Kevin R. Webster

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway

Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer. [Mol Cancer Ther 2009;8(7):1818–27]

Structure-based Design of Pyridone-Aminal eFT508 Targeting Dysregulated Translation by Selective Mitogen-activated Protein Kinase Interacting Kinases 1 and 2 (MNK1/2) Inhibition.

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone-aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Abstract 596: eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, Toll-like receptors and T cell receptor) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling, the tumor microenvironment and immune cell function. eFT508 is a potent and highly selective inhibitor of both MNK1 and MNK2. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of stress response, and effectors of anti-tumor immune response. Given the importance of MAPK signaling and translational control to immune cell activation and differentiation, the immunological effect of eFT508 was further evaluated in both normal human immune cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on αCD3/αCD28 stimulated IL-2 production, T cell proliferation or T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint receptors, including PD-1 and LAG3. Further evaluation of the mechanism of translational regulation has shown LAG3 mRNA contains specific sequence elements in the 5’-untranslated region (UTR) that confer sensitivity to eFT508. In addition, IL-10 mRNA is destabilized upon treatment with eFT508 leading to significant inhibition of IL-10 production in activated T cells. Furthermore, eFT508 treatment results in upregulation of MHC class II molecules on tumor cells, macrophage and dendritic cells through an IL-10/MARCH1 dependent mechanism. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity, modulation of tumor infiltrating lymphocytes and establishment of immune memory. In addition, combination of eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies results in marked efficacy, significantly increasing the percentage of responder animals. eFT508 is currently under evaluation in two phase I/II clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Craig R. Stumpf, Rajesh Sharma, Ivy N. Hung, Gregory S. Parker, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Vera Huang, Joan Chen, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich. eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity [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 596. doi:10.1158/1538-7445.AM2017-596

Abstract 3133: Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening.

Breast cancer is one of the most common cancer types, with greater than 450,000 deaths reported per year worldwide. Through genome wide sequencing efforts, multiple genetic alterations have been identified, including mutations and amplifications in genes such as v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), GATA binding protein 3 (GATA3), phosphatidylinositol 3-kinase alpha catalytic subunit (PIK3CA) as well as novel genomic rearrangements such as the recently identified MAGI3-AKT3 fusion. Now that breast cancer can be characterized to an unprecedented level, one of the key challenges remaining is to identify and distinguish critical ‘driver’ events responsible for tumor progression, from neutral ‘passenger’ lesions. In order to achieve this, we utilized high resolution aCGH analysis of 50 purified breast cancer samples (made up of Her2+, estrogen receptor positive (ER+) and triple negative tumors with variable responses to SOC regimens), in combination with a Gain-of-Function transformation screen to identify and validate novel breast targets. 158 genomic regions were found to be recurrently amplified, consisting of 759 genes in total. The top 32 focally amplified genes, along with 12 cancer-relevant mutant alleles were prioritized and a library generated utilizing the pTRIPZ-tetracycline regulated inducible lentiviral vector system. These 44 genes were subsequently combined into 16 different target pools (5-13 targets per pool, co-expressing genes that were co-amplified in the same clinical specimen) and evaluated for their ability to transform immortalized breast epithelial MCF10A cells (both wild-type and p53 -/- cells). Through this screening approach, p21-activated kinase 1 (PAK1) was identified, whose kinase activity was required to robustly transform MCF10A cells through regulating multiple signalling pathways including MAPK. Several other putative oncogenes were also identified and will be presented here, including the glycosyltransferse asparagine-linked glycosylation 8 (ALG8). Interestingly, PAK1 and ALG8 are co-amplified in both breast (8%) and ovarian cancers (11%). Our target validation studies have suggested that ALG8 can support PAK1-induced transformation, as dramatic suppression of soft-agar colony growth was seen in co-amplified breast cancer cell lines upon combined siRNA treatment to both targets. Thus, this combined high resolution aCGH profiling and functional screening approach has enabled the successful identification of novel oncogenic targets in breast cancer. Citation Format: Krishna Vasudevan, Axel Hernandez, Zhongwu Lai, Yonghong Xiao, Nin Guan, Carolyn Hardy, Robert Godin, Christopher Denz, Minwei Ye, Elizabeth Lenkiewicz, Stephanie Savage, Michael T. Barrett, Donna Prunkard, Peter Rabinovitch, Mark Basik, Ewa Przybytkowski, Kevin Webster, Michael Zinda, Emma-Louise Jenkins. Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2013-3133

Abstract 969: Amplifications, gene fusions, and therapeutic targets in triple negative and refractory ER+ and HER2+ breast cancers

Breast cancer is known to be heterogeneous and choices of targeted therapy are still limited for refractory estrogen receptor positive (ER+), refractory HER2+, and triple negative (TNBC) breast cancer. In order to identify novel drug targets that are either focally amplified or involved in fusion or translocation events in such disease segments, we applied high resolution DNA copy number profiling and whole genome sequencing to a set of DNA content sorted breast cancer samples. We first applied DNA-content based flow cytometry to purify diploid and aneuploid cells in each of 54 samples. Purified populations of both aneuploid and diploid cells were then subjected to high resolution aCGH profiling. Our initial analysis of high resolution copy number profiling data identified a list of genes focally amplified and harboring break points. We then selected 8 of these samples (four ER+ and four triple negative) for whole genome sequencing to identify their fusion partners. Paired-end sequencing data from HiSeq for each flow sorted sample was aligned against the human genome. A custom analysis workflow based on published software tools and custom developed scripts for computationally predicting structural variation events was implemented. Paired ends that mapped to discontinuous regions in the genome were merged with our aCGH data to identify candidate gene fusions, inversions, and translocations in each sample. Automatic scan and custom examination for pair-ends mapped to discontinuous genomic regions identified a list of novel gene fusion and translocation candidates. A subset of such fusion candidates showed precise break point position concordance across the NGS and aCGH data and equal copy number amplification of two fusion partners, further strengthening our confidence that such genes are indeed involved in fusion and translocation events. These candidates include transcription factors (e.g. ZNF492), kinases (e.g. DYRK1A), and phosphatases (e.g. PTPRG). Some of these fusion genes are known to be involved in fusion in other diseases or tumor types but have not been reported in breast cancer, such as AUTS2, which is reported as translocation partner in autism and mental retardation patients and B-cell ALL, and MECOM (aka EVI1/MDS1), which is fused with AML1 in AML patients. Others are completely novel such as BICD1-ZNF492. In conclusion, we have successfully identified a list of fusions genes in ER+ refractory and triple negative breast cancer patients by combining high resolution aCGH profiling of DNA copy number analysis and whole genome sequencing. The next step is to validate the expression of these fusion proteins and their functional relevance to breast cancer. The driver fusion genes identified can provide potential therapeutic targets for ER+ refractory and TN breast cancer patients. 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 969. doi:1538-7445.AM2012-969