Background and rationale Systemic immunoglobulin light chain amyloidosis (AL) is a rare plasma cell dyscrasia where monoclonal light chains misfold, aggregate and form amyloid deposits in target organs, leading to multi-system organ dysfunction. The underlying bone-marrow residing plasma cell clone is usually small and poorly proliferating, and peculiar biologic and molecular features set it apart from multiple myeloma (MM). Expression of amyloidogenic light chains induces ER stress and renders AL plasma cells particularly dependent on the proteo-catabolic activity of the ubiquitin proteasome system (UPS). The proteasome inhibitor bortezomib forms the cornerstone of therapy for transplant-ineligible patients. Yet, a significant proportion of AL patients fail to respond to bortezomib-based combinations, especially those cases that exhibit the t(11;14) translocation, which is seen in >40% of AL patients. Recent work has shown that deubiquitinating enzymes (DUBs), which act upstream of the proteasome itself to modulate proteasomal degradation of polyubiquitinated proteins, may be a novel therapeutic target in different types of cancer, including MM. Whether this is a valid therapeutic target in AL is presently unknown. Materials and methods We used the t(11;14)-positive MM cell lines U266 and KMS-12 and the AL amyloidosis cell line ALMC-2. Cell line authentication was performed through immunophenotyping and FISH-based verification of X/Y chromosomes and t(11;14) translocation status. For the ALMC-2 cell line, the expressed light chain gene sequence was verified. For studies on primary cells, CD138-positive and, as control, CD138-negative cells were derived from bone marrow of therapy-naïve patients with AL amyloidosis at diagnosis using immunomagnetic purification. Additional controls included peripheral blood mononuclear cells from healthy subjects. For co-culture experiments, mesenchymal stromal cells were obtained from bone marrow of healthy subjects. Drug-induced accumulation of polyubiquitinated proteins was verified by Western blotting. Therapeutic effect on cell metabolism and cell viability was analyzed using CellTiter Glo, trypan blue and/or Annexin V-propidium iodide assays, respectively. Results We tested the therapeutic effect of b-AP15, a recently reported small molecule inhibitor of the deubiquitinating enzymes USP14 and UCHL5. Exposure to b-AP15 led to a dose-dependent decline in cell metabolism and cell viability both in the t(11;14)-positive MM cell lines U266 and KMS-12 and in the AL cell line ALMC-2 (IC50 values between 100 and 200 nM). Mechanistic studies with the ALMC-2 cell line showed that b-AP15-mediated inhibition of USP14/UCHL5 resulted in a progressive accumulation of polyubiquitinated proteins, followed by induction of apoptosis. The therapeutic effect of b-AP15 was maintained when ALMC-2 cells were co-cultured with primary bone-marrow derived mesenchymal stromal cells. Treatment of primary bone marrow-derived CD138+ plasma cells from AL patients n=9 patients, of which 4 patients with t(11;14)-positive plasma cell clones with nanomolar concentrations of b-AP15 for 24h significantly decreased their metabolic activity, without markedly affecting matched CD138- fractions and PBMCs from normal healthy donors, suggesting specific anti-AL activity and a favorable therapeutic index for b-AP15. Conclusions Our preclinical data showing efficacy of b-AP15 in AL disease models validate targeting DUBs upstream of the proteasome in the context of the UPS to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in AL.
Contesto e razionale Lamiloidosi sistemica da catene leggere (AL) è una rara discrasia plasmacellulare in cui le catene leggere monoclonali mal ripiegate, saggregano e formano depositi di amiloide in diversi tessuti bersaglio, conducendo ad un danno dorgano multi-sistemico. Il sottostante clone plasmacellulare, localizzato nel midollo osseo, è solitamente piccolo, scarsamente proliferante e possiede peculiari caratteristiche biologiche e molecolari tali da distinguerlo da quello del mieloma multiplo (MM). Laccumulo di catene leggere conduce a stress del reticolo endoplasmatico, rendendo lattività di catabolismo proteico del sistema ubiquitina-proteasoma cruciale per le plasmacellule amiloidogeniche. Linibitore del proteasoma, bortezomib, rappresenta una pietra miliare nel trattamento dei pazienti non candidati al trapianto di midollo osseo. Nonostante ciò, una significativa percentuale di pazienti, soprattutto se portatori della traslocazione t(11;14), non risponde a regimi a base di bortezomib. Studi recenti hanno mostrato che gli enzimi deubiquitinanti, i quali agiscono a monte del proteasoma, potrebbero rappresentare dei potenziali bersagli terapeutici in numerosi tumori, incluso il MM. Ci siamo pertanto occupati della valutazione del ruolo delle deubiquitinasi come target farmacologico nellamiloidosi AL. Materiali e metodi Sono state utilizzate due linee cellulari di mieloma multiplo (U266 e KMS-12) e una di amiloidosi AL (ALMC-2). Per gli studi su cellule primarie, sono state impiegate cellule CD138 positive e negative, come controllo, ottenute mediante purificazione immunomagnetica da prelievi di midollo osseo effettuati allatto della diagnosi di amiloidosi AL in pazienti non precedentemente trattati. Come ulteriore controllo ci si è avvalsi di cellule mononucleate da sangue periferico di donatori sani. Per gli esperimenti di co-coltura, sono state isolate cellule stromali mesenchimali dallaspirato midollare di soggetti sani. Laccumulo di proteine poli-ubiquitinate è stato