RESUMO
Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle-specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S-phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post-transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS-multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B-LIN28B axis.
Assuntos
Endopeptidases , MicroRNAs , Mieloma Múltiplo , Proteínas Proto-Oncogênicas c-myc , Proteínas de Ligação a RNA , Ciclo Celular , Linhagem Celular Tumoral , Endopeptidases/genética , Humanos , MicroRNAs/genética , Mieloma Múltiplo/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas de Ligação a RNA/genética , Ubiquitinas/metabolismoRESUMO
BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.
Assuntos
Desenho de Fármacos , Proteólise , Proteínas Proto-Oncogênicas c-bcl-2 , Ubiquitina-Proteína Ligases , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Humanos , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologia , Sulfonamidas/síntese química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Quimera de Direcionamento de ProteóliseRESUMO
Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. We investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1α, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis.
Assuntos
Inibidores da Angiogênese , Talidomida , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/síntese química , Talidomida/farmacologia , Talidomida/química , Talidomida/análogos & derivados , Talidomida/síntese química , Humanos , Relação Estrutura-Atividade , Estrutura Molecular , Halogenação , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos Imunológicos/uso terapêutico , Agentes de Imunomodulação/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Ubiquitina-Proteína Ligases/genética , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Elementos Facilitadores Genéticos/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Mieloma Múltiplo/genéticaRESUMO
Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.
Assuntos
Gemtuzumab/administração & dosagem , Leucemia Mieloide Aguda , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea , Intervalo Livre de Doença , Feminino , Gemtuzumab/efeitos adversos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Nucleofosmina , Estudos Prospectivos , Recidiva , Fatores de Risco , Taxa de SobrevidaRESUMO
BACKGROUND: This report summarizes three phase I studies evaluating volasertib, a polo-like kinase inhibitor, plus azacitidine in adults with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, or acute myeloid leukemia. METHODS: Patients received intravenous volasertib in 28-day cycles (dose-escalation schedules). In Part 1 of 1230.33 (Study 1; NCT01957644), patients received 250-350 mg volasertib on day (D)1 and D15; in Part 2, patients received different schedules [A, D1: 170 mg/m2; B, D7: 170 mg/m2; C, D1 and D7: 110 mg/m2]. In 1230.35 (Study 2; NCT02201329), patients received 200-300 mg volasertib on D1 and D15. In 1230.43 (Study 3; NCT02721875), patients received 110 mg/m2 volasertib on D1 and D8. All patients in Studies 1 and 2, and approximately half of the patients in Study 3, were scheduled to receive subcutaneous azacitidine 75 mg/m2 on D1-7. RESULTS: Overall, 22 patients were treated (17 with MDS; 12 previously untreated). Across Studies 1 and 2 (n = 21), the most common drug-related adverse events were hematological (thrombocytopenia [n = 11]; neutropenia [n = 8]). All dose-limiting toxicities were grade 4 thrombocytopenia. The only treated patient in Study 3 experienced 18 adverse events following volasertib monotherapy. Studies 1 and 2 showed preliminary activity (objective response rates: 25 and 40%). CONCLUSIONS: The safety of volasertib with azacitidine in patients with MDS was consistent with other volasertib studies. All studies were terminated prematurely following the discontinuation of volasertib for non-clinical reasons by Boehringer Ingelheim; however, safety information on volasertib plus azacitidine are of interest for future studies in other diseases.
