Konstantinos Tsaparikos

PF-06463922, an ALK/ROS1 Inhibitor, Overcomes Resistance to First and Second Generation ALK Inhibitors in Preclinical Models

We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.

Combination of 4-1BB Agonist and PD-1 Antagonist Promotes Antitumor Effector/Memory CD8 T Cells in a Poorly Immunogenic Tumor Model

Chen, Lee, and colleagues compared the antitumor activity of anti-PD-1 in combination with anti-4-1BB versus with anti-LAG-3 and showed in syngeneic, poorly immunogenic mouse tumor models that the combination with anti-4-1BB elicited superior and well-tolerated tumor inhibition that did not require vaccine. Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti–4-1BB/anti–PD-1 combination with that of the anti–PD-1/anti–LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti–PD-1 and anti–4-1BB concomitantly, while combining anti–PD-1 with anti–LAG-3 led to a modest degree of tumor suppression. The activity of the anti–4-1BB/anti–PD-1 combination was dependent on IFNγ and CD8+ T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8+ T cells by anti–4-1BB/anti–PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8+/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8+ CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti–4-1BB/anti–PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients. Cancer Immunol Res; 3(2); 149–60. ©2014 AACR.

PF-03814735, an Orally Bioavailable Small Molecule Aurora Kinase Inhibitor for Cancer Therapy

The Aurora family of highly related serine/threonine kinases plays a key role in the regulation of mitosis. Aurora1 and Aurora2 play important but distinct roles in the G2 and M phases of the cell cycle and are essential for proper chromosome segregation and cell division. Overexpression and amplification of Aurora2 have been reported in different tumor types, including breast, colon, pancreatic, ovarian, and gastric cancer. PF-03814735 is a novel, potent, orally bioavailable, reversible inhibitor of both Aurora1 and Aurora2 kinases that is currently in phase I clinical trials for the treatment of advanced solid tumors. In intact cells, the inhibitory activity of PF-03814735 on the Aurora1 and Aurora2 kinases reduces levels of phospho-Aurora1, phosphohistone H3, and phospho-Aurora2. PF-03814735 produces a block in cytokinesis, resulting in inhibition of cell proliferation and the formation of polyploid multinucleated cells. Although PF-03814735 produces significant inhibition of several other protein kinases, the predominant biochemical effects in cellular assays are consistent with inhibition of Aurora kinases. Once-daily oral administration of PF-03814735 to mice bearing human xenograft tumors produces a reduction in phosphohistone H3 in tumors at doses that are tolerable and that result in significant inhibition of tumor growth. The combination of PF-03814735 and docetaxel in xenograft mouse tumor models shows additive tumor growth inhibition. These results support the clinical evaluation of PF-03814735 in cancer patients. Mol Cancer Ther; 9(4); 883–94. ©2010 AACR.

Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib.

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.

Abstract A277: PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models.

The oncogenic ROS1 gene fusion ( Fig-ROS1 ) was first identified in glioblastoma cells over two decades ago. Recently, ROS1 gene rearrangements were further discovered in a variety of human cancers, including lung adenocarcinoma, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastic tumor, angiosarcoma, and epithelioid hemangioendothelioma, providing additional evidence for ROS1 as an attractive cancer target. The 1st generation Met/ALK/ROS1 inhibitor XALKORI ® (crizotinib) has demonstrated promising clinical response in ROS1 fusion positive NSCLC. But similar to what was seen with acquired ALK secondary resistant mutations in XALKORI refractory patients, a ROS1 kinase domain mutant–ROS1G2032R has been identified in a ROS1 positive NSCLC patient who developed resistance to XALKORI. Therefore, there is an urgent need to develop agents that can overcome this type of resistance. PF-06463922 is a novel, orally available, ATP-competitive small molecule inhibitor of ROS1/ALK with exquisite potency against ROS1 kinase. PF-06463922 inhibited the catalytic activity of recombinant ROS1 with a mean Ki of < 0.005 nM, and inhibited ROS1 autophosphorylation at IC50 values ranging from 0.1 nM to 1 nM cross a panel of cell lines harboring oncogenic ROS1 fusion variants including CD74-ROS1, SLC34A2-ROS1 and Fig-ROS1. PF-06463922 also inhibited cell proliferation and induced cell apoptosis at sub- to low-nanomolar concentrations in the HCC78 human NSCLC cells harboring SLC34A2-ROS1 fusions and the BaF3-CD74-ROS1 cells expressing human CD74-ROS1. In the BaF3 cells engineered to express the XALKORI resistant CD74-ROS1G2032R mutant, PF-06463922 demonstrated nanomolar potency against either ROS1G2032R cellular activity or cell proliferation. In vivo, PF-06463922 demonstrated marked cytoreductive antitumor efficacy at low nanomolar concentration in the NIH3T3 xenograft models expressing human CD74-ROS1 and Fig-ROS1. The antitumor efficacy of PF-06463922 was dose dependent and strongly correlated to inhibition in ROS1 phosphorylation and the downstream signaling molecules pSHP1, pSHP2 and pErk1/2, as well as inhibition of the cell cycle protein Cyclin D1 in tumors. To our knowledge, PF-06463922 is the first reported ROS1 inhibitor that is capable of blocking the resistant ROS1G2032R mutant at predicted pharmacologically relevant concentrations. Our data indicate that PF-06463922 has great potential for treating ROS1 fusion positive cancers including those from patients who relapsed from XALKORI therapy due to acquired ROS1G2032Rmutation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A277. Citation Format: Helen Y. Zou, Lars R. Engstrom, Qiuhua Li, Melissa West Lu, Ruth Wei Tang, Hui Wang, Konstantinos Tsaparikos, Sergei Timofeevski, Justine Lam, Shinji Yamazaki, Wenyue Hu, Hovhannes Gukasyan, Nathan Lee, Ted W. Johnson, Valeria Fantin, Tod Smeal. PF-06463922, a novel ROS1/ALK inhibitor, demonstrates sub-nanomolar potency against oncogenic ROS1 fusions and capable of blocking the resistant ROS1G2032R mutant in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A277.

