Marlena Walls

Evaluation of Selective γ-Secretase Inhibitor PF-03084014 for Its Antitumor Efficacy and Gastrointestinal Safety to Guide Optimal Clinical Trial Design

Aberrant regulation of Notch signaling has been implicated in tumorigenesis. Proteolytic release of the Notch intracellular domain (NICD) by γ-secretase plays a key role in Notch-dependent nuclear signaling. γ-Secretase is an attractive pharmaceutical target for therapeutic intervention in cancer. We describe the potent antitumor effects of PF-03084014, a small molecule that is a reversible, noncompetitive, and selective γ-secretase inhibitor. The ability of PF-03084014 to inhibit γ-secretase activity was shown by the reduction of endogenous NICD levels and by the downregulation of Notch target genes Hes-1 and cMyc in the T-cell acute lymphoblastic leukemia (T-ALL) cell line HPB-ALL. PF-03084014 caused cell growth inhibition of several T-ALL cell lines via cell cycle arrest and induction of apoptosis. PF-03084014 treatment also resulted in robust NICD reduction in HBP-ALL xenograft models. Broad antitumor efficacy at well-tolerated dose levels was observed in six Notch-dependent models. Additional mechanism-of-action studies showed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the antitumor activity of PF-03084014 may be mediated by its direct effects on tumor cell growth or survival. Further studies on PF-03084014–induced gastrointestinal toxicity identified an intermittent dosing schedule that displayed reduced body weight loss and sustained antitumor efficacy. We also showed that glucocorticoids abrogated PF-03084014–induced gastrointestinal toxicity and delayed administration of glucocorticoids did not compromise its protection effect. Collectively, the results show that inhibition of Notch signaling by PF-03084014 while minimizing gastrointestinal toxicity presents a promising approach for development of therapies for Notch receptor–dependent cancers. This compound is being investigated for the treatment of T-ALL and advanced solid tumors in phase I clinical trials. Mol Cancer Ther; 9(6); 1618–28. ©2010 AACR.

Discovery of the Highly Potent PI3K/mTOR Dual Inhibitor PF-04979064 through Structure-Based Drug Design.

PI3K, AKT, and mTOR are key kinases from PI3K signaling pathway being extensively pursued to treat a variety of cancers in oncology. To search for a structurally differentiated back-up candidate to PF-04691502, which is currently in phase I/II clinical trials for treating solid tumors, a lead optimization effort was carried out with a tricyclic imidazo[1,5]naphthyridine series. Integration of structure-based drug design and physical properties-based optimization yielded a potent and selective PI3K/mTOR dual kinase inhibitor PF-04979064. This manuscript discusses the lead optimization for the tricyclic series, which both improved the in vitro potency and addressed a number of ADMET issues including high metabolic clearance mediated by both P450 and aldehyde oxidase (AO), poor permeability, and poor solubility. An empirical scaling tool was developed to predict human clearance from in vitro human liver S9 assay data for tricyclic derivatives that were AO substrates.

Highly Selective and Potent Thiophenes as PI3K Inhibitors with Oral Antitumor Activity

Highly selective PI3K inhibitors with subnanomolar PI3Kα potency and greater than 7000-fold selectivity against mTOR kinase were discovered through structure-based drug design (SBDD). These tetra-substituted thiophenes were also demonstrated to have good in vitro cellular potency and good in vivo oral antitumor activity in a mouse PI3K driven NCI-H1975 xenograft tumor model. Compounds with the desired human PK predictions and good in vitro ADMET properties were also identified. In this communication, we describe the rationale behind the installation of a critical triazole moiety to maintain the intricate H-bonding network within the PI3K receptor leading to both better potency and selectivity. Furthermore, optimization of the C-4 phenyl group was exploited to maximize the compounds mTOR selectivity.

RETRACTED: A novel class of specific Hsp90 small molecule inhibitors demonstrate in vitro and in vivo anti-tumor activity in human melanoma cells

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors. Following an investigation by Pfizer, Figures 2, 5B and 5C appear to be duplications and hence the conclusions in the manuscript cannot be verified. The Authors apologize for this inconvenience.

Targeting Small Cell Lung Cancer Harboring PIK3CA Mutation with a Selective Oral PI3K Inhibitor PF-4989216

