RESUMO
PURPOSE: Estrogen receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR, or PI3K inhibitors has become a central strategy in the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from the ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, and potent small molecule PROteolysis-TArgeting Chimera (PROTAC) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type (WT) and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition (TGI). RESULTS: Vepdegestrant induced ≥90% degradation of wild-type and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in vitro, and achieved substantial TGI (87%-123%) in MCF7 orthotopic xenograft models, better than those of the ET agent fulvestrant (31%-80% TGI). In the hormone independent (HI) mutant ER Y537S patient-derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regression and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant-induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib; the mTOR inhibitor everolimus; and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in vivo compared with fulvestrant, which correlated with improved TGI, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.
Assuntos
Neoplasias da Mama , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Transdução de Sinais , Serina-Treonina Quinases TOR , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Feminino , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/antagonistas & inibidores , Piperazinas/farmacologia , Piperazinas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/administração & dosagem , Receptores de Estrogênio/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacosRESUMO
Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.
Assuntos
Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Doença Aguda , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Cromatina/genética , Dissulfiram/farmacologia , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Dedos de Zinco PHD/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Translocação Genética/efeitos dos fármacos , Translocação Genética/genéticaRESUMO
Malignant transformation is a multistep process that is dictated by the acquisition of multiple genomic aberrations that provide growth and survival advantage. During the post genomic era, high throughput genomic sequencing has advanced exponentially, leading to identification of countless cancer associated mutations with potential for targeted therapy. Mouse models of cancer serve as excellent tools to examine the functionality of gene mutations and their contribution to the malignant process. However, it remains unclear whether the genetic events that occur during transformation are similar in mice and humans. To address that, we chose several transgenic mouse models of hematopoietic malignancies and identified acquired mutations in these mice by means of targeted re-sequencing of known cancer-associated genes as well as whole exome sequencing. We found that mutations that are typically found in acute myeloid leukemia or T cell acute lymphoblastic leukemia patients are also common in mouse models of the respective disease. Moreover, we found that the most frequent mutations found in a mouse model of lymphoma occur in a set of epigenetic modifier genes, implicating this pathway in the generation of lymphoma. These results demonstrate that genetically engineered mouse models (GEMM) mimic the genetic evolution of human cancer and serve as excellent platforms for target discovery and validation.
Assuntos
Modelos Animais de Doenças , Leucemia/genética , Linfoma/genética , Mutação , Animais , Humanos , CamundongosRESUMO
B-1 and B-2 lymphocytes are derived from distinct developmental pathways and represent layered arms of the innate and adaptive immune systems, respectively. In contrast to a majority of murine B-cell malignancies, which stain positive with the B220 antibody, we discovered a novel form of B-cell leukemia in NUP98-PHF23 (NP23) transgenic mice. The immunophenotype (Lin- B220- CD19+ AA4.1+) was identical to that of progenitor (pro) B-1 cells, and VH gene usage was skewed toward 3' V regions, similar to murine fetal liver B cells. Moreover, the gene expression profile of these leukemias was most similar to that of fetal liver pro-B fraction BC, a known source of B-1 B cells, further supporting a pro-B-1 origin of these leukemias. The NP23 pro-B-1 acute lymphoblastic leukemias (ALLs) acquired spontaneous mutations in both Bcor and Janus kinase (Jak) pathway (Jak1/2/3 and Stat5a) genes, supporting a hypothesis that mutations in 3 critical pathways (stem-cell self-renewal, B-cell differentiation, and cytokine signaling) collaborate to induce B-cell precursor (BCP) ALL. Finally, the thymic stromal lymphopoietin (Tslp) cytokine is required for murine B-1 development, and chromosomal rearrangements resulting in overexpression of the TSLP receptor (CRLF2) are present in some patients with high-risk BCP-ALL (referred to as CRLF2r ALL). Gene expression profiles of NP23 pro-B-1 ALL were more similar to that of CRLF2r ALL than non-CRLF2r ALL, and analysis of VH gene usage from patients with CRLF2r ALL demonstrated preferential usage of VH regions used by human B-1 B cells, leading to the suggestion that this subset of patients with BCP-ALL has a malignancy of B-1, rather than B-2, B-cell origin.
