A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.     

Proteasome inhibitor lactacystin augments natural killer cell cytotoxicity of myeloma via downregulation of HLA class I

Modulation of inhibitory and activating natural killer (NK) receptor ligands on tumor cells represents a promising therapeutic approach against cancer, including multiple myeloma (MM). Human leukocyte antigen (HLA) class I molecules, the NK cell inhibitory killer cell immunoglobulin-like receptor (KIR) ligands, are critical determinants of NK cell activity. Proteasome inhibitors have demonstrated significant anti-myeloma activity in MM patients. In this study, we evaluated the effect of proteasome inhibitors on the surface expression of class I in human MM cells. We found that proteasome inhibitors downregulated surface expression of class I in a dose- and time-dependent manner in MM cell line and patient MM cells. No significant changes in the expression of the MHC class I chain-related molecules (MIC) A/B and the UL16-binding proteins (ULBPs) 1–3 were observed. Downregulation of class I by lactacystin (LAC) significantly enhances NK cell-mediated lysis of MM. Furthermore, the downregulation degree of class I was associated with increased susceptibility of myeloma cells to NK cell killing. HLA blocking antibody produced results that were similar to the findings from proteasome inhibitor. Taken together, our data suggest that proteasome inhibitors, possible targeting inhibitory KIR ligand class I on tumor cells, may contribute to the activation of cytolytic effector NK cells in vitro, enhancing their anti-myeloma activity. Our findings provide a rationale for clinical evaluation of proteasome inhibitor, alone or in combination, as a novel approach to immunotherapy of MM.     

Proteasome inhibitors – molecular basis and current perspectives in multiple myeloma

Inhibition of proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, has emerged as a powerful strategy for treatment of multiple myeloma (MM), a plasma cell malignancy. First‐in‐class agent, bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in pre‐clinical and in clinical studies. However, despite its high efficiency, a large proportion of patients do not achieve sufficient clinical response. Therefore, the development of a second‐generation of proteasome inhibitors (PIs) with improved pharmacological properties was needed. Recently, several of these new agents have been introduced into clinics including carfilzomib, marizomib and ixazomib. Further, new orally administered second‐generation PI oprozomib is being investigated. This review provides an overview of main mechanisms of action of PIs in MM, focusing on the ongoing development and progress of novel anti‐proteasome therapeutics.     

A drug repurposing strategy for overcoming human multiple myeloma resistance to standard-of-care treatment

Despite several approved therapeutic modalities, multiple myeloma (MM) remains an incurable blood malignancy and only a small fraction of patients achieves prolonged disease control. The common anti-MM treatment targets proteasome with specific inhibitors (PI). The resulting interference with protein degradation is particularly toxic to MM cells as they typically accumulate large amounts of toxic proteins. However, MM cells often acquire resistance to PIs through aberrant expression or mutations of proteasome subunits such as PSMB5, resulting in disease recurrence and further treatment failure. Here we propose CuET—a proteasome-like inhibitor agent that is spontaneously formed in-vivo and in-vitro from the approved alcohol-abuse drug disulfiram (DSF), as a readily available treatment effective against diverse resistant forms of MM. We show that CuET efficiently kills also resistant MM cells adapted to proliferate under exposure to common anti-myeloma drugs such as bortezomib and carfilzomib used as the first-line therapy, as well as to other experimental drugs targeting protein degradation upstream of the proteasome. Furthermore, CuET can overcome also the adaptation mechanism based on reduced proteasome load, another clinically relevant form of treatment resistance. Data obtained from experimental treatment-resistant cellular models of human MM are further corroborated using rather unique advanced cytotoxicity experiments on myeloma and normal blood cells obtained from fresh patient biopsies including newly diagnosed as well as relapsed and treatment-resistant MM. Overall our findings suggest that disulfiram repurposing particularly if combined with copper supplementation may offer a promising and readily available treatment option for patients suffering from relapsed and/or therapy-resistant multiple myeloma.Subject terms: Myeloma, Cancer therapeutic resistance     

Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

Carfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.Subject terms: Myeloma, Drug development     

Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.     

