IL METABOLISMO MITOCONDRIALE NELLE CELLULE DI MIELOMA MULTIPLO

Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of tumour plasma cells in the bone marrow (BM). The uncontrolled proliferation of terminally differentiated plasma cells is associated with CRAB symptoms, including hypercalcemia, renal impairment, anemia, and osteolytic bone lesions. The bidirectional interaction between MM cells and surrounding cells regulates immune-editing, transforming the microenvironment into a tumor-promoting and immune-suppressive milieu mediating tumor growth, progression, and cell adhesion [2] MM is preceded by an asymptomatic phase (monoclonal gammopathy of unknown significance, MGUS) and (smoldering myeloma, SMM), which are characterized by progressive accumulation of plasma cells, from less to more than 10% in the BM. The survival of MM patients has improved with the introduction of proteasome inhibitors (PI) and immunomodulatory drugs. However, MM remains incurable, and relapses and disease progressions are common among MM patients (1). Malignant cells can use altered pathways and mitochondrial functions, allowing them to escape apoptosis (1). Mitochondria are both bioenergetic organelles and necessary cellular stress sensors that contribute to the tumor microenvironment (2). In addition to ATP production, these organelles also regulate cell death and processes following the generation of reactive oxygen species (ROS) or redox molecules. It is well showed that mitochondria could drive metabolic cancer cell reprogramming. In this regard, the adaptative metabolic changes in MM cells are emerging as the basis for proteasome inhibitor (PI) resistance. Indeed, increased expression of proteins involved in redox and energy metabolism has been found in resistant cells to the first-in-class PI bortezomib (3-5). Cancer cells often display a metabolic adaptation to maintain high ATP levels, increasing the efficiency of mitochondria. Furthermore, many malignant tumor cells can use functional mitochondria depending on mitochondrial respiration (6). PI resistant phenotype shows an increase in mitochondrial biomass and mitochondrial respiration (7). Several aspects of mitochondrial bioenergetics, such as mitochondrial biogenesis and mitochondrial fitness, support the ability to respond to stress, allowing survival under adverse conditions such as chemotherapeutic treatments. The aim of our study is to demonstrate that bortezomib resistance in MM cells is related to mitochondrial energy metabolism and robust antioxidant defences. MM cell strength is associated with a remarkably high mitochondrial activity in mitochondrial dynamism and mitochondrial fitness, which may be a potential target for overcoming BTZ resistance. To test this hypothesis, we first looked at metabolic adaptation to bortezomib (BTZ) exposure of neoplastic plasma cells, disclosing that MM cells have high mitochondrial mass, which can be exchanged with the external microenvironment, and conveys reshaping of both redox and energy metabolism. Mitochondrial functions and energy metabolism are interconnected processes mediating resistance to bortezomib in multiple myeloma cells Our group has recently shown that the protective effect of heme oxygenase-1 (HO-1) on drug-induced cytotoxicity in leukemic cells does not involve its enzymatic byproducts, but rather its nuclear translocation following proteolytic cleavage (8). Interestingly, nuclear HO-1 was implicated as a regulator of DNA repair activities relevant to carcinogenesis (9) and tumor progression (10). HO-1 recruitment, which involves the Toll-like receptor 4 (TLR4) signalling, could protect mitochondria during BTZ-induced ER stress, confirming that this molecular machinery required for BTZ resistance via mitochondria is triggered by a stress-responsive mechanism (Giallongo 2019). Furthermore, it has been shown that the upregulation of HO-1 is involved in promoting mitochondrial biogenesis, permitting intrinsic oxidative stress adaptation. Reactive oxygen species (ROS) adaptation and mitochondrial biogenesis appeared to form a self- amplifying feedback loop in chronic lymphocytic leukemia (CLL) (11). Therefore, both TLR4 and HO-1 may represent targets for a more selective treatment in relapsed/refractory patients. Our data support the