Qingping Zeng

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.

Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy aryl aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a H-bond with Tyr892. Structure activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Disruption of microRNA Biogenesis Confers Resistance to ER Stress-Induced Cell Death Upstream of the Mitochondrion

Global downregulation of microRNAs (miRNAs) is a common feature of human tumors and has been shown to enhance cancer progression. Several components of the miRNA biogenesis machinery (XPO5, DICER and TRBP) have been shown to act as haploinsufficient tumor suppressors. How the deregulation of miRNA biogenesis promotes tumor development is not clearly understood. Here we show that loss of miRNA biogenesis increased resistance to endoplasmic reticulum (ER) stress-induced cell death. We observed that HCT116 cells with a DICER hypomorphic mutation (Exn5/Exn5) or where DICER or DROSHA were knocked down were resistant to ER stress-induced cell death. Extensive analysis revealed little difference in the unfolded protein response (UPR) of WT compared to Exn5/Exn5 HCT116 cells upon ER stress treatment. However, analysis of the intrinsic apoptotic pathway showed that resistance occurred upstream of the mitochondria. In particular, BAX activation and dissipation of mitochondrial membrane potential was attenuated, and there was altered expression of BCL-2 family proteins. These observations demonstrate a key role for miRNAs as critical modulators of the ER stress response. In our model, downregulation of miRNA biogenesis delays ER stress-induced apoptosis. This suggests that disrupted miRNA biogenesis may contribute to cancer progression by inhibiting ER stress-induced cell death.

Pharmacological targeting of MYC-regulated IRE1/XBP1 pathway suppresses MYC-driven breast cancer

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

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 TGFβ2 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.IRE1/XBP-1 activation has a major role in Triple negative breast cancer (TNBC). Here, the authors show that inhibition of IRE1’s RNase activity attenuates autocrine and paracrine signaling of pro-tumorigenic cytokines and synergizes with paclitaxel to confer potent anti-tumor effects in TNBC.

Abstract LB-303: Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Despite the recent advances made in the treatment and management of B cell malignancies, these diseases are not curable and overall survival is limited. Brutons Tyrosine Kinase (BTK) is a member of the Tec family of intracellular kinases first identified for its signaling via the B-cell Receptor (BCR) and its role in the immune system. More recently, BTK was found to play an important role in B cell malignancies and select solid tumors. Preclinical and clinical results with selective irreversible BTK inhibitors provide validation for BTK as a therapeutic target in B cell malignancies. Aiming to leverage the contribution of the BTK signaling pathway to tumor growth, and its role in progression and drug resistance, we have developed a series of relatively selective, reversible, small molecule BTK inhibitors and evaluated their activity in enzyme, cell-based, and in vivo studies. Data obtained with the orally available MKC4659 compound illustrates our findings. In biochemical assays, compound MKC4659 demonstrated a relatively select targeting profile focused on a narrow group of PTKs including significant activity against BTK with IC50 less than 25 nM. In cellular assays the compound demonstrated significant in vitro potency against B cell lymphoma cell lines, inhibiting the growth of several B cell tumor cell lines including ones unresponsive to currently known BTK inhibitors. Importantly, MKC4659 showed a differential effect on B cell lymphoma, with no significant activity detected in control cells lacking detectable BTK expression. In vitro mode of action studies demonstrated that MKC4659 induces apoptosis and PARP cleavage in B cell lymphoma but not in control cells. Assays evaluating the in vitro on-target effect of compounds showed significant inhibition of the B cell receptor-mediated activation of the BTK pathway. In addition to inhibiting the phosphorylation of BTK, MKC4659 inhibited the phosphorylation of PLCγ in several B cell lymphoma cell lines. With in vitro potency demonstrated, and PK and ADMET profiles amenable to in vivo dosing, MKC4659 was evaluated for in vivo efficacy in a xenograft model of B cell lymphoma. In vivo dosing of MKC4659 inhibited growth of DOHH2 xenograft tumors in a dose dependent manner. In summary, our team has identified BTK pathway inhibitors with demonstrated on-target and anti-tumor activity in cellular assays, and efficacy in a preclinical model of B cell malignancy. This effort provides a platform for compound development and evaluation for the treatment of hematologic malignancies. Optimization efforts on the MKC4659 series are ongoing and have yielded potent and drug-like preclinical candidates that are now moving into advanced animal studies. Citation Format: Mary Faris, Uriel M. Malyankar, Victor Tam, Colleen Schweitzer, Diljeet Joea, Alexis Mollard, Bret Stephens, Steven L. Warner, David J. Bearss, Qingping Zeng. Small molecule inhibitor of the BTK pathway disrupts BCR signaling and demonstrates antitumor efficacy in a xenograft model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2013-LB-303