Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Small-molecule inhibitors of the protein methyltransferase SET7/9 identified in a high-throughput screen.

Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.

Mechanism-based screen establishes signalling framework for DNA damage-associated G1 checkpoint response.

DNA damage activates checkpoint controls which block progression of cells through the division cycle. Several different checkpoints exist that control transit at different positions in the cell cycle. A role for checkpoint activation in providing resistance of cells to genotoxic anticancer therapy, including chemotherapy and ionizing radiation, is widely recognized. Although the core molecular functions that execute different damage activated checkpoints are known, the signals that control checkpoint activation are far from understood. We used a kinome-spanning RNA interference screen to delineate signalling required for radiation-mediated retinoblastoma protein activation, the recognized executor of G(1) checkpoint control. Our results corroborate the involvement of the p53 tumour suppressor (TP53) and its downstream targets p21(CIP1/WAF1) but infer lack of involvement of canonical double strand break (DSB) recognition known for its role in activating TP53 in damaged cells. Instead our results predict signalling involving the known TP53 phosphorylating kinase PRPK/TP53RK and the JNK/p38MAPK activating kinase STK4/MST1, both hitherto unrecognised for their contribution to DNA damage G1 checkpoint signalling. Our results further predict a network topology whereby induction of p21(CIP1/WAF1) is required but not sufficient to elicit checkpoint activation. Our experiments document a role of the kinases identified in radiation protection proposing their pharmacological inhibition as a potential strategy to increase radiation sensitivity in proliferating cancer cells.

Targeting the Hsp90 molecular chaperone with novel macrolactams. Synthesis, structural, binding, and cellular studies.

A series of resorcylic acid macrolactams, nitrogen analogues of the naturally occurring macrolactone radicicol, have been prepared by chemical synthesis and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves, as key steps, ring opening of an isocoumarin intermediate, followed by a ring-closing metathesis reaction to form the macrocycle. Subsequent manipulation of the ester group into a range of amides allows access to a range of new macrolactams following deprotection of the two phenolic groups. These new resorcylic acid lactams exhibit metabolic stability greater than that of related lactone counterparts, while co-crystallization of three macrolactams with the N-terminal domain ATP site of Hsp90 confirms that they bind in a similar way to the natural product radicicol and to our previous synthetic lactone analogues. Interestingly, however, in the case of the N-benzylamide, additional binding to a hydrophobic pocket of the protein was observed. In biological assays, the new macrocyclic lactams exhibit a biological profile equivalent or superior to that of the related lactones and show the established molecular signature of Hsp90 inhibitors in human colon cancer cells.

A phase I study of the heat shock protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced solid tumors.

PURPOSE: A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED). PATIENTS AND METHODS: Patients with advanced solid cancers were treated with weekly, intravenous (i.v.) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which ≤ 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual. RESULTS: Twenty-five patients received 17-DMAG (range 2.5-106 mg/m(2)). At 106 mg/m(2) of 17-DMAG 2/4 patients experienced DLT, including one treatment-related death. No DLT occurred at 80 mg/m(2). Common adverse events were gastrointestinal, liver function changes, and ocular. Area under the curve and mean peak concentration increased proportionally with 17-DMAG doses 80 mg/m(2) or less. In peripheral blood mononuclear cells significant (P < 0.05) HSP72 induction was detected (≥ 20 mg/m(2)) and sustained for 96 hours (≥ 40 mg/m(2)). Plasma HSP72 levels were greatest in the two patients who experienced DLT. At 80 mg/m(2) client protein (CDK4, LCK) depletion was detected and tumor samples from 3 of 5 patients confirmed HSP90 inhibition. Clinical activity included complete response (castration refractory prostate cancer, CRPC 124 weeks), partial response (melanoma, 159 weeks), and stable disease (chondrosarcoma, CRPC, and renal cancer for 28, 59, and 76 weeks, respectively). CONCLUSIONS: The recommended phase II dose of 17-DMAG is 80 mg/m(2) weekly i.v.

Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.

Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the 'phosphoryl-transfer' competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase 'active' conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.

CCT241533 is a potent and selective inhibitor of CHK2 that potentiates the cytotoxicity of PARP inhibitors.

CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC(50) of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 µmol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors.

Serum angiogenic profile of patients with glioblastoma identifies distinct tumor subtypes and shows that TIMP-1 is a prognostic factor.