idea that BTZ resistance could not be associated with proteasome activity. As proteasome inhibition activates the unfolded protein response (UPR) and ER stress, regulating mitochondrial morphology (100), we evaluated whether BTZ resistance in U266-R was mediated by increased values of different mitochondrial mass and function parameters. First, by exposure to progressively higher concentrations of BTZ, reaching strength to higher doses of the drug (EC50 value:100 nM), we developed a BTZ-resistant cell line (U266-R) to evaluate resistant clone characteristics, including their metabolic phenotype. Data show that the BTZ was able to induce mitochondrial damage in BTZ-sensitive cell line, while the resistant counterpart required a metabolic rewiring due to a higher concentration of substrates for protein glycosylation (Giallongo, 2020 data in press). Second, we evaluated mitochondrial fitness, and redox balance homeostasis capacity in BTZ- sensitive and resistant cells. We found that in the BTZ-resistant clone (U266-R) mitochondrial biogenesis was increased and mitochondrial dynamics was associated with the acquisition of stronger antioxidant defences. Taken together, these findings have suggested that mitochondrial fitness can induce BTZ resistance in MM cells, representing a possible target for new drug development for BTZ-resistant patients. TLR4 signalling affects mitochondrial fitness and overcomes bortezomib resistance in myeloma plasma cells It has been recently demonstrated that the TLR4 pathway provides a protective effect against BTZ-induced ER stress, and pre-treatment of MM cells with the TLR4 trigger lipopolysaccharide (LPS) significantly reduces BTZ-induced apoptosis (12). The endoplasmic reticulum (ER) and mitochondria are in close contact, which impacts mitochondrial division and fusion dynamics (13), influencing mitochondrial ATP production and apoptosis (12). ER stress-induced apoptosis engages mitochondrial depolarization, cytochrome c release, and its downstream caspase-9 activation. Considering the relationship between PI, ER stress, and mitochondria, in the attempt to elucidate the mechanisms underlying the mitochondria adaptation in response to BTZ exposure, we explored the TLR4 signalling. First, we found that BTZ treatment increased TLR4 expression as a stress-responsive mechanism to protect mitochondria during BTZ-induced mitochondrial depolarization. BTZ-resistant cells improved their bioenergetics, as shown by increase of ECP value and mitochondrial mass, suggesting the cooperation between TLR4 /HO-1axis, the adenylate system (AMP-activated kinases) and cellular redox homeostasis. Second, we hypothesized that MM cells might activate TLR4 signalling after BTZ exposure as a stress-responsive mechanism to protect mitochondria against BTZ-induced apoptosis. Triggering TLR4 was disposable to increase mitochondrial mass in human MM cell lines (HMCLs: U266, MM1.S, OPM2, NCI-H929) and induced up-regulation of mitochondrial biogenesis markers (PGC1a, PRC, and TFAM). After treatment with BTZ for 24h, all HMCLs over-expressed TLR4 signalling, as shown by the consensual up-regulation of MyD88 and MAPK activation. Compared to BTZ-sensitive cells, U266- R had higher levels of TLR4, p-p38, and p-ERK proteins and higher mitochondrial mass. The genetic ablation or selective pharmacological TLR4 inhibition induced cell apoptosis, resulting from the deleterious effects of oxidative stress combined to mitochondrial depolarization, a dramatic drop in mitochondrial respiration, decrease in ATP production, consequent accumulation of AMP and a decreased NAD+/NADH and NADP+/NADPH ratio, and mitophagy (selective autophagy of damaged mitochondria). Third, clinically relevant, to design more efficient treatments for bortezomib-refractory MM patients, the combination of bortezomib and TLR4 inhibitor was selectively cytotoxic for CD138+ plasma cells, preserving healthy cells. Thus, since TLR4 regulates mitochondrial biogenesis and BTZ-resistant cells rely on mitochondrial fitness, a link among TLR4 up-regulation and PI resistance could exist. In conclusion, TLR4 triggering is a potential mechanism of chemotherapy resistance by controlling mitochondrial fitness, and its inhibition could be successful in MM patients to overcome PI resistance.