Purpose: Constitutive activation of phosphoinositide 3-kinase (PI3K) occurs frequently in many human tumors via either gene mutation in the p110α catalytic subunit of PI3K or functional loss of tumor suppressor PTEN. Patients with small-cell lung cancer (SCLC) have very poor prognosis and survival rates such that an effective targeted therapy is in strong demand for these patients. In this study, we characterized the highly selective oral PI3K inhibitor, PF-4989216, in preclinical SCLC models to investigate whether targeting the PI3K pathway is an effective targeted therapy option for SCLCs that harbor a PIK3CA mutation. Experimental Design: A panel of SCLC cell lines with PIK3CA mutation or PTEN loss were treated with PF-4989216 in several in vitro assays, including PI3K pathway signaling, cell viability, apoptosis, cell-cycle progression, and cell transformation. SCLC cell lines that were sensitive in vitro to PF-4989216 were further evaluated by in vivo animal studies to determine the pharmacokinetic/pharmacodynamic relationship and tumor growth inhibition (TGI) by PF-4989216 treatment. Results: PF-4989216 inhibited PI3K downstream signaling and subsequently led to apoptosis induction, and inhibition in cell viability, transformation, and xenograft tumor growth in SCLCs harboring PIK3CA mutation. In SCLCs with PTEN loss, PF-4989216 also inhibited PI3K signaling but did not induce BCL2-interacting mediator (BIM)-mediated apoptosis nor was there any effect on cell viability or transformation. These results implicate differential tumorigenesis and apoptosis mechanisms in SCLCs harboring PIK3CA mutation versus PTEN loss. Conclusions: Our results suggest that PF-4989216 is a potential cancer drug candidate for patients with SCLC with PIK3CA mutation but not PTEN loss. Clin Cancer Res; 20(3); 631–43. ©2013 AACR.

Targeting the mTOR Pathway in Tumor Malignancy

The mammalian target of rapamycin (mTOR) plays a critical role in the regulation of cell growth, proliferation,and metabolism by integrating growth factor stimulation and energy/nutrient input through a complex signaling network.The mTOR kinase is a part of two structurally and functionally distinct multiple protein complexes, mTORC1 and mTORC2. The mammalian target of rapamycin complex 1 (mTORC1) is rapamycin-sensitive and mediates temporal control of cell growth by regulating several cellular processes, such as translation, transcription, and nutrient transport while the mammalian target of rapamycin complex 2 (mTORC2) is in sensitive to rapamycin and is involved in spatial control of cell growth via cytoskeleton regulation. Here we discuss the mechanism of mTOR regulation in tumor malignancy through a variety of signaling pathways and the potential of mTOR inhibitors for the treatment of cancer.

Abstract 4479: Targeting non-small cell lung cancer cells harboring a PIK3CA mutation by a novel and oral PI3K selective inhibitor, PF-4989216

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Aberrant phosphatidylinositol 3-kinase (PI3K)/AKT signaling occurs commonly in cancer. Gene mutation, amplification, and copy number gains of the PI3K catalytic subunit p110α have been shown in a variety of human cancers. The tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) dephosphorylates the 3-phosphoinositides and is frequently mutated, deleted, or down-regulated in many cancers to constitutively activate the PI3K pathway. In response to activation, PI3Ks phosphorylate membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an secondary messenger activating AKT and 3’-phosphoinositide-dependent kinase-1 (PDK1). Phosphorylation of AKT at threonine-308 (T308) by PDK1 and serine-473 (S473) by mTORC2 fully activates the AKT pathway to regulate various cellular processes including cell growth, proliferation, survival, and metabolism. Several inhibitors targeting the PI3K pathway have been developed in preclinical discovery or clinical trials; however, a small molecule inhibitor has not yet been approved for cancer treatment. Most inhibitors have inhibitory activity against both PI3Ks and mTOR kinases. The mTORC1 and mTORC2 complexes control critical pathways regulating cell proliferation, apoptosis, angiogenesis, and metabolism through AKT-dependent and AKT-independent mechanisms. Therefore, agents targeting PI3K with additional mTOR kinase inhibitory activities may carry extra toxicity through the disruption of mTOR function in normal cells. To develop a drug selectively targeting PI3K, we have identified PF-4989216 as a novel potent and selective PI3K inhibitor. In this study, we describe the in vitro and in vivo anti-tumor activity of PF-4989216 in human non-small-cell lung cancer cells (NSCLCs). PF-4989216 inhibits the phosphorylation of PI3K downstream molecules and subsequently leads to inhibition in cell proliferation and xenograft tumor growth in NSCLCs harboring a PIK3CA mutation, including an erlotinib resistant line, NCI-H1975. Although PF-4989216 inhibits the phosphorylation of PI3K downstream molecules in the NSCLCs with PTEN loss (NCI-H1650), PF-4989216 does not sufficiently induce anti-tumor efficacy in NCI-H1650. Our results suggest that there may be a different tumorigenesis mechanism between PIK3CA mutation and PTEN loss in NSCLCs, and indicate that PF-4989216 is a potential cancer drug candidate for erlotinib resistant non-small-cell lung cancer patients with PIK3CA mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2011-4479