Angiogenesis plays a key role in glioblastoma biology and antiangiogenic agents are under clinical investigation with promising results. However, the angiogenic profiles of patients with glioblastoma and their clinical significance are not well understood. Here we characterize the serum angiogenic profile of patients with glioblastoma, and examine the prognostic significance of individual angiogenic factors. Serum samples from 36 patients with glioblastoma were collected on admission and simultaneously assayed for 48 angiogenic factors using protein microarrays. The data were analyzed using hierarchical cluster analysis. Vessel morphology was assessed histologically after immunostaining for the pan-endothelial marker CD31. Tumor samples were also immunostained for tissue inhibitor of metalloproteinase-1 (TIMP-1). Cluster analysis of the serum angiogenic profiles revealed 2 distinct subtypes of glioblastoma. The 2 subtypes had markedly different tumor microvessel densities. A low serum level of TIMP-1 was associated with significantly longer survival independent of patient age, performance status, or treatment. The serum angiogenic profile in patients with glioblastoma mirrors tumor biology and has prognostic value. Our data suggest the serum TIMP-1 level as an independent predictor of survival.

Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930.

AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials.

Discovery of 2-(6-{[(6-fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-3-yl)-N-hydroxypyrimidine-5-carboxamide (CHR-3996), a class I selective orally active histone deacetylase inhibitor.

A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavailability is described. The compounds are potent enzyme inhibitors (IC50 values less than 100 nM), and improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA) through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice, comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r (CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro studies. On the basis of these results, 21r was nominated for clinical development.

Design and synthesis of novel pyrimidine hydroxamic acid inhibitors of histone deacetylases.

Inhibition of histone deacetylase activity represents a promising new modality in the treatment of a number of cancers. A novel HDAC series demonstrating inhibitory activity in cell proliferation assays is described. Optimisation based on the introduction of basic amine linkers to effect good drug distribution to tumour led to the identification of a compound with oral activity in a human colon cancer xenograft study associated with increased histone H3 acetylation in tumour tissue.

Identification by high-throughput screening of viridin analogs as biochemical and cell-based inhibitors of the cell cycle-regulated nek2 kinase.

Nek2 is a serine/threonine protein kinase that localizes to the centrosome and is implicated in mitotic regulation. Overexpression of Nek2 induces premature centrosome separation and nuclear defects indicative of mitotic errors, whereas depletion of Nek2 interferes with cell growth. As Nek2 expression is upregulated in a range of cancer cell lines and primary human tumors, inhibitors of Nek2 may have therapeutic value in cancer treatment. The authors used a radiometric proximity assay in a high-throughput screen to identify small-molecule inhibitors of Nek2 kinase activity. The assay was based on the measurement of the radiolabeled phosphorylated product of the kinase reaction brought into contact with the surface of wells of solid scintillant-coated microplates. Seventy nonaggregating hits were identified from approximately 73,000 compounds screened and included a number of toxoflavins and a series of viridin/wortmannin-like compounds. The viridin-like compounds were >70-fold selective for Nek2 over Nek6 and Nek7 and inhibited the growth of human tumor cell lines at concentrations consistent with their biochemical potencies. An automated mechanism-based microscopy assay in which centrosomes were visualized using pericentrin antibodies confirmed that 2 of the viridin inhibitors reduced centrosome separation in a human tumor cell line. The data presented show that pharmacological inhibition of Nek2 kinase results in the expected phenotype of disruption to centrosome function associated with growth inhibition and further supports Nek2 as a target for cancer drug discovery.

Inhibition of Hsp90 with resorcylic acid macrolactones: synthesis and binding studies.

A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho-toluic acid dianion, esterification, followed by a ring-closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following deprotection of the two phenolic groups. Co-crystallization of one of the new macrolactones with the N-terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90.

A useful approach to identify novel small-molecule inhibitors of Wnt-dependent transcription.

The Wnt signaling pathway is frequently deregulated in cancer due to mutations in genes encoding APC, beta-catenin, and axin. To identify small-molecule inhibitors of Wnt signaling as potential therapeutics, a diverse chemical library was screened using a transcription factor reporter cell line in which the activity of the pathway was induced at the level of Disheveled protein. A series of deconvolution studies was used to focus on three compound series that selectively killed cancer cell lines with constitutive Wnt signaling. Activities of the compounds included the ability to induce degradation of beta-catenin that had been stabilized by a glycogen synthase kinase-3 (GSK-3) inhibitor. This screen illustrates a practical approach to identify small-molecule inhibitors of Wnt signaling that can seed the development of agents suitable to treat patients with Wnt-dependent tumors.

Detection of the ATPase activity of the molecular chaperones Hsp90 and Hsp72 using the TranscreenerTM ADP assay kit.

The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats.

