**4. PI3K inhibitors in cancer therapy**

As aforementioned, PI3K/AKT/mTOR pathway has been implicated in tumorigenesis, promotion of cell survival, angiogenesis, cellular invasion, tumor growth, and the acquisition of chemoresistant phenotype by cancer cells [1]. Currently, more than fifty PI3K/AKT/mTOR axis inhibitors are in different stages of development, with a great number of such inhibitors reaching clinical trials [53]. Analogs of rapamycin (inhibitors of mTORC1), temsirolimus and everolimus, are currently in the lead, having already been approved by the Food and Drug Administration (FDA) as anticancer agents [54-56]. The PI3K/AKT/mTOR pathway inhibitors are summarized in Table 2.

#### **4.1. PI3K inhibitors**

**Figure 1.** Overview of Pl3K/AKT/mTOR signaling pathway and some inhibitors of this pathway in clinical studies. The activation of the PI3K by receptor tyrosine kinases promotes conversion of PIP2 to PIP3. PTEN dephosphorylates PIP3, negatively regulating the PI3K signaling. The phosphorylation and activation of AKT impacts many downstream ef‐

Somatic mutations and/or gains and losses of genes are possible genetic alterations affecting the PI3K/AKT/mTOR pathway in different solid and hematological tumors [33,34]. Indeed, PI3K pathway can be activated by direct upstream signs and can be intrinsically activated due to gain of functional mutations or amplifications in PIK3CA (p110 subunit), mutations in PIK3R (p85 subunit), and mutations or amplifications in one of the AKT isoforms or loss of PTEN [35]. Loss of PTEN via inactivating mutations, due to either copy number loss or homozygous deletions, is associated with both resistance to chemotherapy and reduced

PIK3CA mutations in primary breast tumors have been associated with lymph node metasta‐ ses and overexpression of ER, PR, and HER2 [36]. Furthermore, the presence of activating

fectors, such as mTORCI, and finally leads to multiple cellular processes.

**3. The role of PI3K/AKT/mTOR in cancer**

survival of human patients [3].

132 Updates on Cancer Treatment

PI3K inhibitors can be divided in isoform-specific inhibitors or pan-PI3K inhibitors. pan-PI3K inhibitors target all class IA PI3Ks in tumor cells, whereas isoform-specific inhibitors were developed to decrease toxicity and might be particularly effective in cancers with PIK3CA mutations, for example.

The first-generation of PI3K inhibitors include wortmannin, a fungal metabolite isolated from *Penicillium wortimannin* that irreversibly inhibits p110 by reacting covalently with the catalytic site [57], and LY294002, a synthetic, competitive, and reversible inhibitor of the ATP binding site of PI3K [58]. Both agents achieve significant antiproliferative and pro-apoptotic effects in preclinical *in vitro* and *in vivo* studies. However, unfavorable pharmacokinetic properties, insolubility in water, high levels of toxicity, and lack of selectivity for oncogenic isoforms of Class I PI3K limit its use in clinical trials [59,60]. Although this limiting features for their clinical use, wortmannin and LY294002 have served as important research tool for elucidating diverse signal transduction processes involving PI3K pathway and has spawned a new generation of PI3K inhibitors [61] (Table 2).

Currently, water-soluble wortmannin conjugates are being developed to overcome this issue. PX-866 is a semisynthetic analog of wortmannin with potent, irreversible, pan-class I PI3K inhibitory property against p110-α, p110-δ, and p110-γ enzymes in biochemical assays [62]. In preclinical studies, the compound alone or in combination with chemotherapy (cisplatin), radiotherapy, and targeted cancer drugs (gefitinib) exhibited *in vivo* antitumor activity against numerous mouse xenograft models of human cancers [62,63]. In addition, a phase I study in eighty-four patients with advanced solid tumors showed that PX-866 is well tolerated. The most frequent study drug-related adverse events were gastrointestinal disorders, with diarrhea being the most common [64]. PX-866 is being currently tested in a combination phase I/II studies with cetuximab (NCT01252628) in squamous cell carcinoma of the head and neck (SCCHN) and in metastatic colorectal carcinoma. Furthermore, more two phase I/II studies with PX866 are ongoing: with docetaxel (NCT01204099) in non-small cell lung cancer and SCCHN and in combination with vemurafenib in patients with advanced melanoma (NCT01616199).

