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Suppl Figure 1. Chemical structure and molecular data for TAK-733

Suppl Figure 3. Immunoblot of Effector Proteins and Markers of Apoptosis in Melanoma Cell Lines

Suppl Figure 2. Immunoblot Analysis of Effector Proteins in Melanoma Cell Lines

Suppl Table 1. IC50 Value Matrix

Suppl Figure 5. Immunoblot of Effector Proteins in Tumors from PDTX Mice Treated with TAK-733

Suppl Figure 4. Representative PDTX Model MB1255 Treated with Vehicle or TAK-733

Suppl Table 2. TAK-733 PD/ Conc. Summary, A375 tumor bearing nude mouse

Suppl Table 4. Antitumor Activity of Orally Administered TAK-733 Against A375 Melanoma Tumor Xenografts in Female Athymic Nude Mice

Suppl Figure 6. Genomic Analyses of PDTX MB1374 Treated to Resistance

Supplementary Figure Legends

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

The bromodomain and extra-terminal (BET) family of epigenetic proteins has attracted considerable attention in drug discovery given its involvement in regulating gene transcription. Screening a focused small molecule library based on the bromodomain pharmacophore resulted in the identification of 2-methylisoquinoline-1-one as a novel BET bromodomain-binding motif. Structure guided SAR exploration resulted in >10,000-fold potency improvement for the BRD4-BD1 bromodomain. Lead compounds exhibited excellent potencies in both biochemical and cellular assays in MYC-dependent cell lines. Compound 36 demonstrated good physicochemical properties and promising exposure levels in exploratory PK studies.

Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound 6 (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.

Structure based drug design of a series of novel 1,4-benzoxazin-3-one derived PARP-1 inhibitors are described. The synthesis, enzymatic & cellular activities and pharmacodynamic effects are described. Optimized analogs demonstrated inhibition of poly-ADP-ribosylation in SW620 tumor bearing nude mice through 24h following a single dose.

The structure-based design, synthesis, and biological evaluation of two novel series of potent and selective MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic dihydroindolone core provided compounds with high potency and good drug-like pharmaceutical properties. Further scaffold modification afforded a series of dihydroindolizinone inhibitors, including an orally available advanced preclinical MEK inhibitor with potent in vivo antitumor efficacy.

A novel 5-phenylamino-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione series of MEK inhibitors has been developed using structure-based drug design. Lead optimization of this series led to the discovery of TAK-733. This was advanced to Phase I clinical studies for cancer treatment.

A series of N-(2-amino-5-substituted phenyl)benzamides (3-21) were designed, synthesized and evaluated for their inhibition of HDAC2 and their cytotoxicity in HCT116 cancer cells. Multiple compounds from this series demonstrated time-dependent binding kinetics that is rationalized using a co-complex crystal structure of HDAC2 and N-(4-aminobiphenyl-3-yl)benzamide (6).

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001–0.03). Interestingly, BRAFV600E and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733–mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active. Mol Cancer Ther; 14(2); 317–25. ©2014 AACR.

A novel series of pyrrole inhibitors of MEK kinase has been developed using structure-based drug design. Optimization of the series led to the identification of potent inhibitors with good pharmaceutical properties.

Pitx1 and Pitx2 are highly homologous, bicoid-related transcription factors. Pitx2 was initially identified as the gene responsible for the human Rieger syndrome, an autosomal dominant condition that causes developmental abnormalities. Pitx2 is asymmetrically expressed in the left lateral-plate mesoderm, and mutant mice with laterality defects show altered patterns of Pitx2 expression that correlate with changes in the visceral symmetry (situs). Ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and reverses body rotation in chick and Xenopus embryos. Here we describe the phenotype of Pitx2 gene-deleted mice, characterized by defective body-wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis. Thus, Pitx2 is a transcription factor that encodes 'leftness' of the lung.

