Over the past years, it has become widely accepted that cancer is a multistep genetic disease that arises by the activation of specific oncogenes, inactivation of tumor suppressor genes, and stochastic accumulation of genetic alterations driving tumor progression (Vogelstein and Kinzler, 2004). Despite the genetic and epigenetic complexity observed in cancer, tumor growth and survival can be impaired by the inactivation of a single oncogene. This phenomenon is called “oncogene addiction” (term coined by Weinstein, 2000), and reveals a possible “Achilles heel” within the cancer cell that can be therapeutically exploited (Weinstein, 2000, 2002). The hypothesis of oncogene addiction refers to the observation that a tumor cell, despite several genetic alterations, is dependent on a single oncogenic pathway responsible for sustaining the malignant phenotype. An important implication is that switching off this crucial pathway upon which cancer cells are dependent should have negative effects on cancer while sparing normal cells. Therefore pharmacological inhibition of this crucial pathway cause an “addiction shock,” resulting in the blockade of cell growth or in cell death (Sharma and Settleman, 2007; Janne et al., 2009).

The “addiction paradigm” has been pharmacologically exploited and drugs designed to specifically inhibit mutated proteins have led to what is commonly known as “personalized cancer medicine.” At present only a small subset of anticancer therapies are administered based upon the genetic alterations present in individual tumors (Martini et al., 2012). For example, breast cancer patients with amplification/overexpression of the human epidermal growth factor receptor (EGFR) 2 (HER-2) are selectively sensitive to Trastuzumab and Lapatinib (Stern, 2012), melanomas harboring BRAF V600E mutations to Vemurafenib (Flaherty et al., 2010), non-small cells lung cancers (NSCLC) with mutated EGFR to Erlotinib and Gefitinib (Pallis et al., 2011), and KIT and PDGFRA mutant gastrointestinal stromal tumors (GIST) to Imatinib (Antonescu, 2011).

The phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates several processes in normal cell such as survival, metabolism, and motility and it’s one of the most frequently deregulated pathway in human cancer (Cantley, 2002; Samuels et al., 2004; Liu et al., 2009a). Mutations and/or amplifications of the PI3K catalytic subunits p110α (PIK3CA) and p110β (PIK3CB), the PI3K regulatory subunits p85α (PIK3R1) and p85β (PIK3R2), the PI3K effector AKT (AKT1) are often observed in cancer¹. Moreover, mutations, deletions, or epigenetic changes of negative regulators of PI3K axis (Gewinner et al., 2009; Hollander et al., 2011), such as phosphatase and tensin homolog (PTEN) and inositol polyphosphate-4-phosphatase, type II (INPP4B), may alter sensitivity to chemo- and targeted-therapies (Steelman et al., 2008; Kim et al., 2012).

While class I is the most well characterized of the PI3K to date, the family comprises other two groups; class II and class III. Each class of PI3-kinase has unique preferences for phosphoinositide substrates and produces specific lipid second messengers, responding to a wide variety of signaling molecules.

Class II PI3Ks consists of three members named PI3K-C2α, PI3K-C2β, and PI3K-C2γ. Unlike class I, they lack a regulative subunit and appear to be monomers of high molecular weight, predominantly associated with intracellular membranes (Falasca et al., 2007). PI3K-C2α and PI3K-C2β have a broad tissue distribution and are almost ubiquitously expressed, while PI3K-C2γ displayed a very restricted expression pattern, limited to liver, pancreas, and prostate (Kok et al., 2009). The precise nature of class II substrates and lipid products is still debated. Class II members are thought to act similarly to class III mainly generating PtdIns(3)P in vitro and in vivo (Falasca and Maffucci, 2007), nonetheless, they can also produce PtdIns(3,4)P₂ in vitro (Vanhaesebroeck et al., 2010) and PI3K-C2α has been also reported to produce PtdIns(3,4,5)P₃ in vitro (Gaidarov et al., 2001). Class II PI3Ks are activated downstream of different receptor types including RTKs (EGFR and PDGFR) (Brown et al., 1999; Arcaro et al., 2000, 2002; Falasca and Maffucci, 2007) and GPCRs (Maffucci et al., 2005). Several stimuli promote PI3K-C2α activation such as hormones (insulin) (Brown et al., 1999), chemokines (Turner et al., 1998), and cytokines (TNFα and leptin) (Ktori et al., 2003). Similarly PI3K-C2β is activated by growth factor (EGF) (Arcaro et al., 2002) and phospholipids (LPA) (Maffucci et al., 2005) while at the present there are no study investigating PI3K-C2γ upstream activators. A recent study reported that PI3K-C2α has an essential role in angiogenesis resulting in embryo lethality, impaired endothelial cell signaling, and RhoA activation (Yoshioka et al., 2012).

The increasing understanding of the mechanisms underlying the role of the PI3K pathway in tumorigenesis has encouraged many pharmaceutical companies and academic laboratories to focus their efforts on the development of inhibitors targeting the PI3K signaling pathway at different levels.

In this review, we will discuss the challenges for the development of novel inhibitors to target the PI3K signaling pathway and the binary relationship between PI3K mutations in cancer genotype and personalized medicine.

