RESUMO
BACKGROUND: GSK3368715, a first-in-class, reversible inhibitor of type I protein methyltransferases (PRMTs) demonstrated anticancer activity in preclinical studies. This Phase 1 study (NCT03666988) evaluated safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of GSK3368715 in adults with advanced-stage solid tumors. METHODS: In part 1, escalating doses of oral once-daily GSK3368715 (50, 100, and 200 mg) were evaluated. Enrollment was paused at 200 mg following a higher-than-expected incidence of thromboembolic events (TEEs) among the first 19 participants, resuming under a protocol amendment starting at 100 mg. Part 2 (to evaluate preliminary efficacy) was not initiated. RESULTS: Dose-limiting toxicities were reported in 3/12 (25%) patients at 200 mg. Nine of 31 (29%) patients across dose groups experienced 12 TEEs (8 grade 3 events and 1 grade 5 pulmonary embolism). Best response achieved was stable disease, occurring in 9/31 (29%) patients. Following single and repeat dosing, GSK3368715 maximum plasma concentration was reached within 1 h post dosing. Target engagement was observed in the blood, but was modest and variable in tumor biopsies at 100 mg. CONCLUSION: Based on higher-than-expected incidence of TEEs, limited target engagement at lower doses, and lack of observed clinical efficacy, a risk/benefit analysis led to early study termination. TRIAL REGISTRATION NUMBER: NCT03666988.
Assuntos
Antineoplásicos , Neoplasias , Adulto , Humanos , Antineoplásicos/efeitos adversos , Inibidores Enzimáticos/efeitos adversos , Dose Máxima Tolerável , Neoplasias/patologia , Resultado do TratamentoRESUMO
PAX5, one of nine members of the mammalian paired box (PAX) family of transcription factors, plays an important role in B cell development. Approximately one-third of individuals with pre-B acute lymphoblastic leukemia (ALL) acquire heterozygous inactivating mutations of PAX5 in malignant cells, and heterozygous germline loss-of-function PAX5 mutations cause autosomal dominant predisposition to ALL. At least in mice, Pax5 is required for pre-B cell maturation, and leukemic remission occurs when Pax5 expression is restored in a Pax5-deficient mouse model of ALL. Together, these observations indicate that PAX5 deficiency reversibly drives leukemogenesis. PAX5 and its two most closely related paralogs, PAX2 and PAX8, which are not mutated in ALL, exhibit overlapping expression and function redundantly during embryonic development. However, PAX5 alone is expressed in lymphocytes, while PAX2 and PAX8 are predominantly specific to kidney and thyroid, respectively. We show that forced expression of PAX2 or PAX8 complements PAX5 loss-of-function mutation in ALL cells as determined by modulation of PAX5 target genes, restoration of immunophenotypic and morphological differentiation, and, ultimately, reduction of replicative potential. Activation of PAX5 paralogs, PAX2 or PAX8, ordinarily silenced in lymphocytes, may therefore represent a novel approach for treating PAX5-deficient ALL. In pursuit of this strategy, we took advantage of the fact that, in kidney, PAX2 is upregulated by extracellular hyperosmolarity. We found that hyperosmolarity, at potentially clinically achievable levels, transcriptionally activates endogenous PAX2 in ALL cells via a mechanism dependent on NFAT5, a transcription factor coordinating response to hyperosmolarity. We also found that hyperosmolarity upregulates residual wild type PAX5 expression in ALL cells and modulates gene expression, including in PAX5-mutant primary ALL cells. These findings specifically demonstrate that osmosensing pathways may represent a new therapeutic target for ALL and more broadly point toward the possibility of using gene paralogs to rescue mutations driving cancer and other diseases.
Assuntos
Rim/metabolismo , Osmorregulação , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Células HEK293 , Humanos , Soluções Hipertônicas/farmacologia , Rim/efeitos dos fármacos , Masculino , Camundongos , Mutação , Osmorregulação/efeitos dos fármacos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The importance of epigenetic gene regulatory mechanisms in normal and cancer development is increasingly evident. Genome-wide analyses have revealed the mutation, deletion, and dysregulated expression of chromatin-modifying enzymes in a number of cancers, including hematologic malignancies. Genome-wide studies of DNA methylation and histone modifications are beginning to reveal the landscape of cancer-specific chromatin patterns. In parallel, recent genetic loss-of-function studies in murine models are demonstrating functional involvement of chromatin-modifying enzymes in malignant cell proliferation and self-renewal. Paradoxically, the same chromatin modifiers can, depending on cancer type, be either hyperactive or inactivated. Increasingly, cross talk between epigenetic pathways is being identified. Leukemias carrying MLL rearrangements are quintessential cancers driven by dysregulated epigenetic mechanisms in which fusion proteins containing N-terminal sequences of MLL require few or perhaps no additional mutations to cause human leukemia. Here, we review how recent progress in the field of epigenetics opens potential mechanism-based therapeutic avenues.
Assuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Epigênese Genética , Leucemia Aguda Bifenotípica , Animais , DNA de Neoplasias/genética , Rearranjo Gênico/genética , Estudo de Associação Genômica Ampla , Histona-Lisina N-Metiltransferase , Humanos , Leucemia Aguda Bifenotípica/genética , Leucemia Aguda Bifenotípica/metabolismo , Leucemia Aguda Bifenotípica/patologia , Leucemia Aguda Bifenotípica/terapia , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismoRESUMO
Enhancer of zeste homolog 2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which, in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog, regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized α-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2-EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small-molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte-macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2-EED complex, we pharmacologically modulate an epigenetic 'writer' and suppress PRC2-dependent cancer cell growth.
Assuntos
Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Peptídeos/farmacologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Leucemia/metabolismo , Leucemia/patologia , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Complexo Repressor Polycomb 2/metabolismo , Relação Estrutura-AtividadeRESUMO
A growing body of data suggests the importance of epigenetic mechanisms in cancer. Polycomb repressive complex 2 (PRC2) has been implicated in self-renewal and cancer progression, and its components are overexpressed in many cancers. However, its role in cancer development and progression remains unclear. We used conditional alleles for the PRC2 components enhancer of zeste 2 (Ezh2) and embryonic ectoderm development (Eed) to characterize the role of PRC2 function in leukemia development and progression. Compared with wild-type leukemia, Ezh2-null MLL-AF9-mediated acute myeloid leukemia (AML) failed to accelerate upon secondary transplantation. However, Ezh2-null leukemias maintained self-renewal up to the third round of transplantation, indicating that Ezh2 is not strictly required for MLL-AF9 AML, but plays a role in leukemia progression. Genome-wide analyses of PRC2-mediated trimethylation of histone 3 demonstrated locus-specific persistence of H3K27me3 despite inactivation of Ezh2, suggesting partial compensation by Ezh1. In contrast, inactivation of the essential PRC2 gene, Eed, led to complete ablation of PRC2 function, which was incompatible with leukemia growth. Gene expression array analyses indicated more profound gene expression changes in Eed-null compared with Ezh2-null leukemic cells, including down-regulation of Myc target genes and up-regulation of PRC2 targets. Manipulating PRC2 function may be of therapeutic benefit in AML.
Assuntos
Leucemia/patologia , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proliferação de Células , Imunoprecipitação da Cromatina , Citoproteção , Progressão da Doença , Regulação para Baixo/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Genes Neoplásicos/genética , Loci Gênicos/genética , Genoma/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Leucemia/genética , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Primitive neuroectodermal tumor (PNET) is most common in the second decade of life. Congenital PNET is very rare. Ocular metastasis of PNET is likewise exceedingly rare; with only 5 previously published cases. We report an unusual congenital PNET of the face, which metastasized to subcutis, eyes, and brain. The primary tumor responded to chemotherapy (vincristine/doxorubicin/cyclophosphamide) with metachronous progression of ocular lesions. A therapeutic trial of intraocular bevacizumab showed no efficacy on intraocular lesions. Eventually the patient developed cerebral metastasis, and second line therapy with topotecan/cyclophosphamide was initiated. The tumor progressed and the patient died after acute herniation.
Assuntos
Neoplasias Oculares/secundário , Tumores Neuroectodérmicos Primitivos/congênito , Tumores Neuroectodérmicos Primitivos/patologia , Neoplasias de Tecidos Moles/congênito , Neoplasias de Tecidos Moles/patologia , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Neoplasias Oculares/terapia , Face/patologia , Evolução Fatal , Humanos , Recém-Nascido , Masculino , Tumores Neuroectodérmicos Primitivos/terapia , Neoplasias de Tecidos Moles/terapia , Topotecan/administração & dosagem , Vincristina/administração & dosagemRESUMO
Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.