Targeting the Hsp90 chaperone: synthesis of novel resorcylic acid macrolactone inhibitors of Hsp90.

A series of benzo-macrolactones has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. A new synthesis of these resorcylic acid macrolactone analogues of the natural product radicicol is described in which the key steps are the acylation and ring opening of a homophthalic anhydride to give an isocoumarin, followed by a ring-closing metathesis to form the macrocycle. The methodology has been extended to a novel series of macrolactones incorporating a 1,2,3-triazole ring.

Phase I pharmacokinetic and pharmacodynamic study of LAQ824, a hydroxamate histone deacetylase inhibitor with a heat shock protein-90 inhibitory profile, in patients with advanced solid tumors.

PURPOSE: To determine the safety, maximum tolerated dose, and pharmacokinetic-pharmacodynamic profile of a histone deacetylase inhibitor, LAQ824, in patients with advanced malignancy. PATIENTS AND METHODS: LAQ824 was administered i.v. as a 3-h infusion on days 1, 2, and 3 every 21 days. Western blot assays of peripheral blood mononuclear cell lysates and tumor biopsies pretherapy and posttherapy evaluated target inhibition and effects on heat shock protein-90 (HSP90) client proteins and HSP72. RESULTS: Thirty-nine patients (22 male; median age, 53 years; median Eastern Cooperative Oncology Group performance status 1) were treated at seven dose levels (mg/m(2)): 6 (3 patients), 12 (4 patients), 24 (4 patients), 36 (4 patients), 48 (4 patients), 72 (19 patients), and 100 (1 patient). Dose-escalation used a modified continual reassessment method. Dose-limiting toxicities were transaminitis, fatigue, atrial fibrillation, raised serum creatinine, and hyperbilirubinemia. A patient with pancreatic cancer treated at 100 mg/m(2) died on course one at day 18 with grade 3 hyperbilirubinemia and neutropenia, fever, and acute renal failure. The area under the plasma concentration curve increased proportionally with increasing dose; median terminal half-life ranged from 8 to 14 hours. Peripheral blood mononuclear cell lysates showed consistent accumulation of acetylated histones posttherapy from 24 mg/m(2); higher doses resulted in increased and longer duration of pharmacodynamic effect. Changes in HSP90 client protein and HSP72 levels consistent with HSP90 inhibition were observed at higher doses. No objective response was documented; 3 patients had stable disease lasting up to 14 months. Based on these data, future efficacy trials should evaluate doses ranging from 24 to 72 mg/m(2). CONCLUSIONS: LAQ824 was well tolerated at doses that induced accumulation of histone acetylation, with higher doses inducing changes consistent with HSP90 inhibition.

NVP-AUY922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis.

We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K(d) = 1.7 nmol/L) and proliferation of human tumor cells with GI(50) values of approximately 2 to 40 nmol/L, inducing G(1)-G(2) arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI(50). This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1alpha, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro, WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials.

Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of a novel histone deacetylase inhibitor, LAQ824, in human colon carcinoma cells and xenografts.

The aim of this work was to use phosphorus magnetic resonance spectroscopy ((31)P MRS) to investigate the pharmacodynamic effects of LAQ824, a histone deacetylase (HDAC) inhibitor. Human HT29 colon carcinoma cells were examined by (31)P MRS after treatment with LAQ824 and another HDAC inhibitor, suberoylanilide hydroxamic acid. HT29 xenografts and tumor extracts were also examined using (31)P MRS, pre- and post-LAQ824 treatment. Histone H3 acetylation was determined using Western blot analysis, and tumor microvessel density by immunohistochemical staining of CD31. Phosphocholine showed a significant increase in HT29 cells after treatment with LAQ824 and suberoylanilide hydroxamic acid. In vivo, the ratio of phosphomonoester/total phosphorus (TotP) signal was significantly increased in LAQ824-treated HT29 xenografts, and this ratio was inversely correlated with changes in tumor volume. Statistically significant decreases in intracellular pH, beta-nucleoside triphosphate (beta-NTP)/TotP, and beta-NTP/inorganic phosphate (Pi) and an increase in Pi/TotP were also seen in LAQ824-treated tumors. Tumor extracts showed many significant metabolic changes after LAQ824 treatment, in parallel with increased histone acetylation and decreased microvessel density. Treatment with LAQ824 resulted in altered phospholipid metabolism and compromised tumor bioenergetics. The phosphocholine and phosphomonoester increases may have the potential to act as pharmacodynamic markers for noninvasively monitoring tumor response after treatment with LAQ824 or other HDAC inhibitors.