RESUMO
In this report, we show that expression of a NUP98-PHF23 (NP23) fusion, associated with acute myeloid leukemia (AML) in humans, leads to myeloid, erythroid, T-cell, and B-cell leukemia in mice. The leukemic and preleukemic tissues display a stem cell-like expression signature, including Hoxa, Hoxb, and Meis1 genes. The PHF23 plant homeodomain (PHD) motif is known to bind to H3K4me3 residues, and chromatin immunoprecipitation experiments demonstrated that the NP23 protein binds to chromatin at a specific subset of H3K4me3 sites, including at Hoxa, Hoxb, and Meis1. Treatment of NP23 cells with disulfiram, which inhibits the binding of PHD motifs to H3K4me3, rapidly and selectively killed NP23-expressing myeloblasts; cell death was preceded by decreased expression of Hoxa, Hoxb, and Meis1. Furthermore, AML driven by a related fusion gene, NUP98-JARID1A (NJL), was also sensitive to disulfiram. Thus, the NP23 mouse provides a platform to evaluate compounds that disrupt binding of oncogenic PHD proteins to H3K4me3.
Assuntos
Sítios de Ligação/efeitos dos fármacos , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dissulfiram/farmacologia , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Experimental/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Leucemia Experimental/tratamento farmacológico , Camundongos , Camundongos Transgênicos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
The myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS, and 331 (9.6%) genes hypermethylated in RUNX1 MDS. Thirty-two percent of genes in common between the two models (2/3 NHD13 mice and 2/3 RUNX1 mice) were also hypermethylated in at least two of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressive drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Animais , Células da Medula Óssea , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Síndromes Mielodisplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
The myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML). Although it has been suggested that additional mutations lead to progression of MDS to AML, the causative agent(s) for such mutations remains unclear. Oxidative stress is a potential cause, therefore, we evaluated levels of reactive oxygen species (ROS) in NUP98-HOXD13 (NHD13) transgenic mice, a murine model for MDS. Increased levels of ROS were detected in bone marrow nucleated cells (BMNC) that express CD71, a marker for cell proliferation, as well as immature, lineage negative bone marrow nucleated cells from NHD13 mice. In addition to the increase in ROS, increased DNA double strand breaks and activation of a G2/M phase cell cycle checkpoint were noted in NHD13 BMNC. Finally, using an in vivo assay for mutation frequency, we detected an increased mutation frequency in NHD13 BMNC. These results suggest that oxidative stress may contribute to disease progression of MDS to AML through ineffective repair of DNA damage and acquisition of oncogenic mutations.
Assuntos
Modelos Animais de Doenças , Taxa de Mutação , Síndromes Mielodisplásicas/genética , Proteínas de Fusão Oncogênica/genética , Estresse Oxidativo , Doença Aguda , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Western Blotting , Células da Medula Óssea/metabolismo , Linhagem Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Síndromes Mielodisplásicas/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Myelodysplastic syndrome (MDS) and aplastic anemia (AA) patients both present with symptoms of bone marrow failure. In many AA patients, these features are thought to result from an oligoclonal expansion of cytotoxic T-cells that destroy haematopoietic stem or progenitor cells. This notion is supported by the observation that AA patients respond to immunosuppressive therapy. A fraction of MDS patients also respond well to immunosuppressive therapy suggesting a similar role for cytotoxic T-cells in the etiology of MDS, however the role of cytotoxic T-cells in MDS remains unclear. Mice that express a NUP98-HOXD13 (NHD13) transgene develop a MDS that closely mimics the human condition in terms of dysplasia, ineffective hematopoiesis, and transformation to acute myeloid leukemia (AML). We followed a cohort of NHD13 mice lacking the Rag1 protein (NHD13/Rag1KO) to determine if the absence of lymphocytes might 1) delay the onset and/or diminish the severity of the MDS, or 2) effect malignant transformation and survival of the NHD13 mice. No difference was seen in the onset or severity of MDS between the NHD13 and NHD13/Rag1KO mice. However, NHD13/Rag1KO mice had decreased survival and showed a trend toward increased incidence of transformation to AML compared to the NHD13 mice, suggesting protection from AML transformation by a modest immuno-surveillance effect. In the absence of functional Tcrb signaling in the NHD13/Rag1KO T-cell tumors, Pak7 was identified as a potential Tcrb surrogate survival signal.
