RESUMEN
The introduction of new drugs in the past years has substantially improved outcome in multiple myeloma (MM). However, the majority of patients eventually relapse and become resistant to one or multiple drugs. While the genetic landscape of relapsed/ resistant multiple myeloma has been elucidated, the causal relationship between relapse-specific gene mutations and the sensitivity to a given drug in MM has not systematically been evaluated. To determine the functional impact of gene mutations, we performed combined whole-exome sequencing (WES) of longitudinal patient samples with CRISPR-Cas9 drug resistance screens for lenalidomide, bortezomib, dexamethasone, and melphalan. WES of longitudinal samples from 16 MM patients identified a large number of mutations in each patient that were newly acquired or evolved from a small subclone (median 9, range 1-55), including recurrent mutations in TP53, DNAH5, and WSCD2. Focused CRISPR-Cas9 resistance screens against 170 relapse-specific mutations functionally linked 15 of them to drug resistance. These included cereblon E3 ligase complex members for lenalidomide, structural genes PCDHA5 and ANKMY2 for dexamethasone, RB1 and CDK2NC for bortezomib, and TP53 for melphalan. In contrast, inactivation of genes involved in the DNA damage repair pathway, including ATM, FANCA, RAD54B, and BRCC3, enhanced susceptibility to cytotoxic chemotherapy. Resistance patterns were highly drug specific with low overlap and highly correlated with the treatment-dependent clonal evolution in patients. The functional association of specific genetic alterations with drug sensitivity will help to personalize treatment of MM in the future.
Asunto(s)
Mieloma Múltiple , Preparaciones Farmacéuticas , Sistemas CRISPR-Cas , Humanos , Lenalidomida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Recurrencia Local de NeoplasiaRESUMEN
The bone marrow niche has a pivotal role in progression, survival, and drug resistance of multiple myeloma cells. Therefore, it is important to develop means for targeting the multiple myeloma bone marrow microenvironment. Myeloma-associated macrophages (MAM) in the bone marrow niche are M2 like. They provide nurturing signals to multiple myeloma cells and promote immune escape. Reprogramming M2-like macrophages toward a tumoricidal M1 phenotype represents an intriguing therapeutic strategy. This is especially interesting in view of the successful use of mAbs against multiple myeloma cells, as these therapies hold the potential to trigger macrophage-mediated phagocytosis and cytotoxicity. In this study, we observed that MAMs derived from patients treated with the immunomodulatory drug (IMiD) lenalidomide skewed phenotypically and functionally toward an M1 phenotype. Lenalidomide is known to exert its beneficial effects by modulating the CRBN-CRL4 E3 ligase to ubiquitinate and degrade the transcription factor IKAROS family zinc finger 1 (IKZF1). In M2-like MAMs, we observed enhanced IKZF1 levels that vanished through treatment with lenalidomide, yielding MAMs with a bioenergetic profile, T-cell stimulatory properties, and loss of tumor-promoting capabilities that resemble M1 cells. We also provide evidence that IMiDs interfere epigenetically, via degradation of IKZF1, with IFN regulatory factors 4 and 5, which in turn alters the balance of M1/M2 polarization. We validated our observations in vivo using the CrbnI391V mouse model that recapitulates the IMiD-triggered IKZF1 degradation. These data show a role for IKZF1 in macrophage polarization and can provide explanations for the clinical benefits observed when combining IMiDs with therapeutic antibodies.See related Spotlight on p. 254.
Asunto(s)
Factor de Transcripción Ikaros/metabolismo , Lenalidomida/farmacología , Mieloma Múltiple/inmunología , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Médula Ósea/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/inmunología , Técnicas de Sustitución del Gen , Humanos , Factor de Transcripción Ikaros/antagonistas & inhibidores , Factores Reguladores del Interferón/metabolismo , Lenalidomida/uso terapéutico , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Cultivo Primario de Células , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunología , Microambiente Tumoral/efectos de los fármacos , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/metabolismo , Ubiquitinación/efectos de los fármacos , Adulto JovenRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Factores Inmunológicos/farmacología , Lenalidomida/farmacología , Mieloma Múltiple/tratamiento farmacológico , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Factores Inmunológicos/uso terapéutico , Lenalidomida/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/genética , Mutación Puntual , Proteolisis/efectos de los fármacos , Talidomida/farmacología , Talidomida/uso terapéuticoRESUMEN
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.
Asunto(s)
Enzimas Desubicuitinizantes/genética , Leucemia Mieloide Aguda/genética , Mutación/genética , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Citocinas/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Doxorrubicina/farmacología , Factor Estimulante de Colonias de Granulocitos/genética , Células HEK293 , Humanos , Inflamasomas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , RatonesRESUMEN
Micro-ribonucleic acid-155 (miR-155) is one of the first described oncogenic miRNAs. Although multiple direct targets of miR-155 have been identified, it is not clear how it contributes to the pathogenesis of acute myeloid leukemia. We found miR-155 to be a direct target of Meis1 in murine Hoxa9/Meis1 induced acute myeloid leukemia. The additional overexpression of miR-155 accelerated the formation of acute myeloid leukemia in Hoxa9 as well as in Hoxa9/Meis1 cells in vivo However, in the absence or following the removal of miR-155, leukemia onset and progression were unaffected. Although miR-155 accelerated growth and homing in addition to impairing differentiation, our data underscore the pathophysiological relevance of miR-155 as an accelerator rather than a driver of leukemogenesis. This further highlights the complexity of the oncogenic program of Meis1 to compensate for the loss of a potent oncogene such as miR-155. These findings are highly relevant to current and developing approaches for targeting miR-155 in acute myeloid leukemia.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/etiología , MicroARNs/antagonistas & inhibidores , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/farmacología , Animales , Carcinogénesis/genética , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Ratones , MicroARNs/metabolismoRESUMEN
MicroRNA-155 (miR-155) is an oncogenic miRNA upregulated in various tumor types and leukemias and has been suggested as a potential drug target. Based on our previous work detecting high miR-155 levels in response to Meis1 overexpression in a murine Hox leukemia model, we show here the relationship among HOXA9, MEIS1, and miR-155 levels in MLL-translocated acute myeloid leukemia (AML) patients. Using mouse bone marrow cells transformed by MLL-fusion genes expressing graduated levels of Meis1, we show a positive correlation between miR-155 and Meis1. However, using a miR-155-knockout mouse model, we show that the absence and the depletion of miR-155 have no effect on leukemia formation or progression. We also show for the first time that miR-155 levels are correlated with MLL translocations, but that miR-155 expression is dispensable for the formation of AML and has no effect on leukemia progression.