Research Expertise

Research Interest:
The biology of the endoplasmic reticulum in health and disease

Keywords:
Endoplasmic reticulum (ER); B cell; protein folding and assembly; glycosylation; membrane protein trafficking

Research Details:
The role of the ER stress response in malignant progression of chronic lymphocytic leukemia (CLL) was an under-appreciated research area. We 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, 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 (BTK) inhibitor, 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. Our finding in synergism is important because B-I09 can be used to help ibrutinib to achieve higher cytotoxicity in CLL cells at a lower dose, addressing ibrutinib’s toxicity issue. In addition, we showed that the combination of B-I09 with ibrutinib induced apoptosis not only in CLL cells but also in mantle cell lymphoma and multiple myeloma cells.

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 a photo-labile structural cage group onto D-F07 to impart stimulus-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 the caged derivative by exposure to UV irradiation to emit fluorescence and inhibit IRE-1. Such a novel probe compound is a useful tool to further investigate the roles of the IRE-1/XBP-1 pathway in normal and malignant B 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. Decreased numbers and altered functions of plasma cells were also observed in S729A mice after immunization.

To explore the role of BCR in promoting malignant progression of CLL in mice, we generated hen egg lysozyme (HEL)-specific MD4/Eμ-TCL1 mice. MD4/Eμ-TCL1 mice exhibited significantly shorter survival than Eμ-TCL1 mice. While precancerous B cells in young MD4/Eμ-TCL1 mice recognized HEL, CLL cells developed in older MD4/Eμ-TCL1 mice failed to recognize HEL. Nevertheless, MD4/Eμ-TCL1 CLL cells could be activated by goat F(ab’)2 anti-mouse IgM and respond by BCR signaling, indicating reactivation of a parental Ig gene allele. Notably, CLL-bearing MD4/Eμ-TCL1 mice generated a significantly increased population of CD11b+/Ly6G+ granulocytic cells in the blood, spleens and bone marrow. CD11b+/Ly6G+ granulocytic cells purified from spleens of MD4/Eμ-TCL1 mice suppressed proliferation of CD8+ T cells, qualifying these cells as MDSCs. Increased MDSCs could account for significantly decreased T cells and poor prognosis in CLL-bearing MD4/Eμ-TCL1 mice. Because MD4/Eμ-TCL1 CLL cells also produced large quantities of sIgM, we tested whether sIgM could account for the accumulation of MDSCs by crossing μS-/- mice, which could not produce sIgM, with Eμ-TCL1 mice. The μS-/-/Eμ-TCL1 mice developed significantly lower numbers of MDSCs which were also less capable of suppressing proliferation of T cells, and such mice exhibited significantly longer survival than Eμ-TCL1 mice. We next targeted the synthesis of sIgM by deleting XBP-1s in B cells, because the synthesis of sIgM was controlled by RIDD, which was hyperactivated in response to XBP-1 deficiency. Targeting XBP-1 genetically or pharmacologically (using B-I09) led to decreased levels of sIgM, accompanied by decreased numbers and reduced functions of MDSCs in MD4/Eμ-TCL1 mice. Additionally, μS-/- mice grafted with Lewis lung carcinoma exhibited reduced functions of MDSCs in suppressing T cells, resulting in slower tumor growth. These results established that sIgM produced by B cells could upregulate the functions of MDSCs in tumor-bearing mice to aggravate cancer progression.

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. In addition, while STING agonists have been proposed to be used as adjuvants, our data showing that STING agonists are cytotoxic to B cells suggest that their use as adjuvants to boost antibody production should be further evaluated.

We continue to analyze the functions of ER proteins in malignant progression of leukemia using novel mouse models, in which we selectively delete genes that encode critical ER-resident proteins that support the growth and survival of leukemia. The ultimate goal of our work is to contribute to the design of effective therapeutic approaches that target dysregulated ER functions for patients with leukemia and other malignancies.

Specific laboratory projects:
(1) Investigate IRE-1-interacting proteins to further understand how targeting the IRE-1/XBP-1 pathway can lead to stalled progression of CLL.
(2) Investigate the roles of protein antigen and Toll-like receptor ligands in activating the ER stress response to promote leukemic progression.
(3) Investigate the roles of ER-associated protein degradation in B-cell leukemia.

Laboratory Personnel:
Avery Lee, 4th year PENN CAMB/CB Predoctoral Student

Education

MS (Pharmacology), New York University School of Medicine, 2004
PhD (Pharmacology), New York University School of Medicine, 2006

Specialty Certification

Postgraduate Training

Post-doctoral Fellow, Whitehead Institute for Biomedical Research, Cambridge, MA, 2006-2010

Awards and Honors

Presidential Travel Award, National Sun Yat-Sen University, 1999-1999
Honorary Member of the Phi Tau Phi Scholastic Honor Society, 1999-1999
Young Investigator Travel Award, The Society for Basic Urologic Research, 2004-2004
WIPDA Education Award, Whitehead Institute for Biomedical Research, 2009-2009
Whitehead Appreciation Award, Whitehead Institute for Biomedical Research, 2010-2010
Miles for Moffitt Award, Florida Bank, 2011-2011
American Cancer Society Institutional Research Grant, 2012-2012
Hollis Brownstein Research Grant, Leukemia Research Foundation, 2012-2012

Memberships and Professional Organizations

American Association for Cancer Research, 1999 - 2003
American Society for Cell Biology, 2009 - Present
The European Association for Cancer Research, 2014 - Present

Web Links

Selected Publications