Michael D. Robbins

The CD28 and CTLA-4 Receptors Associate with the Serine/Threonine Phosphatase PP2A

CD28 and CTLA-4 are related members of a family of T lymphocyte cell surface receptors that function to regulate T cell activation. We have found that the cytoplasmic domains of both CTLA-4 and CD28 can associate with members of the PP2A family of serine/threonine phosphatases. The association of PP2A with CD28 was negatively regulated by tyrosine phosphorylation of the CD28 cytoplasmic domain. Inhibition of PP2A activity in Jurkat leukemia T cells by treatment with okadaic acid or by expression of a dominant-negative mutant enhanced T cell activation induced by CD28 engagement. Interactions between cell surface receptors such as CTLA-4 and CD28 and serine/threonine phosphatases may represent a novel mechanism for modulating the intracellular signal transduction pathways associated with cell activation.

Pharmacologic Inhibition of Site 1 Protease Activity Inhibits Sterol Regulatory Element-Binding Protein Processing and Reduces Lipogenic Enzyme Gene Expression and Lipid Synthesis in Cultured Cells and Experimental Animals

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 μM PF-429242 (Bioorg Med Chem Lett 17:4411–4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC50 of 0.5 μM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.

Discovery of PF-04449913, a Potent and Orally Bioavailable Inhibitor of Smoothened.

Inhibitors of the Hedgehog signaling pathway have generated a great deal of interest in the oncology area due to the mounting evidence of their potential to provide promising therapeutic options for patients. Herein, we describe the discovery strategy to overcome the issues inherent in lead structure 1 that resulted in the identification of Smoothened inhibitor 1-((2R,4R)-2-(1H-benzo[d]imidazol-2-yl)-1-methylpiperidin-4-yl)-3-(4-cyanophenyl)urea (PF-04449913, 26), which has been advanced to human clinical studies.

Abstract 4504: PF-04449913, a small molecule inhibitor of Hedgehog signaling, is effective in inhibiting tumor growth in preclinical models

Aberrant activation of the Hedgehog (Hh) signaling pathway has been implicated in several human cancers. Mutations in the Patched (PTCH1) gene are responsible for basal cell nevus syndrome, and are commonly found in sporadic basal cell carcinoma and in medulloblastoma. In this study we evaluated PF-04449913, an inhibitor of the Hh signaling pathway, in a Ptch1+/-p53 mouse model of medulloblastoma and in human patient derived xenograft models. Treatment of Ptch1+/-p53+/- or Ptch1+/-p53-/- medulloblastoma allografts with PF-04449913 produced potent dose-dependent inhibition of Hh pathway activity resulting in stable tumor regression. Using Gli1 transcript levels as a surrogate for Hh pathway activity, the pharmacodynamic effects of PF-04449913 were evaluated in medulloblastoma allografts following single dose and multi dose administrations of compound. PF-04449913 treated medulloblastoma allografts had reduced levels of Gli1 gene expression and down regulation of genes linked to the Hh signaling pathway. PF-04449913 was also effective when combined with a chemotherapeutic agent in a colon patient derived xenograft model and a pancreatic patient derived xenograft model, resulting in 63% and 73% tumor growth inhibition respectively. Collectively, our study demonstrates the therapeutic efficacy of a small molecule inhibitor of Hh pathway in preclinical models of multiple cancer types in either single or combination treatments. 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 4504. doi:10.1158/1538-7445.AM2011-4504

A Mathematical Model for Tumor–Immune Dynamics in Multiple Myeloma

We propose a mathematical model that describes the dynamics of multiple myeloma and three distinct populations of the innate and adaptive immune system: cytotoxic T cells, natural killer cells, and regulatory T cells. The model includes significant biologically- and therapeutically-relevant pathways for inhibitory and stimulatory interactions between these populations. Due to the model complexity, we propose a reduced version that captures the principal biological aspects for advanced disease, while still including potential targets for therapeutic interventions. Analysis of the reduced two-dimensional model revealed details about long-term model behavior. In particular, theoretical results describing equilibria and their associated stability are described in detail. Consistent with the theoretical analysis, numerical results reveal parameter regions for which bistability exits. The two stable states in these cases may correspond to long-term disease control or a higher level of disease burden. This initial analysis of the dynamical system provides a foundation for later work, which will consider combination therapies, their expected outcomes, and optimization of regimens.

Elotuzumab Promotes Self-Engagement of Signaling Lymphocytic Activation Molecule Family Member 7 (SLAMF7) Between Natural Killer (NK) and Multiple Myeloma (MM) Cells to Enhance Cytotoxicity

melphalan regarding efficacy are necessary.

Abstract 2998: Elotuzumab can costimulate NK cell responses by engaging SLAMF7

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, CD319) protein, which is highly and uniformly expressed on multiple myeloma cells. Improved survival has been observed in multiple myeloma patients treated with Elo in combination with immunomodulatory drugs such as dexamethasone and lenalidomide. Previous work showed that the main mechanism of Elo action in vitro is CD16-mediated antibody dependent cellular cytotoxicity (ADCC) via Fc interaction with CD16 (FcγRIIIa) receptor on NK cells. However, SLAMF7 is also expressed on NK cells where it transmits an activating signal. We hypothesized that Elo can directly activate NK cells via SLAMF7 ligation. Utilizing non-fucosylated Elo that has higher affinity to CD16 and Elo mutant that has an Fc region mutation that abrogates binding to CD16, we confirmed that Elo promotes NK cell activation and degranulation in a CD16-dependent manner. Specifically, non-fucosylated Elo had higher potency, whereas Elo mutant did not stimulate degranulation or CD69 expression. To test for co-stimulatory effects of Elo and Elo mutant we measured calcium signaling responses triggered by those antibodies in combination with antibodies to engage the activating receptors NKp46 and NKG2D. Elo or Elo mut alone had no effect on calcium signaling, but when added in combination with multimeric engagement of NKp46 and NKG2D both antibodies co-stimulated calcium signaling responses. We conclude that Elo stimulates NK cells via interaction with CD16, but direct engagement with SLAMF7 can also transduce co-activating signals to potentiate calcium signals generated through NKp46 and NKG2D in a CD16-independent manner. Our results provide evidence that direct engagement with SLAMF7 by Elo has the potential to reduce the threshold of NK cell activation triggered through other activating receptors. Citation Format: Tatiana Pazina, Ashley Mentlik James, Alexander W. MacFarlane, Natalie A. Bezman, Robert F. Graziano, Michael D. Robbins, Adam D. Cohen, Kerry S. Campbell. Elotuzumab can costimulate NK cell responses by engaging SLAMF7 [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 2998. doi:10.1158/1538-7445.AM2017-2998