The differentiation of three anterior pituitary cell types is regulated by the tissue-specific POU domain factor Pit-1, which is initially expressed on Embryonic Day 13.5–14 in mice. The Pit-1 gene remains continuously, highly expressed in the somatotrope, thyrotrope, and lactotrope cells of the adult. Using the Pit-1-defective Snell dwarf as a genetic background, we demonstrate that the Pit-1 gene utilizes distinct enhancers for initial gene activation and for subsequent autoregulation (required for maintenance of expression) and that Pit-1-dependent activation of the distal enhancer can be mediated in the absence of the early enhancer. These two distinct enhancers provide the basis for temporally specific regulation by discrete pituitary-specific factors, events likely to be prototypic for regulation of other classes of genes encoding transcription factors controlling terminal differentiation.

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.

The terminal differentiation of myelinating glia involves complex interactions that culminate in the formation of myelin. The POU domain transcription factor Tst-1/Oct-6/SCIP is expressed transiently during myelination, and we report here that it has a critical role in this developmental process. Deletion of the Tst-1/Oct-6/SCIP gene produces a severe defect in peripheral myelination by arresting Schwann cell maturation before axonal wrapping. Unexpectedly, the activation of major myelin-specific genes appears to be unaffected by the Tst-1/Oct-6/SCIP mutation, demonstrating that multiple, independently regulated events are required for terminal differentiation of Schwann cells. In addition, aberrant differentiation and migration of specific neurons in Tst-1/Oct-6/SCIP mutant homozygotes is associated with a fatal breathing defect, providing a model for investigating the regulation of pulmonary homeostasis.

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes.

We have identified and characterized cDNAs encoding a novel receptor that is a member of a distinct class of seven transmembrane helix, G s coupled receptors. This receptor mediates ligand-dependent stimulation of intracellular cAMP levels in response to physiologic concentrations of corticotropin-releasing factor (CRF) and to the related frog skin peptide, sauvagine. The pattern of CRF receptor mRNA expression in the brain, pituitary gland, and other organs corresponds precisely to that predicted for the classic CRF receptor, suggesting that this receptor serves to mediate the known biological effects of CRF on behavior, stress, and homeostasis. Alternative splicing events generate a second, relatively abundant gene product expressed in a distinct ontogenic pattern. These findings serve to identify the receptor for an important neuropeptide.

A series of N-hydroxy-3-[3-(1-substituted-1H-benzoimidazol-2-yl)-phenyl]-acrylamides (5a–5ab) and N-hydroxy-3-[3-(1,4,5-trisubstituted-1H-imidazol-2-yl)-phenyl]-acrylamides (12a–s) were designed, synthesized, and found to be nanomolar inhibitors of human histone deacetylases. Multiple compounds bearing an N1-piperidine demonstrate EC50s of 20–100 nM in human A549, HL60, and PC3 cells, in vitro and in vivo hyperacetylation of histones H3 and H4, and induction of p21waf. Compound 5x displays efficacy in human tumor xenograft models.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteiniz-ing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1 (ref. 1)f which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report we have identified four CPHD families with homozy-gosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

Despite the importance of myelinating Schwann cells in health and disease, little is known about the genetic mechanisms underlying their development. The POU domain transcription factor pou3f1 (Tst-1, SCIP, Oct-6) is required for the normal differentiation of myelinating Schwann cells, but its precise role requires identification of the genes that it regulates. Here we report the isolation of six genes whose expression is reduced in the absence of pou3f1. Only one of these genes, the fatty acid transport protein P(2), was known previously to be expressed in Schwann cells. The LIM domain proteins cysteine-rich protein-1 (CRP1) and CRP2 are expressed in sciatic nerve and induced by forskolin in cultured Schwann cells, but only CRP2 requires pou3f1 for normal expression. pou3f1 appears to require the claw paw gene product for activation of at least some of its downstream effector genes. Expression of the novel Schwann cell genes after nerve injury suggests that they are myelin related. One of the genes,tramdorin1, encodes a novel amino acid transport protein that is localized to paranodes and incisures. Our results suggest that pou3f1 functions to activate gene expression in the differentiation of myelinating Schwann cells.

