B-cell non-Hodgkin lymphomas (B-NHL) are a heterogeneous group of blood malignancies arising from peripheral B lymphocytes. Among them, a subset of Diffuse Large B-cell Lymphomas (DLBCL) and all Burkitt’s lymphomas (BL) are c-Myc-driven aggressive B-NHL. Although current treatments often provide complete remission, approximately half of the patient’s relapse. Thus, approaches targeting c-Myc may represent a novel therapeutic strategy for aggressive B-NHL. Recently we have demonstrated that activation of the fibroblast growth factor (FGF) signaling is involved in the stabilization of c-Myc protein in different tumor types, FGF or FGF receptor (FGFR) inhibition leading to the proteasomal degradation of c-Myc protein. On this basis, FGF/FGFR blockade may represent a promising strategy to inhibit c-Myc. Data reported in this thesis show that c-Myc positive DLBCL and BL patient-derived samples express high levels of activated/phosphorylated (p)FGFR. Interestingly, FGFR phosphorylation was not observed in c-Myc negative DLBCL samples, suggesting a strong correlation between FGFR activation and c-Myc protein expression. To assess the role of the FGF/FGFR system in c-Myc-driven B-NHL, we tested the effect on lymphoma fitness of the FGF trap molecule NSC12 and the FDA-approved FGFR TK inhibitor Erdafitinib in c-Myc positive DLBCL (RI-1, U2932, SU-DHL--6 and SU-DHL-10) and BL (RAJI, DAUDI) cell lines. All cell lines express several FGFRs and FGF ligands and high levels of pFGFR in the absence of exogenous stimuli, indicating the presence of an autocrine FGF stimulation. In vitro NSC12 or Erdafitinib treatments strongly inhibited FGFR activation and induced the rapid proteasomal degradation of c-Myc protein. This was confirmed by co-treatment with the proteosome inhibitor MG132 that prevented c-Myc degradation. The decrease of c-Myc protein levels was paralleled by reduced tumor cell proliferation and increased ROS-mediated apoptosis in all cell lines tested. By going deeper into the molecular mechanisms involved in c-Myc protein degradation upon FGF/FGFR blockade, we shew that NSC12 affects c-Myc protein stability by reducing c-Myc Ser62 phosphorylation and by increasing the c-Myc Thr58 phosphorylation necessary for c-Myc protein degradation. Moreover, we demonstrated that ERK1/2 is the kinase stabilizing c-Myc while BRD4 is the kinase that phosphorylates c-Myc protein at Thr58 in aggressive B-NHL since its inhibition causes a rescue of c-Myc protein levels. In vivo treatment with NSC12 or Erdafitinib significantly reduced the growth of both DLBCL and BL tumor xenografts. In keeping with in vitro data, immunohistochemical analyses revealed a strong reduction of pFGFR and c-Myc protein levels in treated tumors compared to controls. To note, NSC12 or Erdafitinib in combination with the standard therapy R-CHOP exerted synergistic anti-lymphoma effects on DLBCL cell lines. Altogether, these findings open new therapeutic hints for the treatment of c-MYC-driven aggressive B-NHL.
I linfomi non Hodgkin a cellule B (B-NHL) sono un gruppo eterogeneo di tumori ematologici che originano dai linfociti B periferici. Tra questi, un sottogruppo di linfomi diffusi a grandi cellule B (DLBCL) e tutti i linfomi di Burkitt (BL) sono B-NHL aggressivi guidati dall’oncoproteina c-Myc. Sebbene i trattamenti attuali spesso forniscano una remissione completa, circa la metà dei pazienti presenta una recidiva. Pertanto, gli approcci mirati a inibire c-Myc possono rappresentare una nuova strategia terapeutica per i B-NHL aggressivi. Recentemente abbiamo dimostrato che l'attivazione del segnale indotta dai fattori di crescita dei fibroblasti (FGF) è coinvolta nella stabilizzazione della proteina c-Myc in diversi tipi di tumore; infatti l'inibizione di FGF o dei recettori per FGF (FGFR) porta alla degradazione proteasomale della proteina c-Myc. Su questa base, ipotizziamo che il blocco di FGF/FGFR possa avere un forte impatto sulla crescita e sulla diffusione dei B-NHL aggressivi guidati da c-Myc. Lo scopo di questa tesi è quindi volto a studiare gli effetti dell'inibizione della segnalazione di FGF nei B-NHL aggressivi guidati da c-Myc utilizzando due diversi approcci: (i) una strategia di intrappolamento di FGF a livello extracellulare (utilizzando la small molecule FGF trap NSC12) e (ii) un approccio di inibizione dei recettori per FGF (utilizzando l’inibitore tirosino-chinasico Erdafitinib, approvato dalla FDA). I dati riportati in questa tesi mostrano che i campioni derivati da pazienti DLBCL e BL positivi per c-Myc esprimono alti livelli di (p)FGFR attivato/fosforilato. È interessante notare che la fosforilazione di FGFR non è stata osservata nei campioni DLBCL negativi per c-Myc, suggerendo una forte correlazione tra l'attivazione di FGFR e l'espressione della proteina c-Myc. Per valutare il ruolo del sistema FGF/FGFR nei B-NHL guidati da c-Myc, abbiamo testato l’attività antitumorale di NSC12 ed