RESUMEN
Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos Inmunológicos/uso terapéutico , Agentes Inmunomoduladores/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/genética , Metilación de ADN/efectos de los fármacos , Resistencia a Antineoplásicos , Elementos de Facilitación Genéticos/efectos de los fármacos , Humanos , Intrones/efectos de los fármacos , Mieloma Múltiple/genéticaRESUMEN
Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.
Asunto(s)
GTP Fosfohidrolasas , Mieloma Múltiple , Supervivencia Celular/genética , Genes ras , Humanos , Mieloma Múltiple/genética , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismoAsunto(s)
Evolución Clonal/genética , Dosificación de Gen/fisiología , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteína p53 Supresora de Tumor/genética , Alelos , Proliferación Celular/genética , Progresión de la Enfermedad , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Redes y Vías Metabólicas/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/mortalidad , Mutación , Recurrencia , Análisis de Supervivencia , Células Tumorales CultivadasAsunto(s)
Mieloma Múltiple , Preparaciones Farmacéuticas , Proteínas Adaptadoras Transductoras de Señales , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mutación , Talidomida , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Constitutive phosphatidylinositide 3-kinase (PI3K) signalling has been implicated in multiple myeloma (MM) pathophysiology and is regarded as an actionable target for pharmacological intervention. Isoform-specific PI3K inhibition may offer the most focused treatment approach and could result in greater clinical efficacy and reduced side effects. We therefore performed isoform-specific knockdown of PIK3CA, PIK3CB, PIK3CD, and PIK3CG to analyse their individual contributions to MM cell survival and downstream signalling. In addition, we tested the effectivity of the novel PI3K isoform-specific inhibitors BYL-719 (PIK3CA), TGX-221 (PIK3CB), CAL-101 (PIK3CD), and CAY10505 (PIK3CG). We found the PIK3CA isoform to be of paramount importance for constitutive Akt activity in MM cells, and - in contrast to inhibition of other class I isoforms - only the blockade of PIK3CA was sufficient to induce cell death in a sizeable subgroup of MM samples. Furthermore, pharmacological PIK3CA inhibition in combination treatments of BYL-719 and established anti-myeloma agents resulted in strongly enhanced MM cell death. Our data thus clearly indicate therapeutic potential of PIK3CA inhibitors and support their clinical evaluation in multiple myeloma.
Asunto(s)
Mieloma Múltiple/patología , Fosfatidilinositol 3-Quinasas/fisiología , Antineoplásicos/farmacología , Supervivencia Celular/fisiología , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Mieloma Múltiple/metabolismo , Fosfatidilinositol 3-Quinasas/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , ARN Interferente Pequeño/farmacología , Transducción de SeñalRESUMEN
High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of IGF1R mutations is so far unknown, we investigated the functional impact of IGF1R mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with IGF1RWT, IGF1RD1146N and IGF1RN1129S (Sleeping Beauty), generated CRISPR-Cas9 IGF1R knockouts in the HMCLs U-266 (IGF1RWT) and L-363 (IGF1RD1146N) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. IGF1R knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by IGF1RN1129S in one HMCL, whereby the viability remained unaffected. Expression of IGF1RD1146N reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the IGF1R mutation status. In conclusion, IGF1R mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.
RESUMEN
Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high-risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA) by co-culturing color-labeled genetically modified cell models, we recently showed that mono- and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild-type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment-free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment-related, unlike patient-derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.
