Bernadette Pascual

Effective Targeting of Hedgehog Signaling in a Medulloblastoma Model with PF-5274857, a Potent and Selective Smoothened Antagonist That Penetrates the Blood–Brain Barrier

Inhibition of the Smoothened (Smo) represents a promising therapeutic strategy for treating malignant tumors that are dependent on the Hedgehog (Hh) signaling pathway. PF-5274857 is a novel Smo antagonist that specifically binds to Smo with a Ki of 4.6 ± 1.1 nmol/L and completely blocks the transcriptional activity of the downstream gene Gli1 with an IC50 of 2.7 ± 1.4 nmol/L in cells. This Smo antagonist showed robust antitumor activity in a mouse model of medulloblastoma with an in vivo IC50 of 8.9 ± 2.6 nmol/L. The downregulation of Gli1 is closely linked to the tumor growth inhibition in patched+/− medulloblastoma mice. Mathematical analysis of the relationship between the drugs pharmacokinetics and Gli1 pharmacodynamics in patched+/− medulloblastoma tumor models yielded similar tumor and skin Gli1 IC50 values, suggesting that skin can be used as a surrogate tissue for the measurement of tumor Gli1 levels. In addition, PF-5274857 was found to effectively penetrate the blood–brain barrier and inhibit Smo activity in the brain of primary medulloblastoma mice, resulting in improved animal survival rates. The brain permeability of PF-5274857 was also confirmed and quantified in nontumor-bearing preclinical species with an intact blood–brain barrier. PF-5274857 was orally available and metabolically stable in vivo. These findings suggest that PF-5274857 is a potentially attractive clinical candidate for the treatment of tumor types including brain tumors and brain metastasis driven by an activated Hh pathway. Mol Cancer Ther; 11(1); 57–65. ©2011 AACR.

4-methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors.

Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.

Comparison of dynamic contrast‐enhanced MR, ultrasound and optical imaging modalities to evaluate the antiangiogenic effect of PF‐03084014 and sunitinib

Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high‐frequency ultrasound (HFUS), dynamic contrast enhanced‐MR (DCE‐MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ‐secretase inhibitor PF‐03084014. Sunitinib was tested in parallel for comparison. In the MDA‐MB‐231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF‐03084014. By immunohistochemistry and FITC‐lectin perfusion assays, we showed that the vascular defects upon treatment with PF‐03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down‐stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA‐MB‐231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF‐03084014. A decrease in the tumor vessel function was also observed through contrast‐enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF‐03084014‐induced functional vessel changes measured by suppressing the Ktrans values using DCE‐MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment‐associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF‐03084014 in the tested imaging modalities. In summary, ultrasound and DCE‐MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF‐03084014 and sunitinib.

Preclinical PK/PD modeling and human efficacious dose projection for a glucokinase activator in the treatment of diabetes.

Human Hexokinase IV, or glucokinase (GK), is a regulator of glucose concentrations in the body. It plays a key role in pancreatic insulin secretion as well as glucose biotransformation in the liver, making it a potentially viable target for treatment of Type 2 diabetes. Allosteric activators of GK have been shown to decrease blood glucose concentrations in both animals and humans. Here, the development of a mathematical model is presented that describes glucose modulation in an ob/ob mouse model via administration of a potent GK activator, with the goal of projecting a human efficacious dose and plasma exposure. The model accounts for the allosteric interaction between GK, the activator, and glucose using a modified Hill function. Based on model simulations using data from the ob/ob mouse and in vitro studies, human projections of glucose response to the GK activator are presented, along with dose and regimen predictions to maintain clinically significant decreases in blood glucose in a Type 2 diabetic patient. This effort serves as a basis to build a detailed mechanistic understanding of GK and its role as a therapeutic target for Type 2 diabetes, and it highlights the benefits of using such an approach in a drug discovery setting.

A novel CXCR4 antagonist IgG1 antibody (PF-06747143) for the treatment of hematologic malignancies

The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).

