Abstract B36: Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma

Abstract

Abstract Background: Pancreatic cancer is highly resistant to existing chemotherapy regimens, with only a minority of patients exhibiting a response to standard therapy. One aspect of treatment resistance is based on the cancer stem cell (CSC) hypothesis, which holds that a small population of highly chemoresistant cells is responsible for initiating and sustaining tumor growth. Compounds with activity against this population are highly desired in the adjuvant setting, where failure to sterilize micrometastases not detected at time of operation results in recurrence in nearly all patients. Methods: A high-throughput drug screen (HTS) was performed in an in vitro model of pancreatic cancer stem-like cells (spheroids). Molecules with high potency against both the cancer stem cell-like spheroid and monolayer cell formats were evaluated for efficacy and mechanism of action in a variety of preclinical models of pancreatic cancer. Unbiased global in situ phosphoproteomic profiling using KiNativ technology was used to identify kinase targets impacted by the top hit. Stemness phenotype was interrogated by qRT-PCR of stemness genes, flow cytometry, colony formation assays, and tumor initiation in immunocompromised mice. Results: The top hit from the HTS was a 1H-benzo[d]imidazol-2-amine-based inhibitor of interleukin-2-inducible T-cell kinase (ITK) (NCGC00188382, 1). 1 effectively mediated growth inhibition, blocked migration and invasion, and reduced clonogenicity of cancer cells with an associated decrease in the expression of several CSC-associated stemness markers at non-lethal concentrations (Sox2, Notch1) compared to gemcitabine used at >10-fold concentration. Metastatic burden in both the liver and lung was significantly reduced in mice orthotopically injected with pancreatic CSCs in the treatment group after 6 weeks. Target deconvolution of 1 demonstrated inhibition of multiple select kinase targets. There were 16 targets identified from an initial screening panel of >400 kinases. The targets were further evaluated for the ability to phenocopy the effect of treatment with 1 via shRNA knockdown. Inhibition of the thousand-and-one amino acid kinase 3 (TAOK3), cyclin-dependent kinase 7 (CDK7), aurora B kinase (AURKB), and ephrin B2 receptor (EPHB2) effectively phenocopied the effect of treatment with 1. Concurrent silencing of two or more targets increased apoptosis induced by 1, suggesting intrinsic additivity of the multikinase agent. Enforced overexpression of TAOK3 increased expression of stemness markers, clonogenicity, and tumor initiation in vivo. Conclusion: A potent inhibitor of ITK with a unique target profile and anti-stemness activities was derived from a differential HTS in a pancreatic spheroid CSC model. The 1H-benzo[d]imidazol-2-amine-derivative targets select characteristics attributed to pancreatic CSCs, including cell migration, colony formation, and metastasis formation with concomitant suppression of stemness traits without inducing cell death at the concentrations used. Target deconvolution of the compound identified multiple kinase targets, including TAOK3, a kinase not previously known to be involved in pancreatic tumor biology. Overexpression of TAOK3 results in an enforced CSC phenotype including increased colony formation and tumor initiation in vivo. The observed inhibitory effect of 1 on stemness phenotype at nanomolar concentrations without affecting cell viability or showing toxicity in vivo supports further preclinical development. Citation Format: Taylor Aiken, Yansong Bian, Yaroslav Teper, Lesley Mathews Griner, Rajarshi Guha, Paul Shinn, Hong-Wu Xin, Holger Pflicke, Jian-kang Jiang, Paresma Patel, Steven Rogers, Jeffery Aube, Marc Ferrer, Craig J. Thomas, Udo Rudloff.{Authors}. Preclinical development of a multikinase targeting molecule with activity against the cancer stem cell phenotype in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B36.