Pax6 , a highly conserved member of the paired homeodomain transcription factor family that plays essential roles in ocular, neural, and pancreatic development and effects asymmetric transient dorsal expression during pituitary development, with its expression extinguished before the ventral → dorsal appearance of specific cell types. Analysis of pituitary development in the Small eye and Pax6 −/− mouse mutants reveals that the dorsoventral axis of the pituitary gland becomes ventralized, with dorsal extension of the transcriptional determinants of ventral cell types, particularly PFrk . This ventralization is followed by a marked decrease in terminally differentiated dorsal somatotrope and lactotrope cell types and a marked increase in the expression of markers of the ventral thyrotrope cells and SF-1-expressing cells of gonadotrope lineage. We suggest that the transient dorsal expression of Pax6 is essential for establishing a sharp boundary between dorsal and ventral cell types, based on the inhibition of Shh ventral signals.

Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb.

During development of the mammalian pituitary gland specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse pituitary cell phenotypes from a common precursor, providing in vivo and in vitro evidence that their development involves three sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, the BMP4 signal from the ventral diencephalon, expressing BMP4, Wnt5a, andFGF8, represents a critical dorsal neuroepithelial signal for pituitary organ commitment in vivo. Subsequently, a BMP2 signal emanates from a ventral pituitary organizing center that forms at the boundary of a region of oral ectoderm in which Shh expression is selectively excluded. This BMP2 signal together with a dorsal FGF8 signal, appears to create opposing activity gradients that are suggested to generate overlapping patterns of specific transcription factors underlying cell lineage specification events, whereas Wnt4 is needed for the expansion of ventral pituitary cell phenotypes. In the third phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the hormone-producing pituitary cell types—gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes—appear to be determined, in a ventral-to-dorsal gradient, respectively.

Poly(ADP-ribose) polymerase (PARP) is a critical DNA repair enzyme involved in DNA single-strand break repair via the base excision repair pathway. PARP inhibitors sensitize tumors to DNA-damaging agents and also inhibit the growth of cancers with BRCA1/2 loss. The synthesis, enzymatic, cellular & pharmacodynamic activities of a series of PARP-1 inhibitors with a novel 1,4-benzoxazin-3-one scaffold are described. A small screening fragment was elaborated through structure-based drug design, ultimately producing several vectors for optimization including interaction with a hydrophobic shelf and extension into the back pocket. The synthesis of 40 compounds is outlined. Optimization of the screening fragment improved LLE's to > 5.0. Several of the resulting inhibitors displayed greater than 1000-fold enhancement of temozolimide (TMZ)-mediated DNA damage in cellular assays. Pharmacodynamic effects in SW620 tumor bearing nude mice demonstrated inhibition of poly-ADP-ribosylation through 24 hours following a single dose. PARP-1 Binding Potencies and Efficiency Metrics for Benzoxazinones 2-3CmpdaR1R2R3PARP-1 Kd (uM)PARP-1 LLE2aHHH>500-2dMeHH322.32gMeMeH>500-2bHH6-Formyl1702.02fHH6-Ethyl1002.03aHH6-PhenylNHCH2262.23bHH6-BenzylNHCH2113.53cMeH6-BenzylNHCH25.23.23dHH6- (4-(Me2N)piperidine)CH2153.03eMeH6- (4-(Me2N)piperidine)CH20.0964.63fHH6- (4-(Phenyl)piperidine)CH20.2963.43gMeH6- (4-(Phenyl)piperidine)CH20.0463.73hMeH7- (4-(Phenyl)piperidine)CH2101.43iHH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH20.0154.53jMeH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH20.0034.93kMeH7- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH21000.23lEtH6- (4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine)CH21000.1 Enzymatic and Cellular Activity for Benzoxazinones 10CmpdR1R2R3PARP-1 IC50 (nM)Cell EC50 w/TMZ (uM)Cell EC50 w/o TMZ (uM)Cell PF5010aHHMe2915141.010bHHEt2215422.810cHClEt1841002010dMeHMe1711756.810eMeHEt699410.410fMeHcPr3351002010gOMeHMe29272519.310hOmeHEt23161519.410iOMeHcPr30161621010jMeClMe4914453.210kMeClEt8914473.410lMeClcPr760.0143430010mMeMeMe389788.710nMeMeEt569556.110oMeMecPr52181005.610pOmeMeMe360.06100166710qOMeMeEt320.0144440010rOMeMecPr1470.0277385010sOEtMeMe350.0226130010tOEtMeEt550.02100500010uOEtMecPr410.021005000ABT-888---16316056AZD-2281---60.0616660MK-4827---350.2031160 Citation Format: Anthony R. Gangloff, Jason Brown, Ron De Jong, Philip Erickson, Charles Grimshaw, Mark Hixon, Andy Jennings, Ruhi Kamran, Andre Kiryanov, Shawn O'Connell, Ewan Taylor, Phong Vu. Discovery of novel benzo[b][1,4]oxazin-3(4H)-ones as poly(ADP-Ribose)polymerase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5676. doi:10.1158/1538-7445.AM2013-5676