Gemtuzumab Ozogamicin (GO) Inclusion to Induction Chemotherapy Eliminates Leukemic Initiating Cells and Significantly Improves Survival in Mouse Models of Acute Myeloid Leukemia

Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody-drug conjugate for the treatment of acute myeloid leukemia (AML). Although GO shows a narrow therapeutic window in early clinical studies, recent reports detailing a modified dosing regimen of GO can be safely combined with induction chemotherapy, and the combination provides significant survival benefits in AML patients. Here we tested whether the survival benefits seen with the combination arise from the enhanced reduction of chemoresidual disease and leukemic initiating cells (LICs). Herein, we use cell line and patient-derived xenograft (PDX) AML models to evaluate the combination of GO with daunorubicin and cytarabine (DA) induction chemotherapy on AML blast growth and animal survival. DA chemotherapy and GO as separate treatments reduced AML burden but left significant chemoresidual disease in multiple AML models. The combination of GO and DA chemotherapy eliminated nearly all AML burden and extended overall survival. In two small subsets of AML models, chemoresidual disease following DA chemotherapy displayed hallmark markers of leukemic LICs (CLL1 and CD34). In vivo, the two chemoresistant subpopulations (CLL1+/CD117− and CD34+/CD38+) showed higher ability to self-renewal than their counterpart subpopulations, respectively. CD33 was coexpressed in these functional LIC subpopulations. We demonstrate that the GO and DA induction chemotherapy combination more effectively eliminates LICs in AML PDX models than either single agent alone. These data suggest that the survival benefit seen by the combination of GO and induction chemotherapy, nonclinically and clinically, may be attributed to the enhanced reduction of LICs.

Abstract 2649: Fc-effector function activity of the CXCR4 IgG1 antibody PF-06747143: a novel clinical candidate for the treatment of hematologic malignancies

The chemokine receptor CXCR4 triggers signaling pathways that control cell migration to tissues where its ligand, CXCL12, is highly expressed, including the bone marrow (BM). In hematologic cancers, CXCR4 expression is associated with poor prognosis. CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism of chemotherapy resistance. PF-06747143 is a novel humanized IgG1 therapeutic antibody that binds to CXCR4 and inhibits CXCL12-driven pathways. Human IgG1 antibodies can induce strong cytotoxicity mediated by the antibody Fc-region, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-driven cytotoxicity, while human IgG4 antibodies show minimal or no Fc-driven cytotoxicity. Here we generated an IgG4 version of PF-06747143 (m15-IgG4), which has similar binding to CXCR4 as the IgG1 antibody. We then characterized the role of Fc-driven cytotoxic function, comparing both antibodies in efficacy and safety studies. In an ADCC assay, PF-06747143 showed strong cytotoxicity of non-Hodgkin’s lymphoma (NHL) and acute myeloid leukemia (AML), while m15-IgG4 had no significant cytotoxicity. In a NHL mouse tumor model, the IgG1 CXCR4 antibody resulted in superior tumor growth inhibition, with 50% of mice exhibiting complete tumor regressions, compared to the m15-IgG4 antibody, which had limited activity, with no tumor regressions (p 4 days. Since both antibodies had comparable exposures, the different mobilization duration is likely due to the ability of the IgG1 CXCR4 antibody to reduce the number of mobilized cells via Fc-driven cytotoxic function. Finally, CXCR4 has been shown to play a key role in chemotherapy resistance. In a chemo-resistant PDX AML mouse model, in which the standard of care agents daunorubicin and cytarabine had limited activity, resulting in 30% of tumor cells remaining in the BM post-treatment, we show that combination of PF-06747143 with these chemo agents led to synergistic activity, with tumor burden reduced to 0.3% tumor cells in the BM. In conclusion, PF-06747143 attributes offer potential efficacy- and safety-related advantages over other CXCR4 antagonists currently in development, which do not have Fc-driven cytotoxic activity. PF-06747143 is now being evaluated in a Phase 1 clinical trial in relapsed and refractory AML (NCTID 02954653). Citation Format: Flavia Pernasetti, Shu-Hui Liu, Gu Yin, Bernadette Pascual, Zhengming Yan, Max Hallin, Rolla Yafawi, Cathy Zhang, Connie Fang, Wenlian Wang, Justine Lam, Mary E. Spilker, Eileen Blasi, Brett Simmons, Nanni Huser, Wei-Hsien Ho, Kevin Lindquist, Thomas-Toan Tran, Jyothirmayee Kudaravalli, Jing-Tyan Ma, Gretchen Jimenez, Ishita Barman, Colleen Brown, Sherman-Michael Chin, Maria Costa, David Shelton, Tod Smeal, Valeria R. Fantin. Fc-effector function activity of the CXCR4 IgG1 antibody PF-06747143: a novel clinical candidate for the treatment of hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2649. doi:10.1158/1538-7445.AM2017-2649

