Chih-Chi Andrew Hu

Inhibition of ER stress–associated IRE-1/XBP-1 pathway reduces leukemic cell survival

Activation of the ER stress response is associated with malignant progression of B cell chronic lymphocytic leukemia (CLL). We developed a murine CLL model that lacks the ER stress-associated transcription factor XBP-1 in B cells and found that XBP-1 deficiency decelerates malignant progression of CLL-associated disease. XBP-1 deficiency resulted in acquisition of phenotypes that are disadvantageous for leukemic cell survival, including compromised BCR signaling capability and increased surface expression of sphingosine-1-phosphate receptor 1 (S1P1). Because XBP-1 expression requires the RNase activity of the ER transmembrane receptor IRE-1, we developed a potent IRE-1 RNase inhibitor through chemical synthesis and modified the structure to facilitate entry into cells 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 of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumor-bearing 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 indicate that targeting XBP-1 has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma.

Profiling antibody responses by multiparametric analysis of primary B cells

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (≈103-104). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.

Overexpression of TCL1 activates the endoplasmic reticulum stress response: a novel mechanism of leukemic progression in mice

Chronic lymphocytic leukemia (CLL) represents 30% of adult leukemia. TCL1 is expressed in ~ 90% of human CLL. Transgenic expression of TCL1 in murine B cells (Eμ-TCL1) results in mouse CLL. Here we show for the first time that the previously unexplored endoplasmic reticulum (ER) stress response is aberrantly activated in Eμ-TCL1 mouse and human CLL. This includes activation of the IRE-1/XBP-1 pathway and the transcriptionally up-regulated expression of Derlin-1, Derlin-2, BiP, GRP94, and PDI. TCL1 associates with the XBP-1 transcription factor, and causes the dysregulated expression of the transcription factors, Pax5, IRF4, and Blimp-1, and of the activation-induced cytidine deaminase. In addition, TCL1-overexpressing CLL cells manufacture a distinctly different BCR, as we detected increased expression of membrane-bound IgM and altered N-linked glycosylation of Igα and Igβ, which account for the hyperactive BCR in malignant CLL. To demonstrate that the ER stress-response pathway is a novel molecular target for the treatment of CLL, we blocked the IRE-1/XBP-1 pathway using a novel inhibitor, and observed apoptosis and significantly stalled growth of CLL cells in vitro and in mice. These studies reveal an important role of TCL1 in activating the ER stress response in support for malignant progression of CLL.

XBP-1-Deficient Plasmablasts Show Normal Protein Folding but Altered Glycosylation and Lipid Synthesis

The accumulation of misfolded secreted IgM in the endoplasmic reticulum (ER) of X-box binding protein 1 (XBP-1)-deficient B cells has been held responsible for the inability of such cells to yield plasma cells, through the failure to mount a proper unfolded protein response. LPS-stimulated B cells incapable of secreting IgM still activate the XBP-1 axis normally, as follows: XBP-1 is turned on by cues that trigger differentiation and not in response to accumulation of unfolded IgM, but the impact of XBP-1 deficiency on glycoprotein folding and assembly has not been explored. The lack of XBP-1 compromised neither the formation of functional hen egg lysozyme-specific IgM nor the secretion of free κ-chains. Although XBP-1 deficiency affects the synthesis of some ER chaperones, including protein disulfide isomerase, their steady state levels do not drop below the threshold required for proper assembly and maturation of the Igα/Igβ heterodimer and MHC molecules. Intracellular transport and surface display of integral membrane proteins are unaffected by XBP-1 deficiency. Given the fact that we failed to observe any defects in folding of a variety of glycoproteins, we looked for other means to explain the requirement for XBP-1 in plasma cell development. We observed significantly reduced levels of phosphatidylcholine, sphingomyelin, and phosphatidylinositol in total membranes of XBP-1-deficient B cells, and reduced ER content. Terminal N-linked glycosylation of IgM and class I MHC was altered in these cells. XBP-1 hence has important roles beyond folding proteins in the ER.