Assuntos
Leucemia Mieloide Aguda , Leucemia Mielomonocítica Crônica , Síndromes Mielodisplásicas , Trombocitopenia , Adulto , Azacitidina/uso terapêutico , Ensaios Clínicos Fase I como Assunto , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mielomonocítica Crônica/induzido quimicamente , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Síndromes Mielodisplásicas/induzido quimicamente , Síndromes Mielodisplásicas/tratamento farmacológico , Pteridinas , Trombocitopenia/induzido quimicamenteRESUMO
BACKGROUND: Passive immunization against SARS-CoV-2 limits viral burden and death from COVID-19; however, it poses a theoretical risk of disease exacerbation through antibody-dependent enhancement (ADE). ADE after anti-SARS-CoV2 antibody treatment has not been reported, and therefore the potential risk and promoting factors remain unknown. CASE PRESENTATION: A 75-year-old female was admitted to the emergency room with recurrent, unexplained bruises and leukocytopenia, anemia, and thrombocytopenia. Evaluation of a bone marrow biopsy established the diagnosis of an acute promyelocytic leukemia (APL). SARS-CoV-2 RT-PCR testing of nasal and throat swabs on admission was negative. During the routine SARS-CoV-2 testing of inpatients, our patient tested positive for SARS-CoV-2 on day 14 after admission without typical COVID-19 symptoms. Due to disease- and therapy-related immunosuppression and advanced age conferring a high risk of progressing to severe COVID-19, casirivimab and imdevimab were administered as a preemptive approach. The patient developed immune activation and cytokine release syndrome (CRS) occurring within four hours of preemptive anti-SARS-CoV2 antibody (casirivimab/imdevimab) infusion. Immune activation and CRS were evidenced by a rapid increase in serum cytokines (IL-6, TNFα, IL-8, IL-10), acute respiratory insufficiency, and progressive acute respiratory distress syndrome. DISCUSSION AND CONCLUSION: The temporal relationship between therapeutic antibody administration and the rapid laboratory, radiological, and clinical deterioration suggests that CRS was an antibody-related adverse event, potentially exacerbated by APL treatment-mediated differentiation of leukemic blasts and promyelocytes. This case highlights the need for careful assessment of life-threatening adverse events after passive SARS-CoV-2 immunization, especially in the clinical context of patients with complex immune and hematological landscapes.
Assuntos
COVID-19 , Leucemia Promielocítica Aguda , Síndrome do Desconforto Respiratório , Idoso , Anticorpos Monoclonais Humanizados , COVID-19/complicações , COVID-19/diagnóstico , Teste para COVID-19 , Síndrome da Liberação de Citocina/diagnóstico , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/etiologia , Feminino , Humanos , Leucemia Promielocítica Aguda/complicações , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamento farmacológico , SARS-CoV-2RESUMO
Lenalidomide is a highly effective treatment for myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)), resulting in CK1α degradation. CK1α is encoded by a gene within the common deleted region for del(5q) MDS and haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. We found that mouse cells are resistant to lenalidomide but that changing a single amino acid in mouse Crbn to the corresponding human residue enables lenalidomide-dependent degradation of CK1α. We further demonstrate that minor side chain modifications in thalidomide and a novel analogue, CC-122, can modulate the spectrum of substrates targeted by CRL4(CRBN). These findings have implications for the clinical activity of lenalidomide and related compounds, and demonstrate the therapeutic potential of novel modulators of E3 ubiquitin ligases.
Assuntos
Caseína Quinase I/metabolismo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/fisiopatologia , Talidomida/análogos & derivados , Ubiquitinação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Caseína Quinase I/genética , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Fatores Imunológicos/farmacologia , Células Jurkat , Células K562 , Lenalidomida , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Proteólise/efeitos dos fármacos , Alinhamento de Sequência , Deleção de Sequência , Especificidade da Espécie , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In acute myeloid leukemia, there is growing evidence for splicing pattern deregulation, including differential expression of linear splice isoforms of the commonly mutated gene nucleophosmin (NPM1). In this study, we detect circular RNAs of NPM1 and quantify circRNA hsa_circ_0075001 in a cohort of NPM1 wild-type and mutated acute myeloid leukemia (n=46). Hsa_circ_0075001 expression correlates positively with total NPM1 expression, but is independent of the NPM1 mutational status. High versus low hsa_circ_0075001 expression defines patient subgroups characterized by distinct gene expression patterns, such as lower expression of components of the Toll-like receptor signaling pathway in high hsa_circ_0075001 expression cases. Global evaluation of circRNA expression in sorted healthy hematopoietic controls (n=10) and acute myeloid leukemia (n=10) reveals circRNA transcripts for 47.9% of all highly expressed genes. While circRNA expression correlates globally with parental gene expression, we identify hematopoietic differentiation-associated as well as acute myeloid leukemia subgroup-specific circRNA signatures.