Buparlisib (NVP-BKM120) is an oral highly specific pan-class I PI3K inhibitor with inhibitory property against p110-α, p110-β, p110-δ, and p110-γ enzymes [65]. The compound is also active against activating p110α somatic mutations but does not significantly inhibit the related class III and class IV PI3K kinases. In preclinical cancer studies, buparlisib has shown antiprolifer‐ ative and proapoptotic activity against a panel of 353 cell lines that display different genetic abnormalities that promote PI3K pathway activation [66]. *In vivo* studies have also shown that buparlisib potently inhibits the growth of human xenografts models and behaves synergisti‐ cally when combined with cytotoxic agents such as temozolomide, alkylating agent, and docetaxel, antimitotic drug, or with targeted agents such as HER2 and mitogen-activated protein kinase kinase (MEK) inhibitors [66].

A phase I dose-escalation study in thirty-five patients with advanced-stage solid tumors showed that buparlisib is a safe and well-tolerated drug with favorable pharmacokinetic properties. The major treatment-related adverse events included rash, hyperglycemia, diarrhea, anorexia, mood alteration, nausea, fatigue, pruritus, and mucositis [67]. Importantly, hyperglycemia was more common at higher doses and represents a class effect of the inhibition of PI3K signaling, commonly observed with other PI3K/AKT/mTOR pathway inhibitors [67]. Later, phase I dose-escalation and expansion study of buparlisib was performed in eightythree patients with advanced solid tumors demonstrating that buparlisib was well tolerated up to 100 mg/day and showed preliminary activity in patients with advanced cancers [68]. This subsequently led to the initiation of several clinical trials in multiple cancer types, such as non-small cell lung cancer, prostate cancer, breast cancer, colon cancer, and glioblastoma multiform (GBM).

developed to decrease toxicity and might be particularly effective in cancers with PIK3CA

The first-generation of PI3K inhibitors include wortmannin, a fungal metabolite isolated from *Penicillium wortimannin* that irreversibly inhibits p110 by reacting covalently with the catalytic site [57], and LY294002, a synthetic, competitive, and reversible inhibitor of the ATP binding site of PI3K [58]. Both agents achieve significant antiproliferative and pro-apoptotic effects in preclinical *in vitro* and *in vivo* studies. However, unfavorable pharmacokinetic properties, insolubility in water, high levels of toxicity, and lack of selectivity for oncogenic isoforms of Class I PI3K limit its use in clinical trials [59,60]. Although this limiting features for their clinical use, wortmannin and LY294002 have served as important research tool for elucidating diverse signal transduction processes involving PI3K pathway and has spawned a new generation of

Currently, water-soluble wortmannin conjugates are being developed to overcome this issue. PX-866 is a semisynthetic analog of wortmannin with potent, irreversible, pan-class I PI3K inhibitory property against p110-α, p110-δ, and p110-γ enzymes in biochemical assays [62]. In preclinical studies, the compound alone or in combination with chemotherapy (cisplatin), radiotherapy, and targeted cancer drugs (gefitinib) exhibited *in vivo* antitumor activity against numerous mouse xenograft models of human cancers [62,63]. In addition, a phase I study in eighty-four patients with advanced solid tumors showed that PX-866 is well tolerated. The most frequent study drug-related adverse events were gastrointestinal disorders, with diarrhea being the most common [64]. PX-866 is being currently tested in a combination phase I/II studies with cetuximab (NCT01252628) in squamous cell carcinoma of the head and neck (SCCHN) and in metastatic colorectal carcinoma. Furthermore, more two phase I/II studies with PX866 are ongoing: with docetaxel (NCT01204099) in non-small cell lung cancer and SCCHN and in combination with vemurafenib in patients with advanced melanoma

Buparlisib (NVP-BKM120) is an oral highly specific pan-class I PI3K inhibitor with inhibitory property against p110-α, p110-β, p110-δ, and p110-γ enzymes [65]. The compound is also active against activating p110α somatic mutations but does not significantly inhibit the related class III and class IV PI3K kinases. In preclinical cancer studies, buparlisib has shown antiprolifer‐ ative and proapoptotic activity against a panel of 353 cell lines that display different genetic abnormalities that promote PI3K pathway activation [66]. *In vivo* studies have also shown that buparlisib potently inhibits the growth of human xenografts models and behaves synergisti‐ cally when combined with cytotoxic agents such as temozolomide, alkylating agent, and docetaxel, antimitotic drug, or with targeted agents such as HER2 and mitogen-activated

A phase I dose-escalation study in thirty-five patients with advanced-stage solid tumors showed that buparlisib is a safe and well-tolerated drug with favorable pharmacokinetic properties. The major treatment-related adverse events included rash, hyperglycemia, diarrhea, anorexia, mood alteration, nausea, fatigue, pruritus, and mucositis [67]. Importantly, hyperglycemia was more common at higher doses and represents a class effect of the inhibition of PI3K signaling, commonly observed with other PI3K/AKT/mTOR pathway inhibitors [67].

mutations, for example.

