The Mucolipin TRPML2 Channel Enhances the Sensitivity of Multiple Myeloma Cell Lines to Ibrutinib and/or Bortezomib Treatment

Multiple myeloma (MM) is a haematological B cell malignancy characterised by clonal proliferation of plasma cells and their accumulation in the bone marrow. The aim of the present study is the evaluation of biological effects of Ibrutinib in human MM cell lines alone or in combination with different doses of Bortezomib. In addition, the relationship between the expression of TRPML2 channels and chemosensitivity of different MM cell lines to Ibrutinib administered alone or in combination with Bortezomib has been evaluated. By RT-PCR and Western blot analysis, we found that the Ibrutinib-resistant U266 cells showed lower TRPML2 expression, whereas higher TRPML2 mRNA and protein levels were evidenced in RPMI cells. Moreover, TRPML2 gene silencing in RPMI cells markedly reverted the effects induced by Ibrutinib alone or in combination with Bortezomib suggesting that the sensitivity to Ibrutinib is TRPML2 mediated. In conclusion, this study suggests that the expression of TRPML2 in MM cells increases the sensitivity to Ibrutinib treatment, suggesting for a potential stratification of Ibrutinib sensitivity of MM patients on the basis of the TRPML2 expression. Furthermore, studies in vitro and in vivo should still be necessary to completely address the molecular mechanisms and the potential role of TRPML2 channels in therapy and prognosis of MM patients.

Bortezomib Rescues Ovariectomy-Induced Bone Loss via SMURF-Mediated Ubiquitination Pathway

A balance between bone formation by osteoblasts and bone resorption by osteoclasts is necessary to maintain bone health and homeostasis. As a cancer of plasma cells, multiple myeloma (MM) is accompanied with rapid bone loss and fragility fracture. Bortezomib has been used as a first-line for treating MM for decades. Recently, the potential protection of bortezomib on osteoporosis (OP) is reported; however, the specific mechanism involving bortezomib-mediated antiosteoporotic effect is undetermined. In the present study, we assessed the effects of in vitro bortezomib treatment on osteogenesis and osteoclastogenesis and the protective effect on bone loss in ovariectomized (OVX) mice. Our results indicated that bortezomib treatment increased osteogenic differentiation of MC3T3-E1 cells as evidenced by increased levels of matrix mineralization and osteoblast-specific markers. In bortezomib-treated bone marrow monocytes (BMMs), osteoclast differentiation was suppressed, substantiated by downregulated tartrate-resistant acid phosphatase- (TRAP-) positive multinucleated cells, areas of actin rings, pit formation, and osteoclast-specific genes. Mechanistically, bortezomib exerted a protective effect against OP through the Smad ubiquitination regulatory factor- (SMURF-) mediated ubiquitination pathway. Furthermore, in vivo intraperitoneal injection of bortezomib attenuated the bone microarchitecture in OVX mice. Accordingly, our findings corroborated that bortezomib might have future applications in the treatment of postmenopausal OP.

MCT1 is a Predictive Marker for Lenalidomide Maintenance Therapy in Multiple Myeloma

Biomarkers that predict response to lenalidomide maintenance therapy in patients with multiple myeloma (MM) have remained elusive. We have shown that IMiDs exert anti-MM activity via destabilization of MCT1 and CD147. Here, samples of 654 patients receiving lenalidomide (n=455), thalidomide (n=98) or bortezomib (n=101) maintenance were assessed using gene expression profiling and RNA-sequencing, followed by correlation of MCT1 and CD147 expression with progression-free (PFS) and overall survival (OS) data. Patients with high gene expression levels of MCT1 showed significantly reduced PFS (31.9 vs. 48.2 months in MCT1high vs. MCT1low, P=.03) and OS (75.9 months vs. not reached (NR) months in MCT1high vs. MCT1low; P=.001) in case of lenalidomide maintenance, whereas MCT1 expression had no significant impact on PFS or OS in patients with bortezomib maintenance. We validated the predictive role of MCT1 for IMiD-based maintenance in an independent cohort of patients receiving thalidomide (OS 83.6 months vs. NR in MCT1high vs. MCT1low; P=.03). Functional validation showed that MCT1 overexpression in human MM cell lines significantly reduced efficacy of lenalidomide, while no change was observed upon bortezomib treatment, both in vitro and in an MM xenograft model. Together, we establish MCT1-expression as a predictive marker for response to lenalidomide-based maintenance treatment.

