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Deregulation of the phosphoinositide 3-kinase (PI3K) signaling pathway such as by PTEN loss or PIK3CA mutation occurs frequently in human cancer and contributes to resistance to antitumor therapies. Inhibition of key signaling proteins in the pathway therefore represents a valuable targeting strategy for diverse cancers. PF-04691502 is an ATP-competitive PI3K/mTOR dual inhibitor, which potently inhibited recombinant class I PI3K and mTOR in biochemical assays and suppressed transformation of avian fibroblasts mediated by wild-type PI3K γ, δ, or mutant PI3Kα. In PIK3CA-mutant and PTEN-deleted cancer cell lines, PF-04691502 reduced phosphorylation of AKT T308 and AKT S473 (IC50 of 7.5–47 nmol/L and 3.8–20 nmol/L, respectively) and inhibited cell proliferation (IC50 of 179–313 nmol/L). PF-04691502 inhibited mTORC1 activity in cells as measured by PI3K-independent nutrient stimulated assay, with an IC50 of 32 nmol/L and inhibited the activation of PI3K and mTOR downstream effectors including AKT, FKHRL1, PRAS40, p70S6K, 4EBP1, and S6RP. Short-term exposure to PF-04691502 predominantly inhibited PI3K, whereas mTOR inhibition persisted for 24 to 48 hours. PF-04691502 induced cell cycle G1 arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. Antitumor activity was observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non–small cell lung carcinoma xenografts. In summary, PF-04691502 is a potent dual PI3K/mTOR inhibitor with broad antitumor activity. PF-04691502 has entered phase I clinical trials. Mol Cancer Ther; 10(11); 2189–99. ©2011 AACR.

The phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/mTOR) pathway and the mitogen-activated protein kinase (MAPK) signaling cascades are central facilitators of cellular growth, proliferation, survival and motility within a variety of tumor lineages. To date, several ongoing oncology clinical trials are currently evaluating the benefit of combining PI3K with MEK inhibition. While this dual combination is well founded pre-clinically, investigating potential mechanisms of resistance and identifying novel biological signatures of sensitivity is of great clinical importance. We sought to establish a model of acquired resistance by evaluating the co-treatment of a colorectal carcinoma model with inhibitors of PI3K/mTOR inhibitor (PF-04691502) and MEK (PD-0325901). We can associate the onset of acquired resistance in this model to re-engagement of signaling within the PI3K/mTOR pathway correlative to a loss of PTEN protein expression , relative to parental cells. In addition, next-generation sequencing (NSG) comparing parental versus resistant cells identified a unique somatic “gatekeeper” mutation located in the kinase domain of mTOR. Furthermore, a cell proliferation screen evaluating potential cross resistance was analyzed using a PI3K/mTOR inhibitor with a chemical scaffold distinct from PF-502 (PF-05212384). Treatment of PF-384 as a single agent (IC50 ∼150nM) circumvented the acquired resistance derived from the combination of PF-502+ PD-901 (IC50: ∼2uM and ∼334nM respectfully). Inhibition of growth observed with PF-384 correlated with rescuing of PI3K/mTOR signaling as measured by substrate phosphorylation readouts. Finally, co-crystal structure modeling of mTOR was used to evaluate the potential interaction of PF-502 within the ATP binding pocket and we confirm that this interaction is hindered due to the point mutation identified at mTOR. Furthermore, co-crystal modeling of mTOR with PF-384 illustrates an independence and lack of PF-384 binding to the mutated site and thus substantiates a mechanistic explanation for overcoming PF-502 acquired resistance. In summary, we have identified and characterized a novel mechanism of resistance to the combined treatment of a Pyrimidinone class PI3K/mTOR inhibitor in addition to a MEK inhibitor, and have shown that rescuing of this resistance can be achieved through the use of a Triazine class PI3K/mTOR clinical candidate. Citation Format: Scott J. Garza, Paul Lira, Stephen D. Huang, Hengmiao Cheng, Stephen G. Dann, Todd L. VanArsdale, Valeria Fantin, James Christensen, Julie LC Kan. Identifying a mechanism of acquired resistance to the combined inhibition of PI3K/mTOR and MEK in colorectal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3713. doi:10.1158/1538-7445.AM2014-3713

