Janine Arts

R306465 is a novel potent inhibitor of class I histone deacetylases with broad-spectrum antitumoral activity against solid and haematological malignancies

R306465 is a novel hydroxamate-based histone deacetylase (HDAC) inhibitor with broad-spectrum antitumour activity against solid and haematological malignancies in preclinical models. R306465 was found to be a potent inhibitor of HDAC1 and -8 (class I) in vitro. It rapidly induced histone 3 (H3) acetylation and strongly upregulated expression of p21waf1,cip1, a downstream component of HDAC1 signalling, in A2780 ovarian carcinoma cells. R306465 showed class I HDAC isotype selectivity as evidenced by poor inhibition of HDAC6 (class IIb) confirmed by the absence of downregulation of Hsp90 chaperone c-raf protein expression and tubulin acetylation. This distinguished it from other HDAC inhibitors currently in clinical development that were either more potent towards HDAC6 (e.g. vorinostat) or had a broader HDAC inhibition spectrum (e.g. panobinostat). R306465 potently inhibited cell proliferation of all main solid tumour indications, including ovarian, lung, colon, breast and prostate cancer cell lines, with IC50 values ranging from 30 to 300u2009nM. Haematological cell lines, including acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphoblastic leukaemia, chronic myeloid leukaemia, lymphoma and myeloma, were potently inhibited at a similar concentration range. R306465 induced apoptosis and inhibited angiogenesis in cell-based assays and had potent oral in vivo antitumoral activity in xenograft models. Once-daily oral administration of R306465 at well-tolerated doses inhibited the growth of A2780 ovarian, H460 lung and HCT116 colon carcinomas in immunodeficient mice. The high activity of R306465 in cell-based assays and in vivo after oral administration makes R306465 a promising novel antitumoral agent with potential applicability in a broad spectrum of human malignancies.

Bortezomib Alone or in Combination with the Histone Deacetylase Inhibitor JNJ-26481585: Effect on Myeloma Bone Disease in the 5T2MM Murine Model of Myeloma

The proteasome inhibitor bortezomib (Velcade) is currently approved as second-line treatment of multiple myeloma (MM). MM-related bone disease is one of the most debilitating complications of MM. Besides supportive care with biphosphonates, which have proven efficacy in reducing and delaying skeletal-related events, there is no specific treatment of lytic bone lesions. The present study investigated the effect of bortezomib alone or in combination with a hydroxamate-based histone deacetylase inhibitor, JNJ-26481585 on tumor burden, and MM bone disease in the 5T2MM model. Injection of 5T2MM cells into C57Bl/KaLwRij mice resulted in MM bone disease, characterized by an increase in the percentage osteoclasts, a decrease in osteoblasts, trabecular bone volume, trabecular number, and the development of bone lesions. Treatment of 5T2MM-bearing mice with bortezomib significantly reduced tumor burden, angiogenesis, and MM bone disease. More importantly, the combination of bortezomib with JNJ-26481585 resulted in a more pronounced reduction of osteoclasts and increase of osteoblasts, trabecular bone volume, and trabecular number compared with bortezomib as single agent. These data suggest that bortezomib has bone remodeling properties that can be improved in combination with low dose JNJ-26481585. The study indicates that this combination therapy could be a useful strategy for the treatment of MM patients, especially in those patients with skeletal complications.

Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells

The hdm-2 oncogene is overexpressed in several types of malignancies including osteosarcomas, soft tissue sarcomas and gliomas and hdm-2 has been associated with accelerated tumor formation in both hereditary and sporadic cancers. Among the other key binding partners, hdm-2 forms a complex with the tumor suppressor p53, resulting in a rapid proteasome-mediated degradation of the p53 protein. This positions the hdm-2–p53 complex as an attractive target for the development of anticancer therapy and recently the first small molecule hdm-2 antagonist has been reported. Development of hdm-2 antagonists is currently focused on malignancies containing a wild-type p53 genotype, which is the case in approximately half of human cancer indications. However, hdm-2 has also been implicated in oncogenesis in the absence of p53. We therefore studied the effect of hdm-2 antagonists in p53-deficient human H1299 lung carcinoma cells. The hdm-2 antagonistic peptide caused G1 cell cycle arrest, inhibited colony growth and induced expression of G1 checkpoint regulatory proteins, such as p21waf1,cip1. These data demonstrate that hdm-2 regulates the G1 cell cycle checkpoint in a p53-independent manner, suggesting that hdm-2 antagonists represent a novel class of anticancer therapeutics with broad applicability towards tumors with different p53 genetic backgrounds.

Initial testing of JNJ-26854165 (Serdemetan) by the pediatric preclinical testing program†

JNJ‐26854165 was originally developed as an activator of p53 capable of inducing apoptosis in cancer cell lines. In vitro, JNJ‐26854165 demonstrated cytotoxic activity. The ALL cell line panel had a significantly lower median IC50 (0.85u2009µM) than the remaining cell lines. In vivo JNJ‐26854165 induced significant differences in EFS distribution compared to control in 18 of 37 solid tumors and in 5 of 7 of the evaluable ALL xenografts. Objective responses were observed in 4 of 37 solid tumor xenografts, and 2 of 7 ALL xenografts achieved PR or CR. Responses were noted in xenografts with both mutant and wild‐type p53. Pediatr Blood Cancer 2012;59:329–332.

Abstract A143: A preclinical in vivo model to assess pharmacodynamic inhibition of ACK1.

Introduction: Activated Cdc42-associated kinase (ACK1, TNK2) has been reported to be over-expressed in a range of tumor types including prostate and lung cancer, and has been suggested as a potential novel anticancer target. A number of kinase inhibitors currently in clinical development show inhibition of ACK1 biochemical activity in enzyme assays, yet their ability to inhibit ACK1 in vivo is unknown. This study describes the development of a pharmacodynamic model to evaluate ACK1 target coverage in vivo. Methods: MDA-MB-231 cells were engineered to over-express ACK1 under the control of a doxycycline-inducible promoter (MDA-MB-231-ACK1). MDA-MB-231-ACK1 cells were grown as subcutaneous xenografts in MF-1 nude mice and ACK1 expression induced by dosing doxycycline by oral gavage. Saracatinib/AZD-0530 (100mg/kg qd) or ASP-3026 (50mg/kg bid) were dosed for four consecutive days to reach steady state exposures at approximately MTD. Pharmacodynamic inhibition of ACK1 autophosphorylation was assessed using an in-house MSD assay and confirmed by Western blotting, in order to evaluate target inhibition in vivo. Results: ASP-3026 and saracatinib showed inhibition of cellular pACK1 of MDA-MB-231-ACK1 cells in vitro, with IC50s of 0.12 μM and 1.1 μM, respectively. Using the same cell line in vivo, expression of total and phosphorylated ACK1 was induced by daily oral dosing of doxycycline. We used this model to evaluate PK-PD correlations of ASP-3026 and saracatinib using pACK1 as a marker of target inhibition. Good plasma and tumor exposure of ASP-3026 was achieved at 50mg/kg bid and no drug-drug interactions were observed in combination with doxycycline. ASP-3026 showed 50-80% inhibition of pACK1 in vivo consistent with unbound plasma concentrations in excess of in vitro IC50 and in good agreement with predictions. Good exposure of saracatinib was observed at 100 mg/kg qd with total plasma Cmax of >4μM in excess of maximally tolerated clinical exposures (0.82μM). Tumor exposures of saracatinib were up to 10 x higher than plasma exposures (Cmax >40 μM), and in considerable excess of the cellular IC50, yet no ACK1 inhibition was observed in vivo. Conclusions: A robust PD model was established to evaluate ACK1 inhibition in vivo. Up to 80% inhibition of ACK1 in vivo was observed with ASP-3026, falling to approximately 50% at Cmin, suggesting that this agent does not completely inhibit ACK1 in vivo at steady state. In contrast, saracatinib did not significantly inhibit ACK1 at any of the exposures achieved, suggesting that it has limited in vivo activity against this target. Unbound plasma exposures predicted in vivo PD responses very well, whereas tumor exposures of these agents were not predictive of target inhibition. This model can be incorporated into a screening cascade for prioritising in vivo active ACK1 inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A143. Citation Format: Mark Albertella, Shilina Roman, Mike Briggs, Aaron Cranston, Ralph Graeser, George Hynd, David Jones, Peter Lockey, Akbor Malik, Stephen Price, Susanna Stimpson, Patrizia Tisselli, Janine Arts, Simon Green. A preclinical in vivo model to assess pharmacodynamic inhibition of ACK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A143.