Deubiquitinase USP9X loss Sensitizes Cancer Cells to mTOR Inhibitors through p62 Dysregulation

BackgroundMutations in MTOR and TSC1/2 can only explain part of variability in mTOR inhibitor response. Here, we performed a comprehensive molecular characterization of tumors with high sensitivity to these drugs to uncover novel mechanisms of response.MethodsChromophobe renal cell carcinoma (chRCC) tumors were analyzed by whole-exome sequencing. Rapamycin sensitivity, expression of mTOR pathway effectors, ubiquitylome analyses to identify USP9X substrates, p62 immunoprecipitation and autophagy assessment through immunofluorescence assays for p62 and LC3 were performed in USP9X depleted HeLa and 786-O cells for mechanistic investigation. ResultsWhole-exome sequencing of chRCC patients with high mTOR inhibitor sensitivity, uncovered USP9X deubiquitinase as the only mutated gene shared by these tumors. The clonal characteristics of the mutations, revealed by studying multiple primary and metastatic samples, together with the low USP9X mutation rate in unselected chRCC series, implied a causal link between USP9X and mTOR inhibitor sensitivity. The high sensitivity was recapitulated in vitro, and while no direct effect on mTORC1 was detected, an unbiased ubiquitylome analysis revealed p62 as a direct USP9X target. Bortezomib treatment, which also led to p62 ubiquitination, increased rapamycin effect. Finally, immunofluorescence assays for p62 and LC3 confirmed dysregulated autophagy in USP9X depleted cells, supporting a synthetic lethal interaction between rapamycin-induced autophagy via mTOR-axis and USP9X loss.ConclusionsOur study uncovers USP9X as a potential novel marker of sensitivity to mTOR inhibitors and suggests the inhibition of this gene as a clinically exploitable strategy able to increase sensitivity to these drugs through a novel p62 regulatory mechanism.

DKK1 activates noncanonical NF-κB signaling via IL-6–induced CKAP4 receptor in multiple myeloma

Proteasome inhibitors, such as bortezomib (BTZ), represent the key elements in chemotherapy regimens for multiple myeloma (MM), whereas acquired chemoresistance and ultimately relapse remain a major obstacle. In the current study, we screened differently expressed cytokines in bortezomib-resistant MM cells and found that Dickkopf-1 (DKK1) level was remarkably augmented, whereas CD138 level was significantly suppressed. DKK1 in vitro specifically enhanced the resistance of myeloma cells to bortezomib treatment, and excessive DKK1 drove CD138 downregulation via inhibition of canonical Wnt signaling. Notably, DKK1 mainly induced drug resistance in MM cells via the receptor of CKAP4. Mechanistically, CKAP4 transduced DKK1 signal and evoked NF-κB pathway through recruiting and preventing cullin associated and neddylation dissociated 1 from hampering the assembly of E3 ligase-mediated ubiquitination of IκBα. In addition, we found that interleukin-6 (IL-6) stimulated CKAP4 expression to generate drug resistance, and disturbance of DKK1-CKAP4 axis improved sensitivity to BTZ treatment of MM and attenuated bone destruction in a mouse model. Collectively, our study revealed the previously unidentified role of DKK1 in myeloma drug resistance via Wnt signaling dependent and independent manners, and clarified the importance of antagonism of DKK1-IL-6 loop in bone marrow microenvironment.

