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Supplementary Figure 3 from The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

Supplementary Figure 1 from The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

The development of small-molecule activators of p53 is currently focused on malignancies containing a wild-type p53 genotype, which is present in most leukemias. JNJ-26854165 is one such p53-activating agent, but its mechanism of action remains to be elucidated. Here, we report the effects of JNJ-26854165 in acute leukemias. JNJ-26854165 treatment induced p53-mediated apoptosis in acute leukemia cells with wild-type p53, in which p53 rapidly drives transcription-independent apoptosis followed by activation of a transcription-dependent pathway. JNJ-26854165 accelerated the proteasome-mediated degradation of p21 and antagonized the transcriptional induction of p21 by p53. Interestingly, JNJ-26854165 induced S-phase delay and upregulated E2F1 expression in p53 mutant cells, resulting in apoptosis preferentially of S-phase cells. E2F1 knockdown blocked apoptosis induced by JNJ-26854165 in p53 mutant cells. Apoptotic activity of JNJ-26854165 against primary acute leukemia cells was maintained in leukemia/stroma cocultures, unlike doxorubicin, which has reduced cytrotoxicity in coculture systems. JNJ-26854165 synergizes with 1-β-arabinofuranosylcytosine or doxorubicin to induce p53-mediated apoptosis. Our data suggest that JNJ-26854165 may provide a novel therapeutic approach for the treatment of acute leukemias. The presence of p53-independent apoptotic activity in addition to p53-mediated apoptosis induction, if operational in vivo, may prevent the selection of p53 mutant subclones during therapy. Mol Cancer Ther; 9(9); 2545–57. ©2010 AACR.

Supplementary Tables 1-3 from The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy.Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors.Results:In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L).Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

Supplementary Figure 2 from The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

Supplementary Figure Legends from The Novel Tryptamine Derivative JNJ-26854165 Induces Wild-Type p53- and E2F1-Mediated Apoptosis in Acute Myeloid and Lymphoid Leukemias

Supplementary Data from JNJ-26481585, a Novel Second-Generation Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

Conventional models for preclinical drug screening offer poor predictive value for patient response, causing high attrition rates of new agents in the clinic. HUB’s proprietary Patient-Derived Organoid (PDO) Technology enables long-term expansion of primary patient material to generate ‘mini organs in a dish’ that can be used as patient avatars, thus bringing every “patient in the lab®”. We present the world’s first NMIBC PDO biobank comprising 50 PDO models from transurethral resection of bladder (TURB) biopsies collected at three hospitals. Clinical and pathological characterization of the PDOs confirmed the representation in the biobank of a range of tumor stages and differentiation grades, as well as patient treatment and follow-up. The genomic analyses of PDOs revealed mutations in known BC driver genes and genomic instability, a hallmark of cancer. Finally, transcriptomic analyses confirmed gene expression profiles familiar to BC and characterized gene fusions. Next, we successfully screened the biobank on a panel of two reference compounds for the treatment of bladder cancer and seven candidate drugs, indicating potential new leads for further development of some of the compounds. The work presented here shows the feasibility of building PDO biobanks representing a specific patient population, suitable for improving the drug development pipeline for the next generation of cancer drugs. Citation Format: Lars-Eric Fielmich, Annemarie Buijs, Daniele Mori, Bas Viergever, Nihed Draoui, Anna Schepers, Mariana M. Costa e Silva, Mathijs Scholtus, Tokameh Mahmoudi, Tahlita Zuiverloon, Merel Derksen, Paul Gavine, Sylvia F. Boj, Richard Meijer, Onno Kranenburg, Janine Arts, Robert Vries. Developing a patient-derived organoid biobank, suitable for large scale drug screenings [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 189.