Assuntos
Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , RNA/análise , Estudos de Casos e Controles , Expressão Gênica , Humanos , Nucleofosmina , Splicing de RNA , RNA CircularAssuntos
Caseína Quinase Ialfa/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Modelos Animais de Doenças , Hematopoese/efeitos dos fármacos , Humanos , Camundongos , Células Tumorais Cultivadas , Quinase Ativadora de Quinase Dependente de CiclinaAssuntos
Anemia Macrocítica/genética , Mutação , Síndromes Mielodisplásicas/genética , Proteína Fosfatase 2C/genética , Cariótipo Anormal , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Evolução Clonal , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genes p53 , Humanos , Lenalidomida/farmacologia , Lenalidomida/uso terapêutico , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Proteína Fosfatase 2C/fisiologia , Estudos RetrospectivosRESUMO
Mutations in the nucleophosmin 1 (NPM1) gene are considered a founder event in the pathogenesis of acute myeloid leukemia (AML). To address the role of clonal evolution in relapsed NPM1-mutated (NPM1mut) AML, we applied high-resolution, genome-wide, single-nucleotide polymorphism array profiling to detect copy number alterations (CNAs) and uniparental disomies (UPDs) and performed comprehensive gene mutation screening in 53 paired bone marrow/peripheral blood samples obtained at diagnosis and relapse. At diagnosis, 15 aberrations (CNAs, n = 10; UPDs, n = 5) were identified in 13 patients (25%), whereas at relapse, 56 genomic alterations (CNAs, n = 46; UPDs, n = 10) were detected in 29 patients (55%) indicating an increase in genomic complexity. Recurrent aberrations acquired at relapse included deletions affecting tumor suppressor genes (ETV6 [n = 3], TP53 [n = 2], NF1 [n = 2], WT1 [n = 3], FHIT [n = 2]) and homozygous FLT3 mutations acquired via UPD13q (n = 7). DNMT3A mutations (DNMT3Amut) showed the highest stability (97%). Persistence of DNMT3Amut in 5 patients who lost NPM1mut at relapse suggests that DNMT3Amut may precede NPM1mut in AML pathogenesis. Of note, all relapse samples shared at least 1 genetic aberration with the matched primary AML sample, implying common ancestral clones. In conclusion, our study reveals novel insights into clonal evolution in NPM1mut AML.
Assuntos
Evolução Clonal/genética , DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Adulto , Idoso , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 9 , Impressões Digitais de DNA , DNA Metiltransferase 3A , Feminino , Deleção de Genes , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/epidemiologia , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , Recidiva , Fatores de Risco , Adulto JovemRESUMO
Acute myeloid leukemia (AML) is characterized by molecular heterogeneity. As commonly altered genomic regions point to candidate genes involved in leukemogenesis, we used microarray-based comparative genomic hybridization and single nucleotide polymorphism profiling data of 391 AML cases to further narrow down genomic regions of interest. Targeted resequencing of 1000 genes located in the critical regions was performed in a representative cohort of 50 AML samples comprising all major cytogenetic subgroups. We identified 120 missense/nonsense mutations as well as 60 insertions/deletions affecting 73 different genes (â¼ 3.6 tumor-specific aberrations/AML). While most of the newly identified alterations were nonrecurrent, we observed an enrichment of mutations affecting genes involved in epigenetic regulation including known candidates like TET2, TET1, DNMT3A, and DNMT1, as well as mutations in the histone methyltransferases NSD1, EZH2, and MLL3. Furthermore, we found mutations in the splicing factor SFPQ and in the nonclassic regulators of mRNA processing CTCF and RAD21. These splicing-related mutations affected 10% of AML patients in a mutually exclusive manner. In conclusion, we could identify a large number of alterations in genes involved in aberrant splicing and epigenetic regulation in genomic regions commonly altered in AML, highlighting their important role in the molecular pathogenesis of AML.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Leucemia Mieloide Aguda/genética , Mutação , Splicing de RNA/genética , Hibridização Genômica Comparativa , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.