134 Updates on Cancer Treatment

PI3K inhibitors [61] (Table 2).

(NCT01616199).

protein kinase kinase (MEK) inhibitors [66].

BASALT-1, an ongoing phase II trial (NCT01297491), is investigating the efficacy of singleagent buparlisib in patients with metastatic non-small cell lung cancer with PI3K pathway activation. Furthermore, phase Ib/II is under evaluation in patients with advanced non-small cell lung cancer of different histotype, testing buparlisib in combination with other targeted agents such as everolimus (NCT01470209), erlotinib (NCT01487265), MEK inhibitor (NCT01363232), or in combination with standard chemotherapeutic drugs, such as docetaxel (NCT01911325), gemcitabine, and cisplatin (NCT01971489) and carboplatin and paclitaxel (NCT01820325).

At present, several active, not recruiting, and recruiting clinical trials are being conducted in all the biological subsets of breast cancer, including combinations with endocrine therapy, anti-HER2 agents, poly (ADP-ribose) polymerase (PARP) inhibitors, and chemotherapy with buparlisib. Two large phase III studies (BELLE-2 and BELLE-3) (NCT01610284, NCT01633060) are investigating the combination of buparlisib plus fulvestrant in postmenopausal women with hormone receptor-positive/HER2-negative breast cancer after failure of aromatase inhibitor alone or aromatase inhibitor plus mTOR inhibitor treatment, respectively. Another ongoing clinical study is BELLE-4, a placebo-controlled phase II trial of buparlisib with paclitaxel in the first-line treatment of HER2-negative metastatic breast cancer (NCT01572727). Buparlisib has also been evaluated in a phase II study of paclitaxel plus trastuzumab in HER2 overexpressing breast cancer (NCT01816594).

Pilaralisib (XL147) is an oral pan-class I PI3K inhibitor (α, β, γ, and δ) through reversible, competitive inhibition with ATP for p110-α, -δ, -γ, and -β enzymes [69]. *In vitro* tests revealed that pilaralisib inhibits the formation of PIP3 in the membrane and phosphorylation of AKT and S6K-1 in multiple tumor cell lines with diverse genetic alterations in PI3K pathway [70]. Moreover, in mouse xenograft models, oral administration of pilaralisib results in significant tumor growth inhibition and combination with chemotherapeutic agents improved the growth-inhibitory effect observed with the single agents [71]. Based on this preclinical rationale, pilaralisib has been evaluated in phase I/II clinical trials.

In a phase I dose-escalation trial of sixty-nine patients with advanced solid tumors, pilaralisib was tolerable at doses associated with PI3K pathway inhibition, and the most frequent drugrelated adverse events included dermatologic toxicities, diarrhea, nausea, and decreased appetite [72]. However, a phase I dose-escalation study of pilaralisib with erlotinib in patients with solid tumors showed that combination had limited antitumor activity with moderate inhibition of PI3K, MAPK and EGFR pathways [73]. Moreover, phase I/II study of pilaralisib in combination with trastuzumab or trastuzumab plus paclitaxel in trastuzumab-refractory HER2-positive metastatic breast cancer related that no responses were observed in patients treated with pilaralisib plus trastuzumab while clinical activity was observed in paclitaxel arm [74]. Additional clinical evaluation of this PI3K inhibitor is ongoing in phase I/II studies (NCT01587040).

Pictilisib (GDC-0941) is another potent, selective, and orally bioavailable inhibitor of pan-class I PI3K. In biochemical assays, pictilisib demonstrates selectivity over a large panel of protein kinases and PI3K family kinases, including mTOR and DNA-dependent protein kinase (DNA-PK) [75]. Interestingly, pictilisib induces apoptosis in a subset of human tumor cell lines and potently inhibited tumor growth in xenograft models, including those with mutations in PI3K, PTEN, and K-Ras [76]. Significant *in vivo* antitumor activity has also been observed when administered orally in combination with other anticancer drugs, for example, docetaxel and MEK inhibitor U0126 [77-80].

In a first-in-human phase I study of pictilisib in sixty patients with advanced solid tumors, the most frequently reported drug-related adverse events were nausea, fatigue, and rash [81]. Importantly, one patient with V600E BRAF-mutant melanoma and another with platinumrefractory ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response [81]. Pictilisib is currently under evaluation in several phase I/II clinical trials, mainly in non-small cell lung cancer and breast cancer (NCT01918306, NCT01740336, NCT01493843, and NCT00974584).