Lattice light sheet microscopy real-time imaging reveals novel insights in leukocyte transendothelial migration. Janine J.G. Arts1,2,+, Eike K. Mahlandt2,+, Jos van Rijssel1, Simon Tol1, Satya Khuon3, John M. Heddleston3,4, Eric Wait3, Teng-Leong Chew3, Joachim Goedhart2, Jaap D. van Buul1,2,* +Authors contributed equally Affiliations: 1Molecular Cell Biology Lab at Dept. Molecular Hematology, Sanquin Research and Landsteiner Laboratory, Amsterdam, the Netherlands; 2Leeuwenhoek Centre for Advanced Microscopy (LCAM), section Molecular Cytology at Swammerdam Institute for Life Sciences (SILS) at University of Amsterdam, Amsterdam, the Netherlands; 3Advanced Imaging Center at Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA; 4Microscopy Facility at the Cleveland Clinic Florida Research and Innovation Center, Port St. Lucie, FL, USA *Correspondence should be addressed to: Jaap D. van Buul Molecular Cell Biology Lab, Dept. Molecular Hematology Sanquin Research and Landsteiner Laboratory Academic Medical Center at the University of Amsterdam Plesmanlaan 125, 1066CX Amsterdam, the Netherlands Phone: +31205121219 E-mail: j.vanbuul@sanquin.nl

Background Gene expression-based profiling of colorectal cancer (CRC) can be used to identify four molecularly homogeneous consensus molecular subtype (CMS) groups with unique biologic features. However, its applicability to colorectal premalignant lesions remains unknown. Patients and methods We assembled the largest transcriptomic premalignancy dataset by integrating different public and proprietary cohorts of adenomatous and serrated polyps from sporadic (N = 311) and hereditary (N = 78) patient populations and carried out a comprehensive analysis of carcinogenesis pathways using the CMS random forest (RF) classifier. Results Overall, transcriptomic subtyping of sporadic and hereditary polyps revealed CMS2 and CMS1 subgroups as the predominant molecular subtypes in premalignancy. Pathway enrichment analysis showed that adenomatous polyps from sporadic or hereditary cases (including Lynch syndrome) displayed a CMS2-like phenotype with WNT and MYC activation, whereas hyperplastic and serrated polyps with CMS1-like phenotype harbored prominent immune activation. Rare adenomas with CMS4-like phenotype showed significant enrichment for stromal signatures along with transforming growth factor-β activation. There was a strong association of CMS1-like polyps with serrated pathology, right-sided anatomic location and BRAF mutations. Conclusions Based on our observations made in premalignancy, we propose a model of pathway activation associated with CMS classification in colorectal carcinogenesis. Specifically, while adenomatous polyps are largely CMS2, most hyperplastic and serrated polyps are CMS1 and may transition into other CMS groups during evolution into carcinomas. Our findings shed light on the transcriptional landscape of premalignant colonic polyps and may help guide the development of future biomarkers or preventive treatments for CRC.

Prevention is an essential component of cancer eradication. Next-generation sequencing of cancer genomes and epigenomes has defined large numbers of driver mutations and molecular subgroups, leading to therapeutic advances. By comparison, there is a relative paucity of such knowledge in premalignant neoplasia, which inherently limits the potential to develop precision prevention strategies. Studies on the interplay between germ-line and somatic events have elucidated genetic processes underlying premalignant progression and preventive targets. Emerging data hint at the immune system’s ability to intercept premalignancy and prevent cancer. Genetically engineered mouse models have identified mechanisms by which genetic drivers and other somatic alterations recruit inflammatory cells and induce changes in normal cells to create and interact with the premalignant tumor microenvironment to promote oncogenesis and immune evasion. These studies are currently limited to only a few lesion types and patients. In this Perspective, we advocate a large-scale collaborative effort to systematically map the biology of premalignancy and the surrounding cellular response. By bringing together scientists from diverse disciplines (e.g., biochemistry, omics, and computational biology; microbiology, immunology, and medical genetics; engineering, imaging, and synthetic chemistry; and implementation science), we can drive a concerted effort focused on cancer vaccines to reprogram the immune response to prevent, detect, and reject premalignancy. Lynch syndrome, clonal hematopoiesis, and cervical intraepithelial neoplasia which also serve as models for inherited syndromes, blood, and viral premalignancies, are ideal scenarios in which to launch this initiative.