Subtilase cytotoxin cleaves newly synthesized BiP and blocks antibody secretion in B lymphocytes

Shiga-toxigenic Escherichia coli (STEC) use subtilase cytotoxin (SubAB) to interfere with adaptive immunity. Its inhibition of immunoglobulin secretion is both rapid and profound. SubAB favors cleavage of the newly synthesized immunoglobulin heavy chain–binding protein (BiP) to yield a C-terminal fragment that contains BiP’s substrate-binding domain. In the absence of its regulatory nucleotide-binding domain, the SubAB-cleaved C-terminal BiP fragment remains tightly bound to newly synthesized immunoglobulin light chains, resulting in retention of light chains in the endoplasmic reticulum (ER). Immunoglobulins are thus detained in the ER, making impossible the secretion of antibodies by SubAB-treated B cells. The inhibitory effect of SubAB is highly specific for antibody secretion, because other secretory proteins such as IL-6 are released normally from SubAB-treated B cells. Although SubAB also causes BiP cleavage in HepG2 hepatoma cells, (glyco)protein secretion continues unabated in SubAB-exposed HepG2 cells. This specific block in antibody secretion is a novel means of immune evasion for STEC. The differential cleavage of newly synthesized versus “aged” BiP by SubAB in the ER provides insight into the architecture of the ER compartments involved.

IgG1+ ovalbumin-specific B-cell transnuclear mice show class switch recombination in rare allelically included B cells

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1+ ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1−/− mice have exclusively IgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3′ of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM+IgG1+ double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3.

Synthesis of novel tricyclic chromenone-based inhibitors of IRE-1 RNase activity.

Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase ER stress sensor that is activated in response to excessive accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stress stimuli. Activation of IRE-1 RNase function exerts a cytoprotective effect and has been implicated in the progression of cancer via increased expression of the transcription factor XBP-1s. Here, we describe the synthesis and biological evaluation of novel chromenone-based covalent inhibitors of IRE-1. Preparation of a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was achieved via a Duff formylation that is attended by an unusual cyclization reaction. Biological evaluation in vitro and in whole cells led to the identification of 30 as a potent inhibitor of IRE-1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines.

Regulated IRE1-dependent mRNA decay sets the threshold for dendritic cell survival

The IRE1–XBP1 signalling pathway is part of a cellular programme that protects against endoplasmic reticulum (ER) stress, but also controls development and survival of immune cells. Loss of XBP1 in splenic type 1 conventional dendritic cells (cDC1s) results in functional alterations without affecting cell survival. However, in mucosal cDC1s, loss of XBP1 impaired survival in a tissue-specific manner—while lung cDC1s die, intestinal cDC1s survive. This was not caused by differential activation of ER stress cell-death regulators CHOP or JNK. Rather, survival of intestinal cDC1s was associated with their ability to shut down protein synthesis through a protective integrated stress response and their marked increase in regulated IRE1-dependent messenger RNA decay. Furthermore, loss of IRE1 endonuclease on top of XBP1 led to cDC1 loss in the intestine. Thus, mucosal DCs differentially mount ATF4- and IRE1-dependent adaptive mechanisms to survive in the face of ER stress.

Total synthesis and structural revision of lucentamycin A.

Lucentamycin A is a marine-derived peptide natural product harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed structure of lucentamycin A and the core Emp residue have recently been called into question through synthesis. Here, we report the first total synthesis of lucentamycin A, which confirms that the ethylidene substituent in Emp bears an E geometry, in contrast to the originally assigned Z configuration. Synthesis of the desired (E)-Emp subunit required the implementation of a novel strategy starting from Garners aldehyde.

IRE1α RNase–dependent lipid homeostasis promotes survival in Myc-transformed cancers

Myc activation is a primary oncogenic event in many human cancers; however, these transcription factors are difficult to inhibit pharmacologically, suggesting that Myc-dependent downstream effectors may be more tractable therapeutic targets. Here, we show that Myc overexpression induces endoplasmic reticulum (ER) stress and engages the inositol-requiring enzyme 1&agr; (IRE1&agr;)/X-box binding protein 1 (XBP1) pathway through multiple molecular mechanisms in a variety of c-Myc– and N-Myc–dependent cancers. In particular, Myc-overexpressing cells require IRE1&agr;/XBP1 signaling for sustained growth and survival in vitro and in vivo, dependent on elevated stearoyl-CoA-desaturase 1 (SCD1) activity. Pharmacological and genetic XBP1 inhibition induces Myc-dependent apoptosis, which is alleviated by exogenous unsaturated fatty acids. Of note, SCD1 inhibition phenocopies IRE1&agr; RNase activity suppression in vivo. Furthermore, IRE1&agr; inhibition enhances the cytotoxic effects of standard chemotherapy drugs used to treat c-Myc–overexpressing Burkitt’s lymphoma, suggesting that inhibiting the IRE1&agr;/XBP1 pathway is a useful general strategy for treatment of Myc-driven cancers.