Pamela Whalen

Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

Cancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC(50) of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC(50) of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100x selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC(50) of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity.

Development and validation of an alpha fetoprotein immunoassay using Gyros technology.

Circulating alpha fetoprotein (AFP) is a diagnostic and prognostic biomarker for hepatocellular carcinoma (HCC) with potential utility as a pharmacodynamic endpoint in rodent tumor models. This application is limited, however, by low sample volumes, highlighting the need for sensitive, sample-sparing biomarker assay methods. In order to improve the utility of AFP as an oncology biomarker, we developed a method for AFP using the Gyrolab™, an automated microimmunoassay platform. Commercially available antibodies were screened to identify optimal combinations that were then used in a multi-factorial design of experiments (DOE) to optimize reaction conditions. Analytical validation included assessments of accuracy and precision (A&P), and dilutional linearity/hook effect, as well as reagent and sample stability. The method is reliable, with total error, a measure of accuracy and precision, less than 30% for all concentrations tested. AFP concentrations were measurable in diseased mice and undetectable in normal mice. Therefore, this novel, low volume AFP immunoassay is suitable for pre-clinical drug development, where its miniaturized format facilitates serial sampling in rodent models of cancer.

Abstract 1958: Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of cancers and a small molecule inhibitor of Smoothened (SMO), Vismodegib, has been approved for the treatment of basal cell carcinoma, a disease frequently driven by Hh pathway signaling due to pathway mutations. SMO dependent Hh signaling has also been implicated in models of myeloid leukemia, primarily CML, where genetic loss of SMO or pharmacological inhibition limits disease progression in part through targeting the leukemic stem cell (LSC). Outside of BCR-ABL driven leukemia the role of Hh signaling and impact of SMO inhibition on disease progression and the LSC remains unclear. To explore the role of Hedgehog pathway signaling and interrogate responses to SMO inhibition in AML, a panel of primary AML patient-derived models was utilized to examine responses to PF-04449913, an oral small molecule SMO inhibitor currently in early phase clinical trials targeting myeloid malignancies. AML patient samples were characterized for Hh pathway expression levels and screened for responses to PF-04449913. Ex vivo treatment of AML bone marrow cells showed PF-04449913 was capable of inhibiting Hh pathway activity, reducing expression of key LSC regulators and decreasing populations of cells expressing LSC markers. Use of AML xenotransplant models to assess in vivo responses to PF-04449913 as single agent and in combination with Cytarabine have shown potential for combination efficacy of the two agents in select models suggesting patient selection strategies may be critical for SMO inhibitor-based therapies in AML. Citation Format: Amy Jackson-Fisher, Pamela Whalen, Mark Elliott, Melissa McMahon, Enhong Chen, Xianxian Zheng, Mark Ozeck, Donghui Huang, Paul Lira, Joseph Lee, Cathy Zhang, Justine Lam, Mary Spilker, Shibing Deng, Patrick Lappin, Penny Venne, Cynthia Heinlein, Annelie Schairer, Karen McLachlan, Todd VanArsdale. Interrogating hedgehog pathway and smoothened inhibition by PF-04449913 in patient-derived acute myeloid leukemia models. [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 1958. doi:10.1158/1538-7445.AM2014-1958

Abstract A86: Design, synthesis, and SAR of focal adhesion kinase (FAK) inhibitors

Focal adhesion kinase (FAK) is a non‐tyrosine kinase that localizes to focal adhesion plaques. It is activated in response to intergin binding to cellular ligands and when phosphorylated inhibits anoikis allowing for anchorage independent cell growth. Recent studies have shown increased FAK expression and phosphorylation status in many types of invasive and aggressive human tumors strongly suggesting FAK is a possible target for anticancer chemotherapy. Literature, in house HTS and de novo studies identified 2, 4‐diaminopyrimidines as potent FAK inhibitors. Early SAR efforts quickly determined that smaller substituents, particularly CF3, were optimal in the C5 position. Parallel medicinal chemistry strategies were executed for the C2 and C4 positions. These studies suggested that substituted aryl and fused heteroaryl groups at the C2 position in conjunction with substituted phenyl and heterocycles at the C4 position imparted optimum activity and metabolic stability. Inhibitor‐FAK co‐crystal structures were utilized to guide in the SAR strategy around the 2, 4‐diaminopyrimidine template which afforded several lead compounds. The team9s effort culminated in the advancement of PF‐562,271 as a potent and reversible inhibitor of FAK (kinase IC50 of 2 nM and cell IC50 of 5 nM) that is > 100x selective against a long list of non‐target kinases. In summary, detailed SAR studies were executed on the 2, 4‐diaminopyrimidine templates that produced potent inhibitors of FAK with improved ADME properties, and identified a novel and potent series of FAK inhibitors that are selective against most other kinases and have shown activity in clinical trials. This poster will present design, synthesis, challenging chemistry, optimization, and complete inhibitor chemical structures of lead analogs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A86.

