Increased expression of the tight junction protein TJP1/ZO-1 is associated with upregulation of TAZ-TEAD activity and an adult tissue stem cell signature in carfilzomib-resistant multiple myeloma cells and high-risk multiple myeloma patients

Abstract

Tight junction protein 1 (TJP1) has recently been proposed as a biomarker to identify multiple myeloma (MM) patients most likely to respond to bortezomib- and carfilzomib-based proteasome inhibitor regimens. Herein we report increased expression of TJP1 during the adaptive response mediating carfilzomib resistance in the LP-1/Cfz MM cell line. Moreover, increased TJP1 expression delineated a subset of relapsed/refractory MM patients on bortezomib-based therapy sharing an LP-1/Cfz-like phenotype characterized by activation of interacting transcriptional effectors of the Hippo signaling cascade (TAZ and TEAD1) and an adult tissue stem cell signature. siRNA-mediated knockdown of TJP1 or TAZ/TEAD1 partially sensitized LP-1/Cfz cells to carfilzomib. Connectivity Map analysis identified translation inhibitors as candidate therapeutic agents targeting this molecular phenotype. We confirmed this prediction by showing that homoharringtonine (omacetaxine mepesuccinate) - the first translation inhibitor to be approved by the U.S. Food and Drug Administration - displayed potent cytotoxic activity on LP-1/Cfz cells. Homoharringtonine treatment reduced the levels of TAZ and TEAD1 as well as the MM-protective proteins Nrf2 and MCL1. Thus, our data suggest the importance of further studies evaluating translation inhibitors in relapsed/refractory MM. On the other hand, use of TJP1 as a MM biomarker for proteasome inhibitor sensitivity requires careful consideration.

Keywords: Nrf2; TJP1/ZO-1; WWTR1/TAZ-TEAD1; cancer stem cell-related features; carfilzomib; homoharringtonine; multiple myeloma; proteasome inhibitors; translation inhibitors.

Figure 1. TJP1 protein levels are increased in carfilzomib-resistant LP-1/Cfz cells

Western blot analysis showing significantly higher TJP1 levels in LP-1/Cfz compared to parental LP-1 cells. Note that TJP1 levels are also increased in doxorubicin-resistant RPMI-8226/Dox40 compared to parental RPMI-8226 cells.

Figure 2. Increased expression of TJP1 is associated with upregulation of TAZ-TEAD activity and adult stem cell genes in LP-1/Cfz cells and TT3 protocol patients with relapsed/refractory disease (GEO: GSE31161)

A. GSEA enrichment plots of the Cordenonsi YAP Conserved Signature for triplicate samples of LP-1/Cfz versus parental LP-1 cells (top), for TT3 patients with increased TJP1 levels at relapse (middle), and for TT3 patients with decreased TJP1 levels at relapse (bottom). B. GSEA enrichment plots of the Wong Adult Tissue Stem Module for triplicate samples of LP-1/Cfz versus parental LP-1 cells (top), for TT3 patients with increased TJP1 levels at relapse (middle), and for TT3 patients with decreased TJP1 levels at relapse (bottom). NES, normalized enrichment score.

Figure 3. TAZ-TEAD1 activation in LP-1/Cfz cells confers resistance to carfilzomib

A. TAZ levels are increased in LP-1/Cfz (Cfz) versus parental LP-1 cells (1.6 ± 0.3-fold, n = 10, P < 0.01). Cells were pretreated with MG-132 (15 μM) for 18 hours to prevent proteasomal degradation. B. TEAD1 levels are increased in LP-1/Cfz (Cfz) versus parental LP-1 cells (2.2 ± 0.3-fold, n = 10, P < 0.01). Cells were pretreated with MG-132 (15 μM) for 18 hours to prevent proteasomal degradation. C. siRNA-mediated knockdown of TAZ slightly sensitized LP-1/Cfz cells to carfilzomib. Consistent with low YAP1 levels in LP-1/Cfz cells, siRNA-mediated knockdown of YAP1 in concert with TAZ did not meaningfully enhance sensitization of the cells to the drug. LP-1/Cfz cells were significantly sensitized to carfilzomib by combined siRNA-mediated knockdown of TAZ and TEAD1. No effects were observed on parental LP-1 cells under any of the conditions. Cells were treated with carfilzomib (12.5 nM) for 48 hours after transient transfection and cell viability was determined by alamarBlue assay. *, P = 0.04 vs negative siRNA control (siNeg, n = 3). D, E. siRNA-mediated knockdown of TAZ and TEAD1 in LP-1/Cfz cells was accompanied by decreased TAZ (40 ± 15% decrease) (D) and TEAD1 (57 ± 6% decrease) (E) levels.