RESUMEN
The heat shock transcription factor 1 (HSF1) has recently been reported to promote malignant transformation and growth. Here we provide experimental evidence for a role of HSF1 in the pathogenesis of multiple myeloma (MM). Immunohistochemical analyses revealed that HSF1 was overexpressed in half of the investigated MM samples, including virtually all cases with extramedullary manifestations or anaplastic morphology. HSF1 function was inhibited either by siRNA-mediated knockdown or pharmacologically through treatment with triptolide. Both approaches caused depletion of HSF1, lowered the constitutively high expression of a multitude of protective HSPs (such as HSP90, HSP70, HSP40 and HSP27), induced apoptosis in human MM cells in vitro, and strongly reduced MM tumour growth in vivo. Furthermore, we observed that treatment-induced upregulation of HSPs after proteasome or HSP90 inhibition was critically dependent on HSF1. Importantly, the apoptotic effects of the HSP90 inhibitor NVP-AUY922 or the proteasome inhibitor bortezomib were strongly enhanced in combination with triptolide, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Collectively, our data indicate that inhibition of HSF1 affects multiple protective HSPs and might therefore represent a therapeutic strategy - in particular in combination with proteasome or HSP90 inhibitors.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Mieloma Múltiple/tratamiento farmacológico , Factores de Transcripción/fisiología , Animales , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Diterpenos/farmacología , Regulación hacia Abajo , Compuestos Epoxi/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/metabolismo , Humanos , Inmunohistoquímica , Isoxazoles/farmacología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Fenantrenos/farmacología , Células Plasmáticas/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Pirazinas/farmacología , ARN Interferente Pequeño/farmacología , Resorcinoles/farmacología , Factores de Transcripción/antagonistas & inhibidores , Trasplante HeterólogoRESUMEN
We have recently shown that approximately half of primary multiple myeloma (MM) samples display constitutive Akt activity, which disposes them for sensitivity to Akt inhibition. The Akt pathway counts among the signaling conduits for oncogenic RAS and activating mutations of K- and N-RAS frequently occur in MM. We therefore analyzed the relation between RAS mutation and Akt dependency in biopsies and CD138-purified cells from MM patients (n = 65) and the function of oncogenic RAS for MM cell survival in a range of MM cell lines with differing RAS status. Whereas RAS mutations do not predict Akt dependency, oncogenic RAS retains an important role for MM cell survival. Knockdown of either K- or N-RAS strongly decreased the viability of MM cells that harbored the respective oncogenic isoform, whereas ablation of wild-type RAS isoforms had little or no effect. Silencing of oncogenic RAS did not affect the Akt pathway, again indicating lack of a direct link. Combined inhibition of RAS and Akt strongly enhanced MM cell death. These data suggest that oncogenic RAS and Akt may independently contribute to MM cell survival. Targeting of both pathways could provide an attractive therapeutic strategy for patients with oncogenic RAS and dysregulated Akt signaling.
Asunto(s)
Genes ras , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Supervivencia Celular , Cartilla de ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas , Mieloma Múltiple/patología , Mieloma Múltiple/terapia , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
Despite therapeutic advances multiple myeloma remains largely incurable, and novel therapeutic concepts are needed. The Hsp90-chaperone is a reasonable therapeutic target, because it maintains oncogenic signaling of multiple deregulated pathways. However, in contrast to promising preclinical results, only limited clinical efficacy has been achieved through pharmacological Hsp90 inhibition. Because Hsp70 has been described to interact functionally with the Hsp90-complex, we analyzed the suitability of Hsp72 and Hsp73 as potential additional target sites. Expression of Hsp72 and Hsp73 in myeloma cells was analyzed by immunohistochemical staining and western blotting. Short interfering RNA-mediated knockdown or pharmacological inhibition of Hsp72 and Hsp73 was performed to evaluate the role of these proteins in myeloma cell survival and for Hsp90-chaperone function. Furthermore, the role of PI3K-dependent signaling in constitutive and inducible Hsp70 expression was investigated using short interfering RNA-mediated and pharmacological PI3K inhibition. Hsp72 and Hsp73 were frequently overexpressed in multiple myeloma. Knockdown of Hsp72 and/or Hsp73 or treatment with VER-155008 induced apoptosis of myeloma cells. Hsp72/Hsp73 inhibition decreased protein levels of Hsp90-chaperone clients affecting multiple oncogenic signaling pathways, and acted synergistically with the Hsp90 inhibitor NVP-AUY922 in the induction of death of myeloma cells. Inhibition of the PI3K/Akt/GSK3ß pathway with short interfering RNA or PI103 decreased expression of the heat shock transcription factor 1 and down-regulated constitutive and inducible Hsp70 expression. Treatment of myeloma cells with a combination of NVP-AUY922 and PI103 resulted in additive to synergistic cytotoxicity. In conclusion, Hsp72 and Hsp73 sustain Hsp90-chaperone function and critically contribute to the survival of myeloma cells. Translation of Hsp70 inhibition into the clinic is therefore highly desirable. Treatment with PI3K inhibitors might represent an alternative therapeutic strategy to target Hsp70.