verificato mediante Western blotting. Gli effetti terapeutici su metabolismo e vitalità cellulare sono stati indagati, rispettivamente, con i seguenti saggi: CellTiter Glo, trypan blue e/o Annessina V/ioduro di propidio. Risultati Abbiamo testato gli effetti terapeutici di b-AP15, un inibitore degli enzimi deubiquitinanti USP14 e UCHL5. Lesposizione a questo farmaco ha condotto ad una riduzione dose-dipendente del metabolismo e della vitalità cellulare descritto in entrambe le linee cellulari di MM, U266 e KMS-12, e in quella di amiloidosi AL, ALMC-2 (con valori di IC50 compresi tra 100 e 200 nM). Linibizione di USP14 e UCHL5 mediante b-AP15 è risultata inoltre in un progressivo accumulo di proteine poli-ubiquitinate causanti linnesco dellapoptosi cellulare. Lefficacia di b-AP15 sulla linea ALMC-2 è stata dimostrata anche in presenza di cellule stromali mesenchimali di provenienza midollare. Il trattamento con concentrazioni nanomolari di b-AP15 per 24 ore comporta una significativa riduzione dellattività metabolica delle plasmacellule CD138+ derivate dal midollo di pazienti con amiloidosi AL - n=9 pazienti, di cui 4 positivi per la traslocazione t(11;14) non altrettanto rilevabile invece nella frazione CD138- e nelle cellule mononucleate da sangue periferico di volontari sani, suggerendo la specificità dellattività anti-tumorale di b-AP15. Conclusioni I nostri dati preclinici dimostrano lefficacia di b-AP15 in modelli di amiloidosi AL, validando luso delle deubiquitinasi come bersaglio di terapie per superare la resistenza agli inibitori del proteasoma e aprendo la strada alla valutazione in ambito clinico degli inibitori di USP14/UCHL5.
Targeting Deubiquitylating Enzymes USP14 and UCHL5 in Systemic Immunoglobulin Light Chain (AL) Amyloidosis (Valutazione del Ruolo delle Deubiquitinasi USP14 e UCHL5 come Potenziali Bersagli Terapeutici nell'Amiloidosi Sistemica da Catene Leggere (AL))
VALSECCHI, FRANCESCA
2018/2019
Abstract
Background and rationale Systemic immunoglobulin light chain amyloidosis (AL) is a rare plasma cell dyscrasia where monoclonal light chains misfold, aggregate and form amyloid deposits in target organs, leading to multi-system organ dysfunction. The underlying bone-marrow residing plasma cell clone is usually small and poorly proliferating, and peculiar biologic and molecular features set it apart from multiple myeloma (MM). Expression of amyloidogenic light chains induces ER stress and renders AL plasma cells particularly dependent on the proteo-catabolic activity of the ubiquitin proteasome system (UPS). The proteasome inhibitor bortezomib forms the cornerstone of therapy for transplant-ineligible patients. Yet, a significant proportion of AL patients fail to respond to bortezomib-based combinations, especially those cases that exhibit the t(11;14) translocation, which is seen in >40% of AL patients. Recent work has shown that deubiquitinating enzymes (DUBs), which act upstream of the proteasome itself to modulate proteasomal degradation of polyubiquitinated proteins, may be a novel therapeutic target in different types of cancer, including MM. Whether this is a valid therapeutic target in AL is presently unknown. Materials and methods We used the t(11;14)-positive MM cell lines U266 and KMS-12 and the AL amyloidosis cell line ALMC-2. Cell line authentication was performed through immunophenotyping and FISH-based verification of X/Y chromosomes and t(11;14) translocation status. For the ALMC-2 cell line, the expressed light chain gene sequence was verified. For studies on primary cells, CD138-positive and, as control, CD138-negative cells were derived from bone marrow of therapy-naïve patients with AL amyloidosis at diagnosis using immunomagnetic purification. Additional controls included peripheral blood mononuclear cells from healthy subjects. For co-culture experiments, mesenchymal stromal cells were obtained from bone marrow of healthy subjects. Drug-induced accumulation of polyubiquitinated proteins was verified by Western blotting. Therapeutic effect on cell metabolism and cell viability was analyzed using CellTiter Glo, trypan blue and/or Annexin V-propidium iodide assays, respectively. Results We tested the therapeutic effect of b-AP15, a recently reported small molecule inhibitor of the deubiquitinating enzymes USP14 and UCHL5. Exposure to b-AP15 led to a dose-dependent decline in cell metabolism and cell viability both in the t(11;14)-positive MM cell lines U266 and KMS-12 and in the AL cell line ALMC-2 (IC50 values between 100 and 200 nM). Mechanistic studies with the ALMC-2 cell line showed that b-AP15-mediated inhibition of USP14/UCHL5 resulted in a progressive accumulation of polyubiquitinated proteins, followed by induction of apoptosis. The therapeutic effect of b-AP15 was maintained when ALMC-2 cells were co-cultured with primary bone-marrow derived mesenchymal stromal cells. Treatment of primary bone marrow-derived CD138+ plasma cells from AL patients n=9 patients, of which 4 patients with t(11;14)-positive plasma cell clones with nanomolar concentrations of b-AP15 for 24h significantly decreased their metabolic activity, without markedly affecting matched CD138- fractions and PBMCs from normal healthy donors, suggesting specific anti-AL activity and a favorable therapeutic index for b-AP15. Conclusions Our preclinical data showing efficacy of b-AP15 in AL disease models validate targeting DUBs upstream of the proteasome in the context of the UPS to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in AL.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/21962