Treatment Induced Cytotoxic T-Cell Modulation in Multiple Myeloma Patients

The biology of plasma cell dyscrasias (PCD) involves both genetic and immune-related factors. Since genetic lesions are necessary but not sufficient for Multiple Myeloma (MM) evolution, several authors hypothesized that immune dysfunction involving both B and T cell counterparts plays a key role in the pathogenesis of the disease. The aim of this study is to evaluate the impact of cornerstone treatments for Multiple Myeloma into immune system shaping. A large series of 976 bone marrow samples from 735 patients affected by PCD was studied by flow analysis to identify discrete immune subsets. Treated MM samples displayed a reduction of CD4+ cells (p<0.0001) and an increase of CD8+ (p<0.0001), CD8+/DR+ (p<0.0001) and CD3+/CD57+ (p<0.0001) cells. Although these findings were to some extent demonstrated also following bortezomib treatment, a more pronounced cytotoxic polarization was shown after exposure to autologous stem cell transplantation (ASCT) and Lenalidomide (Len) treatment. As a matter of fact, samples of patients who received ASCT (n=110) and Len (n=118) were characterized, towards untreated patients (n=138 and n=130, respectively), by higher levels of CD8+ (p<0.0001 and p<0.0001, respectively), CD8+/DR+ (p=0.0252 and p=0.0001, respectively) and CD3+/CD57+ cells (p<0.0001 and p=0.0006, respectively) and lower levels of CD4+ lymphocytes (p<0.0001 and p=0.0005, respectively). We demonstrated that active MM patients are characterized by a relevant T cell modulation and that most of these changes are therapy-related. Current Myeloma treatments, notably ASCT and Len treatments, polarize immune system towards a dominant cytotoxic response, likely contributing to the anti-Myeloma effect of these regimens.

Bortezomib for anti-NMDAR encephalitis following daclizumab treatment in a patient with multiple sclerosis

BackgroundDaclizumab is an anti-CD25 monoclonal antibody developed for the treatment of relapsing remitting multiple sclerosis, which was withdrawn worldwide in March 2018, due to emerging serious immune-mediated systemic andcentral nervous system adverse events. We report a case of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis occurring 14 weeks after stopping daclizumab, which responded to the proteasome inhibitor bortezomib.MethodsFollowing lack of effective clinical response to first line (corticosteroid, plasma exchange, intravenous immunoglobulin) and second line (rituximab) treatments, bortezomib therapy was commenced. The patient received six cycles of bortezomib treatment.ResultsClinical improvement was noted 4 weeks after the first of six cycles of bortezomib and the patient experienced sustained clinical improvement.ConclusionOur case provides further class IV evidence of the use of bortezomib therapy for treatment refractory anti-NMDAR encephalitis.

Prevalence of Chemotherapy-Induced Peripheral Neuropathy in Multiple Myeloma Patients and its Impact on Quality of Life: A Single Center Cross-Sectional Study

Bortezomib is a pivotal drug for the management of multiple myeloma. However, bortezomib is a neurotoxic anticancer drug responsible for chemotherapy-induced peripheral neuropathy (CIPN). CIPN is associated with psychological distress and a decrease of health-related quality of life (HRQoL), but little is known regarding bortezomib-related CIPN. This single center, cross-sectional study assessed the prevalence and severity of sensory/motor CIPN, neuropathic pain and ongoing pain medications, anxiety, depression, and HRQoL, in multiple myeloma patients after the end of bortezomib treatment. Paper questionnaires were sent to patients to record the scores of sensory and motor CIPNs (QLQ-CIPN20), neuropathic pain (visual analogue scale and DN4 interview), anxiety and depression (HADS), the scores of HRQoL (QLQ-C30 and QLQ-MY20) and ongoing pain medications. Oncological data were recorded using chemotherapy prescription software and patient medical records. The prevalence of sensory CIPN was 26.9% (95% CI 16.7; 39.1) among the 67 patients analyzed and for a mean time of 2.9 ± 2.8 years since the last bortezomib administration. The proportion of sensory CIPN was higher among patients treated by intravenous and subcutaneous routes than intravenous or subcutaneous routes (p = 0.003). QLQ-CIPN20 motor scores were higher for patients with a sensory CIPN than those without (p < 0.001) and were correlated with the duration of treatment and the cumulative dose of bortezomib (coefficient: 0.31 and 0.24, p = 0.01 and 0.0475, respectively), but not sensory scores. Neuropathic pain was screened in 44.4% of patients with sensory CIPN and 66.7% of them had ongoing pain medications, but none were treated with duloxetine (recommended drug). Multivariable analysis revealed that thalidomide treatment (odds-ratio: 6.7, 95% CI 1.3; 35.5, p = 0.03) and both routes of bortezomib administration (odds-ratio: 13.4, 95% CI 1.3; 139.1, p = 0.03) were associated with sensory CIPN. Sensory and motor CIPNs were associated with anxiety, depression, and deterioration of HRQoL. Sensory CIPN was identified in a quarter of patients after bortezomib treatment and associated with psychological distress that was far from being treated optimally. There is a need to improve the management of patients with CIPN, which may include better training of oncologists regarding its diagnosis and pharmacological treatment.

Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

Development of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.