Since PI3K/AKT/mTOR axis has been classified among the most frequently activated pathway in cancer, members of the cascade represent an attractive target for cancer therapeutics (Miled et al., 2007). The activation of the PI3K signaling pathway contributes to several aspects of tumorigenesis as tumor development, progression, invasiveness, and metastasis formation. A number of molecules targeting members of the PI3K axis have been developed and evaluated in preclinical studies as well as in clinical trials (Figure 1). Based on pharmacokinetics properties and isoform selectivity for the ATP binding site, PI3K inhibitors have been classified into different groups (Table 1).

The first group encompasses inhibitors able to bind all class I PI3Ks (pan inhibitors), and in particular PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ. Wortmannin and LY294002, the first two prototype PI3K inhibitors, represented for a long time a useful tool in the study of PI3K function in cellular processes, given their effectiveness at low concentration (nM). Nevertheless, given their poor pharmacokinetic properties and lack of selectivity, these compounds have limited their therapeutic potential. The availability of the crystal structure of the p110 isoform-specific catalytic subunits gave a boost to the development of new PI3K inhibitors (Vadas et al., 2011). Therefore, several novel compounds have been further developed in order to improve pharmacokinetic profiles, to increase target specificity and to minimize toxicity. At the present, several promising pan-PI3K inhibitors are under development and evaluation in clinical trials for cancer therapy. These molecules predominantly display cytostatic effects with consequent G1 phase arrest in vitro and favorable anticancer effects in vivo.

Lately, a second group of PI3K inhibitors has been developed to overcome the toxicity displayed by the treatment with pan-PI3K inhibitors. They are characterized by greater selective activity (isoform-specific) and several molecules are currently under evaluation in preclinical and clinical studies.

On the other hand, since PI3K and mTOR share several structural similarities, many chemical compounds, under evaluation in clinical trials, are able to inhibit both catalytic subunits. This third group of inhibitors is termed “dual PI3K/mTOR” and they have the advantage of inhibiting not only all class I isoforms but also mTORC1 and mTORC2 thus having a strongest effectiveness in switching off the PI3K signaling pathway.

Given the growing interest in inhibiting PI3K signaling pathway, the drug development landscape is becoming increasingly crowded and highly competitive, so that several pharmaceutical companies, such as Novartis, Sanofi-Aventis, Roche/Genentech, Bayer, and GlaxoSmithKline, are currently in competition to bring their own inhibitors in the market. On the basis of these considerations, we will describe and discuss results for the most important PI3K inhibitors currently in clinical trial.

Several PI3K inhibitors have progressed through early clinical safety and dose-escalation studies to phase I clinical trials on solid tumors. This allowed to define the treatment efficacy and to identify patient population that will benefit from PI3K inhibitor administration. Furthermore, a few number of compounds are now progressing to phase Ib expansion cohort and phase II single agent efficacy studies.

Several early reports were presented in the last 3 years revealing safety and some preliminary activity data about the use of class I PI3K and dual mTOR/PI3K inhibitors in patients.

Although the majority of PI3K inhibitors are under development for the treatment of solid tumors, hematological malignancies also represent a therapeutic area of interest, especially for isoform-selective PI3K inhibitors. Constitutive activation of class I PI3K isoform has been identified in high percentage of acute and chronic leukemia. In addition, in some cases leukemias cell display overexpression of PI3Kα, PI3Kβ, and PI3Kγ. Conversely, the expression of PI3Kδ has been found up-regulated in acute myelogenous leukemia (AML) and in a subset of promyelocytic leukemia (APL). AML comprises a heterogeneous group of tumors characterized by uncontrolled proliferation of hematopoietic precursors thus leading to accumulation of blast cells in the bone marrow. These blasts are blocked in their differentiation program at different stage of maturation. This blast accumulation causes a progressive failure in the hematopoiesis process, which in turn leads to anemia, neutropenia, and thrombocytopenia (Smith et al., 2004). The standard therapeutic approach for the treatment of AML is based on high-dose chemotherapy, nonetheless the prognosis of AML remains poor with a 5-year survival rate in a 15–30% of patients. A recent evidence demonstrated that up-regulation of the PI3K/AKT/mTOR axis is a common feature in AML. Consequently, several pan-PI3K and dual PI3K/mTOR inhibitors, in particular BKM120 and BEZ235, are undergoing phase I clinical development to assess safety, dose, and preliminary efficacy in patients with advanced leukemias, relapsed or refractory acute lymphoblastic and myelocytic leukemia (see text footnote 2).

Several studies demonstrated that treatment with the PI3Kδ selective inhibitor, EC87114, inhibits AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells (Sujobert et al., 2005; Billottet et al., 2006). In this context, PI3Kδ inhibitors represent a promising therapeutic treatment for AML without producing undesirable side effects that are conversely expected for other pan-PI3K inhibitors (Di Nicolantonio et al., 2010). Additionally, since PI3Kδ is expressed in leukocytes and plays a key role in B-cell signaling (Jou et al., 2002; Bilancio et al., 2006), it represents an interesting target in B-cells malignancies. Mice with deleted or kinase dead PI3Kδ exhibit B-cell defects, such as lack of B1 lymphocytes, decreased number of mature B-cell and impaired antibody production. The absence of PI3Kδ in B-cell leads also to a reduction in AKT phosphorylation and decreased PtdIns(3,4,5)P₃ levels (Okkenhaug and Vanhaesebroeck, 2003). All these data support the use of PI3Kδ inhibitors in B-cell malignancies including the chronic lymphocytic leukemia (CLL).