Assuntos
Vigilância Imunológica , Síndromes Mielodisplásicas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Modelos Animais de Doenças , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Humanos , Leucemia Experimental/etiologia , Leucemia Experimental/imunologia , Leucemia Experimental/patologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Síndromes Mielodisplásicas/etiologia , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/terapia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Quinases Ativadas por p21/imunologiaRESUMO
Structural chromosomal rearrangements of the Nucleoporin 98 gene (NUP98), primarily balanced translocations and inversions, are associated with a wide array of hematopoietic malignancies. NUP98 is known to be fused to at least 28 different partner genes in patients with hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia in blast crisis, myelodysplastic syndrome, acute lymphoblastic leukemia, and bilineage/biphenotypic leukemia. NUP98 gene fusions typically encode a fusion protein that retains the amino terminus of NUP98; in this context, it is important to note that several recent studies have demonstrated that the amino-terminal portion of NUP98 exhibits transcription activation potential. Approximately half of the NUP98 fusion partners encode homeodomain proteins, and at least 5 NUP98 fusions involve known histone-modifying genes. Several of the NUP98 fusions, including NUP98-homeobox (HOX)A9, NUP98-HOXD13, and NUP98-JARID1A, have been used to generate animal models of both lymphoid and myeloid malignancy; these models typically up-regulate HOXA cluster genes, including HOXA5, HOXA7, HOXA9, and HOXA10. In addition, several of the NUP98 fusion proteins have been shown to inhibit differentiation of hematopoietic precursors and to increase self-renewal of hematopoietic stem or progenitor cells, providing a potential mechanism for malignant transformation.
Assuntos
Neoplasias Hematológicas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Animais , Transformação Celular Neoplásica/metabolismo , Neoplasias Hematológicas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Poro Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Inversão de Sequência , Transcrição Gênica , Translocação GenéticaRESUMO
The activation of genes important to acute lymphoblastic leukemia (ALL) may be evidenced by somatically acquired chromosomal translocations found recurrently in different patient subgroups. It is for this reason that research efforts have focused on the molecular dissection of recurring chromosomal rearrangements. However, even though a large number of leukemia-causing genes have been identified, the genetic basis of many ALL cases remains unknown. We and others have reasoned that novel translocations found in the leukemic cells of ALL patients may mark the location of more frequent gene rearrangements that are otherwise hidden submicroscopically within normal or complex karyotypes. Towards this end, we here describe the first reported association of a t(5;10)(q22;q24) with adult ALL. Fluorescence in situ hybridization (FISH) and Southern blot hybridization studies have eliminated likely involvement of the candidate genes APC and MCC on chromosome 5, and PAX2, TLX1, and NFKB2 on chromosome 10. Results further suggest that the breakpoint on chromosome 5 lies centromeric of APC and the chromosome 10 breakpoint is centromeric of PAX2. The genomic regions disrupted by this t(5;10)(q22;q24) have not previously been associated with leukemia.
Assuntos
Cromossomos Humanos Par 5 , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética , Células da Medula Óssea/patologia , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , Humanos , Masculino , Metáfase , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
BACKGROUND: Chromosome band 10q24 is a gene-rich domain and host to a number of cancer, developmental, and neurological genes. Recurring translocations, deletions and mutations involving this chromosome band have been observed in different human cancers and other disease conditions, but the precise identification of breakpoint sites, and detailed characterization of the genetic basis and mechanisms which underlie many of these rearrangements has yet to be resolved. Towards this end it is vital to establish a definitive genetic map of this region, which to date has shown considerable volatility through time in published works of scientific journals, within different builds of the same international genomic database, and across the differently constructed databases. RESULTS: Using a combination of chromosome and interphase fluorescent in situ hybridization (FISH), BAC end-sequencing and genomic database analysis we present a physical map showing that the order and chromosomal orientation of selected genes within 10q24 is CEN-CYP2C9-PAX2-HOX11-NFKB2-TEL. Our analysis has resolved the orientation of an otherwise dynamically evolving assembly of larger contigs upstream of this region, and in so doing verifies the order and orientation of a further 9 cancer-related genes and GOT1. This study further shows that the previously reported human papillomavirus type 6a DNA integration site HPV6AI1 does not map to 10q24, but that it maps at the interface of chromosome bands 14q13.3-q21.1. CONCLUSIONS: This revised map will allow more precise localization of chromosome rearrangements involving chromosome band 10q24, and will serve as a useful baseline to better understand the molecular aetiology of chromosomal instability in this region. In particular, the relocation of HPV6AI1 is important to report because this HPV6a integration site, originally isolated from a tonsillar carcinoma, was shown to be rearranged in other HPV6a-related malignancies, including 2 of 25 genital condylomas, and 2 of 7 head and neck tumors tested. Our finding shifts the focus of this genomic interest from 10q24 to the chromosome 14 site.