Inhibition of the ubiquitin-proteasome pathway through the use of proteasome inhibitors (PIs) has been validated by our group and others as a successful strategy against multiple myeloma that has improved patient outcomes. However, these agents are currently used without patient selection, as no biomarkers have been validated that identify patients most or least likely to benefit. Also, drug resistance emerges in the vast majority through largely undefined mechanisms, and limits the activity of further therapy based on PIs. There is therefore an urgent need to identify such biomarkers, especially if they could also represent novel therapeutic targets to achieve resensitization.
We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. In addition, using the Lentiviral GeneNet™ small hairpin (sh) RNA Library, we performed genome-wide RNA interference (RNAi) to identify genes whose knockdown conferred resistance. Genes of interest were subjected to further validation using myeloma cell lines,primary samples, murine models, and using clinically annotated GEP databases. These studies were supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma.
Bortezomib resistance was associated with decreased expression of TJP1 by GEP studies of isogenic bortezomib-resistant and -sensitive cell lines. TJP1 was also identified as a chemoresistance factor by RNA interference designed to detect genes that conferred a survival advantage after drug treatment. Suppression of TJP1 using shRNAs in RPMI 8226 and U266 myeloma cell lines with high TJP1 expression reduced sensitivity to both bortezomib and carfilzomib. Conversely, its over-expression in MOLP-8 cells, which had low TJP1 levels, conferred enhanced sensitivity to both PIs. Also, forced expression of TJP1 in bortezomib-resistant RPMI 8226 cells that had lost endogenous TJP1 levels restored drug sensitivity. In these resistant cells, TJP1 promoter hypermethylation was found, and treatment with decitabine restored both TJP1 expression, and sensitivity to bortezomib or carfilzomib. GEP studies showed TJP1 suppression was associated with enhanced expression of MHC class II region genes, including PSMB8 and PSMB9. This was mirrored at the protein level by enhanced PSMB8 and PSMB9 protein by Western blotting. As a result, TJP1 suppression was associated with increased activity of the chymotrypsin-like activity of the proteasome, while TJP1 overexpression reduced proteasome activity. A link between TJP1 and PSMB8 and 9 was supported by studies showing that TJP1 influenced activity of EGFR and STAT3 and indeed, EGFR inhibition with erlotinib enhanced PI sensitivity. Consistent with a role for TJP1 in vivo, treatment of mice with bortezomib showed a greater reduction of myeloma growth in tumors with high TJP1 expression compared with isogenic lines with low TJP1 expression. Finally, analysis of the Millennium Pharmaceuticals database of bortezomib studies in the relapsed and relapsed/refractory settings showed high TJP1 expression was associated with a greater likelihood of responding to bortezomib (p<0.0002), and a longer median overall survival (OS)(p=0.008). In addition, in the Total Therapy (TT) databases, higher TJP1 expression was associated with a better progression-free and OS in both TT3a (p=0.004 and <0.0001, respectively), and TT3b (p=0.001 and <0.0001).
Taken together, these data support the hypothesis that TJP1 modulates PI sensitivity in myeloma through effects on the proteasome’s protein turnover capacity, thereby tilting the load versus capacity balance in favor of cell death. Also, they indicate that strategies targeting TJP1 signaling should be studied as approaches to overcome both primary and secondary resistance. Finally, they support the possibility that TJP1 could be a useful biomarker to identify patients who are most likely to benefit from PI-based therapies, and prospective studies to validate this further are currently underway.
Celgene: Consultancy, Research Funding, Speakers Bureau; Onyx: Research Funding, Speakers Bureau. Orlowski:Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Resverlogix: Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity’s Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.