Abstract A15: High-Content screen for inhibitors of cell migration in cancer metastasis using adenoviral knock-down

Enhanced cell migration is a hallmark of metastatic cancer cells. The propensity of cancer cells to close an open wound in a cell monolayer is thought to predict this ability. Using our adenoviral shRNA knockdown library we have established a high-throughput wound healing assay to identify novel genes involved in cell migration. A 96-pin tool was designed to apply a constant mechanical scratch-wound in the cellular monolayer. We used transmitted light imaging for segmentation and quantification of the scratch wound that remained open. Genes whose knockdown inhibit cell migration can be identified by their effect on the open wound. We demonstrated that two knockdown constructs targeting a known player in motility, CXCR4, inhibit wound healing, thereby validating our approach. Using this wound healing assay we have identified a number of novel genes associated with cancer cell motility. These targets have consecutively been validated for their role in 3-D invasion using Boyden chambers. As our adenoviral knockdown libraries focus on druggable targets, these validated targets can quickly be employed to generate small-molecule compounds or antibody therapeutics targeting cancer metastasis. Citation Format: Remko de Pril, Annemarie Lekkerkerker, Desire van Steenhoven, Ilhem Maghrani, Tim Perera, Janine Arts, David Fischer, Richard Janssen. High-Content screen for inhibitors of cell migration in cancer metastasis using adenoviral knock-down [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A15.

Abstract 5443: Rational combinations with the HDAC inhibitor JNJ-26481585: Prostate cancer and taxane therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnWe have recently reported the identification and characterization of JNJ-26481585, as a “second generation” oral pan-HDAC inhibitor. The compound has shown potent in vitro activity against solid and hematological tumor cell lines and significant induction of apoptosis was observed, consistent with the potency of JNJ-26481585 for Class I HDACs. In dose response to JNJ-26481585, western blot analysis of A2780 cell extracts confirmed modulation of HDAC1-3 substrates and also HDAC6 substrates. Inhibition of HDAC6, the deacetylase for tubulin and Hsp90, results in depletion of oncogenic Hsp90 client proteins, decreased cell motility, and potentiation of therapeutics such as taxanes. We hypothesized that rational combinations with taxanes and JNJ-26481585 could result in enhanced tumor cell killing and describe our observations from the combination of JNJ-26481585 in PC3-M prostate tumors.nnCombinations of JNJ-26481585 and taxanes were performed by co-administration or 24h separation of agents in A549, NCI-H460, NCI-H1373, NCI-H1703, NCI-H322 and NCI H-1650 lung cell lines; breast cell lines, MDA-MB-231, T47D, BT474 and MCF-7 cells; and prostate cell lines PC3M, DU-145, 22Rv1 and LnCap. We will present data on the outcome of these combinations, including additive and synergistic effects.nnThe in vitro data encouraged us to explore the effects of the compound in vivo and in prostate models where we already had excellent single agent activity for JNJ-26481585 in DU-145, PC-3 and the PC-3M orthotopic model (significant T/C values in each model and at doses substantially below MTD). In PC3-M xenograft models, the combination of 1.25, 5 or 10mpk JNJ-26481585 (Q2D) and taxotere (Q7D), was more effective than either agent alone and resulted in dose dependent tumor regression and substantial extension of time to relapse. We then selected the more challenging PC-3M orthotopic mouse model to further define the contribution to efficacy from the excellent tissue distribution of our agent. Comparing 5mpk taxotere (Q7D), with 1 or 3mpk of JNJ-26481585 (QD) or the combination of agents, the activity of taxotere alone was surpassed by either dose of HDAC inhibitor alone and substantial tumor growth delay was observed from the combination of 3mpk JNJ-26481585 and taxotere; activity greater than predicted from the in vitro experiments.nnFinally, although HDAC6 inhibition could directly explain potentiation of taxotere by JNJ-26481585 in vitro, we believe the substantial effects in vivo may be due to the anti-angiogenic effect observed for our agent. We present also our data showing anti-angiogenic effects of JNJ-26481585 in PC3-M tumors, consistent with this hypothesis. Our data support the further use of more complex models, reflective of human disease in preclinical assessment and also support further exploration of this agent in rational combinations with SOC agents. JNJ-26481585 is currently in Phase I clinical trials.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5443.