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 IC(50) (0.85 µM) 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.

The development of small-molecule activators of p53 is currently focused on malignancies containing a wild-type p53 genotype, which is present in most leukemias. JNJ-26854165 is one such p53-activating agent, but its mechanism of action remains to be elucidated. Here, we report the effects of JNJ-26854165 in acute leukemias. JNJ-26854165 treatment induced p53-mediated apoptosis in acute leukemia cells with wild-type p53, in which p53 rapidly drives transcription-independent apoptosis followed by activation of a transcription-dependent pathway. JNJ-26854165 accelerated the proteasome-mediated degradation of p21 and antagonized the transcriptional induction of p21 by p53. Interestingly, JNJ-26854165 induced S-phase delay and upregulated E2F1 expression in p53 mutant cells, resulting in apoptosis preferentially of S-phase cells. E2F1 knockdown blocked apoptosis induced by JNJ-26854165 in p53 mutant cells. Apoptotic activity of JNJ-26854165 against primary acute leukemia cells was maintained in leukemia/stroma cocultures, unlike doxorubicin, which has reduced cytrotoxicity in coculture systems. JNJ-26854165 synergizes with 1-β-arabinofuranosylcytosine or doxorubicin to induce p53-mediated apoptosis. Our data suggest that JNJ-26854165 may provide a novel therapeutic approach for the treatment of acute leukemias. The presence of p53-independent apoptotic activity in addition to p53-mediated apoptosis induction, if operational in vivo, may prevent the selection of p53 mutant subclones during therapy. Mol Cancer Ther; 9(9); 2545–57. ©2010 AACR.

Summary Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti-cancer agents and may be useful to enhance the therapeutic efficiency of established anti-myeloma treatments. This study preclinically evaluated the effects of the ‘second generation’ pan-HDAC inhibitor JNJ-26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n = 42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2-family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl-1 depletion and Hsp72 induction were the most reliable features observed in JNJ-26481585-treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ-26481585 with anti-myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC-inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.

Histone deacetylase (HDAC) inhibitors have been shown to induce cell cycle arrest, terminal differentiation, and apoptosis in a broad spectrum of human tumors and animal xenograft models. JNJ-26481585 is a hydroxamic acid derivative, second-generation pan-HDAC inhibitor that has demonstrated high potency in preclinical studies. In the current study, we demonstrated that JNJ-26481585 has antileukemia and molecular activity in leukemia cell lines and primary human leukemia cells. We also observed a synergistic effect between treatment with decitabine and JNJ-26481585. In conclusion, JNJ-26481585 is a potent second-generation pan-HDAC inhibitor with activity in human leukemia, and it is currently in clinical development.

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy. Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors. Results: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L). Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

A series of pyrimidyl-5-hydroxamic acids was prepared for evaluation as inhibitors of histone deacetylase (HDAC). Amino-2-pyrimidinyl can be used as a linker to provide HDAC inhibitors of good enzymatic potency.

