Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.

Discovery of Orally Bioavailable Ligand Efficient Quinazolindiones as Potent and Selective Tankyrases Inhibitors.

We report herein the discovery of quinazolindiones as potent and selective tankyrase inhibitors. Elucidation of the structure-activity relationship of the lead compound 1g led to truncated analogues that have good potency in cells, pharmacokinetic (PK) properties, and excellent selectivity. Compound 21 exhibited excellent potencies in cells and proliferation studies, good selectivity, in vitro activities, and an excellent PK profile. Compound 21 also inhibited H292 xenograft tumor growth in nude mice. The synthesis, biological, pharmacokinetic, in vivo efficacy studies, and safety profiles of compounds are presented.

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet Bispecific Antibody, Induces Receptor Downmodulation and Antitumor Activity by Monocyte/Macrophage Trogocytosis.

Small molecule inhibitors targeting mutant EGFR are standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo antitumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR, and this activity is associated with receptor downregulation. Despite these robust antitumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation, leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo antitumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-mediated antitumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

The Combined Effect of FGFR Inhibition and PD-1 Blockade Promotes Tumor-Intrinsic Induction of Antitumor Immunity.

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a Clinical Candidate for the Treatment of IBS.

Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.

Characterization of In Vivo Resistance to Osimertinib and JNJ-61186372, an EGFR/Met Bispecific Antibody, Reveals Unique and Consensus Mechanisms of Resistance.

Approximately 10% of non-small cell lung cancer (NSCLC) patients in the United States and 40% of NSCLC patients in Asia have activating epidermal growth factor receptor (EGFR) mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be cotargeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry-based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy-resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third-generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase (SFK) substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as SFK signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages. Mol Cancer Ther; 16(11); 2572-85. ©2017 AACR.

Discovery and Pharmacological Characterization of JNJ-42756493 (Erdafitinib), a Functionally Selective Small-Molecule FGFR Family Inhibitor.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010-20. ©2017 AACR.

Fc-mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced killing of lung cancer cells.

Epidermal growth factor receptor (EGFR) mutant non-small cell lung cancers acquire resistance to EGFR tyrosine kinase inhibitors through multiple mechanisms including c-Met receptor pathway activation. We generated a bispecific antibody targeting EGFR and c-Met (JNJ-61186372) demonstrating anti-tumor activity in wild-type and mutant EGFR settings with c-Met pathway activation. JNJ-61186372 was engineered with low fucosylation (<10 %), resulting in enhanced antibody-dependent cell-mediated cytotoxicity and FcγRIIIa binding. In vitro and in vivo studies with the single-arm EGFR or c-Met versions of JNJ-61186372 identified that the Fc-activity of JNJ-61186372 is mediated by binding of the anti-EGFR arm and required for inhibition of EGFR-driven tumor cells. In a tumor model driven by both EGFR and c-Met, treatment with Fc-silent JNJ-61186372 or with c-Met single-arm antibody reduced tumor growth inhibition compared to treatment with JNJ-61186372, suggesting that the Fc function of JNJ-61186372 is essential for maximal tumor inhibition. Moreover in this same model, downregulation of both EGFR and c-Met receptors was observed upon treatment with Fc-competent JNJ-61186372, suggesting that the Fc interactions are necessary for down-modulation of the receptors in vivo and for efficacy. These Fc-mediated activities, in combination with inhibition of both the EGFR and c-Met signaling pathways, highlight the multiple mechanisms by which JNJ-61186372 combats therapeutic resistance in EGFR mutant patients.

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer.

BACKGROUND: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. METHODS: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). RESULTS: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. CONCLUSIONS: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.

A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.

Dabrafenib; preclinical characterization, increased efficacy when combined with trametinib, while BRAF/MEK tool combination reduced skin lesions.

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF(V600E) kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAF(V600E)-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.

Discovery of Dabrafenib: A Selective Inhibitor of Raf Kinases with Antitumor Activity against B-Raf-Driven Tumors.

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf(V600E) mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf(V600E) human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.

Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations.

