and optimization of a new series of benzimidazole- and benzoxazole-pyrimidones as small molecular mass PI3Kβ-selective inhibitors. Starting with compound 5 obtained from a one-pot reaction via a novel intermediate 1, medicinal chemistry optimization led to the discovery of compound 8, which showed a significant activity and selectivity for PI3Kβ and adequate in vitro pharmacokinetic properties. The X-ray costructure of compound 8 in PI3Kδ showed key interactions and structural features supporting the observed PI3Kβ isoform selectivity. Compound 8 achieved sustained target modulation and tumor growth delay at well tolerated doses when administered orally to SCID mice implanted with PTEN-deficient human tumor xenografts.

Moving Towards Precision Urologic Oncology: Targeting Enzalutamide-resistant Prostate Cancer and Mutated Forms of the Androgen Receptor Using the Novel Inhibitor Darolutamide (ODM-201)

Darolutamide (ODM-201) is a novel androgen receptor (AR) antagonist with a chemical structure distinctly different from currently approved AR antagonists that targets both wild-type and mutated ligand binding domain variants to inhibit AR nuclear translocation. Here, we evaluate the activity of darolutamide in enzalutamide-resistant castration resistant prostate cancer (CRPC) as well as in AR mutants detected in patients after treatment with enzalutamide, abiraterone, or bicalutamide. Darolutamide significantly inhibited cell growth and AR transcriptional activity in enzalutamide-resistant MR49F cells in vitro, and led to decreased tumor volume and serum prostate-specific antigen levels in vivo, prolonging survival in mice bearing enzalutamide-resistant MR49F xenografts. Moreover, darolutamide inhibited the transcriptional activity of AR mutants identified in the plasma of CRPC patients progressing on traditional therapies. In particular, darolutamide significantly inhibited the transcriptional activity of the F877L, H875Y/T878A, F877L/T878A, and the previously unreported T878G AR mutants, that transform enzalutamide into a partial agonist. In silico cheminformatics computer modeling provided atomic level insights confirming darolutamide antagonist effect in F877L and T878G AR mutants. In conclusion, our results provide a rationale for further clinical evaluation of darolutamide in enzalutamide-resistant CRPC, in particular in combination with circulating tumor DNA assays that detect AR mutants sensitive to darolutamide, in a precision oncology setting. PATIENT SUMMARY In this study we evaluated the novel drug darolutamide in preclinical models of prostate cancer. We found that darolutamide delays growth of enzalutamide-resistant prostate cancer, in particular in cells with mutated forms of the androgen receptor after previous treatment. Our data supports further evaluation of darolutamide in clinical trials.

Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to pro-apoptotic agents

Non-small cell lung cancer (NSCLC) has the highest incidence of cancer-related death worldwide and a high medical need for more effective therapies. Small-molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1, I-BET762 and OTX-015 are active in a wide range of different cancer types, including lung cancer. Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. Here we evaluated the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptosis response. Using a panel of 12 KRAS-mutated NSCLC models, we found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with downregulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. In the H1373 xenograft model, treatment with JQ1 significantly reduced tumor growth and downregulated the expression of c-Myc. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and we found the expression of the two key apoptosis regulators FLIP and XIAP to be highly BET dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Further we showed that combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether, these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment. They also demonstrate that BET inhibition primes NSCLC cells for induction of apoptosis and that a combination with pro-apoptotic compounds represents a valuable strategy to overcome treatment resistance.

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.

Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4.

Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.Interdependence between YAP/TAZ and BRD4 drives transcriptional addiction in cancer cells and determines sensitivity to BET inhibition.

Abstract 321: Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models

The DNA damage response (DDR) system consists of complex signalling pathways that secure the integrity of the genome in eukaryotic cells. DDR pathway activation follows recognition of DNA damage and results in cell cycle arrest, suppression of general translation, induction of DNA repair, cell survival or even cell death. Proteins that directly recognize aberrant DNA structures recruit and activate kinases of the DDR, such as ATR (ataxia telangiectasia and Rad3-related). ATR responds to a broad spectrum of DNA damages, including double-strand breaks (DSB) and lesions derived from interference with DNA replication as well as increased replication stress. Therefore, inhibition of ATR kinase activity could be the basis for a novel anti-cancer therapy in tumors with increased DNA damage, deficiency in DDR or replication stress. The potential of combining ATR kinase inhibitor with DNA damage inducing or DNA repair compromising anti-cancer therapeutics was studied in preclinical tumor models. We assessed the novel ATR kinase inhibitor (ATRi) BAY 1895344 in combination with external beam radiation therapy (EBRT), poly ADP ribose polymerase (PARP) inhibition or anti-androgen (AA) therapy. In cellular anti-proliferation assays as well as in tumor xenograft studies we could demonstrate synergistic activity of BAY 1895344 in combination treatment with the PARP inhibitor AZD-2281 in the homologous recombination (HR) defective breast cancer model MDA-MB-436, and with the non-steroidal AA darolutamide in the hormone-dependent prostate cancer model LAPC-4. Strong synergistic anti-tumor activity of BAY 1895344 could be further demonstrated in combination with EBRT inducing long-lasting tumor growth inhibition in the colorectal cancer xenograft model LOVO. The mechanism-based potential of combining DNA damage induction by EBRT with ATRi BAY 1895344 suggests a potential new treatment option for radiation therapy-resistant patients. Furthermore, the inhibition of parallel DDR pathways, as a combination of ATRi BAY 1895344 with a PARP inhibitor, indicates novel treatment opportunities in breast cancer patients with homologous recombination deficiencies, as does the synergism of BAY 1895344 and AA darolutamide therapy in hormone-dependent prostate cancer patients. BAY 1895344 is currently under clinical investigation in patients with advanced solid tumors and lymphomas (NCT03188965). Citation Format: Antje Margret Wengner, Gerhard Siemeister, Ulrich Luecking, Julien Lefranc, Kirstin Meyer, Eleni Lagkadinou, Bernard Haendler, Pascale Lejeune, Dominik Mumberg. Synergistic activity of the ATR inhibitor BAY 1895344 in combination with DNA damage inducing and DNA repair compromising therapies in preclinical tumor models [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 321.