Asunto(s)
Proteínas del Choque Térmico HSC70/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas del Choque Térmico HSP72/biosíntesis , Proteínas HSP90 de Choque Térmico/fisiología , Mieloma Múltiple/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/genética , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas HSP70 de Choque Térmico/genética , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/patologíaRESUMEN
The globus pallidus (GP) of primates is divided conventionally into distinct internal and external parts. The literature repeats since 1930 the opinion that the homolog of the primate internal pallidum in rodents is the hypothalamic entopeduncular nucleus (embedded within fiber tracts of the cerebral peduncle). To test this idea, we explored its historic fundaments, checked the development and genoarchitecture of mouse entopeduncular and pallidal neurons, and examined relevant comparative connectivity data. We found that the extratelencephalic mouse entopeduncular structure consists of four different components arrayed along a dorsoventral sequence in the alar hypothalamus. The ventral entopeduncular nucleus (EPV), with GABAergic neurons expressing Dlx5&6 and Nkx2-1, lies within the hypothalamic peduncular subparaventricular area. Three other formations-the dorsal entopeduncular nucleus (EPD), the prereticular entopeduncular nucleus (EPPRt ), and the preeminential entopeduncular nucleus (EPPEm )-lie within the overlying paraventricular area, under the subpallium. EPD contains glutamatergic neurons expressing Tbr1, Otp, and Pax6. The EPPRt has GABAergic cells expressing Isl1 and Meis2, whereas the EPPEm population expresses Foxg1 and may be glutamatergic. Genoarchitectonic observations on relevant areas of the mouse pallidal/diagonal subpallium suggest that the GP of rodents is constituted as in primates by two adjacent but molecularly and hodologically differentiable telencephalic portions (both expressing Foxg1). These and other reported data oppose the notion that the rodent extratelencephalic entopeduncular nucleus is homologous to the primate internal pallidum. We suggest instead that all mammals, including rodents, have dual subpallial GP components, whereas primates probably also have a comparable set of hypothalamic entopeduncular nuclei. Remarkably, there is close similarity in some gene expression properties of the telencephalic internal GP and the hypothalamic EPV. This apparently underlies their notable functional analogy, sharing GABAergic neurons and thalamopetal connectivity.
Asunto(s)
Globo Pálido , Roedores , Animales , Ratones , Núcleo Entopeduncular , Hipotálamo , Primates , Neuronas GABAérgicas , Factores de Transcripción/genética , Proteínas del Tejido Nervioso , Factores de Transcripción ForkheadRESUMEN
So-called RAS-dependent pathways, such as those signalling via mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)/Akt, are implicated in proliferation and survival of multiple myeloma (MM) cells. However, the effects of their combined blockade and its potential therapeutic utility for the treatment of RAS-mutated MM have not systematically been analysed. Here, we tested the functional consequences of single versus combined inhibition of the MEK/MAPK and PI3K/Akt pathways in a large series of primary MM samples (n = 55) and MM cell lines (n = 11). Additionally, the anti-myeloma activity of different treatments was analysed with respect to the RAS mutation status. PI3K/Akt blockade was generally more pro-apoptotic than blockade of MEK/MAPK both in cell lines and in primary MM samples. Simultaneous blockade of both pathways led to significantly enhanced anti-myeloma activity in 75% of primary MM samples, whereas the remainder was largely resistant. Resistance to combination blockade was exclusively observed in RAS wildtype cases, whereas sensitivity was noted in RAS wildtype and in RAS mutated MM. These results suggest that oncogenic RAS is a predictor of sensitivity to combination treatment with PI3K/Akt and MEK/MAPK inhibitors and that such an approach might therefore be beneficial for this genetically well-defined subgroup of MM patients.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Genes ras , Humanos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mieloma Múltiple/genética , Mutación , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
We present here a thorough and complete analysis of mouse P0-P140 prethalamic histogenetic subdivisions and corresponding nuclear derivatives, in the context of local tract landmarks. The study used as fundamental material brains from a transgenic mouse line that expresses LacZ under the control of an intragenic enhancer of Dlx5 and Dlx6 (Dlx5/6-LacZ). Subtle shadings of LacZ signal, jointly with pan-DLX immunoreaction, and several other ancillary protein or RNA markers, including Calb2 and Nkx2.2 ISH (for the prethalamic eminence, and derivatives of the rostral zona limitans shell domain, respectively) were mapped across the prethalamus. The resulting model of the prethalamic region postulates tetrapartite rostrocaudal and dorsoventral subdivisions, as well as a tripartite radial stratification, each cell population showing a characteristic molecular profile. Some novel nuclei are proposed, and some instances of potential tangential cell migration were noted.
