RAF-Mutant Melanomas Differentially Depend on ERK2 Over ERK1 to Support Aberrant MAPK Pathway Activation and Cell Proliferation.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. IMPLICATIONS: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Genome-wide association analysis identifies genetic correlates of immune infiltrates in solid tumors.

Therapeutic options for the treatment of an increasing variety of cancers have been expanded by the introduction of a new class of drugs, commonly referred to as checkpoint blocking agents, that target the host immune system to positively modulate anti-tumor immune response. Although efficacy of these agents has been linked to a pre-existing level of tumor immune infiltrate, it remains unclear why some patients exhibit deep and durable responses to these agents while others do not benefit. To examine the influence of tumor genetics on tumor immune state, we interrogated the relationship between somatic mutation and copy number alteration with infiltration levels of 7 immune cell types across 40 tumor cohorts in The Cancer Genome Atlas. Levels of cytotoxic T, regulatory T, total T, natural killer, and B cells, as well as monocytes and M2 macrophages, were estimated using a novel set of transcriptional signatures that were designed to resist interference from the cellular heterogeneity of tumors. Tumor mutational load and estimates of tumor purity were included in our association models to adjust for biases in multi-modal genomic data. Copy number alterations, mutations summarized at the gene level, and position-specific mutations were evaluated for association with tumor immune infiltration. We observed a strong relationship between copy number loss of a large region of chromosome 9p and decreased lymphocyte estimates in melanoma, pancreatic, and head/neck cancers. Mutations in the oncogenes PIK3CA, FGFR3, and RAS/RAF family members, as well as the tumor suppressor TP53, were linked to changes in immune infiltration, usually in restricted tumor types. Associations of specific WNT/beta-catenin pathway genetic changes with immune state were limited, but we noted a link between 9p loss and the expression of the WNT receptor FZD3, suggesting that there are interactions between 9p alteration and WNT pathways. Finally, two different cell death regulators, CASP8 and DIDO1, were often mutated in head/neck tumors that had higher lymphocyte infiltrates. In summary, our study supports the relevance of tumor genetics to questions of efficacy and resistance in checkpoint blockade therapies. It also highlights the need to assess genome-wide influences during exploration of any specific tumor pathway hypothesized to be relevant to therapeutic response. Some of the observed genetic links to immune state, like 9p loss, may influence response to cancer immune therapies. Others, like mutations in cell death pathways, may help guide combination therapeutic approaches.

Discovery of imidazo[1,2-a]-pyridine inhibitors of pan-PI3 kinases that are efficacious in a mouse xenograft model.

Alterations in PI3K/AKT signaling are known to be implicated with tumorigenesis. The PI3 kinases family of lipid kinases has been an attractive therapeutic target for cancer treatment. Imidazopyridine compound 1, a potent, selective, and orally available pan-PI3K inhibitor, identified by scaffold morphing of a benzothiazole hit, was further optimized in order to achieve efficacy in a PTEN-deleted A2780 ovarian cancer mouse xenograft model. With a hypothesis that a planar conformation between the core and the 6-heteroaryl ring will allow for the accommodation of larger 5'-substituents in a hydrophobic area under P-loop, SAR efforts focused on 5'-alkoxy heteroaryl rings at the 6-position of imidazopyridine and imidazopyridazine cores that have the same dihedral angle of zero degrees. 6'-Alkoxy 5'-aminopyrazines in the imidazopyridine series were identified as the most potent compounds in the A2780 cell line. Compound 14 with 1,1,1-trifluoroisopropoxy group at 6'-position demonstrated excellent potency and selectivity, good oral exposure in rats and in vivo efficacy in A2780 tumor-bearing mouse. Also, we disclose the X-ray co-crystal structure of one enantiomer of compound 14 in PI3Kα, confirming that the trifluoromethyl group fits nicely in the hydrophobic hot spot under P-loop.

Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor.

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.

Structure guided optimization of a fragment hit to imidazopyridine inhibitors of PI3K.

PI3 kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. The PI3 Kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe how the potency of a benzothiazole fragment hit was quickly improved based on structural information and how this early chemotype was further optimized through scaffold hopping. This effort led to the identification of a series of 2-acetamido-5-heteroaryl imidazopyridines showing potent in vitro activity against all class I PI3Ks and attractive pharmacokinetic properties.

Identification and characterization of NVP-BKM120, an orally available pan-class I PI3-kinase inhibitor.

Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer.

Synthesis and in Vitro and in Vivo Evaluation of Phosphoinositide-3-kinase Inhibitors.

Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided optimization of these pyrimidines with a focus on replacing the phenol moiety, while maintaining potent target inhibition and improving in vivo properties. A series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit PI3K, were discovered. Within this series a compound, 17, was identified with suitable pharmacokinetic (PK) properties, which allowed for the establishment of a PI3K PK/pharmacodynamic-efficacy relationship as determined by in vivo inhibition of AKT(Ser473) phosphorylation and tumor growth inhibition in a mouse A2780 tumor xenograft model.

Identification of NVP-BKM120 as a Potent, Selective, Orally Bioavailable Class I PI3 Kinase Inhibitor for Treating Cancer.

Phosphoinositide-3-kinases (PI3Ks) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. Herein we describe the structure guided optimization of a series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines where the pharmacokinetic properties were improved by modulating the electronics of the 6-position heterocycle, and the overall druglike properties were fine-tuned further by modification of the 4-position substituent. The resulting 2,4-bismorpholino 6-heterocyclic pyrimidines are potent class I PI3K inhibitors showing mechanism modulation in PI3K dependent cell lines and in vivo efficacy in tumor xenograft models with PI3K pathway deregulation (A2780 ovarian and U87MG glioma). These efforts culminated in the discovery of 15 (NVP-BKM120), currently in Phase II clinical trials for the treatment of cancer.

Identification and structure-activity relationship of 2-morpholino 6-(3-hydroxyphenyl) pyrimidines, a class of potent and selective PI3 kinase inhibitors.

PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.

Class IA phosphatidylinositol 3-kinase: from their biologic implication in human cancers to drug discovery.

BACKGROUND: A substantial number of epidemiologic and experimental studies support an important role for Class I phosphatidylinositol 3-kinases (PI3Ks) in the biology of human cancer. OBJECTIVE: This article reviews the authors' understanding of the role of Class IA PI3K in the biology of human cancers and present discovery efforts to identify and develop inhibitors of this class of lipid kinases. METHODS: Books, journals, databases and websites have been searched to find the latest information on the subject. RESULTS/CONCLUSIONS: In spite of the progress made over the past few years, more studies are still needed to better understand the biology of this pathway, its interaction(s) with other signaling cascades, and the role of the individual paralogs and PI3Kalpha mutants in human cancer. From a drug discovery perspective, medicinal chemistry efforts have led to the discovery of new pan-PI3K and isoform selective inhibitors with improved specificity, potency and pharmaceutical properties. Phase I clinical studies have been initiated with some of these PI3K inhibitors and the efficacy and therapeutic index of this new generation of anticancer agents is eagerly awaited.