Abstract 1489: Function of Rho kinases on HCC tumor growth and metastasis

The Rho/ROCK (Rho-associated protein kinase) pathway has been found to be frequently upregulated in hepatocellular carcinoma (HCC). Clinico-pathological research has shown that increased RhoA and ROCK2 expression are associated with the presence of tumor microsatellite formation. RhoA is over expressed in 69% of HCC, and high RhoA levels are correlated with poor survival. ROCK2 protein level is upregulated in 54% of HCC, and its expression is associated with intrahepatic metastasis. The ROCK2 knockdown by short hairpin (sh)RNA leads to significant reduction of tumor formation. These observations suggest that Rho/ROCK pathway could be a potential therapeutic target for the treatment of liver cancer. Down-regulation of the Rho/ROCK pathway may inhibit tumor metastasis. However, the role of ROCK1 in HCC metastasis is still not clear. In order to dissect out the roles of ROCK1 and ROCK2 in liver tumor formation and metastasis, we used the shRNAmir- lentiviral system to knockdown ROCK1, ROCK2, and ROCK1/ROCK2 in HCC cell lines. The RNA and protein levels of ROCK1 and ROCK2 were significantly reduced in HCC cell lines that expressed shRNAs targeting ROCK1 and ROCK2. Orthotopically implanted HCC cells carrying shRNA targeted at ROCK1 and ROCK2 either alone or in combination were examined for tumor induction in mice. Our data showed that tumor growth was significantly reduced in those mice inoculated with cells expressing shRNAs. In addition, fewer tumor nodules as compared to the control (Non-target shRNA control) group were seen in those mice. Using qRT-PCR, we confirmed that the decreased ROCK activity was associated with the reduction of ROCK1 and ROCK2 RNA levels in mouse liver samples. Conclusion: our results indicate that both ROCK1 and ROCK2 are important for HCC tumor growth. Moreover, we demonstrated that ROCK1 played a more significant role than ROCK2 in HCC intrahepatic metastasis. 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 1489. doi:10.1158/1538-7445.AM2011-1489

Abstract 4424: Antitumor activities of targeted therapeutic agents in preclinical models of gastric cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Gastric cancer (GC) is one of the most prevalent cancer types in Asia, and the 2nd most common cause of cancer deaths worldwide. Although considerable advances have been made with the use of improved surgical techniques and chemotherapy regimens, the prognosis for most GC patients remains poor. Therefore there is an urgent medical need for novel, effective targeted therapeutic agents for GC. One of the key drivers for a successful drug discovery program is utilization of predictive preclinical models that represent the patients of interest. Through internal efforts and collaborations we have developed subcutaneous (SC), orthotopic, syngeneic and patient-derived xenograft (PDX) models for target validation and pharmacological studies. Here we report the establishment of various cell line-derived mouse models of GC, and their validation with targeted therapeutic agents. In SC models of MKN-74, NCI-N87 and MKN-45, sunitinib malate significantly inhibited tumor growth. To establish orthotopic GC model, luciferase-expressing MKN-74 and NCI-N87 cells were first SC implanted in athymic nude mice. Four weeks after in vivo propagation, tumor fragments (1 mm3) were surgically implanted to the stomach wall in the greater curvature region. Primary tumor growth and spontaneous metastasis were monitored by bioluminescent imaging. Sunitinib malate treatment inhibited primary tumor growth but the effect on metastasis was inconclusive. Furthermore, PF-299804, an orally available irreversible pan-HER tyrosine kinase inhibitor, inhibited in vivo growth of NCI-N87 (with ERBB2 amplification), but not of AGS (without ERBB2 amplification). Both sunitinib malate and PF-299804 were well tolerated by the animals during the course of the studies. These results support anti-angiogenesis and as a valid approach for effective treatment of GC, and preclinically demonstrated the importance of patient selection for targeted agents. 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 4424. doi:10.1158/1538-7445.AM2011-4424