The endoplasmic reticulum (ER) stress response is frequently hyperactivated in cancer due to the accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stimuli associated with tumorigenesis. Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase that governs one of the three arms of the ER stress response through its regulation of the transcription factor XBP-1s. IRE-1 controls the expression of XBP-1s through its specific mRNA splicing activity, causing a frame-shift in translation. The resulting 54-kDA XBP-1s protein translocates into the nucleus and regulates ER stress response genes, contributing to normal B cell function and differentiation. Since gene copy number amplifications and aberrant protein expression are hallmarks of cancer, IRE-1 serves a critical cytoprotective function that allows tumor cells to evade stress- induced apoptosis. Building on our recent genetic and pharmacological validation of the IRE-1/XBP-1 pathway as a therapeutic target in CLL, we initiated an SAR campaign around novel tricyclic chromenones that form a stable and specific covalent complex with lysine 907 in the RNase domain of IRE-1. C-B06 (and its prodrug derivative, B-I09) potently inhibits XBP-1 mRNA splicing and suppresses XBP-1s expression in intact cells. Moreover, C-B06 and B-I09 are remarkably selective IRE-1 RNase antagonists that inhibit CLL cell growth in an XBP-1-dependant manner and reduce tumor burden in a transgenic mouse model of chronic lymphocytic leukemia (CLL) without imposing toxicity. Although a limited number of non-electrophilic allosteric inhibitors of IRE-1 RNase activity have also been reported, they have thus far not shown the ability to inhibit tumor cell growth in vitro on in vivo. This raises the possibiity that covalent inhibition of the IRE-1 RNase domain is required for anticancer efficacy. Here, we propose to use E-TCL1 mouse CLL cells, human CLL cell lines, and freshly isolated patient samples to guide the development of covalent IRE-1 RNase inhibitors as novel antileukemic agents. Moreover, we will assess the importance of specific covalent IRE-1 inhibition by comparing the activities of IRE-1 inhibitors with distinct mechanisms-of-action.
In Aim 1, we will use structure- based drug design and chemical synthesis to generate a library of optimized C-B06 analogues for evaluation in a FRET-suppression IRE-1 RNase assay, and carry out counterscreening with potent leads. Compounds will then be tested for their ability to block XBP-1s expression in intact cells.
In Aim 2, we will determine the cytotoxicity of potent and selective covalent IRE-1 RNase inhibitors with purified E-TCL1 CLL cells, CLL cell lines, and freshly isolated primary patient samples collected at our Cancer Center. We will establish induction of apoptosis, XBP-1-dependant cytotoxicity, and the effect of our inhibitors on transcriptional targets downstream of XBP-1s. We will also carry out the above studies with existing allosteric antagonists to investigate whether reversible covalent inhibition is critical for the efficacy of IE-1 RNase inhibitors.

Public Health Relevance

This proposal seeks to develop new covalent inhibitors of IRE-1, a protein whose hyperactivation results in the accelerated progression of chronic lymphocytic leukemia. Our research plan will employ structure-based drug design, chemical synthesis, and mechanism-of action studies to elucidate the determinants of anticancer efficacy within this class of compounds.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA199553-02
Application #
9139426
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Soyombo-Shoola, Abigail Adebisi
Project Start
2015-09-07
Project End
2017-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of South Florida
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
069687242
City
Tampa
State
FL
Country
United States
Zip Code
33612
Schutt, Steven D; Wu, Yongxia; Tang, Chih-Hang Anthony et al. (2018) Inhibition of the IRE-1?/XBP-1 pathway prevents chronic GVHD and preserves the GVL effect in mice. Blood Adv 2:414-427
Xie, Hong; Tang, Chih-Hang Anthony; Song, Jun H et al. (2018) IRE1? RNase-dependent lipid homeostasis promotes survival in Myc-transformed cancers. J Clin Invest 128:1300-1316
Tang, Chih-Hang Anthony; Chang, Shiun; Paton, Adrienne W et al. (2018) Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization. J Cell Biol 217:1739-1755
Tang, Chih-Hang; Chang, Shiun; Hashimoto, Ayumi et al. (2018) Secretory IgM Exacerbates Tumor Progression by Inducing Accumulations of MDSCs in Mice. Cancer Immunol Res 6:696-710
Tavernier, Simon J; Osorio, Fabiola; Vandersarren, Lana et al. (2017) Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival. Nat Cell Biol 19:698-710
Tang, Chih-Hang Anthony; Zundell, Joseph A; Ranatunga, Sujeewa et al. (2016) Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells. Cancer Res 76:2137-52