Uniquely amongst the major tumor types, the premalignant state in colorectal cancer (CRC) is readily detectable and diagnosed. Indeed, a multi-step process of CRC genesis was defined by the seminal work of Vogelstein et al, who described the mutational events leading from adenoma to adenocarcinoma. In addition to genetic aberrations, recent evidence has highlighted the importance of the gut microbiome and associated inflammation in predicting CRC progression. To date however, these insights have not led to significant changes in the treatment paradigm because efforts continue to be focused on the later stages of disease. We believe CRC presents an exceptional opportunity for disease interception but to design effective interventional strategies, the exact sequence of molecular events underlying progression needs to be better defined and understood. To address these needs, we have assembled a clinically annotated sample database encompassing all stages of CRC (healthy colon, adjacent mucosa, adenomas, high-grade dysplasia, primary CRC and liver metastases), including samples from the conventional, microsatellite stable subtype, as well as from the serrated, microsatellite instable pathway. The progression status of each sample was characterized using standard pathology criteria. In addition, molecular progression was determined by targeted mutation profiling and targeted copy number profiling, as well as genome-wide expression profiling. This analysis confirms previous observations of early mutational events at the adenoma stage, including known tumor suppressor and oncogene driver mutations, e.g. KRAS G12 and G13 are mutated in 15% of the conventional adenomas but not in sessile serrated adenomas, as well as in 23% of the colorectal tumors. Copy number aberrations were observed at the adenoma and carcinoma stage, but with a lower prevalence then somatic mutations. Furthermore, genome wide expression analysis indicates that several pathways known to be affected in colorectal cancer are already disregulated at the adenoma stage. These pathways include Wnt signaling, mucosal barrier defects, bile acid metabolism, and several immune respons genes, again fingerpointing at the interplay between local inflammation, the microbiome, and epithelial events. In addition, we observed that while genetic events are very dissimilar between the serrated/MSI and conventional/MSS pathway, the transcriptional regulation has many similarities, indicating at a possibility at targeting these disease subtypes using the same therapeutics. These initial findings provide rational avenues to intercept CRC at the adenoma stage and efforts are now focused on exploring the added role of the colonic microbiota and immune system modulation. A more comprehensive and integrated view of the changes associated with disease initiation will lead to the identification of new paths for prevention, interception and cure. Citation Format: Joke Reumers, Liesbeth Van Wesenbeeck, Eric Ciamporcero, Gerald Chu, Stan Gaj, Emanuele Palescandolo, Carl Van Hove, Karin Verstraeten, Gary Borzillo, Dianna Wu, Pieter Peeters, Janine Arts. Characterisation of molecular events across the colorectal cancer progression axis [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 4388. doi:10.1158/1538-7445.AM2017-4388

Estrogens have been shown to be essential for maintaining a sufficiently high bone mineral density and ER alpha expression has been demonstrated in bone cells. Recently, a novel estrogen receptor, estrogen receptor beta (ERbeta) has been identified. Here we demonstrate that also ERbeta is expressed in human osteoblasts, and that ER alpha and ERbeta are differentially expressed during human osteoblast differentiation. ERbeta mRNA expression increased gradually during osteoblast culture, resulting in an average increase of 9.9+/-5.3 fold (mean+/-S.D., n=3) at day 21 (mineralization phase) as compared to day 6 (proliferation phase). In contrast, ER alpha mRNA expression levels increased only slightly until day 10 (2.3+/-1.7 fold) and then remained constant. The observed differential regulation of ER alpha and beta is suggestive for an additional functional role of ERbeta to ER alpha in bone metabolism.

Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR-alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids.

Serdemetan (JNJ-26854165) is a novel tryptamine compound with antiproliferative activity in various p53 wild-type (WT) tumor cell lines. We investigated its potential as radiosensitizer using four human cancer cell lines: H460, A549, p53-WT-HCT116, and p53-null-HCT116. Serdemetan inhibited clonogenic survival in all cell lines, but in a lower extent in p53-null-HCT116. In the combination studies, Serdemetan treatment at 0.25μM in H460 and at 5μM in A549 cells resulted in a sensitivity-enhancement ratio of 1.18 and 1.36, respectively. At 2Gy, surviving fractions were 0.72 and 0.97 for p53-WT HCT116 and p53-null cells exposed to 0.5μM of Serdemetan, respectively (p