Combined gemcitabine and CHK1 inhibitor treatment induces apoptosis resistance in cancer stem cell-like cells enriched with tumor spheroids from a non-small cell lung cancer cell line

Evaluating the effects of novel drugs on appropriate tumor models has become crucial for developing more effective therapies that target highly tumorigenic and drug-resistant cancer stem cell (CSC) populations. In this study, we demonstrate that a subset of cancer cells with CSC properties may be enriched into tumor spheroids under stem cell conditions from a non-small cell lung cancer cell line. Treating these CSC-like cells with gemcitabine alone and a combination of gemcitabine and the novel CHK1 inhibitor PF-00477736 revealed that PF-00477736 enhances the anti-proliferative effect of gemcitabine against both the parental and the CSC-like cell populations. However, the CSC-like cells exhibited resistance to gemcitabine-induced apoptosis. Collectively, the spheroid-forming CSC-like cells may serve as a model system for understanding the mechanism underlying the drug resistance of CSCs and for guiding the development of better therapies that can inhibit tumor growth and eradicate CSCs.

Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621.

The c-Met proto-oncogene is a multifunctional receptor tyrosine kinase that is stimulated by its ligand, hepatocyte growth factor (HGF), to induce cell growth, motility and morphogenesis. Dysregulation of c-Met function, through mutational activation or overexpression, has been observed in many types of cancer and is thought to contribute to tumor growth and metastasis by affecting mitogenesis, invasion, and angiogenesis. We identified human monoclonal antibodies that bind to the extracellular domain of c-Met and inhibit tumor growth by interfering with ligand-dependent c-Met activation. We identified antibodies representing four independent epitope classes that inhibited both ligand binding and ligand-dependent activation of c-Met in A549 cells. In cells, the antibodies antagonized c-Met function by blocking receptor activation and by subsequently inducing downregulation of the receptor, translating to phenotypic effects in soft agar growth and tubular morphogenesis assays. Further characterization of the antibodies in vivo revealed significant inhibition of c-Met activity (≥ 80% lasting for 72–96 h) in excised tumors corresponded to tumor growth inhibition in multiple xenograft tumor models. Several of the antibodies identified inhibited the growth of tumors engineered to overexpress human HGF and human c-Met (S114 NIH 3T3) when grown subcutaneously in athymic mice. Furthermore, lead candidate antibody CE-355621 inhibited the growth of U87MG human glioblastoma and GTL-16 gastric xenografts by up to 98%. The findings support published pre-clinical and clinical data indicating that targeting c-Met with human monoclonal antibodies is a promising therapeutic approach for the treatment of cancer.

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

Abstract 4863: PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors

Tumors use tryptophan-catabolizing enzymes such as Indoleamine 2,3-dioxygenase-1 (IDO-1) to induce an immunosuppressive microenvironment. IDO-1 expression is upregulated in many cancers and described to be a resistance mechanism to immune checkpoint therapies. IDO-1 is induced in response to inflammatory stimuli such as IFN-a and promotes immune tolerance through the catabolism of tryptophan and accumulation of tryptophan catabolites including kynurenine. IDO-1 activity leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As such, IDO1 is a nexus for the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology. We have identified and characterized a new IDO-1 inhibitor. PF-06840003 is a highly selective orally bioavailable IDO-1 inhibitor. PF-06840003 reversed IDO-1-induced T-cell anergy in vitro. In vivo, PF-06840003 reduced intratumoral kynurenine levels in mice by >80% and inhibited tumor growth in multiple preclinical syngeneic models in mice, in combination with immune checkpoint inhibitors. PF-0684003 has favorable predicted human pharmacokinetic properties, including a predicted t1/2 of 16-19 hours. These studies highlight the strong potential of PF-06840003 as a clinical candidate in Immuno-Oncology. Citation Format: Joseph Tumang, Bruno Gomes, Martin Wythes, Stefano Crosignani, Patrick Bingham, Pauline Bottemanne, Helene Cannelle, Sandra Cauwenberghs, Jenny Chaplin, Deepak Dalvie, Sofie Denies, Coraline De Maeseneire, Peter Folger, Kim Frederix, Jie Guo, James Hardwick, Ken Hook, Katti Jessen, Erick Kindt, Marie-Claire Letellier, Kai-Hsin Liao, Wenlin Li, Karen Maegley, Reece Marillier, Nichol Miller, Brion Murray, Romain Pirson, Julie Preillon, Virginie Rabolli, Chad Ray, Stephanie Scales, Jay Srirangam, Jim Solowiej, Nicole Streiner, Vince Torti, Konstantinos Tsaparikos, Paolo Vicini, Gregory Driessens, Manfred Kraus. PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors. [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 4863.