NSCLCs with EGFR mutations, such as a deletion in exon 19 (del E746-A750) or point mutation in exon 21 (L858R), are sensitive to TKI (gefitinib and erlotinib) treatments. However, acquired resistance to TKI therapy occurs in patients after 12-14 months. Approximately 50% of these resistant patients have developed secondary (gatekeeper) T790M mutation in EGFR; many other resistance mechanisms also have been described including a PIK3CA mutation and PTEN deletion. In this study, we characterize a dual PI3K/mTOR inhibitor, PF-4979064, in combination with a pan-ErbB inhibitor, PF-299804 (dacomitinib) in erlotinib resistant NSCLC lines NCI-H1975 (harboring EGFR and PIK3CA mutation) and NCI-H1650 (harboring EGFR and PTEN deletion). As a clinically relevant concentration of PF-299804 only induces moderate anti-tumor activity in both H1975 and H1650 cells, PF-4979064 was added in a variety of cellular and animal studies to determine whether the combination was able to achieve a synergistic induction of anti-tumor activity. Results demonstrate that the combination of PF-4979064 and PF-299804 lead to a synergistic inhibition in cell proliferation and xenograft tumor growth in both the NCI-H1975 and NCI-H1650 models. Our results further suggest that there is a rationale to examine a potential clinical development plan combining the dual PI3K/mTOR inhibitor (PF-4979064) and dacomitinib (PF-299804) in NSCLC patients who have developed resistance to erlotinib and have a concomitant PIK3CA mutation or PTEN deletion. Citation Format: Pramod P. Mehta, Sangita M. Baxi, Marlena Walls, Stella Chen, Hengmiao Cheng, Gang Li, Tod Smeal, Min-Jean Yin. Targeting both PI3K/mTOR and EGFR pathways leads to synergistic anti-tumor activity in erlotinib resistant non-small-cell lung cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4467. doi:10.1158/1538-7445.AM2013-4467

The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Ki's of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50's of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.

PF-04691502 is a dual inhibitor of both PI3K and mTOR, inhibits PI3K signaling in cancer cell lines, and exhibits in vitro and in vivo anti-proliferative activity in PI3K-pathway driven cell lines. It is a nanomolar inhibitor of all 4 isoforms of the catalytic subunit of PI3K and of both TORC1 and TORC2. Anti-cancer activity of the inhibitor is hypothesized to be through inhibition of survival, proliferative, and anti-apoptotic processes. Its predicted human bioavalibility is roughly 60% and it will soon be entering phase 1 studies. To provide an improved predictability of this compound and its PD markers in the clinic we correlated PK, PD (phosphoproteins and FDG-PET imaging) and tumor growth inhibition in a single model. Since this inhibitor exhibits an IC50 of 37 nM in the PC3 prostate cell line (PTEN null) in vitro we chose this cell line for a xenograft model. Three groups (Vehicle, 5 and 7.5 mg/kg.) were dosed p.o. daily and subsequently evaluated. Tissues were harvested on day 7 and 13 at 4.5 hr post-dose. FDG-PET imaging was completed on days 1, 3, 7 and 13 at 3 hr post-dose. First, negative correlations were established between blood levels of PF-04691502 and the following tumor phosphoprotein epitopes: pS6, pAKTS473 and pAKTT308. The R2 values for these proteins were −0.3, −0.4 and −0.4, respectively. P-values of the correlations are all significant for all three proteins. Second, all PD markers were positively correlated with TGI. When correlating FDG-PET and TGI, day 7 has the best correlation and an R2 of 0.3 while days 3 and 13 have poorer correlations (R2 of 0.04 and 0.13, respectively). Although the R2 isn't high for day 7, the positive trend between image and TGI is statistically significant. When correlating PD markers and TGI, R2 values for pS6, pAKTS473 and pAKTT308 were 0.6, 0.8 and 0.6, respectively. In conclusion, the data from this single model indicates PD phosphoprotein markers are more reflective of TGI than FDG-PET imaging. These results will be applied to estimating the size of the cohort recruited for mechanistic evaluation in phase 1 trials of PF-04691502. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3492.