Asunto(s)
Mapeo Cromosómico/métodos , Proteínas de Homeodominio/genética , Operón Lac/genética , Tálamo/embriología , Animales , Animales Recién Nacidos , Femenino , Expresión Génica , Proteínas de Homeodominio/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Tálamo/crecimiento & desarrollo , Tálamo/metabolismo , Pez CebraRESUMEN
Pharmacological inhibitors of histone deacetylases (HDACs) are currently being developed and tested as anti-cancer agents and may be useful to enhance the therapeutic efficiency of established anti-myeloma treatments. This study preclinically evaluated the effects of the 'second generation' pan-HDAC inhibitor JNJ-26481585 on human multiple myeloma (MM) cells from established cell lines and primary MM samples (n=42). Molecular responses in both groups of MM cells included histone acetylation, a shift in Bcl2-family members towards proapoptotic bias, attenuation of growth and survival pathway activity and Hsp72 induction. Mcl-1 depletion and Hsp72 induction were the most reliable features observed in JNJ-26481585-treated primary MM samples. The drug alone effectively induced myeloma cell death at low nanomolar concentrations. In vitro combination of JNJ-26481585 with anti-myeloma therapeutic agents generally resulted In effects close to additivity. In view of the favourable activity of this novel HDAC-inhibitor towards primary myeloma cells further evaluation in a clinical setting is warranted.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Mieloma Múltiple/patología , Acetilación/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Histonas/metabolismo , Humanos , Mieloma Múltiple/metabolismo , Células Tumorales CultivadasRESUMEN
Alternative splicing of transcripts from many cancer-associated genes is believed to play a major role in carcinogenesis as well as in tumor progression. Alternative splicing of one such gene, the neural cell adhesion molecule CD56 (NCAM), impacts the progression, inadequate therapeutic response, and reduced total survival of patients who suffer from numerous malignant neoplasms. Although previous investigations have determined that CD56 exists in three major isoforms (CD56(120kD), CD56(140kD), and CD56(180kD)) with individual structural and functional properties, neither the expression profiles nor the functional relevance of these isoforms in malignant tumors have been consistently investigated. Using new quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) strategies and novel CD56 isoform-specific antibodies, CD56(140kD) was shown to be exclusively expressed in a number of highly malignant CD56(+) neoplasms and was associated with the progression of CD56(+) precursor lesions of unclear malignant potential. Moreover, only CD56(140kD) induced antiapoptotic/proliferative pathways and specifically phosphorylated calcium-dependent kinases that are relevant for tumorigenesis. We conclude, therefore, that the specific detection of CD56 isoforms will help to elucidate their individual functions in the pathogenesis and progression of malignant neoplasms and may have a positive impact on the development of CD56-based immunotherapeutic strategies.