Il mieloma multiplo (MM) è una neoplasia delle cellule B che richiede segnali del microambiente infiammatorio per la sopravvivenza e la proliferazione cellulare. Nonostante i miglioramenti negli strumenti farmacologici, il MM rimane oggi incurabile principalmente a causa della resistenza ai farmaci. L'inibitore del proteasoma Bortezomib (BTZ) è emerso come un farmaco efficace per il trattamento del mieloma multiplo anche se molti pazienti ricadono dalla terapia BTZ. Il presente studio ha nella prima parte esama le vie metaboliche alla base dell'acquisizione della resistenza al BTZ nel mieloma multiplo. Abbiamo utilizzato due diversi cloni di linee cellulari di mieloma multiplo che mostravano sensibilità diverse a BTZ (U266-S e U266-R) e li abbiamo confrontati in termini di profilo metabolico, fitness mitocondriale e capacità di omeostasi dell'equilibrio redox. I nostri risultati hanno mostrato che il clone resistente alla BTZ (U266-R) presentava un aumento dei derivati UDP glicosilati rispetto alle cellule sensibili alla BTZ (U266-S), suggerendo così anche attività più elevate della via biosintetica dell'esosamina (HBP), regolando non solo la proteina O - e N-glicosilazione ma anche funzioni mitocondriali. In particolare, l'U266-R ha mostrato una maggiore biogenesi mitocondriale e dinamiche mitocondriali associate a difese antiossidanti più forti. Inoltre, l'U266-R ha mantenuto una concentrazione significativamente più alta di substrati per la glicosilazione delle proteine rispetto all'U266, in particolare per UDP-GlcNac, suggerendo così ulteriormente l'importanza della glicosilazione nella risposta farmacologica BTZ. Inoltre, l'U266-R trattato con BTZ ha mostrato rapporti ATP / ADP e livelli di ECP significativamente più alti e ha anche mostrato una maggiore fitness mitocondriale e risposta antiossidante. Nella seconda parte lo studio mira a indagare l'implicazione del recettore Toll-like 4 (TLR4) come potenziale meccanismo di resistenza a bortezomib (BTZ) in relatiozione al metabolosmo mitocondriale. Abbiamo scoperto che l'attivazione di TLR4 ha indotto la biogenesi mitocondriale e una maggiore massa mitocondriale nelle linee cellulari MM umane. Inoltre, la segnalazione TLR4 è stata attivata dopo l'esposizione a BTZ ed è stata aumentata nelle cellule U266 (U266-R) resistenti a BTZ. Una combinazione di BTZ con TAK-242, un inibitore selettivo di TLR4, ha superato la resistenza ai farmaci attraverso la generazione di stress ossidativo più elevato ed esteso, forte depolarizzazione mitocondriale e grave compromissione della forma mitocondriale che a sua volta ha causato crisi energetica cellulare e mitofagia attivata e apoptosi. Abbiamo ulteriormente confermato l'efficacia di una combinazione TAK-242 / BTZ nelle plasmacellule di pazienti affetti da mieloma refrattario. Coerentemente, l'inibizione di TLR4 ha aumentato la depolarizzazione mitocondriale indotta da BTZ, ripristinando la risposta farmacologica. Presi insieme, questi risultati indicano che la segnalazione TLR4 agisce come un meccanismo di risposta allo stress proteggendo i mitocondri durante l'esposizione a BTZ, sostenendo il metabolismo mitocondriale e promuovendo la resistenza ai farmaci. In conclusione, i nostri risultati suggeriscono che il metabolismo mitocondriale guida la resistenza al BTZ nel mieloma multiplo, rappresentando così un possibile bersaglio per lo sviluppo di nuovi farmaci per i pazienti resistenti alla BTZ. Inoltre l'inibizione di TLR4 potrebbe quindi essere un possibile obiettivo nei pazienti con MM refrattario per superare la resistenza BTZ.