Non‐lethal proteasome inhibition activates pro‐tumorigenic pathways in multiple myeloma cells

Multiple myeloma (MM) is a haematological malignancy being characterized by clonal plasma cell proliferation in the bone marrow. Targeting the proteasome with specific inhibitors (PIs) has been proven a promising therapeutic strategy and PIs have been approved for the treatment of MM and mantle‐cell lymphoma; yet, while outcome has improved, most patients inevitably relapse. As relapse refers to MM cells that survive therapy, we sought to identify the molecular responses induced in MM cells after non‐lethal proteasome inhibition. By using bortezomib (BTZ), epoxomicin (EPOX; a carfilzomib‐like PI) and three PIs, namely Rub999, PR671A and Rub1024 that target each of the three proteasome peptidases, we found that only BTZ and EPOX are toxic in MM cells at low concentrations. Phosphoproteomic profiling after treatment of MM cells with non‐lethal (IC₁₀) doses of the PIs revealed inhibitor‐ and cell type‐specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokine/chemokine profiling revealed the increased secretion of immunosuppressive pro‐tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T cell chemoattractant chemokines (CXCL10). These findings indicate that MM cells that survive treatment with therapeutic PIs shape a pro‐tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse.     

High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma

In multiple myeloma (MM), malignant plasma cells produce large amounts of antibodies and have highly active protein translational machinery. It is not known whether regulation of the abundance and aminoacylation (charging) of transfer RNA (tRNA) takes place in myeloma cells to accommodate for the increased amount of protein translation. Using tRNA-specific microarrays, we demonstrate that tRNA levels are significantly elevated in MM cell lines compared to normal bone marrow cells. We furthermore show that the addition of the proteasome inhibitor, bortezomib (Velcade™, PS-341) results in decreased charging levels of tRNAs, in particular those coding for hydrophobic amino acids. These results suggest that tRNA properties are altered in MM to accommodate for its increased need for protein translation, and that proteasome inhibition directly impacts protein synthesis in MM through effects on tRNA charging.     

Multitargeted therapies for multiple myeloma

Multiple myeloma (MM) comprises 1% of all malignancies and 10% of all hematological malignancies. MM is a malignancy of plasma cells in the bone marrow where complex and dynamic interactions with the bone marrow microenvironment lead to tumor progression, skeletal destruction and angiogenesis. Despite the discovery of several novel treatments against MM, including the proteasome inhibitor bortezomib, it is considered to be an incurable disease with an average 4–5 years overall survival.     

Molecular mechanisms of bortezomib resistant adenocarcinoma cells

Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.     

The fatty acid transport function of fatty acid-binding proteins

The intracellular fatty acid-binding proteins (FABPs) comprise a family of 14-15 kDa proteins which bind long-chain fatty acids. A role for FABPs in fatty acid transport has been hypothesized for several decades, and the accumulated indirect and correlative evidence is largely supportive of this proposed function. In recent years, a number of experimental approaches which more directly examine the transport function of FABPs have been taken. These include molecular level in vitro modeling of fatty acid transfer mechanisms, whole cell studies of fatty acid uptake and intracellular transfer following genetic manipulation of FABP type and amount, and an examination of cells and tissues from animals engineered to lack expression of specific FABPs. Collectively, data from these studies have provided strong support for defining the FABPs as fatty acid transport proteins. Further studies are necessary to elucidate the fundamental mechanisms by which cellular fatty acid trafficking is modulated by the FABPs.     

Novel cell line models to study mechanisms and overcoming strategies of proteasome inhibitor resistance in multiple myeloma

Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.     

