Wnt inhibitors reduce the unfolded protein response and enhance bortezomib-induced cell death in multiple myeloma

Abstract

The Wnt pathway is one of the main regulators of cell growth and is essential for embryonic development and tissue homeostasis.1 Dysregulation of the Wnt pathway is involved in the pathogenesis of many types of carcinomas and is associated with certain hematological cancers, including multiple myeloma (MM).2-4 Several studies, primarily using cell lines and mouse models, reported that MM plasma cells (PCs) carry a functional Wnt/β-catenin signaling pathway that promotes cell survival.3-7 Despite new and successful treatment options, MM remains incurable because of clinical, genetic, and transcriptomic heterogeneity, which ultimately results in therapy resistance and relapse. Thus, an unmet need exists to find new therapeutic targets and improve responses to approved drugs. In the canonical Wnt pathway, Wnt ligands bind and activate Frizzled receptors and LRP5/6 coreceptors to recruit components of the β-catenin destruction complex.1,3 Subsequent stabilization of nonphosphorylated β-catenin results in the transcription of Wnt target genes. Here, we targeted 2 pathway components to inhibit Wnt signaling in MM cells. Inhibition of tankyrase promotes degradation of β-catenin owing to stabilization of Axin, which is the concentration-limiting component of the β-catenin destruction complex.8 Inhibition of porcupine, an enzyme required for the secretion of all human Wnt ligands, was shown to reduce Wnt signaling in vitro and in a mouse model.9 We demonstrate that targeting Wnt signaling promotes cell death of patient-derived MM cells and downregulates genes involved in the unfolded protein response (UPR). Subsequently, we show that Wnt inhibitors enhance bortezomib (BTZ)-induced cell death in primary MM cells. First, we tested the functionality of tankyrase inhibitor (TNKSi) XAV939 and porcupine inhibitor (PORCi) C59 in MM cell lines. Single-drug exposure in MM1.S blocked β-catenin upregulation by Wnt3a-conditioned medium in case of TNKSi and downregulated β-catenin expression in case of PORCi (Figure 1A). Treatment with TNKSi caused only moderate apoptosis in MM1.S and L363, ranging from 0.2% to 3.9% specific apoptosis at 10 to 40 μM TNKSi and 3.9% to 29.1% specific apoptosis at 5 to 10 μM PORCi (supplemental Figure 1A). The inhibitor combination induced more than additive cell death in MM1.S, with specific apoptosis increasing to 57.1% for 20 μM TNKSi and 10 μM PORCi (supplemental Figure 1A,B). A significant impact on the number of viable cells was observed after 7 days of treatment (supplemental Figure 1C). We also demonstrated a significant reduction in transcriptional Wnt reporter activity after treatment with both inhibitors (Figure 1B). In addition to MM1.S and L363, the combination of Wnt inhibitors was effective in MM cell lines INA-6 and KMS12- PE (supplemental Figure 1D). In summary, these data show that the Wnt pathway can be inhibited by a combination of TNKSi and PORCi and affects cell survival in MM cell lines. The Wnt pathway is one of the main regulators of cell growth and is essential for embryonic development and tissue homeostasis.1 Dysregulation of the Wnt pathway is involved in the pathogenesis of many types of carcinomas and is associated with certain hematological cancers, including multiple myeloma (MM).2-4 Several studies, primarily using cell lines and mouse models, reported that MM plasma cells (PCs) carry a functional Wnt/β-catenin signaling pathway that promotes cell survival.3-7 Despite new and successful treatment options, MM remains incurable because of clinical, genetic, and transcriptomic heterogeneity, which ultimately results in therapy resistance and relapse. Thus, an unmet need exists to find new therapeutic targets and improve responses to approved drugs. In the canonical Wnt pathway, Wnt ligands bind and activate Frizzled receptors and LRP5/6 coreceptors to recruit components of the β-catenin destruction complex.1,3 Subsequent stabilization of nonphosphorylated β-catenin results in the transcription of Wnt target genes. Here, we targeted 2 pathway components to inhibit Wnt signaling in MM cells. Inhibition of tankyrase promotes degradation of β-catenin owing to stabilization of Axin, which is the concentration-limiting component of the β-catenin destruction complex.8 Inhibition of porcupine, an enzyme required for the secretion of all human Wnt ligands, was shown to reduce Wnt signaling in vitro and in a mouse model.9 We demonstrate that targeting Wnt signaling promotes cell death of patient-derived MM cells and downregulates genes involved in the unfolded protein response (UPR). Subsequently, we show that Wnt inhibitors enhance bortezomib (BTZ)-induced cell death in primary MM cells. First, we tested the functionality of tankyrase inhibitor (TNKSi) XAV939 and porcupine inhibitor (PORCi) C59 in MM cell lines. Single-drug exposure in MM1.S blocked β-catenin upregulation by Wnt3a-conditioned medium in case of TNKSi and downregulated β-catenin expression in case of PORCi (Figure 1A). Treatment with TNKSi caused only moderate apoptosis in MM1.S and L363, ranging from 0.2% to 3.9% specific apoptosis at 10 to 40 μM TNKSi and 3.9% to 29.1% specific apoptosis at 5 to 10 μM PORCi (supplemental Figure 1A). The inhibitor combination induced more than additive cell death in MM1.S, with specific apoptosis increasing to 57.1% for 20 μM TNKSi and 10 μM PORCi (supplemental Figure 1A,B). A significant impact on the number of viable cells was observed after 7 days of treatment (supplemental Figure 1C). We also demonstrated a significant reduction in transcriptional Wnt reporter activity after treatment with both inhibitors (Figure 1B). In addition to MM1.S and L363, the combination of Wnt inhibitors was effective in MM cell lines INA-6 and KMS12- PE (supplemental Figure 1D). In summary, these data show that the Wnt pathway can be inhibited by a combination of TNKSi and PORCi and affects cell survival in MM cell lines.