The ubiquitin ligase Mdm2 targets the p53 tumor suppressor protein for proteasomal degradation. Mutating phosphorylation sites in the central domain of Mdm2 prevents p53 degradation, although it is still ubiquitylated, indicating that Mdm2 has a post-ubiquitylation function for p53 degradation. We show that Mdm2 associates with several subunits of the 19S proteasome regulatory particle in a ubiquitylation-independent manner. Mdm2 furthermore promotes the formation of a ternary complex of itself, p53, and the proteasome. Replacing phosphorylation sites within the central domain with alanines reduced the formation of the ternary complex. The C-terminus of Mdm2 was sufficient for interaction with the proteasome despite an additional proteasome binding site in the Mdm2 N-terminus. In addition to binding to the proteasome, the C-terminus of Mdm2 bound to the central domain, possibly competing with, and therefore blocking, Mdm2/proteasome interaction. We propose that Mdm2 facilitates, or at least enhances, the association of p53 with the proteasome and that phosphorylation of the central domain of Mdm2 regulates this process.

Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti-cancer agents and may be useful to enhance the therapeutic efficiency of established anti-myeloma treatments. This study preclinically evaluated the effects of the 'second generation' pan-HDAC inhibitor JNJ-26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n=42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2-family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl-1 depletion and Hsp72 induction were the most reliable features observed in JNJ-26481585-treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ-26481585 with anti-myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC-inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Multiple myeloma (MM) is a B cell malignancy, which often remains incurable due to the development of drug resistance governed by the bone marrow (BM) micro-environment. Novel treatment strategies are therefore urgently needed. In this study we evaluated the anti-MM activity of JNJ-26481585, a novel "second generation" pyrimidyl-hydroxamic acid based histone deacetylase inhibitor (HDACi), using the syngeneic murine 5TMM model of multiple myeloma. In vitro, JNJ-26481585 induced caspase cascade activation and up-regulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 towards cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity which can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC50 of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase- dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

Abstract —We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1α (IL-1α) in the human hepatoma cell line HepG2. PMA, serum, and IL-1α induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1α) increase in PAI-1 mRNA, peaking after ≈4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1α on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the −489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate–response element (TRE; between −58 and −50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1α. In line with these findings, IL-1α poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK) 1,2 activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1α. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 α only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1α in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three CC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and Hela cells differed in the exact G residues protected by nuclear proteins, in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non-stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and > 20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation. Chemicals/CAS: CCAAT-Enhancer-Binding Proteins; CTF-1 transcription factor; DNA Primers; DNA-Binding Proteins; Endopeptidases, EC 3.4.-; NFI Transcription Factors; Nuclear Proteins; Oncogene Proteins; Oncogene Proteins, Fusion; Tetradecanoylphorbol Acetate, 16561-29-8; Tissue Plasminogen Activator, EC 3.4.21.68; Transcription Factors; USP6 protein, human, EC 3.4.99.

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.

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.