Abstract 5441: Preclinical assessment of the HDAC inhibitor JNJ-26481585: potent in vivo activity across a broad spectrum of human tumor xenografts

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnJNJ-26481585 is a promising “second generation” oral pan-HDAC inhibitor. Recently, we have published data describing the identification and characterization of the molecule as a potent inhibitor of HDAC1 with a favorable pharmacodynamic profile. Subsequent analysis has shown that the compound has pan-HDAC inhibitory activity and demonstrates effective inhibition of tumor cell growth across a broad spectrum of solid and hematological tumor cell lines. Recent data has also shown that in vivo JNJ-26481585 alone, or in combination with bortezomib, is an effective agent in myeloma. Herein we describe the activity of JNJ-26481585 against a broad range of preclinical solid tumor xenograft models, at doses substantially below MTD and with different schedules of administration in order to address the clinical utility of this agent.nnSignificant growth inhibition was achieved when dosing JNJ-26481585 at MTD with T/C in tumors of; A2780 ovarian tumor xenograft (3%), ras-mutant colon (HCT-116), (7%), MDA-MB-231 breast (38%), DU-145 prostate (11%) and lung H460, A549 and ras mutant NCI-H1373 (29%, 15% and 32% respectively). In the A2780 model, activity of JNJ-26481585 was superior to that of five other HDAC inhibitors tested. This class leading activity was observed at MTD and optimal route of administration of all compounds and importantly, we also observed significant activity of JNJ-26481585 at concentrations as low as 12.5% of MTD (T/C = 35%). Both class leading activity and significant tumor growth inhibition at concentrations substantially below MTD was also observed in A549, DU145 and HCT116 tumor xenografts. Furthermore, in HCT116, in line with taxotere in prostate models, JNJ-26481585 was shown to be more effective than the SOC, 5-FU. The HCT116 model was used further to address the potential of the compound for clinical utility by exploring alternative dosing schedules to that of QD IP dosing. 3 days on/4 days off, 1 week on/ 1 week off, Q2D and Q7D were all investigated alongside QD dosing and observed to inhibit tumor growth in all cases; Q7D was the only alternative schedule not to achieve efficacy equivalent to that with QD dosing.nnFinally, data indicating greater activity or equivalence with the SOC suggested that JNJ-26481585 could be used in those indications, thus potency was investigated in drug resistant patient material. In both taxol resistant NSCLC and doxorubicin resistant breast cancer cultures, JNJ-26481585 dramatically inhibited proliferation. We conclude that in preclinical models, JNJ-26481585 was effective at reducing the tumor size to the greatest extent of all HDAC agents studied. Potent antitumoral activity was also observed at doses substantially below MTD and from alternative dosing schedules, suggesting that this could be a promising agent for clinical development in such indications. JNJ-26481585 is currently in Phase I clinical trials.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5441.