The gene apparently responsible for a heritable form of murine pituitary-dependent dwarfism (Ames dwarf, df) has been positionally cloned, identifying a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (Prop-1). The df phenotype results from an apparent failure of initial determination of the Pit-1 lineage required for production of growth hormone, prolactin or thyroid-stimulating hormone, resulting in dysmorphogenesis and failure to activate Pit-1 gene expression. These results imply that a cascade of tissue-specific regulators is responsible for the determination and differentiation of specific cell lineages in pituitary organogenesis.

THE neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1–6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene7 that constitute, with Brn-3.0 (refs 1–3,8,9), the class IV POU-domain transcription factors10. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences1,2 and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain1–6,8,9, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

MEK and Aurora A kinase inhibitors (AAK) have profound impacts on cell cycle progression. Inhibition of MEK leads to defects in cell-cycle progression followed by cell cycle arrest and/or apoptosis. These cell-cycle progression defects may in part be due to the role of MEK in the DNA replication or damage checkpoint responses through positive regulation of DNA repair mechanisms. AAK inhibition leads to chromosome congression and segregation defects leading to DNA damage followed by apoptosis or senesence. The overlapping biological effects of inhibition of AAK and MEK kinases in cell cycle progression and chromosomal integrity raise the possibility that inhibiting both targets would provide added benefit over the inhibition of either target alone. The in vivo antitumor activity of TAK-733, an investigational potent, selective, non-ATP-competitive allosteric inhibitor of MEK, in combination with alisertib, an investigational potent, selective, reversible, ATP-competitive inhibitor of Aurora A kinase, was examined in experimental human solid tumor xenograft models including NSCLC (NCI H23 [KRAS and LKB1 mutations]), CRC (SW620 [KRAS, APC, p53 mutations]), and pancreatic cancer (Panc 1 and Capan 1 [KRAS mutations] and BxPC-3 [No MAPK mutations]) models in immunocompromised mice. The previously established maximally efficacious doses for the single agents dosed once daily (QD) orally (PO) (10 mg/kg TAK-733 and 30-mg/kg alisertib) were examined. A lower dose of 20 mg/kg alisertib was also examined in the event the maximally efficacious dose of each agent was not tolerated in combination. The once daily (QD) concurrent oral administration of TAK-733 and alisertib resulted in additive to synergistic antitumor activity and in prolonged inhibition of tumor regrowth after terminating treatment compared to single agent treatment in all xenograft models examined. Concurrent administration of TAK-733 and alisertib daily for 21 days was well tolerated. Treatment with TAK-733 alone and in combination with alisertib clearly inhibited phosphorylated ERK and produced a slight increase in phosphorylated histone H3. The results from the nonclinical models examined demonstrate considerable improvement in nonclinical antitumor activity over either single agent alone and provide a biological rationale for clinical evaluation of a TAK-733/alisertib combination in patients with advanced malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3739. doi:1538-7445.AM2012-3739