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2011-1361

We have shown that the oral pan-HDAC inhibitor, JNJ-26481585, is a potent inhibitor of in vitro growth in a broad panel of solid and hematological tumor cell lines and western blot analysis confirmed modulation of HDAC1, 3 and 6 substrates (Arts et al, 2009). Inhibition of HDAC1 and 3 is associated with transcriptional suppression of target genes and regulation of tumor growth; modulation of HDAC6, impacting tubulin and Hsp90, results in depletion of oncogenic Hsp90 client proteins and potentiates tubulin acetylation with paclitaxel (Marcus et al, 2005; Valenzuela-Fernandez et al, 2008; Ai et al, 2009). We hypothesized that combinations between taxanes and JNJ-26481585 could result in enhanced tumor cell killing and describe our observations from such combinations in PC3-M prostate xenografts as a model of castrate resistant disease. We have previously shown that JNJ-26481585 has significant activity in xenograft models of prostate cancer, including PC3-M. In comparative studies in mice, JNJ-26481585 has favorable tissue distribution, achieving higher levels of exposure than panobinostat and vorinostat in tissues including e.g. prostate (2-fold and 3-fold, respectively); a sustained PD response is also observed in lung and skin (suppression of Ki67 for 24h; elevation of AcH3). We further demonstrate that the levels of JNJ-26481585 observed in orthotopically implanted PC3-M tumors are superior to those of panobinostat or vorinostat (5-fold and 13-fold, respectively). Thus we selected this orthotopic model in order to define further how the distribution of JNJ-26481585 to target tissue contributes to efficacy. We found that the activity of taxotere alone was surpassed by that of JNJ-26481585 alone (tumor growth delay (TGD) of 5d for taxotere, and 15d for JNJ-26481585; vs vehicle) and significant TGD was observed from the combination of the agents (with 1mpk JNJ-26481585 = 20d; with 3mpk JNJ-26481585 = beyond study end). The role of HDAC inhibition in the stability and regulation of the androgen receptor has recently been described as dependent on both HDAC1, 3 and 6 activities (Welsbie et al, 2009; Ai et al, 2009), which gives further support to our rationale for combining agents with JNJ-26481585. We hypothesized that JNJ-26481585 would be an efficacious agent when combined with abiraterone, an inhibitor of androgen synthesis. In LnCAP and VCaP prostate cancer cells, we showed by western blot analysis that exposure to JNJ-26481585 resulted in a dose dependent reduction in AR levels and profiling on Affymetrix microarrays also showed modulation of the expression of AR responsive genes e.g. KLKs and TMPRSS2. Consistent with a sustained PD response in tissues, we see continued reduction in AR levels and expression of AR dependent genes up to 24h after removal of compound. We continue to investigate the combination of JNJ-26481585 and abiraterone in vivo as our data provide strong support for the combination in prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2606. doi:10.1158/1538-7445.AM2011-2606

We 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. Combinations 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. The 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. Finally, 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. Citation 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.

Background: The ubiquitin-proteasome pathway has been validated as a target for NHL with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of anti-apoptotic heat shock proteins, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor. Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule JNJ-26854165, an inhibitor of HDM-2-function, in both p53 wt and mut cell line models. Results: Treatment of wt p53 NHL cell lines with JNJ-26854165 induced a dose- and time-dependent inhibition of proliferation, with an IC50 in the 0.02–0.3 μM range. Cell death, which was typically seen within 48 hours of HDM-2 inhibition, occurred through induction of type I PCD, as judged by the appearance of increased staining with Annexin V and activation of caspase 3. While cell lines with mut p53 were generally less sensitive than their wt p53 counterparts, JNJ-26854165 remained potent, with an IC50 in the 0.05–0.6 μM range. The latter cell lines showed a longer kinetics of death, with PCD being seen within 72 hours of drug exposure. Notably, in these mut p53 cell lines, very little Annexin V staining or caspase 3 activation was seen, consistent with a minor role for type I PCD. Instead, mut p53 cell lines demonstrated an increased content of acidic vacuoles by acridine orange staining, increased expression of Beclin 1 and Sequestosome 1/p62, and conversion of microtubule-associated protein 1 light chain 3 form I to form II, consistent with activation of type II PCD, or autophagy. Also, electron microscopy showed an increased presence of autophagosomes and autolysosomes, further supporting the activation of this pathway. Combinations of JNJ-26854165 with other agents, including rapamycin, doxorubicin, and an inhibitor of Bcl 2, showed enhanced anti-proliferative effects in a sequence-dependent fashion, which were greatest when the chemotherapeutic preceded the HDM-2 inhibitor. Combination index analysis revealed that these interactions met criteria for synergy. Conclusions: Inhibition of the function of HDM-2 using JNJ-26854165 is a promising approach that is effective against both wt and mut p53 models by activating type I and type II PCD, respectively. The effectiveness of JNJ-26854165 was enhanced in combination with currently used chemotherapeutics in a sequence specific manner, providing a rationale for translation of this novel approach into the clinic.

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. [Cancer Res 2009;69(13):5307–11]