Abstract LB-277: Induction chemotherapy induces enrichment of leukemic stem cells in PDX models of acute myeloid leukemia

Human leukemic stem cells may be one of the leading causes for resistance or treatment failure in acute myeloid leukemia patients. To gain insights into the mechanisms underlying chemo-resistance of AML disease, we evaluated the biomarkers and the pharmacological properties of minimal residual disease post induction therapy. The PDX models were established by iv injecting acute myeloid leukemia patient cells in NOD/SCID/Il2rg(-/-) mice (NSG). The disease progression was tracked by the AML counts in peripheral blood and bone marrow via FACS analysis. In the PDX models, daunorubicin/cytarabine (DA) chemotherapy showed early antileukemic efficacy through apoptosis induction and antiproliferation. However, treatment showed minimal survival benefits and all treated leukemic mice relapsed due to the minimal residual disease (MRD). FACS analysis showed that DA therapy induced enrichment of CD34+ and CLL1+/CD117- cells in the MRD of the BM0407 and BM2407 AML PDX models, respectively. Subsequently, we performed self-renewal functional test by in vivo reimplantation of sorted CD34+ and CLL1+/CD117- cells. The limiting delusion analysis showed that the chemo- resistant CD34+ and CLL1+/CD117- cells are much more tumorigenic than their counterpart CD34- and CLL1+/CD117+ cells, respectively. This work provides insights into the mechanism of chemo-resistance and helps identifying new strategies to improve AML therapy. Citation Format: Cathy C. Zhang, Zhengming Yan, Bernadette Pascual, Stephen Huang, Qing Zong, Mark Elliot, Patrick Lappin. Induction chemotherapy induces enrichment of leukemic stem cells in PDX models of acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-277.

Abstract 4535: CENP-E and c-Myc expression identifies a sensitive population of gastric cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Centromere-associated protein E (CENP-E) is expressed during mitosis and plays an essential role in establishing and maintaining stable connections between mitotic chromosomes and the microtubules of the spindle. Previous preclinical studies have shown that inhibition of CENP-E function by small interfering RNA, blocking antibodies or small molecule inhibitors arrested cell cycle before metaphase, and could lead to cell death. In a number of cancers, CENP-E is frequently overexpressed in tumor tissues compared with their normal counterparts. In this study, we showed that CENP-E is overexpressed in about 60% of gastric cancer (intestinal and diffuse-type) tissues. In addition, c-Myc up-regulation was observed in more than 50% of the same samples. Overall, about 40% of all gastric tumor samples harbor both CENP-E and c-Myc upregulation. Using a panel of small molecule inhibitors of CENP-E, we evaluated in vitro anti-proliferation effect in a panel of gastric cancer cell lines. Our data suggested that cells with higher levels of c-Myc transcript were more sensitive to CENP-E inhibition. This preliminary study indicated that c-Myc amplification and/or over-expression could be a potential biomarker for selecting gastric cancer patients more likely to respond to CENP-E inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4535. doi:1538-7445.AM2012-4535

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