The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin inhibitor (mTOR) pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. NVP-BEZ235 is an imidazo [4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. In cellular settings using human tumor cell lines, this molecule is able to effectively and specifically block the dysfunctional activation of the PI3K pathway, inducing G 1 arrest. The cellular activity of NVP-BEZ235 translates well in in vivo models of human cancer. Thus, the compound was well tolerated, displayed disease stasis when administered orally, and enhanced the efficacy of other anticancer agents when used in in vivo combination studies. Ex vivo pharmacokinetic/pharmacodynamic analyses of tumor tissues showed a time-dependent correlation between compound concentration and PI3K/Akt pathway inhibition. Collectively, the preclinical data show that NVP-BEZ235 is a potent dual PI3K/mTOR modulator with favorable pharmaceutical properties. NVP-BEZ235 is currently in phase I clinical trials.

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110A catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110A yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110A mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110A isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition. (Cancer Res 2005; 65(23): 10992-11000)

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 PI3Ka 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 concentrationdependent 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. Mol Cancer Ther; 11(2); 317-28. Ó2011 AACR.

Somatic PIK3CA mutations are frequently found in solid tumors, raising the hypothesis that selective inhibition of PI3Ka may have robust efficacy in PIK3CA-mutant cancers while sparing patients the side-effects associated with broader inhibition of the class I phosphoinositide 3-kinase (PI3K) family. Here, we report the biologic properties of the 2-aminothiazole derivative NVP-BYL719, a selective inhibitor of PI3Ka and its most common oncogenic mutant forms. The compound selectivity combined with excellent drug-like properties translates to dose-and time-dependent inhibition of PI3Ka signaling in vivo, resulting in robust therapeutic efficacy and tolerability in PIK3CA-dependent tumors. Novel targeted therapeutics such as NVP-BYL719, designed to modulate aberrant functions elicited by cancer-specific genetic alterations upon which the disease depends, require well-defined patient stratification strategies in order to maximize their therapeutic impact and benefit for the patients. Here, we also describe the application of the Cancer Cell Line Encyclopedia as a preclinical platform to refine the patient stratification strategy for NVP-BYL719 and found that PIK3CA mutation was the foremost positive predictor of sensitivity while revealing additional positive and negative associations such as PIK3CA amplification and PTEN mutation, respectively. These patient selection determinants are being assayed in the ongoing NVP-BYL719 clinical trials.

Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85␣ gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85␣, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85␣ represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.Phosphoinositide (PI) 3-kinase plays a pivotal role in the metabolic and mitogenic actions of insulin and insulin-like growth factor 1 (IGF-1) (9, 43). Following IGF-1 and insulin stimulation, the tyrosine-phosphorylated pYMXM and pYXXM motifs in the insulin receptor substrate (IRS) proteins bind to class Ia PI 3-kinase, thereby increasing its activity (2, 43). The class Ia PI 3-kinases are dimers composed of a p110 catalytic subunit and a regulatory subunit with SH2 domains which can interact with IRS proteins (17, 52). At least eight isoforms of the regulatory subunits derived from three distinct genes have been identified. p85␣ and p85␤ represent the full-length versions of the regulatory subunits and contain an SH3 domain, a bcr homology (BH) domain flanked by two proline-rich domains, two SH2 domains (referred to as the amino-terminal and carboxy-terminal SH2 domains), and an inter SH2 domain containing the p110 binding region (35). The shorter versions of the regulatory subunits, AS53 (also known as p55␣) (1, 23) and p50␣ (15,24), are splicing variants derived from the same gene encoding p85␣ (Pik3r1) (15). They share the common amino-terminal SH2-inter SH2-carboxy-terminal SH2 structure with p85␣ but lack the amino-terminal half containing the SH3 domain, amino-terminal proline-rich domain, and BH domain, and in its place they have unique amino-terminal sequences consisting of 34 and 6 amino acids, respectively. Another small version of the regulatory subunit, p55 PIK , has a homologous structure with AS53/p55␣ but is encoded by a different gene (36). Of these isoforms, p85␣ is predominantly and ubiquitously expressed in most tissues and is thought to be the major response pathway for most stimuli (35, 43). The spliced variants, AS53 and p50␣, may have differing levels of potency for PI 3-kinase signaling (1, 24, 50) and appear to play specific roles in some selected tissues (1, 15, 24) or in particular states of insulin resistance (26).To elucidate the physiological roles of the ...

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.kinase inhibitor ͉ small molecule ͉ PI3K ͉ breat cancer ͉ mTOR