Regulation of CD38 on Multiple Myeloma and NK Cells by Monoclonal Antibodies

CD38 is highly expressed on multiple myeloma (MM) cells and plays a role in regulating tumor generation and development. CD38 monoclonal antibodies (mAbs) have been used as an effective therapy for MM treatment by various mechanisms, including complement-dependent cytotoxic effects, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, enzymatic modulation, and immunomodulation. Although CD38 mAbs inhibit the proliferation and survival of MM cells, there are substantial side effects on antitumoral NK cells. The NK-mediated immune response needs to be further evaluated to minimize the adverse effects of NK cell loss. The killing effect of CD38 mAbs on CD38^high NK cells should be minimized and the potential combination of CD38^low/- NK cells and CD38 mAbs should be maximized to better benefit from their therapeutic efficacy against MM. CD38 mAb effects against MM can be maximized by combination therapies with immunomodulatory imide drugs (IMiDs), proteasome inhibitors (PIs), anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) antibodies, or cellular therapies for the treatment of MM, especially in patients with relapsed or refractory MM (R/R MM) and drug-resistant MM.     

Pirh2 mediates the sensitivity of myeloma cells to bortezomib via canonical NF-κB signaling pathway

Clinical success of the proteasome inhibitor established bortezomib as one of the most effective drugs in treatment of multiple myeloma (MM). While survival benefit of bortezomib generated new treatment strategies, the primary and secondary resistance of MM cells to bortezomib remains a clinical concern. This study aimed to highlight the role of p53-induced RING-H2 (Pirh2) in the acquisition of bortezomib resistance in MM and to clarify the function and mechanism of action of Pirh2 in MM cell growth and resistance, thereby providing the basis for new therapeutic targets for MM. The proteasome inhibitor bortezomib has been established as one of the most effective drugs for treating MM. We demonstrated that bortezomib resistance in MM cells resulted from a reduction in Pirh2 protein levels. Pirh2 overexpression overcame bortezomib resistance and restored the sensitivity of myeloma cells to bortezomib, while a reduction in Pirh2 levels was correlated with bortezomib resistance. The levels of nuclear factorkappaB (NF-κB) p65, pp65, pIKBa, and IKKa were higher in bortezomib-resistant cells than those in parental cells. Pirh2 overexpression reduced the levels of pIKBa and IKKa, while the knockdown of Pirh2 via short hairpin RNAs increased the expression of NF-κB p65, pIKBa, and IKKa. Therefore, Pirh2 suppressed the canonical NF-κB signaling pathway by inhibiting the phosphorylation and subsequent degradation of IKBa to overcome acquired bortezomib resistance in MM cells.     

Historic overview of studies on fatty acid-binding proteins

Summary Fatty acid-binding proteins (FABPs) were first identified in the cytosol of rat intestinal mucosa during studies on the regulation of intestinal fatty acid uptake. The subsequent finding of FABP activity in the cytosol of many other tissues initially was believed to reflect a single protein. However, the FABPs are now recognized as products of an ancient gene family comprised of at least 9 structurally related, soluble intracellular members, a number of which exhibit high-affinity binding of long-chain fatty acids. Despite recent insights into regulation and tissue-specific expression suggesting FABPs to subserve diverse roles, their precise biological functions remain to be elucidated.     

Characterization and binding properties of fatty acid-binding proteins from human, pig, and rat heart

Fatty acid-binding proteins (FABPs) were isolated from the cytosols of hearts of man, pig, and rat by gel filtration and anion-exchange chromatography. The heart FABPs had a Mr of about 15,000 (pig, rat) and 15,500 (man); pI values were 5.2, 4.9, and 5.0 for human, pig, and rat heart, respectively. In contrast to liver FABPs, tryptophan was present in the heart FABPs. Binding characteristics for long-chain fatty acids determined with the radiochemical Lipidex assay were comparable for all three proteins. Heart FABPs also bind palmitoyl-CoA and -carnitine with an affinity comparable to that for palmitic acid. Other ligands investigated, heme, bilirubin, cholesterol, retinoids, and prostaglandins, could not compete with oleic acid for binding by human heart FABP. Binding parameters of FABP for oleic acid from multilamellar liposomes were comparable to those from the Lipidex binding assay. Immunological interspecies cross-reactivity with antisera against the heart FABPs was much higher between man and pig than between rat and man or pig. None of the antisera reacted with liver FABPs. The IgG fraction of anti-human heart FABP serum inhibited fatty acid binding to human heart FABP.