Given the increasing interest in inhibiting PI3Kδ for the treatment of hematopoietic malignancies, several inhibitors are currently under evaluation in preclinical and in clinical trials. Presently, the most promising PI3Kδ inhibitor is represent by CAL-101 (Calistoga Pharmaceuticals/Gilead Sciences), an orally available selective inhibitor with an IC50 of 2.5 nM for PI3Kδ and 820, 565, 89 nM for PI3Kα, PI3Kβ, PI3Kγ respectively (Lannutti et al., 2011). At the present, CAL-101 is undergoing in preclinical and clinical development in a variety of lymphoid malignancies thanks to its high selectivity. Phase I studies, on patients with relapsed or refractory hematologic malignancies, including non-Hodgkin lymphoma (NHL), CLL, and AML, revealed a very favorable toxicity profile and pharmacokinetics during a week of CAL-101 oral administration (Fruman and Rommel, 2011). The predominant toxicity, caused by high serum transaminases, was observed in 21% of patients at doses of 200 and 350 mg. However, this side effect resulted to be reversible disappearing with a temporary discontinuation of the drug (Fruman and Rommel, 2011). In this study, 57 patients were treated with different dosages, corresponding to 50, 100, 200, and 350 mg. Clinical responses were seen at all dose levels resulting in 50% of reduction in lymphadenopathy and around 6 months of stable disease. In a successive phase I clinical trial CAL-101 was administered orally one or two times per day for a cycle of 28 day in 54 patients with CLL. After treatment with CAL-101, 26% of patients achieved a PR with 80% of patients showing reduced lymphadenopathy by ≥50% (Di Nicolantonio et al., 2010; Fruman and Rommel, 2011).

Pharmacological inhibitors selectively targeting class II PI3Ks have not been described yet. In particular, in vitro PI3K-C2α is refractory to inhibition by LY294002 and Wortmannin (Virbasius et al., 1996; Domin et al., 1997), two very well-known PI3Ks inhibitors. However, emerging evidence suggests that class II PI3Ks may have crucial role in different types of tumor independently from class I PI3K activity.

As PI3K inhibitors progress into trials focusing on their clinical efficacy, it is critical to identify genomic determinants of response and to stratify the patient population that will most likely benefit from the treatment (Weigelt and Downward, 2012).

Historically rapalogs were the first PI3K pathway inhibitors tested in clinical trials for cancer therapy and currently preclinical models and early clinical data suggested that PIK3CA mutations may predict sensitivity to treatment with PI3K/AKT/mTOR inhibitors (Engelman et al., 2008; Ihle et al., 2009). In particular, it has been shown that patients with advanced malignancies carrying PIK3CA pathway aberrations, showed high percentage of response to rapalogs and/or PI3K pan-inhibitor (Janku et al., 2011b; Moroney et al., 2011). Moreover, PI3K pathway aberrations due to PIK3CA mutations or PTEN loss correlate with increase response to Doxorubicin, Bevacizumab, and Temsirolimus in patients with advanced gynecologic and breast malignancies (Moroney et al., 2011). Likewise, ovarian cancers with coexisting PIK3CA and MAPK pathway mutations are sensitive to PI3K inhibition, whereas CRC with the same repertoire of mutations are resistant (Di Nicolantonio et al., 2010; Janku et al., 2011a, 2012). The identification of feedback loops leading to MAPK activation, upon PI3K inhibition, underscores the potential of a combined therapeutic approach with PI3K and MAPK inhibitors (Carracedo et al., 2008; Laplante and Sabatini, 2012). According to these findings, it has been proposed to incorporate predictive biomarkers during the clinical drug development process from phase I studies onward in order to enrich trials with patients more likely to respond to a given targeted therapy and to increase the chances of drug registration (Carden et al., 2010). For the guidance and prioritization of predictive biomarker candidates in early clinical trials, results derived from the study of preclinical models are of primary importance to develop accurate companion diagnostic tools.

In summary, although available dataset showed that a high percentage of tumors harboring PIK3CA will likely benefit from inhibition of the PI3K pathway, a substantial proportion of patients with PIK3CA activating mutations may be de novo resistant to these agents. On the other hand, not all the patients with PIK3CA mutations are sensitive to PI3K inhibitors and not all the patients with wt PIK3CA/PTEN tumors are responsive. Moreover, while the majority of the studies have restricted the analysis on the PIK3CA and PTEN status, also occurring in other members of the pathway, such as mTOR activating mutations, or INPP4B loss of function, may play a role in the response to inhibitors. Therefore, the possibility to target PI3K signaling pathway in cancer requires deeper investigation, in order to identify additional biomarkers and to improve therapeutic strategies in the clinic.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.