Erdafitinib in linee cellulari c-Myc positive di DLBCL (RI -1, U2932, SU-DHL-6 e SU-DHL-10) e di BL (RAJI, DAUDI). Tutte le linee cellulari esprimono diversi FGFR e FGF e alti livelli di pFGFR in assenza di stimoli esogeni, indicando la presenza di una stimolazione autocrina mediata da FGF. I trattamenti in vitro con NSC12 o Erdafitinib hanno fortemente inibito l’attivazione di FGFR e hanno indotto la rapida degradazione proteosomale della proteina c-Myc. Ciò è stato confermato dal co-trattamento con l'inibitore del proteosoma MG132 che ha impedito la degradazione di c-Myc. Meccanicisticamente, il blocco di FGF/FGFR influisce sulla stabilità della proteina c-Myc riducendo la fosforilazione di c-Myc in Ser62 e aumentando la fosforilazione di c-Myc in Thr58 necessaria per la degradazione della proteina c-Myc. Ciò si ottiene attraverso l'inibizione della chinasi ERK1/2 (che fosforila c-Myc in Ser62) e l'attivazione della chinasi BRD4 (che fosforila c-Myc in Thr58). La diminuzione dei livelli di proteina c-Myc è stata accompagnata da una ridotta proliferazione delle cellule tumorali e da un aumento dell'apoptosi mediata dai ROS. È importante sottolineare che il trattamento in vivo con NSC12 o Erdafitinib ha ridotto significativamente la crescita di xenotrapianti tumorali sia DLBCL che BL. Da notare che NSC12 o Erdafitinib in combinazione con la terapia standard R-CHOP hanno potenziato gli effetti anti-linfoma sulle linee cellulari DLBCL e sulle cellule purificate da pazienti DLBCL.
c-Myc oncoprotein is stabilized by FGFR activation: therapeutic potential of FGF/FGFR inhibitors in c-Myc-driven B-cell non-Hodgkin Lymphoma / Faletti, Jessica. - (2025 Jan 24).
c-Myc oncoprotein is stabilized by FGFR activation: therapeutic potential of FGF/FGFR inhibitors in c-Myc-driven B-cell non-Hodgkin Lymphoma
Faletti, Jessica
2025-01-24
Abstract
B-cell non-Hodgkin lymphomas (B-NHL) are a heterogeneous group of blood malignancies arising from peripheral B lymphocytes. Among them, a subset of Diffuse Large B-cell Lymphomas (DLBCL) and all Burkitt’s lymphomas (BL) are c-Myc-driven aggressive B-NHL. Although current treatments often provide complete remission, approximately half of the patient’s relapse. Thus, approaches targeting c-Myc may represent a novel therapeutic strategy for aggressive B-NHL. Recently we have demonstrated that activation of the fibroblast growth factor (FGF) signaling is involved in the stabilization of c-Myc protein in different tumor types, FGF or FGF receptor (FGFR) inhibition leading to the proteasomal degradation of c-Myc protein. On this basis, FGF/FGFR blockade may represent a promising strategy to inhibit c-Myc. Data reported in this thesis show that c-Myc positive DLBCL and BL patient-derived samples express high levels of activated/phosphorylated (p)FGFR. Interestingly, FGFR phosphorylation was not observed in c-Myc negative DLBCL samples, suggesting a strong correlation between FGFR activation and c-Myc protein expression. To assess the role of the FGF/FGFR system in c-Myc-driven B-NHL, we tested the effect on lymphoma fitness of the FGF trap molecule NSC12 and the FDA-approved FGFR TK inhibitor Erdafitinib in c-Myc positive DLBCL (RI-1, U2932, SU-DHL--6 and SU-DHL-10) and BL (RAJI, DAUDI) cell lines. All cell lines express several FGFRs and FGF ligands and high levels of pFGFR in the absence of exogenous stimuli, indicating the presence of an autocrine FGF stimulation. In vitro NSC12 or Erdafitinib treatments strongly inhibited FGFR activation and induced the rapid proteasomal degradation of c-Myc protein. This was confirmed by co-treatment with the proteosome inhibitor MG132 that prevented c-Myc degradation. The decrease of c-Myc protein levels was paralleled by reduced tumor cell proliferation and increased ROS-mediated apoptosis in all cell lines tested. By going deeper into the molecular mechanisms involved in c-Myc protein degradation upon FGF/FGFR blockade, we shew that NSC12 affects c-Myc protein stability by reducing c-Myc Ser62 phosphorylation and by increasing the c-Myc Thr58 phosphorylation necessary for c-Myc protein degradation. Moreover, we demonstrated that ERK1/2 is the kinase stabilizing c-Myc while BRD4 is the kinase that phosphorylates c-Myc protein at Thr58 in aggressive B-NHL since its inhibition causes a rescue of c-Myc protein levels. In vivo treatment with NSC12 or Erdafitinib significantly reduced the growth of both DLBCL and BL tumor xenografts. In keeping with in vitro data, immunohistochemical analyses revealed a strong reduction of pFGFR and c-Myc protein levels in treated tumors compared to controls. To note, NSC12 or Erdafitinib in combination with the standard therapy R-CHOP exerted synergistic anti-lymphoma effects on DLBCL cell lines. Altogether, these findings open new therapeutic hints for the treatment of c-MYC-driven aggressive B-NHL.File | Dimensione | Formato | |
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