One strategy to achieve significant pathway inhibition clinically with tolerable adverse effect profile is the use of isoform-specific PI3K inhibitors. As aforementioned, each isoform has distinct role in normal physiological processes and disease (Table 1). PI3K catalytic subunit p110α is predominantly responsible for mediating growth factor signaling from receptor tyrosine kinases and is a frequent genetic driver (*PIK3CA* mutations) in several cancers [82]. However, p110α is dispensable for PI3K pathway activation in tumors lacking PTEN. Thus, these cells depend largely on p110β to activate the pathway [82,83]. Preclinical tests showed that p110β-selective inhibitors had a significantly greater activity in cell lines with PTEN null than in those with PTEN intact, although, some PTEN-intact cell lines were sensitive and a number of cells lines lacking PTEN were resistant [84]. GSK-2636771 is a PI3K p110β-selective inhibitor currently in phase I studies in subjects with advanced solid tumors with PTEN deficiency (NCT01458067). Moreover, PI3Kδ is predominantly expressed in leukocytes and control immune responses [85]. Idelalisib (CAL-101), a highly specific PI3Kδ inhibitor, was the first isoform-specific PI3K inhibitors approved for cancer treatment [86].

Alpelisib (NVP-BYL719) is an oral inhibitor that selectively targets PI3K p110α equipotent against the wild type and the most common somatic mutations of p110α [87]. NVP-BYL719 has been the first PI3Kα-selective inhibitor to enter in clinical trials after positive preclinical investigations. *In vivo* studies have demonstrated dose-dependent antitumor activity of NVP-BYL719 in PIK3CA-mutant or PIK3CA-amplified tumor xenograft models, such as ovarian, breast, and head and neck cancers [88, 89]. Preliminary results of phase I study performed in patients with advanced solid tumors carrying *PIK3CA* gene alterations demonstrated that NVP-BYL719 has a favorable safety profile with manageable toxicities, as hyperglycemia, nausea, diarrhea, decreased appetite, vomiting, and fatigue [90]. To date, more than fifteen clinical trial is ongoing in order to evaluate the combination of NVP-BYL719 with several agents, such conventional cytotoxic drugs (paclitaxel, cisplatin, and irinotecan) and target drugs (cetuximab, olaparib, and trastuzumab) in a subset of cancers (NCT02051751, NCT01822613, NCT01602315, NCT01623349, and NCT02167854)

Taselisib (GDC-0032) is a PI3K inhibitor with higher affinity for mutated PI3Kα with reduced inhibitory activity against PI3Kβ [91]. Preclinical studies show that taselisib has enhanced activity against PI3Kα isoform mutant cancer cell lines [92]. In an ongoing phase I study, taselisib has been well tolerated with hyperglycemia and fatigue being the dose-limiting toxicities [93]. This selectivity profile and excellent pharmacokinetic properties allowed fewer clinical studies with GDC-0032. Currently, several clinical studies are ongoing to evaluate the combination of taselisib with endocrine therapy, trastuzumab, and conventional chemother‐ apy in breast cancer (NCT02285179, NCT02390427, and NCT01862081). In addition, a phase I study is currently ongoing in taselisib with CDK4/6 inhibitor, palbociclib, in advanced solid tumors and breast cancer (NCT02389842).

Idelalisib was approved in 2014 in the United States and European Union for the treatment of three indolent B-cell neoplasms: relapsed chronic lymphocytic leukemia, in combination with rituximab, relapsed follicular B-cell non-Hodgkin's lymphoma, and relapsed small lympho‐ cytic lymphoma (as monotherapy) [94]. In lymphoid cell lines and primary patient samples, idelalisib abrogates PI3K/AKT/mTOR signaling and promotes apoptosis [95,96]. The first phase I trial in healthy volunteers established the bioavailability and safety of idelalisib [97]. Another phase I study in patients with relapsed/refractory mantle cell lymphoma reported the most common adverse events, which includes diarrhea, nausea, pyrexia, fatigue, rash, upper respiratory infection, pneumonia and alanine transaminase, or aspartate transaminase elevations [98]. To date, about twenty-five clinical trials are ongoing with idelalisib. A phase I/II trial studies aimed evaluated idelalisib in combination with lenalidomide and rituximab in patients with relapsed or refractory mantle cell lymphoma (NCT01838434). In addition, idelalisib is being evaluated in combination with rituximab in adults with previously treated indolent non-Hodgkin lymphoma (NCT01732913).