Assuntos
Aberrações Cromossômicas , Fatores de Ligação ao Core/genética , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Doença Aguda , Adulto , Criança , Deleção Cromossômica , Variações do Número de Cópias de DNA , Humanos , Leucemia Mieloide/patologia , Perda de Heterozigosidade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , Polimorfismo de Nucleotídeo Único , RecidivaRESUMO
Multiple myeloma (MM) is a heterogeneous malignancy of plasma cells. Despite improvement in the prognosis of MM patients after the introduction of many new drugs in the past decades, MM remains incurable since most patients become treatment-resistant. Cyclin-dependent kinase 6 (CDK6) is activated in many types of cancer and has been associated with drug resistance in MM. However, its association with disease stage, genetic alterations, and outcome has not been systematically investigated in large cohorts. Here, we analyzed CDK6 expression using immunohistochemistry in 203 formalin-fixed paraffin-embedded samples of 146 patients and four healthy individuals. We found that 61.5% of all MM specimens express CDK6 at various levels. CDK6 expression increased with the progression of disease with a median of 0% of CDK6-positive plasma cells in monoclonal gammopathy of undetermined significance (MGUS) (n = 10) to 30% in newly diagnosed MM (n = 78) and up to 70% in relapsed cases (n = 55). The highest median CDK6 was observed in extramedullary myeloma (n = 12), a highly aggressive manifestation of MM. Longitudinal analyses revealed that CDK6 is significantly increased in lenalidomide-treated patients but not in those who did not receive lenalidomide. Furthermore, we observed that patients who underwent lenalidomide-comprising induction therapy had significantly shorter progression-free survival when their samples were CDK6 positive. These data support that CDK6 protein expression is a marker for aggressive and drug-resistant disease and describes a potential drug target in MM.
RESUMO
Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.
Assuntos
Antígeno de Maturação de Linfócitos B , Quimiocina CXCL12 , Imunoterapia Adotiva , Células Matadoras Naturais , Mieloma Múltiplo , Receptores CXCR4 , Receptores de Antígenos Quiméricos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Humanos , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Antígeno de Maturação de Linfócitos B/imunologia , Antígeno de Maturação de Linfócitos B/metabolismo , Antígeno de Maturação de Linfócitos B/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Imunoterapia Adotiva/métodos , Quimiocina CXCL12/metabolismo , Linhagem Celular Tumoral , Citotoxicidade ImunológicaRESUMO
Multiple myeloma (MM) is a plasma cell malignancy of the bone marrow. Despite therapeutic advances, MM remains incurable, and better risk stratification as well as new therapies are therefore highly needed. The proteome of MM has not been systematically assessed before and holds the potential to uncover insight into disease biology and improved prognostication in addition to genetic and transcriptomic studies. Here we provide a comprehensive multiomics analysis including deep tandem mass tag-based quantitative global (phospho)proteomics, RNA sequencing, and nanopore DNA sequencing of 138 primary patient-derived plasma cell malignancies encompassing treatment-naive MM, plasma cell leukemia and the premalignancy monoclonal gammopathy of undetermined significance, as well as healthy controls. We found that the (phospho)proteome of malignant plasma cells are highly deregulated as compared with healthy plasma cells and is both defined by chromosomal alterations as well as posttranscriptional regulation. A prognostic protein signature was identified that is associated with aggressive disease independent of established risk factors in MM. Integration with functional genetics and single-cell RNA sequencing revealed general and genetic subtype-specific deregulated proteins and pathways in plasma cell malignancies that include potential targets for (immuno)therapies. Our study demonstrates the potential of proteogenomics in cancer and provides an easily accessible resource for investigating protein regulation and new therapeutic approaches in MM.
RESUMO
Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.