XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS.

Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRASG12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRASG12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRASG12D -mutated pre-B ALL.

Targeting of BCR-ABL1 and IRE1α induces synthetic lethality in Philadelphia-positive acute lymphoblastic leukemia.

BCR-ABL1-positive acute lymphoblastic leukemia (ALL) cell survival is dependent on the inositol-requiring enzyme 1 alpha (IRE1α) branch of the unfolded protein response. In the current study, we have focused on exploring the efficacy of a simultaneous pharmacological inhibition of BCR-ABL1 and IRE1α in Philadelphia-positive (Ph+) ALL using tyrosine kinase inhibitor (TKI) nilotinib and the IRE1α inhibitor MKC-8866. The combination of 0.5 µM nilotinib and 30 µM MKC-8866 in Ph+ ALL cell lines led to a synergistic effect on cell viability. To mimic this dual inhibition on a genetic level, pre-B-cells from conditional Xbp1+/fl mice were transduced with a BCR-ABL1 construct and with either tamoxifen-inducible cre or empty vector. Cells showed a significant sensitization to the effect of TKIs after the induction of the heterozygous deletion. Finally, we performed a phosphoproteomic analysis on Ph+ ALL cell lines treated with the combination of nilotinib and MKC-8866 to identify potential targets involved in their synergistic effect. An enhanced activation of p38 mitogen-activated protein kinase α (p38α MAPK) was identified. In line with this findings, p38 MAPK and, another important endoplasmic reticulum-stress-related kinase, c-Jun N-terminal kinase (JNK) were found to mediate the potentiated cytotoxic effect induced by the combination of MKC-8866 and nilotinib since the targeting of p38 MAPK with its specific inhibitor BIRB-796 or JNK with JNK-in-8 hindered the synergistic effect observed upon treatment with nilotinib and MKC-8866. In conclusion, the identified combined action of nilotinib and MKC-8866 might represent a successful therapeutic strategy in high-risk Ph+ ALL.

Publisher Correction: Pharmacological targeting of the unfolded protein response for disease intervention.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pharmacological targeting of the unfolded protein response for disease intervention.

Accumulation of unfolded proteins at the endoplasmic reticulum (ER) is a salient attribute of many human diseases including obesity, liver disorders, cancer, diabetes and neurodegeneration. To restore ER proteostasis, cells activate the unfolded protein response (UPR), a signaling pathway that imposes adaptive programs or triggers apoptosis of damaged cells. The UPR is critical to sustain the normal function of specialized secretory cells (i.e., pancreatic ß cells and B lymphocytes) and to control the production of lipids and cholesterol in the liver. In the context of disease, adaptive UPR responses have been linked to the growth of solid tumors, whereas chronic ER stress contributes to cell dysfunction in brain diseases, metabolic syndromes, among other conditions. Here we discuss recent developments in the design and optimization of novel compounds to manipulate UPR signaling and their efficacy in various disease models.

Blockade of XBP1 splicing by inhibition of IRE1α is a promising therapeutic option in multiple myeloma.

Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response. Inositol-requiring enzyme 1α (IRE1α) is activated to splice X-box binding protein 1 (XBP1) mRNA, thereby increasing XBP1s protein, which in turn regulates genes responsible for protein folding and degradation during the unfolded protein response. In this study, we examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM using a small-molecule IRE1α endoribonuclease domain inhibitor MKC-3946. MKC-3946 triggered modest growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Importantly, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG, even in the presence of bone marrow stromal cells or exogenous IL-6. Both bortezomib and 17-AAG induced ER stress, evidenced by induction of XBP1s, which was blocked by MKC-3946. Apoptosis induced by these agents was enhanced by MKC-3946, associated with increased CHOP. Finally, MKC-3946 inhibited XBP1 splicing in a model of ER stress in vivo, associated with significant growth inhibition of MM cells. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential therapeutic option in MM.

Correction: Dastghaib et al. Simvastatin Induces Unfolded Protein Response and Enhances Temozolomide-Induced Cell Death in Glioblastoma Cells. Cells 2020, 9, 2339.

In the original publication [...].

Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease.

Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.

