Data are representative of 3 indie experiments
Data are representative of 3 indie experiments. to target the IRE-1/XBP-1 pathway. Treatment of CLL cells with this inhibitor (B-I09) mimicked XBP-1 deficiency, including upregulation of IRE-1 expression and compromised BCR signaling. Moreover, B-I09 treatment did not affect the transport Quinestrol of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumorCbearing mice suppressed leukemic progression by inducing apoptosis and did not cause systemic toxicity. Additionally, B-I09 and ibrutinib, an FDA-approved BTK inhibitor, synergized to induce apoptosis in B cell leukemia, lymphoma, and multiple myeloma. These data show that targeting XBP-1 has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma. Introduction The functional role of the ER stress response in mature B cell leukemia or lymphoma has been largely overlooked because leukemia and lymphoma cells do not expand their ER as do multiple myeloma (MM) cells. Recently, chronic lymphocytic leukemia (CLL), the most common adult leukemia, was shown to require activation of the ER stress response for survival (1). The IRE-1/XBP-1 pathway represents the most conserved ER stress-response pathway. IRE-1 contains a luminal stress-sensor domain name and a cytoplasmic kinase/RNase domain name (Supplemental Physique 1; supplemental material available online with this short article; doi:10.1172/JCI73448DS1). The RNase domain name is critical for the function of IRE-1 because it splices 26 nucleotides from your mRNA, causing a frame shift in translation (2C4). The spliced mRNA encodes a functional 54-kDa XBP-1s transcription factor. The role of XBP-1 in malignancy has not been validated by genetic deletion of the gene in mice. Thus, we deleted the gene from B cells of E-TCL1 transgenic mice (E-TCL1, herein referred to as XBP-1KO/E-TCL1), arguably the best CLL mouse model to date (5, 6). The E-TCL1 mouse model is usually clinically relevant because TCL1 expression is found Quinestrol in 90% of human CLL cases (1, 7). E-TCL1 mice develop leukemia with all clinical features of aggressive human CLL (6, 8) and have been used repeatedly for preclinical drug assessments (9C16). Using XBP-1KO/E-TCL1 mice, we examine the role of the IRE-1/XBP-1 pathway in tumor progression. Quinestrol While most transcription factors remain undruggable, the specific activation mechanism of XBP-1 renders IRE-1 an attractive target for therapeutic intervention. Although chemical screens have led to the identification of inhibitors of the IRE-1 RNase activity (17C20), there is a need to develop novel small molecules with improved cellular and in vivo efficacy. We synthesized and evaluated novel tricyclic chromenone inhibitors of IRE-1 RNase activity that potently suppress the expression of XBP-1 and induce apoptosis. We also decided the bioavailability and pharmacokinetics of our lead inhibitor, B-I09, and showed that B-I09, when administered as a single agent, effectively induces leukemic regression without causing systemic Rabbit polyclonal to ND2 toxicity in CLL-bearing E-TCL1 mice. Since the inhibition of the IRE-1/XBP-1 pathway compromises B cell receptor (BCR) signaling, we tested for any potential synergistic effect between B-I09 and the Brutons tyrosine kinase (BTK) inhibitor ibrutinib. Our results demonstrate the effectiveness of targeting both the IRE-1/XBP-1 and BCR signaling pathways to induce apoptosis in human B cell leukemia, lymphoma, and MM cells. Results XBP-1KO/E-TCL1 mice develop leukemia significantly more slowly than XBP-1WT/E-TCL1 mice. To investigate how the loss of XBP-1 can counter malignant progression of leukemia, we crossed B cellCspecific XBP-1KO mice (= 5 in each age group). (F) CD5+B220+ CLL cells purified from spleens of XBP-1WT/E-TCL1 and XBP-1KO/E-TCL1 mice were lysed to analyze for the expression of indicated proteins. Data shown in immunoblots are representative of 3 impartial experiments. (G) Spleens from 12-month-old age-matched XBP-1WT/E-TCL1 and XBP-1KO/E-TCL1 littermates and a WT mouse. (H) Kaplan-Meier analysis of overall survival of XBP-1KO/E-TCL1 mice (= 18). Four mice from your XBP-1KO/E-TCL1 group were censored (circled in reddish), as they were removed for other studies. XBP-1Cdeficient E-TCL1 CLL cells exhibit compromised BCR signaling. Constitutive BCR activation is usually a critical survival transmission for CLL cells (22, 23). To understand how the loss of XBP-1 may contribute to the slower progression of leukemia in E-TCL1 mice, we purified CLL cells from XBP-1WT/E-TCL1 and XBP-1KO/E-TCL1 littermates (Supplemental Physique 2, B and C), cultured them in LPS for 3 days, activated the BCR using F(ab)2 anti-mouse IgM, and lysed the cells. Cell lysates were immunoblotted for phospho-Syk and phospho-BTK because Syk and BTK are crucial BCR signaling molecules for CLL survival (22, 23). Compared with XBP-1WT/E-TCL1 CLL cells, XBP-1KO/E-TCL1 CLL cells are defective in Syk and BTK phosphorylation upon activation of the BCR (Physique ?(Figure2A).2A). Unlike naive normal B cells, XBP-1WT/E-TCL1 CLL cells synthesize significantly increased amounts of secretory forms of IgM and release them into culture medium in the absence of any activation (Physique ?(Physique2,2, B and C). The lack of XBP-1.