RESUMO
Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , ProteostaseRESUMO
Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/administração & dosagem , Spliceossomos/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Feminino , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Oligopeptídeos/administração & dosagem , Splicing de RNA/efeitos dos fármacos , Spliceossomos/genética , Spliceossomos/metabolismo , Spliceossomos/microbiologiaAssuntos
ADP-Ribosil Ciclase 1/metabolismo , Anticorpos Monoclonais/uso terapêutico , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Mieloma Múltiplo/metabolismo , Plasmócitos/metabolismoRESUMO
A major driver of multiple myeloma (MM) is thought to be aberrant signaling, yet no kinase inhibitors have proven successful in the clinic. Here, we employed an integrated, systems approach combining phosphoproteomic and transcriptome analysis to dissect cellular signaling in MM to inform precision medicine strategies. Unbiased phosphoproteomics initially revealed differential activation of kinases across MM cell lines and that sensitivity to mammalian target of rapamycin (mTOR) inhibition may be particularly dependent on mTOR kinase baseline activity. We further noted differential activity of immediate downstream effectors of Ras as a function of cell line genotype. We extended these observations to patient transcriptome data in the Multiple Myeloma Research Foundation CoMMpass study. A machine-learning-based classifier identified surprisingly divergent transcriptional outputs between NRAS- and KRAS-mutated tumors. Genetic dependency and gene expression analysis revealed mutated Ras as a selective vulnerability, but not other MAPK pathway genes. Transcriptional analysis further suggested that aberrant MAPK pathway activation is only present in a fraction of RAS-mutated vs wild-type RAS patients. These high-MAPK patients, enriched for NRAS Q61 mutations, have inferior outcomes, whereas RAS mutations overall carry no survival impact. We further developed an interactive software tool to relate pharmacologic and genetic kinase dependencies in myeloma. Collectively, these predictive models identify vulnerable signaling signatures and highlight surprising differences in functional signaling patterns between NRAS and KRAS mutants invisible to the genomic landscape. These results will lead to improved stratification of MM patients in precision medicine trials while also revealing unexplored modes of Ras biology in MM.
Assuntos
Perfilação da Expressão Gênica , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Transdução de Sinais , Transcriptoma , Biologia Computacional/métodos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Aprendizado de Máquina , Masculino , Anotação de Sequência Molecular , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mutação , Medicina de Precisão/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
The myeloma bone marrow microenvironment promotes proliferation of malignant plasma cells and resistance to therapy. Activation of JAK/STAT signaling is thought to be a central component of these microenvironment-induced phenotypes. In a prior drug repurposing screen, we identified tofacitinib, a pan-JAK inhibitor Food and Drug Administration (FDA) approved for rheumatoid arthritis, as an agent that may reverse the tumor-stimulating effects of bone marrow mesenchymal stromal cells. Herein, we validated in vitro, in stromal-responsive human myeloma cell lines, and in vivo, in orthotopic disseminated xenograft models of myeloma, that tofacitinib showed efficacy in myeloma models. Furthermore, tofacitinib strongly synergized with venetoclax in coculture with bone marrow stromal cells but not in monoculture. Surprisingly, we found that ruxolitinib, an FDA approved agent targeting JAK1 and JAK2, did not lead to the same anti-myeloma effects. Combination with a novel irreversible JAK3-selective inhibitor also did not enhance ruxolitinib effects. Transcriptome analysis and unbiased phosphoproteomics revealed that bone marrow stromal cells stimulate a JAK/STAT-mediated proliferative program in myeloma cells, and tofacitinib reversed the large majority of these pro-growth signals. Taken together, our results suggest that tofacitinib reverses the growth-promoting effects of the tumor microenvironment. As tofacitinib is already FDA approved, these results can be rapidly translated into potential clinical benefits for myeloma patients.