The role of the ER stress response in malignant progression of CLL is still an under-appreciated research area because CLL cells do not exhibit dramatic ER expansion like that in multiple myeloma cells. My laboratory established that ER stress response is critically important for the survival of human CLL, and for the malignant progression of CLL in mice. We chose to use the Eµ-TCL1 mouse model to study B-cell leukemia because ~90% of human CLL patients expressed the TCL1 protein, and the overexpression of TCL1 in B cells led to the development of CLL in mice. We showed that TCL1 oncoprotein associated with XBP-1 and turned on vital ER proteins to support leukemic growth. We further examined the role of the IRE-1/XBP-1 pathway by genetically deleting the XBP-1 gene from CLL cells of Eµ-TCL1 mice, and showed significantly slower progression of CLL in the XBP-1KO/Eµ-TCL1 mice. To translate our research into potential therapeutics for CLL patients, we set up a high throughput in vitro screening platform to look for effective inhibitors that could block the IRE-1/XBP-1 pathway. In collaboration with Dr. Juan R. Del Valle at University of Notre Dame, we synthesized and published a number of compounds that could inhibit this pathway. B-I09 was developed as a specific inhibitor with high potency and efficacy to suppress the expression of XBP-1. B-I09 clearly suppressed activation of the IRE-1/XBP-1 pathway as evidenced by the decreased mRNA and protein levels of XBP-1 in intact cells. B-I09 specifically targeted mouse CLL cells in vivo by inducing apoptosis. We also discovered that genetic deficiency of XBP-1 compromised the B cell receptor (BCR) signaling, a crucial survival signal for CLL. To test whether pharmacological inhibition of XBP-1 could enhance the effect of inhibitors to the BCR signaling, we combined B-I09 with the FDA-approved Bruton’s Tyrosine Kinase inhibitor (BTKi), ibrutinib, to treat human CLL cells. A true and strong synergistic effect in pharmacology was established using the Chou-Talalay combination index method. Such results established that B-I09 could decelerate the growth of CLL either as a single agent or in combination with ibrutinib. In addition, the combination of B-I09 with ibrutinib induced apoptosis not only in CLL cells but also in mantle cell lymphoma and multiple myeloma cells. Our finding in synergism is important because B-I09 can be used to help ibrutinib or other BTKi to achieve higher cytotoxicity in cancer cells at a lower dose, addressing BTKi’s toxicity issue.
We recently developed a novel fluorescent tricyclic chromenone inhibitor, D-F07, in which we incorporated a 9-methoxy group onto the chromenone core to enhance its potency and masked the aldehyde to achieve long-term efficacy. Protection of the aldehyde as a 1,3-dioxane acetal led to restoration of strong fluorescence emitted by the coumarin chromophore, enabling D-F07 to be tracked in cultured cells and in vivo. Importantly, chemical modifications of the hydroxy group adjacent to the aldehyde could stabilize the 1,3-dioxane acetal, allowing precise control of inhibitory activity. We installed photo-labile, ROS-sensitive, and thiol-reactive structural cage groups onto D-F07 to impart stimuli-responsive biological activities and stabilize the 1,3-dioxane acetal prodrug moiety through perturbing chromenone electron density. We demonstrated that D-F07 could be generated from these caged derivatives by exposure to UV irradiation, hydrogen peroxide or glutathione, respectively, to emit fluorescence and inhibit IRE-1. These novel probe compounds are useful tools to further investigate the roles of the IRE-1/XBP-1 pathway in normal and malignant B cells. In addition, our structural tailoring strategies can be applied to other salicylaldehyde-based compounds to achieve spatiotemporal control of their biological activities.
We discovered the interaction of the IRE-1/XBP-1 pathway with STING (stimulator of interferon genes), an ER-resident transmembrane protein critical for cytoplasmic DNA sensing and production of type I interferons to defend our body from viral, bacterial and parasitic invasions. We discovered that the IRE-1/XBP-1 pathway was downstream of STING because IRE-1- or XBP-1-deficient cells failed to respond to STING activation by producing interferons, while the IRE-1/XBP-1 pathway could be activated normally in cells missing STING. Our further investigation on the response of XBP-1-proficient and XBP-1-deficient B cells to STING agonists led us to discover that STING agonists were cytotoxic specifically to normal and malignant B cells but not to other types of cells, including fibroblasts, melanoma, hepatoma and Lewis lung carcinoma cells. STING agonists potently induced mitochondria-mediated apoptosis in normal and malignant B cells while inducing production of interferons in other cell types. The agonists clearly induced apoptosis through STING because no cytotoxicity was observed in B cell lymphoma and multiple myeloma cells in which the STING gene was deleted with zinc finger nucleases. Different from fibroblasts, melanoma, hepatoma and Lewis lung carcinoma cells, normal and malignant B cells were not capable of degrading STING after stimulations with STING agonists, suggesting that prolonged activation of STING in B cells could engage apoptotic machineries. Transient activation of the IRE-1/XBP-1 pathway could protect agonist-stimulated malignant B cells from cytotoxicity, indicating a critical survival role of the IRE-1/XBP-1 pathway in B cell malignancies. Injections of the STING agonist, 3’3’-cGAMP, induced apoptosis and regression of CLL in Eµ-TCL1 mice and resulted in the prolonged survival of syngeneic mice grafted with multiple myeloma. Importantly, injections of 3’3’-cGAMP suppressed the growth of multiple myeloma in immunodeficient NSG mice. Thus, other than the potential use of STING agonists in boosting anti-tumor immune response, these agonists can directly target B cell malignancies.