The IRE1 and PERK arms of the unfolded protein response promote survival of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma, is associated with a low 5-year survival and harsh treatment side effects, underscoring an urgent need for therapy. The unfolded protein response (UPR) is activated in response to endoplasmic reticulum (ER) stress, where three ER stress receptors, IRE1, PERK and ATF6, aim to restore cellular homeostasis. The UPR is pro-tumourigenic in many cancers. In this study, we investigate basal UPR activity in RMS. Basal activation of IRE1 and PERK was observed in RMS cell lines, which was diminished upon addition of the IRE1 RNase inhibitor, MKC8866, or PERK inhibitor, AMGEN44. UPR inhibition caused a reduction in cell viability, cell proliferation and inhibition of long-term colony formation in both subtypes of RMS. Alveolar RMS (ARMS) subtype was highly sensitive to IRE1 inhibition, whereas embryonal RMS (ERMS) subtypes responded more markedly to PERK inhibition. Further investigation revealed a robust activation of senescence upon UPR inhibition. For the first time, the UPR is implicated in RMS biology and phenotype, and inhibition of UPR signalling reduces cell growth, suggesting that the UPR may be a promising target in RMS.

Simvastatin Induces Unfolded Protein Response and Enhances Temozolomide-Induced Cell Death in Glioblastoma Cells.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor with a very poor survival rate. Temozolomide (TMZ) is the common chemotherapeutic agent used for GBM treatment. We recently demonstrated that simvastatin (Simva) increases TMZ-induced apoptosis via the inhibition of autophagic flux in GBM cells. Considering the role of the unfolded protein response (UPR) pathway in the regulation of autophagy, we investigated the involvement of UPR in Simva-TMZ-induced cell death by utilizing highly selective IRE1 RNase activity inhibitor MKC8866, PERK inhibitor GSK-2606414 (PERKi), and eIF2α inhibitor salubrinal. Simva-TMZ treatment decreased the viability of GBM cells and significantly increased apoptotic cell death when compared to TMZ or Simva alone. Simva-TMZ induced both UPR, as determined by an increase in GRP78, XBP splicing, eukaryote initiation factor 2α (eIF2α) phosphorylation, and inhibited autophagic flux (accumulation of LC3ß-II and inhibition of p62 degradation). IRE1 RNase inhibition did not affect Simva-TMZ-induced cell death, but it significantly induced p62 degradation and increased the microtubule-associated proteins light chain 3 (LC3)ß-II/LC3ß-I ratio in U87 cells, while salubrinal did not affect the Simva-TMZ induced cytotoxicity of GBM cells. In contrast, protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibition significantly increased Simva-TMZ-induced cell death in U87 cells. Interestingly, whereas PERK inhibition induced p62 accumulation in both GBM cell lines, it differentially affected the LC3ß-II/LC3ß-I ratio in U87 (decrease) and U251 (increase) cells. Simvastatin sensitizes GBM cells to TMZ-induced cell death via a mechanism that involves autophagy and UPR pathways. More specifically, our results imply that the IRE1 and PERK signaling arms of the UPR regulate Simva-TMZ-mediated autophagy flux inhibition in U251 and U87 GBM cells.

Correction: Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Local intracerebral inhibition of IRE1 by MKC8866 sensitizes glioblastoma to irradiation/chemotherapy in vivo.

Glioblastoma multiforme (GBM) is the most severe primary brain cancer. Despite an aggressive treatment comprising surgical resection and radio/chemotherapy, patient's survival post diagnosis remains short. A limitation for success in finding novel improved therapeutic options for such dismal disease partly lies in the lack of a relevant animal model that accurately recapitulates patient disease and standard of care. In the present study, we have developed an immunocompetent GBM model that includes tumor surgery and a radio/chemotherapy regimen resembling the Stupp protocol and we have used this model to test the impact of the pharmacological inhibition of the endoplasmic reticulum (ER) stress sensor IRE1, on treatment efficacy.

Stress chaperone GRP78/BiP confers chemoresistance to tumor-associated endothelial cells.