Asunto(s)
Antígeno CD56/biosíntesis , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Neoplasias/genética , Anticuerpos , Especificidad de Anticuerpos , Apoptosis/fisiología , Biomarcadores de Tumor/análisis , Western Blotting , Antígeno CD56/inmunología , Línea Celular Tumoral , Progresión de la Enfermedad , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Although the phosphatidylinositide 3-kinase (PI3K)/Akt pathway has been reported to contribute to the malignant growth of multiple myeloma (MM), the true relevance of Akt kinases for this disease is still unclear. In particular, functional analyses in primary tumor cells and genetic target validation experiments are missing. Here, we used combined functional and molecular analyses to determine the importance of Akt activity in a large panel of primary MM samples and in MM cell lines. Akt down-regulation with isoform-specific siRNA constructs or with an Akt1/2-specific pharmacologic inhibitor strongly induced apoptosis in approximately half of the primary MM samples analyzed. Sensitivity to Akt inhibition strongly correlated with the activation status of Akt as determined by immunohistochemistry, phospho-Akt-specific flow cytometry, and Western analysis. Additional blockade of the MAPK and the IL-6R/STAT3 pathways was often not sufficient to decrease the viability of MM cells resilient to Akt inhibition. Taken together, these experiments led to the identification of 2 myeloma subgroups: Akt-dependent and Akt-independent MM.
Asunto(s)
Células de la Médula Ósea/citología , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/clasificación , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Apoptosis , Línea Celular Tumoral , Citometría de Flujo , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Células Madre/citologíaRESUMEN
Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase ß, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/enzimología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos Alquilantes , Línea Celular Tumoral , Reparación del ADN , Progresión de la Enfermedad , Femenino , Humanos , Melfalán , Ratones SCID , Neoplasias Experimentales , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS. However, there is still no final consent on whether KRAS-mutations are associated with disease outcome. Specifically, no data exist on whether KRAS-mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRASp.G12C entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRASp.G12A and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRASWT, KRASp.G12A, KRASp.A146T, and KRASp.A146V were overexpressed in HEK293 cells and the KRASWT MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS-mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.
RESUMEN
The 90 kD heat shock protein (Hsp90) molecular chaperone sustains multiple components of oncogenic pathways and has recently emerged as a therapeutic target that is now being clinically tested in a number of malignancies. In order to address formulation issues and to deal with possible resistance mechanisms against small molecule Hsp90 inhibitors, a range of compounds based on different molecular scaffolds are now being developed. The present study preclinically tested the effects of the novel 2-aminothienopyrimidine class Hsp90 inhibitor NVP-BEP800, which is suitable for oral formulations, on multiple myeloma cells from established cell lines and on a larger cohort (n = 40) of primary myeloma samples. The drug effectively and specifically killed the majority of primary myeloma cells in coculture with bone marrow stromal cells and reliably entailed molecular consequences of Hsp90 blockade - such as survival pathway breakdown and client protein depletion - in multiple myeloma cells from cell lines as well as from patients. Collectively, the properties of this novel drug support clinical testing in multiple myeloma.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Mieloma Múltiple/patología , Pirimidinas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Células de la Médula Ósea/patología , Muerte Celular/efectos de los fármacos , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Mieloma Múltiple/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Células del Estroma/patología , Células Tumorales CultivadasRESUMEN
Experimental data on resistance mechanisms of multiple myeloma (MM) to ixazomib (IXA), a second-generation proteasome inhibitor (PI), are currently lacking. We generated MM cell lines with a 10-fold higher resistance to IXA as their sensitive counterparts, and observed cross-resistance towards the PIs carfilzomib (CFZ) and bortezomib (BTZ). Analyses of the IXA-binding proteasome subunits PSMB5 and PSMB1 show increased PSMB5 expression and activity in all IXA-resistant MM cells, and upregulated PSMB1 expression in IXA-resistant AMO1 cells. In addition, sequence analysis of PSMB5 revealed a p.Thr21Ala mutation in IXA-resistant MM1.S cells, and a p.Ala50Val mutation in IXA-resistant L363 cells, whereas IXA-resistant AMO1 cells lack PSMB5 mutations. IXA-resistant cells retain their sensitivity to therapeutic agents that mediate cytotoxic effects via induction of proteotoxic stress. Induction of ER stress and apoptosis by the p97 inhibitor CB-5083 was strongly enhanced in combination with the PI3Kα inhibitor BYL-719 or the HDAC inhibitor panobinostat suggesting potential therapeutic strategies to circumvent IXA resistance in MM. Taken together, our newly established IXA-resistant cell lines provide first insights into resistance mechanisms and overcoming treatment strategies, and represent suitable models to further study IXA resistance in MM.