Recent results from clinical trials with the BRAF inhibitors GSK2118436 (dabrafenib) and PLX4032 (vemurafenib) have shown encouraging response rates; however, the duration of response has been limited. To identify determinants of acquired resistance to GSK2118436 and strategies to overcome the resistance, we isolated GSK2118436 drug-resistant clones from the A375 BRAF(V600E) and the YUSIT1 BRAF(V600K) melanoma cell lines. These clones also showed reduced sensitivity to the allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor GSK1120212 (trametinib). Genetic characterization of these clones identified an in-frame deletion in MEK1 (MEK1(K59del)) or NRAS mutation (NRAS(Q61K) and/or NRAS(A146T)) with and without MEK1(P387S) in the BRAF(V600E) background and NRAS(Q61K) in the BRAF(V600K) background. Stable knockdown of NRAS with short hairpin RNA partially restored GSK2118436 sensitivity in mutant NRAS clones, whereas expression of NRAS(Q61K) or NRAS(A146T) in the A375 parental cells decreased sensitivity to GSK2118436. Similarly, expression of MEK1(K59del), but not MEK1(P387S), decreased sensitivity of A375 cells to GSK2118436. The combination of GSK2118436 and GSK1120212 effectively inhibited cell growth, decreased ERK phosphorylation, decreased cyclin D1 protein, and increased p27(kip1) protein in the resistant clones. Moreover, the combination of GSK2118436 or GSK1120212 with the phosphoinositide 3-kinase/mTOR inhibitor GSK2126458 enhanced cell growth inhibition and decreased S6 ribosomal protein phosphorylation in these clones. Our results show that NRAS and/or MEK mutations contribute to BRAF inhibitor resistance in vitro, and the combination of GSK2118436 and GSK1120212 overcomes this resistance. In addition, these resistant clones respond to the combination of GSK2126458 with GSK2118436 or GSK1120212. Clinical trials are ongoing or planned to test these combinations.

Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212.

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers, sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors, RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines, co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines, transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition, a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines, with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6, whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines, acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall, this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors.

Inhibition of Polo-like kinase 1 prevents the growth of metastatic breast cancer cells in the brain.

Few therapeutic strategies exist for the treatment of metastatic tumor cells in the brain because the blood-brain barrier (BBB) limits drug access. Thus the identification of molecular targets and accompanying BBB permeable drugs will significantly benefit brain metastasis patients. Polo-like kinase 1 (Plk1) is an attractive molecular target because it is only expressed in dividing cells and its expression is upregulated in many tumors. Analysis of a publicly available database of human breast cancer metastases revealed Plk1 mRNA expression was significantly increased in brain metastases compared to systemic metastases (P = 0.0018). The selective Plk1 inhibitor, GSK461364A, showed substantial uptake in normal rodent brain. Using a breast cancer brain metastatic xenograft model (231-BR), we tested the efficacy of GSK461364A to prevent brain metastatic colonization. When treatment was started 3 days post-injection, GSK461364A at 50 mg/kg inhibited the development of large brain metastases 62% (P = 0.0001) and prolonged survival by 17%. GSK461364A sensitized tumor cells to radiation induced cell death in vitro. Previously, it was reported that mutations in p53 might render tumor cells more sensitive to Plk1 inhibition; however, p53 mutations are uncommon in breast cancer. In a cohort of 41 primary breast tumors and matched brain metastases, p53 immunostaining was increased in 61% of metastases; 44% of which were associated with primary tumors with low p53. The data suggest that p53 overexpression occurs frequently in brain metastases and may facilitate sensitivity to Plk1 inhibition. These data indicate Plk1 may be a new druggable target for the prevention of breast cancer brain metastases.

GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition.

PURPOSE: Despite their preclinical promise, previous MEK inhibitors have shown little benefit for patients. This likely reflects the narrow therapeutic window for MEK inhibitors due to the essential role of the P42/44 MAPK pathway in many nontumor tissues. GSK1120212 is a potent and selective allosteric inhibitor of the MEK1 and MEK2 (MEK1/2) enzymes with promising antitumor activity in a phase I clinical trial (ASCO 2010). Our studies characterize GSK1120212' enzymatic, cellular, and in vivo activities, describing its unusually long circulating half-life. EXPERIMENTAL DESIGN: Enzymatic studies were conducted to determine GSK1120212 inhibition of recombinant MEK, following or preceding RAF kinase activation. Cellular studies examined GSK1120212 inhibition of ERK1 and 2 phosphorylation (p-ERK1/2) as well as MEK1/2 phosphorylation and activation. Further studies explored the sensitivity of cancer cell lines, and drug pharmacokinetics and efficacy in multiple tumor xenograft models. RESULTS: In enzymatic and cellular studies, GSK1120212 inhibits MEK1/2 kinase activity and prevents Raf-dependent MEK phosphorylation (S217 for MEK1), producing prolonged p-ERK1/2 inhibition. Potent cell growth inhibition was evident in most tumor lines with mutant BRAF or Ras. In xenografted tumor models, GSK1120212 orally dosed once daily had a long circulating half-life and sustained suppression of p-ERK1/2 for more than 24 hours; GSK1120212 also reduced tumor Ki67, increased p27(Kip1/CDKN1B), and caused tumor growth inhibition in multiple tumor models. The largest antitumor effect was among tumors harboring mutant BRAF or Ras. CONCLUSIONS: GSK1120212 combines high potency, selectivity, and long circulating half-life, offering promise for successfully targeting the narrow therapeutic window anticipated for clinical MEK inhibitors.

Discovery of a Highly Potent and Selective MEK Inhibitor: GSK1120212 (JTP-74057 DMSO Solvate).

Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.

Pooled shRNA screen for sensitizers to inhibition of the mitotic regulator polo-like kinase (PLK1).

RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.

Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)6°°(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.