Paul D. Lira

Absence of the P2X7 receptor alters leukocyte function and attenuates an inflammatory response

When challenged with extracellular ATP, leukocytes respond and activate processes attributed to the P2X7 receptor (P2X7R), an unusual ligand-gated ion channel. To prove P2X7R involvement, blood samples from P2X7R-deficient mice were characterized. Monocytes and lymphocytes associated with wild-type blood responded to ATP and underwent volume/shape changes and shed L-selectin. In contrast, leukocytes from P2X7R-deficient animals demonstrated no change in physical properties or L-selectin expression following ATP challenge. Blood stimulated with LPS or ATP individually generated minimal quantities of the leaderless polypeptide IL-1β, but sequential treatment of wild-type, but not P2X7R-deficient, blood with LPS and ATP yielded large amounts of cell-free cytokine. Based on these differences, wild-type and P2X7R-deficient animals were compared following induction of monoclonal anti-collagen-induced arthritis. Ab-treated wild-type animals subsequently challenged with LPS developed inflamed, swollen paws; their joint cartilage demonstrated lesions, loss of proteoglycan content, and the presence of collagen degradation products. P2X7R-deficient animals subjected to the same challenge were markedly less affected; both the incidence and severity of disease were reduced. These data indicate that ATP does act via the P2X7R to affect leukocyte function and that the P2X7R can serve as an important component of an in vivo inflammatory response.

Impaired Invariant Chain Degradation and Antigen Presentation and Diminished Collagen-Induced Arthritis in Cathepsin S Null Mice

Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness.

CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases

The chemokines CCL3 and CCL5, as well as their shared receptor CCR1, are believed to play a role in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, multiple sclerosis, and transplant rejection. In this study we describe the pharmacological properties of a novel small molecular weight CCR1 antagonist, CP-481,715 (quinoxaline-2-carboxylic acid [4(R)-carbamoyl-1(S)-(3-fluorobenzyl)-2(S),7-dihydroxy-7-methyloctyl]amide). Radiolabeled binding studies indicate that CP-481,715 binds to human CCR1 with a Kd of 9.2 nm and displaces 125I-labeled CCL3 from CCR1-transfected cells with an IC50 of 74 nm. CP-481,715 lacks intrinsic agonist activity but fully blocks the ability of CCL3 and CCL5 to stimulate receptor signaling (guanosine 5′-O-(thiotriphosphate) incorporation; IC50 = 210 nm), calcium mobilization (IC50 = 71 nm), monocyte chemotaxis (IC50 = 55 nm), and matrix metalloproteinase 9 release (IC50 = 54 nm). CP-481,715 retains activity in human whole blood, inhibiting CCL3-induced CD11b up-regulation and actin polymerization (IC50 = 165 and 57 nm, respectively) on monocytes. Furthermore, it behaves as a competitive and reversible antagonist. CP-481,715 is >100-fold selective for CCR1 as compared with a panel of G-protein-coupled receptors including related chemokine receptors. Evidence for its potential use in human disease is suggested by its ability to inhibit 90% of the monocyte chemotactic activity present in 11/15 rheumatoid arthritis synovial fluid samples. These data illustrate that CP-481,715 is a potent and selective antagonist for CCR1 with therapeutic potential for rheumatoid arthritis and other inflammatory diseases.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity

Abstract4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation

We report how rotational variations in transmembrane (TM) helix interactions participate in the activity states of the thrombopoietin receptor (TpoR), a type 1 cytokine receptor that controls the production of blood platelets. We also explore the mechanism of small‐molecule agonists that do not mimic the natural ligand. We show, by a combination of cysteine cross‐linking, alanine‐scanning mutagenesis, and computational simulations, that the TpoR TM dimerizes strongly and can adopt 3 different stable, rotationally related conformations, which may correspond to specific states of the full‐length receptor (active, inactive, and partially active). Thus, our data suggest that signaling and inactive states of the receptor are related by receptor subunit rotations, rather than a simple monomer‐dimer transition. Moreover, results from experiments with and without agonists in vitro and in cells allow us to propose a novel allosteric mechanism of action for a class of small molecules, in which they activate TpoR by binding to the TM region and by exploiting the rotational states of the dimeric receptor. Overall, our results support the emerging view of the participation of mutual rotations of the TM domains in cytokine receptor activation.—Matthews, E. E., Thévenin, D., Rogers, J. M., Gotow, L., Lira, P. D., Reiter, L. A., Brissette, W. H., Engelman, D. M. Thrombopoietin receptor activation: transmembrane helix dimerization, rotation, and allosteric modulation. FASEB J. 25, 2234–2244 (2011). www.fasebj.org

Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment.

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10-11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.

The identification of orally bioavailable thrombopoietin agonists.

Recently, we disclosed a series of potent pyrimidine benzamide-based thrombopoietin receptor agonists. Unfortunately, the structural features required for the desired activity conferred physicochemical properties that were not favorable for the development of an oral agent. The physical properties of the series were improved by replacing the aminopyrimidinyl group with a piperidine-4-carboxylic acid moiety. The resulting compounds possessed favorable in vivo pharmacokinetic properties, including good bioavailability.

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

Analysis of the role of the HIF hydroxylase family members in erythropoiesis

HIF (hypoxia-inducible factor) hydroxylases have been implicated in EPO (erythropoietin) production and erythropoiesis. Here we examined the role of each of the three EGLN family members and the HIF asparaginyl hydroxylase FIH (factor inhibiting HIF) in EPO production. We examined the effect of inhibiting individual as well as combinations of HIF hydroxylases with RNAi. We found that inhibition of EGLN1 (egl nine homolog 1) as well as other members of the EGLN family (EGLN2 and EGLN3) led to accumulative EPO production in vitro. We then knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. Among all the in vitro and in vivo studies, no or minimal VEGF (vascular endothelial growth factor) mRNA or protein stimulation resulted from inhibition of EGLN1.

Abstract 76: Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We have developed and validated a 109 AML associated gene panel NGS assay based on sequence capture technologies. The overall assay sensitivity was 99.%, and overall assay specificity was 100% when sequenced with reference “gold” standard NA12878. Assay analytical accuracy was evaluated with diluted cell line samples harboring mutations in this gene panel, all diluted mutations with minor allele frequencies were detected, analytical limit of detection (LOD) is < 5%, and assay reproducibility is 96.8%. The assay also demonstrated robustness in sequencing 10 commercial AML patient samples (whole blood and bone marrow) and detecting mutations. Therefore, the AML gene panel deep sequencing assay is sensitive and accurate and is ready to be used for preclinical and clinical studies. We subsequently sequenced 15 AML patient derived xenograft (PDX) models with this assay. These models were also deep sequenced with a 409 comprehensive cancer gene panel based on multiplex amplicon technologies. The mutation profiles of each model will be summarized in this presentation using two sequencing platforms. By analysis of the mutation profile of each model, sub-clones of mutations were revealed in the early passages of these models. Two of these models with FLT-3 mutations were later treated with Quizartinib and Crenolanib separately, the treated mouse models were profiled with these two targeted gene panels; clonal evolution of mutations in these models treated with FLT-3 inhibitors was documented. Deep sequencing with target gene panel is a powerful tool to decipher the mutation sub-clones in the early passages of PDX models, and understand the acquired resistance of drug treatment. Citation Format: Stephen Huang, Paul Lira, Kai Wang, Cathy Zhang, Amy Jackson-Fisher, Keith Ching, Paul Rejto. Molecular profiling of AML patient derived xenograft models with deep sequencing using a 109 AML associated gene panel and a 409 gene comprehensive cancer panel. [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 76. doi:10.1158/1538-7445.AM2015-76