Figure 4. Prognostic value of TJP1 expression in MM patient survival outcomes

KaplanMeier survival plots of 47 MM patients in the high-risk PR subgroup (GEO: GSE2658) created using PROGgeneV2: A. Cordenonsi YAP Conserved Signature minus (left) and plus (right) TJP1 coexpression. B. Leading edge subset (top 50 genes) of the LP-1/Cfz Wong Adult Tissue Stem Module minus (left) and plus (right) TJP1 coexpression. C. WWTR1/TAZ expression (left) plus coexpression of TEAD1, ITGB5, CRIM1 and TJP1 (right). Median gene expression values were used as bifurcation points.

Figure 5. Gene Ontology terms and canonical pathways involving cell contacts are enriched in LP-1/Cfz cells and TT3 protocol patients (GEO: GSE31161) with increased TJP1 levels at relapse

A. Bar graphs showing the top 10 gene sets out of 580 total gene sets in the MSigDB C5.CC Gene Ontology Cellular Component collection that overlap with the Cordenonsi YAP Conserved Signature. P values were calculated using the hypergeometric distribution. See also Table S1: GO gene sets. B. GSEA enrichment plots of the GO Anchoring Junction gene set for triplicate samples of LP-1/Cfz versus parental LP-1 cells (top), for TT3 patients with increased TJP1 levels at relapse (middle), and for TT3 patients with decreased TJP1 levels at relapse (bottom). NES, normalized enrichment score. C. Heat map of the leading edge subset of genes in the GO Anchoring Junction gene set upregulated in LP-1/Cfz (Cfz) versus parental LP-1 cells (triplicate samples). Probe sets for TJP1, ITGB5 and RND3 are highlighted. See text for details. D. Bar graphs showing the top 10 gene sets out of 1329 total gene sets in the MSigDB C2.CP Canonical pathways collection that overlap with the GO Anchoring Junction gene set. Note that 5 out of the 10 gene sets involve translational control mechanisms. P values were calculated using the hypergeometric distribution. See also Table S1: C2.CP Canonical pathways.

Figure 6. Enriched MSigDB gene sets in MM patients enrolled on the TT3 protocol with high TJP1 levels at relapse

A. Heat map of TJP1 neighbors for 30 TT3 relapsed samples in the GEO: GSE31161 data set stratified on the basis of TJP1 202011_at probe set levels. Asterisks indicate TJP1-up patients (see Table S1: GSE31161, TT3 samples). B. Enrichment plots of selected gene sets containing genes whose expression is highly correlated with TJP1 expression. NES, normalized enrichment score.

Figure 7. Subcellular localization and knockdown of TJP1 in LP-1/Cfz cells

A. LP-1/Cfz and parental LP-1 cells were cultured on poly-L-lysine-coated microscope slides. Cells were fixed and labeled with anti-TJP1 (Alexa Fluor 488, green) or anti-TAZ (Alexa Fluor 568, red) antibodies. Cell nuclei were stained with TOTO-3 (blue). B. siRNA-mediated knockdown of TJP1 mRNA was accompanied by decreased TJP1 levels (64 ± 4% decrease; n = 3). C. Knockdown of TJP1 mRNA using two specific siRNAs (siTJP1 #1, siTJP1 #2) sensitizes LP-1/Cfz cells to carfilzomib. Cells were cultured on fibronectin-coated plates and treated with carfilzomib (6 nM) for 48 hours after transient transfection; cell viability was determined by alamarBlue assay. *, P = 0.03 vs negative siRNA control (siNeg, n = 3).

Figure 8. Cytotoxic activity of translation inhibitors against LP-1/Cfz cells

A. Cells were treated with the indicated concentrations of emetine (top) or homoharringtonine (bottom) for 48 hours and cell viability was determined by alamarBlue assay. B. Treatment with emetine (0.3 μM) or homoharringtonine (30 nM) for 18 hours decreases TAZ (top) and TEAD1 (bottom) levels in LP-1/Cfz cells. C. Treatment with emetine (0.3 μM) or homoharringtonine (30 nM) for 18 hours decreases Nrf2 (top) and MCL1 (bottom) levels in LP-1/Cfz cells.