Abstract 2594: Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR

Activating mutations in EGFR confer constitutive activity providing the oncogenic drive in EGFR-mutant NSCLC. First and 2nd generation EGFR tyrosine kinase inhibitors (TKIs) are effective drugs in this setting, but are constrained by dose-limiting toxicities attributed to inhibition of wild type (WT) EGFR and by drug resistance caused, in the majority of cases, via a T790M secondary mutation in EGFR. We report the pharmacology of a novel irreversible 3rd generation EGFR TKI active against EGFR with activating and T790M mutations, but sparing WT EGFR. Our novel 3rd generation EGFR TKI was studied in a variety of in vitro and in vivo models to determine its inhibitory potencies on different EGFR variants, pharmacokinetics (PK), antitumor efficacy, exposure-response relationships, mechanism of action, and predicted human efficacious dose. In enzyme and cell assays, our compound is a highly potent inhibitor of EGFR double mutants (L858R/T790M and Del/T790M) and EGFR activating mutants (L858R and Del), but a weak inhibitor of WT EGFR (26-fold margin over mutant target potencies). Effects on downstream signaling and function indicate the underlying mechanism of the compound is direct inhibition of EGFR, with subsequent inhibition of downstream signaling that results in apoptosis and viable cell decline. In xenograft mouse models, the compound demonstrates tumor growth inhibition and regression at well-tolerated doses in models driven by EGFR double mutants and EGFR activating mutants. The antitumor efficacy is dose-dependent and strongly correlates with inhibition of EGFR phosphorylation and EGFR-mediated downstream signaling, and induction of apoptosis. Plasma concentrations assumed to be sufficient for efficacy (Ceff) were defined using a mathematical model incorporating the plasma levels of the compound, the associated inhibitory effects on EGFR phosphorylation, and the antitumor efficacy in the double and activating mutant xenograft models. Ceff was in agreement across several models and was used with in vitro human PK properties to calculate required human dose. While our compound possesses a similar profile as other recently disclosed 3rd generation EGFR TKIs, this molecule is distinguished by better potency on the activating mutants and by the widest potency margin on WT EGFR. Given that the target potencies and WT margins of 3rd generation EGFR TKIs have been sufficient for tolerated clinical efficacy in preliminary results, it can be inferred that our compound will have similar promise in the clinic. These results support our compound as a novel EGFR TKI with an inhibitory profile and favorable drug-like properties that suggest utility for treating patients with NSCLC with EGFR activating and resistance mutations. Citation Format: Mike Zientek, Sangita Baxi, Henry Cheng, Valeria Fantin, Jun Li Feng, Allison Given, Zelanna Goldberg, Jie Guo, Michelle Hemkens, John Kath, Jennifer Lafontaine, Gary Li, Pramod Mehta, Brion Murray, Sajiv Nair, Simon Planken, Chad Ray, Yuli Wang, Manli Shi, Anand Sistla, Tod Smeal, Greg Stevens, Wei Tan, Paolo Vicini, Marlena Walls, Liu Yang, Min-Jean Yin, Scott L. Weinrich. Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2015-2594

Abstract 4467: Targeting both PI3K/mTOR and EGFR pathways leads to synergistic anti-tumor activity in erlotinib resistant non-small-cell lung cancers.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Abstract 3578: A novel and selective 3-phosphoinositide-dependent kinase-1 inhibitor, PF-5177624, blocks insulin-like growth factor-1 induced tumorigenesis in breast cancer cells

Phosphatidylinositol 3-kinases (PI3Ks) phosphorylate membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an important secondary messenger recruiting and activating proteins that contain a pleckstrin homology (PH) domain including AKT and 3’-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a 63-kDa Ser/Thr kinase with a catalytic domain near its N-terminus and a pleckstrin homology domain at its C-terminus. The PH domain is necessary for targeting PDK1 to the plasma membrane to phosphroylate the T-loop sites of numerous substrates such as AKT at the residue threonine-308 (T308). The phosphorylation at both T308 and the serine-473 residue by mTORC2, fully activates AKT to induce the downstream signaling pathways that are important for tumor progression. PDK1 also has been shown to phosphorylate p70S6K, isoforms of PKCs, and many other kinase substrates. The oncogenic activity of aberrant PI3K pathway signaling through PDK1 has been extensively validated. PDK1 is highly expressed in a majority of human breast cancers and cell lines, and elevated phosphorylation of PDK1 was associated with PIK3CA mutations in human breast tumors and cell lines. Therefore, targeting PDK1 in the PI3K/AKT pathway may provide an opportunity for breast cancer treatment. In this study, we demonstrate that insulin-like growth factor-1 (IGF-1) stimulates PDK1 activity and a specific and potent PDK1 inhibitor, PF-5177624, inhibits IGF-1 stimulated downstream signaling in breast cancer cells, including a reduction in phosphorylation of both AKT (T308) and p70S6K. The inhibition of PDK1 activity through the IGF-1 axis is sufficient to induce in vitro anti-tumor activity in breast cancer cells such that PF-5177624 inhibits cell proliferation and cell transformation in breast cancer cells. Our data suggest that a specific and potent PDK1 inhibitor (PF-5177624) is likely to have antitumor activity in breast cancer cells and can be developed as a cancer drug for breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3578. doi:10.1158/1538-7445.AM2011-3578