Blocking of Transient Receptor Potential Vanilloid1 (TRPV1) Promotes Lysosomal Destabilization and Enhances Bortezomib-Induced ER Stress and Cell Death Via HSP70 and LAMP3 Down-Regulation: Novel Therapeutic Target for Multiple Myeloma
Authors: Beider Katia , Voevoda Valeria , Bitner Hanna , Rosenberg Evgenia , Sirovsky Yaarit , Magen Hila , Ostrovsky Olga , Shimoni Avichai , Bromberg Zohar , Peled Amnon , Nagler Arnon 
Published: April 2021

Abstract
Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Acquired or de novo resistance to current anti-MM therapy remains a major treatment obstacle. Novel new therapies are thus in need. Recent data have highlighted the contribution of Ca2+channels in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. As no data is available evaluating TRPV1 in MM, the aim of the current study was to evaluate its possible role in MM.
Results: Elevated levels of TRPV1 transcript was detected in MM cell lines (n=8) and BM aspirates from MM patients (n=24) in comparison to normal BM (n=5). AMG9810 a specific antagonist of TRPV1, significantly reduced the viability of MM cell lines (n=8) and primary CD138+ cells (n=6),in a time- and dose-dependent manner (p&lt;0.01) and induced apoptosis manifested by phosphatidylserine externalization, loss of mitochondrial membrane potential (ψm), caspase 3 cleavage and DNA fragmentation. AMG9810-triggered apoptosis could be partially blocked by inhibition of calpains and cathepsins, indicating the role of lysosomal rapture in AMG9810-mediated cell death. Indeed, treatment with TRPV1 antagonist induced rapid lysosomal acidification and increased the number of acidic vesicles (detected by acridine orange stain). The acidic vesicles appeared as early as 1 hour post exposure to AMG9810 preceding the mitochondrial destabilization and apoptosis, thus suggesting that TRPV1 blockade induces lysosomal-induced cell death in MM. Furthermore, TRPV1 inhibition with AMG9810 completely suppressed the pro-survival AKT/mTOR pathway and significantly reduced the levels of anti-apoptotic factors BCL-2 and BCL-XL. Combining AMG9810 with the proteasome inhibitor bortezomib (Bort) induced synergistic cell death in both native and Bort-resistant cells (CI&lt;0.4). Moreover, TRPV1 inhibition successfully overcame the CXCR4-mediated protection from Bort provided by BM stromal cells. This finding suggests that the TRPV1 channel may regulate the activity of CXCR4 chemokine receptor in MM cells affecting the MM-microenvironment interactions. In accordance, the TRPV1 antagonist AMG8910 prevented the responsiveness of CXCR4-expressing MM cells to CXCL12 stimulation, decreased the phosphorylation of signaling mediators like Erk1/2 and AKT and suppressed cell migration, while TRPV1 activator capsaicin promoted the CXCR4-mediated signaling and migration. Gene and protein expression analysis were next performed to delineate the molecular mechanisms underlying the observed synergism between Bort and AMG9810. Bort treatment resulted in robust induction of endoplasmic reticulum (ER) stress genes including the increase in pro-apoptotic factors ATF4, CHOP and GADD34. Compensatory unfolded protein response (UPR) was activated as well, with increase in chaperons HSP27, HSP70, HSP90, and lysosomal chaperon LAMP3 known to stabilize lysosome, protecting cells against lysosomal membrane permeabilization (LMP) and subsequent cell death. AMG9810 further increased ER stress, elevating CHOP and GADD34 expression, while significantly reducing both basal and Bort-increased levels of HSP70 and LAMP3, thus overcoming the protective response to Bort treatment and prompting lethal LMP. Finally, combining Bort with AMG9810 resulted in significantly reduced ROS that was correlated with impaired mitochondria and increased MM apoptosis, suggesting that dissipation of intracellular ROS may be involved in AMG9810-promoted cytotoxicity.
Conclusions: Altogether, our data indicate that TRPV1 is implicated in MM cell survival, proliferation, migration, microenvironment interactions and stress response. TRPV1 inhibition by AMG9810 inhibits CXCR4-mediated migration and stromal protection, synergizes with Bort, amplifies ER stress, targets cytoprotective HSP70 and LAMP3, destabilizes lysosome, impairs mitochondria and promotes MM cell death. These results unravel the mechanism mediating the strong synergistic anti-MM activity of Bort in combination with TRPV1 inhibition which may be translated into the clinic.

Disclosures
Peled: Biokine: Consultancy; Biosight: Consultancy.