Bone Pain Induced by Multiple Myeloma Is Reduced by Targeting V-ATPase and ASIC3

Abstract

Multiple myeloma (MM) patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model which employs JJN3 human MM cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ SN sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons co-cultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Further, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Lastly, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP which was refractory to zoledronic acid. Overall, our results show that osteoclasts and MM cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Further, they present a mechanistic rationale for targeting ASIC3 on neurons along with the MM-induced acidic bone microenvironment as a strategy to relieve MMBP in patients.

Differential Pain‐Related Behaviors and Bone Disease in Immunocompetent Mouse Models of Myeloma

“…Our results are in agreement with previously published reports of bone marrow denervation in a mouse model of osteosarcoma; here, sarcoma‐inoculated mice also showed a decrease in limb use scores that were accompanied by multiple neurochemical changes in the cell bodies of the sensory neurons, potentially as a consequence of tumor‐induced bone marrow denervation. Interestingly, the only available report describing the sensory innervation of myeloma‐bearing femurs shows neuronal sprouting parallel to disease development, which has also been described in other models of cancer‐induced bone pain . Although it cannot be ruled out that myeloma‐inoculated mice also presented nerve sprouting prior to spinal compression and paraplegia development, our results suggest that pain‐related behaviors were present during the last 4 to 5 experimental days, suggesting that the denervation process had already been initiated in parallel to the development of pain‐related behavior.…”

“…Although many preclinical models of MM are available, (36) currently only one study by Hiasa and colleagues (14) has addressed pain-related behaviors, but their results are mainly based on a xenograft myeloma model, which uses immunocompromised mice. However, mounting evidence has shown that the immune system plays a pivotal role in the transition from acute to chronic pain, (41,42) suggesting that the validity of xenograft models is compromised.…”

“…Although several preclinical models of MM have been developed over the years with the goal of understanding disease etiology and identifying novel drug targets, only one xenograft mouse model of MM has been evaluated in terms of myelomainduced bone pain. (14) Xenograft models, however, rely on the use of immunosuppressed mice, whereas MM is a plasma cell cancer and mounting evidence suggests that the immune system plays a pivotal role in chronic pain development. (15)(16)(17)(18) Thus, there is a clear need for a superior model of myeloma-induced bone pain that integrates the immune component.…”

Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

“…While the control treatment, supplemented with NGF, did not produce a significant effect in the MFR, the DRG treated with secretome from resorbing osteoblasts (BS) presented a highly significative effect. Previous reports have shown that medium acidification and the molecules released by osteoclasts and matrix itself, upon matrix degradation, contribute to the activation of nociceptors on the nerve terminals (Hiasa et al, 2017; Yoneda et al, 2015a). Here we show that the secretome from mature, non-resorbing, osteoclasts, also contribute to the electrical activation and signal propagation across sensory neurons.…”

Naked mole-rat acid-sensing ion channel 3 forms nonfunctional homomers, but functional heteromers

“…Understanding the structure-function of ASIC3 is of particular interest because there is substantial evidence supporting involvement of ASIC3 in pain (9,(23)(24)(25)(26)(27)(28), as well itch (29), mechanosensation (23,30) and anxiety (31). Although ASIC3 expression was initially thought to be restricted to the peripheral nervous system (and hence its original name, dorsal root ganglia acid-sensing ion channel, DRASIC (32)), we and others have demonstrated that ASIC3 is also expressed in both the spinal cord and numerous brain regions (33,34), which makes it even more important to understand how ASIC3 functions because any potential drug that targets ASIC3 for the treatment of pain may also produce side effects with the central nervous system.…”