The tumor vasculature is essential for tumor growth and survival and is a key target for anticancer therapy. Glioblastoma multiforme, the most malignant form of brain tumor, is highly vascular and contains abnormal vessels, unlike blood vessels in normal brain. Previously, we showed that primary cultures of human brain endothelial cells, derived from blood vessels of malignant glioma tissues (TuBEC), are physiologically and functionally different from endothelial cells derived from nonmalignant brain tissues (BEC) and are substantially more resistant to apoptosis. Resistance of TuBEC to a wide range of current anticancer drugs has significant clinical consequences as it represents a major obstacle toward eradication of residual brain tumor. We report here that the endoplasmic reticulum chaperone GRP78/BiP is generally highly elevated in the vasculature derived from human glioma specimens, both in situ in tissue and in vitro in primary cell cultures, compared with minimal GRP78 expression in normal brain tissues and blood vessels. Interestingly, TuBEC constitutively overexpress GRP78 without concomitant induction of other major unfolded protein response targets. Resistance of TuBEC to chemotherapeutic agents such as CPT-11, etoposide, and temozolomide can be overcome by knockdown of GRP78 using small interfering RNA or chemical inhibition of its catalytic site. Conversely, overexpression of GRP78 in BEC rendered these cells resistant to drug treatments. Our findings provide the proof of principle that targeting GRP78 will sensitize the tumor vasculature to chemotherapeutic drugs, thus enhancing the efficacy of these drugs in combination therapy for glioma treatment.

Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

The inflammasome is a multiprotein complex assembled in response to Pathogen Associated Molecular Patterns (PAMPs) and Danger Associated Molecular Patterns (DAMPs). Inflammasome activation occurs through a two-step mechanism, with the first signal facilitating priming of inflammasome components while the second signal triggers complex assembly. Once assembled, the inflammasome recruits and activates pro-caspase-1, which in turn processes pro-interleukin (IL)-18 and pro-IL-1ß into their bio-active forms. Owing to its key role in the regulation of innate immune responses, the inflammasome has emerged as a therapeutic target for the treatment of inflammatory conditions. In this study we demonstrate that IRE1α, a key component of the Unfolded Protein Response, contributes to assembly of the NLRP3 inflammasome. Blockade of IRE1α RNase signaling lowered NLRP3 inflammasome assembly, caspase-1 activation and pro-IL-1ß processing. These results underscore both the importance and potential therapeutic relevance of targeting IRE1α signaling in conditions of excessive inflammasome formation.

IRE1α-XBP1s pathway promotes prostate cancer by activating c-MYC signaling.

Activation of endoplasmic reticulum (ER) stress/the unfolded protein response (UPR) has been linked to cancer, but the molecular mechanisms are poorly understood and there is a paucity of reagents to translate this for cancer therapy. Here, we report that an IRE1α RNase-specific inhibitor, MKC8866, strongly inhibits prostate cancer (PCa) tumor growth as monotherapy in multiple preclinical models in mice and shows synergistic antitumor effects with current PCa drugs. Interestingly, global transcriptomic analysis reveal that IRE1α-XBP1s pathway activity is required for c-MYC signaling, one of the most highly activated oncogenic pathways in PCa. XBP1s is necessary for optimal c-MYC mRNA and protein expression, establishing, for the first time, a direct link between UPR and oncogene activation. In addition, an XBP1-specific gene expression signature is strongly associated with PCa prognosis. Our data establish IRE1α-XBP1s signaling as a central pathway in PCa and indicate that its targeting may offer novel treatment strategies.

Infliction of proteotoxic stresses by impairment of the unfolded protein response or proteasomal inhibition as a therapeutic strategy for mast cell leukemia.

The intensity and duration of endoplasmic reticulum (ER) stress converts the unfolded protein response (UPR) from an adaptive into a terminal response. The first regulates homeostasis, the latter triggers apoptosis. Cells that rapidly proliferate and possess developed secretory capabilities, such as leukemia cells, depend on an efficiently operating UPR to maintain proteostasis. Activation of terminal UPR by either blockade of adaptive UPR or exaggeration of ER stress has been explored as a novel approach in cancer therapy. For mast cell leukemia (MCL) the efficacy of both approaches, by utilizing the KITV560G,D816V-positive MCL cell line HMC-1.2, was investigated. We show that HMC-1.2 cells display a tonic activation of the IRE1α arm of the UPR, which constitutively generates spliced XBP1. Inhibition of IRE1α by different types of inhibitors (MKC-8866, STF-083010, and KIRA6) suppressed proliferation at concentrations needed for blockade of IRE1α-mediated XBP1 splicing. At higher concentrations, these inhibitors triggered an apoptotic response. Blocking the proteasome by bortezomib, which confers an exaggerated UPR, resulted in a marked cytotoxic response. Bortezomib treatment also caused activation of the kinase JNK, which played a pro-proliferative and anti-apoptotic role. Hence, the combination of bortezomib with a JNK inhibitor synergized to induce cell death. In summary, the UPR can be addressed as an effective therapeutic target against KITD816V-positive MCL.

Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy.

Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFß2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.