MEK has emerged as an attractive target for pharmacologic intervention in cancer as the RAS/RAF/MEK/ERK cascade has proven to play a central role in the signaling required for cellular transformation and proliferation. Inhibition of MEK impacts a diverse array of additional cellular events, including differentiation, apoptosis, and angiogenesis. Identification of an orally active inhibitor of MEK which shows significant efficacy as a mono-therapy for the treatment of cancer would be ideal. TAK-733 is a potent non-ATP competitive allosteric inhibitor of MEK1/2 with IC50 values for the inhibition of MEK1/2 signaling of 2-5 nM at high (400 μM) and low (10 μM) ATP concentrations. TAK-733 was selective for mammalian MEK-1/2 in a panel of 71 enzyme assays. These included phosphodiesterases, 17 kinases (IC50 values >25 µM), cyclases, and 14 other enzymes (IC50 values >10 µM). Using an MTS (a tetrazolium salt) viability assay, TAK-733 was found to inhibit proliferation of numerous cancer cell lines expressing BRAF, KRAS, NRAS, PI3K, MEK1, EGFR mutations, cMET amplification, and wild type MAPK signaling with EC50 values ranging from 2 to 90 nM. A quantitative PK/PD relationship was established in the HT-29 human colorectal carcinoma murine xenograft model. This model showed a range of PD activity (ie, from no effect to maximum effect) which correlated well with tumor and plasma TAK-733 concentrations. The relationship between TAK-733 plasma concentration (PK) and target modulation (PD) was evaluated by modeling percent inhibition of tumor ERK phosphorylation and plasma TAK-733 concentrations at 4 hours postdose, using an inhibitory sigmoidal concentration response model. The plasma EC50 was calculated to be 45 ng/mL. TAK-733 exhibited broad-spectrum in vivo antitumor activity against a panel of human tumor xenografts with and without mutations in the MAPK pathway. These xenografts were of colon (COLO 205/ BRAF V600E and HT-29/ BRAF V600E/PI3K P449T), NSCLC (A549/ KRAS G12S, PC-9/ EGFRΔE746-A750, NCI-H1975/ EGFR L858R T790M), AML (HL-60/ NRAS), cholangio-carcinoma (EGI-1/ KRAS G12D) and melanoma (A375/ BRAF V600E) origins. A high incidence of tumor regressions was observed in this collection of cancer models. Collectively, preclinical pharmacology studies indicate TAK-733 is a specific inhibitor of MEK with the potential to significantly impair growth of a large number of cancers relying on the MAPK signaling cascade for growth and survival. and provides a strong rationale for study of this agent in 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 2523.

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type–specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding–independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding–dependent and –independent actions of abundant determining factors contribute to generate distinct cell phenotypes.

Axon pathfinding relies on the ability of the growth cone to detect and interpret guidance cues and to modulate cytoskeletal changes in response to these signals. We report that the murine POU domain transcription factor Brn-3.2 regulates pathfinding in retinal ganglion cell (RGC) axons at multiple points along their pathways and the establishment of topographic order in the superior colliculus. Using representational difference analysis, we identified Brn-3.2 gene targets likely to act on axon guidance at the levels of transcription, cell–cell interaction, and signal transduction, including the actin-binding LIM domain protein abLIM. We present evidence that abLIM plays a crucial role in RGC axon pathfinding, sharing functional similarity with its C. elegans homolog, UNC-115. Our findings provide insights into a Brn-3.2-directed hierarchical program linking signaling events to cytoskeletal changes required for axon pathfinding.

Characterization of Brn-3.0 and identification of a highly related member (Brn-3.1) of the class IV POU-domain family suggest potential roles of Brn-3.0 in the development of retinal ganglion cells and sensory neurons, as well as potential roles in the pituitary gland and the immune system. Brn-3.0 is expressed in the pituitary gland and in a corticotroph cell line. A functional DNA response element has been identified in the proopiomelanocortin promoter. In contrast to previously described mammalian POU-domain proteins, Brn-3.0 binds relatively ineffectively to known octamer DNA motifs, but instead binds with high affinity to a distinct set of DNA elements, functioning as a transcriptional activator. Brn-3.0, Brn-3.1, and the Drosophila tI-POU share an N-terminal region of homology, referred to as the "POU-IV box," which is similar to a conserved functional domain in the c-myc gene family.