Abstract A028: In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models

Darolutamide is a novel, high-affinity androgen receptor (AR) antagonist currently in clinical phase 3 studies for nonmetastatic castration-resistant prostate cancer and metastatic hormone-sensitive prostate cancer. Its efficacy and mode of action were analyzed in detail using two different prostate cancer models. In vitro efficacy was determined by measuring cell proliferation with alamarBlue® and spheroid formation with the ImageJ program, following treatment of single-cell suspensions in a 3D culture model. Gene expression was determined with RT2 profiler arrays and by real-time PCR, AR protein levels by ELISA and AR or RNA polymerase II binding to DNA by ChIP-qPCR. In vivo efficacy studies were performed following subcutaneous injection of LAPC-4 cells into noncastrated, testosterone-supplemented SCID male mice. Treatment started on day 26 post tumor inoculation at a mean tumor range of 137-150 mm3. Tumor size was measured with calipers and converted in volume using the formula: (length x width2)/2. For gene expression analysis, RNA was extracted from tumors using the RNeasy® Plus Mini Kit. Treatment of androgen-stimulated VCaP or LAPC-4 prostate cancer cells with darolutamide, diastereomers (S,R)- and (S,S)-darolutamide and the main circulating metabolite keto-darolutamide led to a superior inhibition of proliferation, demonstrating a continuous high pressure on AR. A strong reduction of spheroid formation and dose-dependent reduction of androgen-stabilized AR protein levels were observed in both cell lines. There was marked downregulation in expression of androgen target genes such as KLK3/PSA, TMPRSS2, and FKBP5 with reduced binding of the AR to the KLK3/PSA upstream regulatory region. Furthermore, we observed a reduction of RNA polymerase II recruitment. In vivo efficacy studies revealed a significant reduction of tumor growth when darolutamide was dosed orally, daily or twice daily, compared to castrated, untreated mice. At day 56, the ΔT/C was 25% for the 100 mg/kg bi-daily dose and 23% for the 200 mg/kg once-daily dose. All the treatment schedules were well tolerated, as indicated by the limited body weight loss observed (-3% maximal mean body weight loss). Gene expression analysis of the tumors at the end of treatment showed a 50% reduction of FKBP5 transcript levels for the highest treatment doses. In conclusion, darolutamide shows a consistent high pressure on AR. Significant antitumor efficacy was observed in vitro and in vivo in prostate cancer models. RNA-seq and ChIP-seq studies are currently ongoing for the determination of the complete transcription profiles and AR binding pattern of treated tumor models. Altogether, these data further support the evaluation of darolutamide in clinical studies for prostate cancer. Conflict of Interest: All authors are employees and/or shareholders of Bayer. Darolutamide is jointly developed by Bayer and Orion. Citation Format: Tatsuo Sugawara, Simon J. Baumgart, Kristin Reichert, Pascale Lejeune, Bernard Haendler. In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A028.

Abstract 4749: New benzazepine BET-inhibitors with improved oral bioavailability

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, binds to acetylated histone tails via its two bromodomains BD1 and BD2. It forms a complex with the positive transcription elongation factor b which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation. An essential role of BRD4 in cell proliferation and cancer growth has been reported in several recent studies. The benzodiazepine JQ-1 is a strong antagonist of the binding of BRD4 to acetylated histone tails and has been used to elucidate the functions of the BET protein family members. JQ-1 is active in vivo in several therapeutic models but reveals a poor pharmacokinetic profile with high clearance and low oral bioavailability in animal studies. We investigated the benzodiazepine core of JQ-1 with the aim of optimization of oral bioavailability. Several possible core variations were identified that kept overall cellular activity but increased metabolic stability. The benzazepine BAY6356 was selected as a potent BET inhibitor with an improved overall pharmacokinetic profile and oral bioavailability between 60 and 100% in mouse, rat and dog. The strong antiproliferative activity observed in vitro in acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines was confirmed in vivo in the MOLM-13 (AML) and MOLP-8 (MM) tumor models implanted in SCID mice. Daily oral treatment at the Maximal Tolerated Dose of 30 mg/kg led to strong tumor reduction in MOLM-13 (17% T/C on day 13 post tumor implantation) and in MOLP-8 (4% T/C on day 19 post tumor implantation) xenografts (T/C≤ 40% = active, T/C≤10% = highly active, according to NCI criteria). In the MOLM-13 model, c-Myc down-regulation was shown in vivo. Furthermore, for a same total dose, intermittent dosing every other day or twice a week in the MOLP-8 model proved to be as active as daily dosing, demonstrating exposure-driven efficacy. These favorable preclinical data support the evaluation of BAY6356 for further development. Citation Format: Norbert Schmees, Bernard Haendler, Pascale Lejeune, Antje Stresemann, Roland Neuhaus, Stephan Siegel, Amaury Ernesto Fernandez-Montalvan, Hilmar Weinmann, Volker Gekeler. New benzazepine BET-inhibitors with improved oral bioavailability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4749. doi:10.1158/1538-7445.AM2014-4749