Emerging evidences suggest that cancer stem cells (CSC) may be critically responsible for tumor initiation, progression, metastasis, and drug resistance. It becomes important to ask whether anti-cancer agents are able to target the tumor-initiating subpopulation in relevant CSC models. In this study, we first established xenograft tumors in NOD/SCID mice from a colorectal cancer patient specimen and demonstrated that CD133/EpCAM-expressing CSC population was highly tumorigenic. We then sought to propagate the CSC population under a serum-free condition. In culture, the tumor cells formed non-adherent, 3-dimensional spheroids, a fraction of which retained expression of the CSC markers. When exposed to a serum-containing medium, tumor spheroid cells differentiated into epithelial-like adherent cells with an increase in cell proliferation rate. In comparison with the differentiated progeny, tumor spheroid cells exhibited resistance to the standard-of-care agent oxaliplatin and, in limiting dilution assays in mice, displayed substantially higher tumorigenic potential. In contrast to the tumors originated from the differentiated cells, tumor spheroid cell-derived tumors recapitulated not only the CSC frequency marked by CD133/EpCAM expression, but also the histological characters of the original tumor. Similarly, only were the fragments of spheroid cell-derived xenograft tumors capable of regenerating highly proliferative tumors in secondary transplantation. Thus, the tumor spheroid culture is indeed enriched of drug resistant, self-renewing, and tumor-initiating CSC populations. Mutation profiling of frequently mutated oncogenes using Sequenom OncoCarta™ panel identified a mutation in the kinase domain of PIK3CA (H1047R) in the cultured CSCs. This mutation has been reported present in a large number of colon cancer patients and likely functions as an oncogene (Samuels et al., Science 304:554; 2004). We further demonstrated that a dual mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitor (PF-04691502) exhibited a more potent effect on inhibition of in vitro proliferation of the mutated CSCs compared to the chemotoxic agent oxaliplatin. Collectively, our findings suggest that CSC models provide a novel avenue to drug sensitivity and efficacy studies. The well-characterized CSC model systems may assist in the development of more effective therapy against the subpopulation of tumors driven by the CSCs bearing specific mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4483.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway plays a critical role in the regulation of cell growth, proliferation and survival in cancer. In non-small cell lung carcinomas cells (NSCLCs), the PI3K/AKT/mTOR pathway is frequently activated through either the amplification/mutation of PIK3CA (which encodes the p110α subunit of PI3K), or the functional loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In addition, the PI3K/AKT/mTOR pathway plays a critical role downstream of epidermal growth factor receptor (EGFR) in a defined NSCLC population; in particular, those harboring the EGFR mutations. Dual inhibition of PI3K and mTOR therefore represents a high therapeutic value for NSCLC patients. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in biochemical, cell, and animal studies. In this study, we show PF-04691502 inhibits phosphorylation of AKT (at both S473 and T308) and S6 ribosomal protein (S6RP) in a variety of NSCLC lines harboring a PIK3CA mutation or EGFR mutation, subsequently resulting in anti-proliferative activity in the NSCLC lines. Furthermore, we demonstrate that PF-04691502 is capable of inducing tumor growth inhibition in four NSCLC xenograft models including an Erlotinib resistant line, NCI-H1975. In these murine xenograft models, decreased phosphorylation of AKT, p70S6K, and S6RP was observed in treated tumors in a manner consistent with the drug levels achieved. Therefore, PF-04691502 exhibits potent inhibition in the PI3K/mTOR axis and leads to both in vitro and in vivo anti-tumor activity in NSCLCs. Moreover, our data suggests that PF-04691502 may have clinical benefit to NSCLC patients including those found to be resistant to Erlotinib treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4473.