RESUMO
The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are approved drugs for the treatment of multiple myeloma. IMiDs induce cereblon (CRBN) E3 ubiquitin ligase-mediated ubiquitination and degradation of Ikaros transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), which are essential for multiple myeloma. However, because of a single amino acid substitution of valine to isoleucine in mouse CRBN at position 391, mice are not susceptible to IMiD-induced degradation of neosubstrates. Here, we report that expression of human CRBN or the CrbnI391V mutant enables IMiD-induced degradation of IKZF1 and IKZF3 in murine MOPC.315.BM.Luc.eGFP and 5T33MM multiple myeloma cells. Accordingly, lenalidomide and pomalidomide decreased cell viability in a dose-dependent fashion in murine multiple myeloma cells expressing CrbnI391V in vitro. The sensitivity of murine cells expressing CrbnI391V to IMiDs highly correlated with their dependence on IKZF1. After transplantation, MOPC.315.BM.Luc.eGFP cells expressing murine CrbnI391V induced multiple myeloma in mice, and treatment with lenalidomide and pomalidomide significantly delayed tumor growth. This straightforward model provides a proof-of-concept for studying the effects of IMiDs in multiple myeloma in mice, which allows for in vivo testing of IMiDs and other CRBN E3 ligase modulators.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fatores Imunológicos/farmacologia , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligases/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Fatores Imunológicos/uso terapêutico , Lenalidomida/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/genética , Mutação Puntual , Proteólise/efeitos dos fármacos , Talidomida/farmacologia , Talidomida/uso terapêuticoRESUMO
BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.
Assuntos
Enzimas Desubiquitinantes/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Citocinas/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Doxorrubicina/farmacologia , Fator Estimulador de Colônias de Granulócitos/genética , Células HEK293 , Humanos , Inflamassomos/genética , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Small-molecule heterobifunctional degraders can effectively control protein levels and are useful research tools. We assembled proteolysis targeting chimeras (PROTACs) from a cereblon (CRBN) and a von-Hippel-Lindau (VHL) ligase ligand and demonstrated a PROTAC-induced heterodimerization of the two E3 ligases leading to unidirectional and efficient degradation of CRBN.
Assuntos
Ligantes , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dimerização , Humanos , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Proteólise , Bibliotecas de Moléculas Pequenas/química , Talidomida/análogos & derivados , Talidomida/química , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.
