The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL

Abstract

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond

“…Indeed, the SH2 domain of growth factor receptor-binding domain 2 fused to either the RING domain of Cbl or U-box domain from carboxyl terminus of Hsc70-interacting protein (CHIP) is sufficient to downregulate EGFR in lung cancer cells (Lee et al, 2014;Zhong et al, 2015). Similarly, a SH2 domain fused to the U-box domain of CHIP downregulates breakpoint cluster region protein-Abelson murine leukemia viral oncogene homolog 1 kinase and inhibits tumor growth of chronic myeloid leukemia cells in mouse xenografts (Ru et al, 2016).…”

Regulatory Molecules and Corresponding Processes of BCR-ABL Protein Degradation

“…Non-endogenous chimeric E3 ligases can be constructed by fusing the adaptor domain that can bind to the target protein with a domain that has E3 catalytic activity. SH2-U-box and SH2-RING are two E3 ligases artificially synthesized based on CHIP and c-CBL respectively, and they both can target the BCR-ABL protein for degradation 39. SH2-U-box consists of SH2 domain and U-box domain, and SH2-RING is composed of SH2 domain and RING domain.…”

“…Both two E3 ligases can promote the wild-type and mutant BCR-ABL proteins ubiquitination and degradation, and the effect of SH2-U-box is stronger than that of SH2-RING. Furthermore, it has been proved that SH2-U-box could inactivate BCR-ABL-dependent signaling pathways in cell and could induce BCR-ABL-dependent cell growth inhibition 39.…”

Análise de mutações do domínio BCR-ABL quinase em pacientes com leucemia mielóide crônica refratários ao tratamento com mesilato de imatinibe

“…Dentre esses mecanismos de escape terapêutico, as mutações pontuais no domínio quinase (DQ) do BCR-ABL1 são as mais envolvidas na falha clínica ao imatinibe, responsável por cerca de 20% dos casos de resistência adquirida, apresentando-se com maior frequência a T315I, Y253F e E255K [6][7][8] . Estas mutações dispõem de variações de sensibilidade, tanto para os ITK de primeira geração, quanto para os de segunda geração -nilotinibe e dasatinibe -prejudicando e reduzindo o efeito desses inibidores em seu alvo terapêutico.…”

“…Extração de DNAO DNA das amostras de sangue dos pacientes foi extraído com o reagente TRIzol ® , de acordo com as instruções do fabricante (Life Technologies, Carlsbad, CA, USA). de Mutações Ponto Resistentes A detecção de mutação pontual foi baseada em polimorfismo de nucleotídeo único (SNP), através da presença das variantes de resistência que não respondem ao tratamento com IM e que são mais frequentemente relatados na literatura[6][7][8][9][10][11][12][13] , apresentados na Tabela 2. -Definições de resposta ao tratamento com base na expressão gênica do BCR-ABL1, de acordo com a Escala Internacional (IS), proposta pela European LeukemiaNet13 .…”