To understand how activation of STING caused apoptosis in B cells, we decided to generate a novel STING V154M mouse model to investigate the role of constitutively activated STING in B cells. By examining LPS-stimulated plasmablasts from STING V154M mice, we discovered that activated STING caused rapid ER-associated degradation of the BCR (but not secretory IgM or class I and class II MHC molecules), leading to significantly decreased BCR on the cell surface and less capability of these plasmablasts to conduct BCR signaling. Since continuous BCR signaling is critical for B cells to differentiate into plasma cells in vivo, we decided to immunize STING V154M mice with a T-independent antigen, TNP-Ficoll, and our data indeed showed that these mice responded to the single TNP-Ficoll immunization by producing significantly decreased TNP-specific plasma cells and antibodies. We further generated B cell-specific STINGKO mice. By activating the BCR of STING-deficient B cells and plasmablasts, we showed that these cells responded with more robust BCR signaling than their STING-proficient counterparts. By immunizing B cell-specific STINGKO mice with the same T-independent antigen, we found that these mice produced significantly increased TNP-specific plasma cells and antibodies. Altogether, our results revealed the B cell-intrinsic roles of STING in negatively regulating BCR signaling and antibody production in plasma cells.
IRE-1 splices the mRNA of XBP-1 or engages regulated IRE-1-dependent decay (RIDD) of other mRNAs. It was unclear how IRE-1 RNase activity was regulated to perform the two functions. Upon XBP-1 deficiency, IRE-1 switched to perform RIDD. We examined IRE-1 in XBP-1-deficient B cells and discovered that IRE-1 was phosphorylated at S729. We generated an anti-phospho-S729 antibody and confirmed that S729 was indeed phosphorylated in XBP-1-deficient B cells. Compared with pharmacological ER stress inducers or TLR ligands, subtilase cytotoxin (SubAB) produced by Shiga-toxigenic E. coli (STEC) had an unusual capability in causing rapid and strong phosphorylation of IRE-1 at S729 and triggering B cells to express XBP-1s. To assess the function of S729 of IRE-1, we generated S729A knock-in mice. Compared with wild-type B cells, B cells carrying the S729A mutation similarly responded to LPS by expressing XBP-1s, but LPS-stimulated S729A plasmablasts completely failed to respond to additional ER stress. To evaluate the roles of S729 and the kinase domain of IRE-1 in regulating RIDD, we crossed mice carrying S729A mutation or ΔIRE-1 (missing the kinase domain) with B cell-specific XBP-1-deficient mice to trigger RIDD. RIDD was evidenced by the decreased mRNA levels of secretory immunoglobulin (Ig) μ heavy chains as well as other RIDD substrates in XBP-1-deficient B cells. As expected, RIDD was blocked in S729A/XBP-1KO and ΔIRE1/XBP-1KO B cells. While deleting the kinase function of IRE-1 blocked both XBP-1 splicing and RIDD, mutating S729 only blocked RIDD, highlighting a critical role of S729 in regulating RIDD. Since SubAB could efficiently trigger phosphorylation of IRE-1 at S729, we further demonstrated that exposure to SubAB caused RIDD of the mRNA of secretory Ig μ heavy chains, highlighting a novel mechanism used by STEC to dismantle the capability of B cells to produce sIgM. Importantly, RIDD could regulate the numbers and functions of plasma cells in immunized mice.
Finally, my laboratory actively collaborates with other research groups to investigate the roles of the ER stress response in graft-versus-host disease, ovarian cancer, Myc-transformed lymphoma, CD19-targeted immunotherapy, lupus, dendritic cell biology, natural killer cell biology, and Karposi’s sarcoma-associated herpesvirus (KSHV) latency. Our collaborations have led to the publication of other equally exciting stories!