Assuntos
Terapia Genética/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/genética , Animais , Linhagem da Célula , Cães , Vetores Genéticos , Retroviridae/genéticaRESUMO
INTRODUCTION: Daratumumab is a CD38-targeting monoclonal antibody that has demonstrated clinical benefit for multiple myeloma. Daratumumab inhibition of CD38, which is expressed on immune cell populations and cardiomyocytes, could potentially affect cardiac function. This QTc substudy of the phase 2 CENTAURUS study investigated the potential effect of intravenous daratumumab monotherapy on QTc prolongation and other electrocardiogram (ECG) parameters, including concentration-QTc effect modeling. METHODS: Patients had intermediate- or high-risk smoldering multiple myeloma. Patients with QT interval corrected by Fridericia's formula (QTcF) > 470 ms, QRS interval ≥ 110 ms, or PR interval ≥ 200 ms were excluded. Triplicate ECGs were collected at screening, Dose 1, and Dose 8. Analyses of on-treatment ECGs were conducted with a time-matched baseline (primary analysis). By time-point, pharmacokinetic-pharmacodynamic (PK/PD), and outlier analyses were conducted. RESULTS: Of 123 patients in CENTAURUS, 31 were enrolled in the QTc substudy. Daratumumab produced a small increase in heart rate (5-12 beats per minute) of unclear significance. There was a small but clinically insignificant effect on QTc, as measured by both time-matched time-point and PK/PD analyses. The primary analysis demonstrated a maximum mean increase in QTcF of 9.1 ms (90% 2-sided upper confidence interval [CI], 14.1 ms). The primary PK/PD analysis predicted a maximum QTcF increase of 8.5 ms (90% 2-sided upper CI, 13.5 ms). No patient had an abnormal U wave, a new QTcF > 500 ms, or > 60 ms change from baseline for QTcF. CONCLUSION: Analysis of ECG intervals and concentration-QTc relationships showed a small but clinically insignificant effect of daratumumab. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02316106.
Assuntos
Mieloma Múltiplo Latente , Anticorpos Monoclonais/efeitos adversos , Eletrocardiografia , Frequência Cardíaca , HumanosRESUMO
Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.
Assuntos
Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Leucemia Mieloide Aguda/metabolismo , Biossíntese de Proteínas , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteína de Leucina Linfoide-Mieloide/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
Current guidelines for smoldering multiple myeloma (SMM) recommend active monitoring until the onset of multiple myeloma (MM) before initiating treatment or enrollment in a clinical trial. Earlier intervention may delay progression to MM. In CENTAURUS, 123 patients with intermediate-risk or high-risk SMM were randomly assigned to daratumumab 16 mg/kg intravenously on extended intense (intense), extended intermediate (intermediate), or short dosing schedules. At the prespecified primary analysis (15.8-month median follow-up), the complete response (CR) rates (co-primary endpoint) were 2.4%, 4.9%, and 0% for intense, intermediate, and short dosing, respectively; the co-primary endpoint of CR rate >15% was not met. Progressive disease (PD)/death rates (number of patients who progressed or died divided by total duration of progression-free survival [PFS] in patient-years; co-primary endpoint) for intense, intermediate, and short dosing were 0.055 (80% confidence interval [CI], 0.014-0.096), 0.102 (80% CI, 0.044-0.160), and 0.206 (80% CI, 0.118-0.295), respectively, translating to a median PFS ≥24 months in all arms (P < 0.0001, <0.0001, and =0.0213, respectively). With longer follow-up (median follow-up, 25.9 months), CR rates were 4.9%, 9.8%, and 0% for intense, intermediate, and short dosing, respectively. PD/death rates for intense, intermediate, and short dosing were 0.059 (80% CI, 0.025-0.092), 0.107 (80% CI, 0.058-0.155), and 0.150 (80% CI, 0.089-0.211), respectively, again translating to a median PFS ≥ 24 months in all arms (P < 0.0001 for all arms). Twenty-four-month PFS rates were 89.9% (90% CI, 78.5-95.4%), 82.0% (90% CI, 69.0-89.9%), and 75.3% (90% CI, 61.1-85.0%) for intense, intermediate, and short dosing, respectively. Pharmacokinetic analyses indicated that intense dosing maintained target-saturating trough concentrations in most patients throughout weekly, every-2-week, and every-4-week dosing periods. No new safety signals were observed. These data provide the basis for an ongoing phase 3 study of daratumumab in SMM.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Mieloma Múltiplo Latente/tratamento farmacológico , Mieloma Múltiplo Latente/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
OBJECTIVE: Recent studies in the mouse model have shown improved engraftment of repopulating cells when cells were administered by intramarrow (IM) vs intravenous (IV) injection. Here we wished to determine if IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model. MATERIALS AND METHODS: We used a competitive repopulation assay to directly compare IM vs IV injection in four baboons. CD34+ autologous bone marrow cells were split into two equal fractions and transduced with either green fluorescent protein (GFP) or yellow fluorescent protein (YFP). Gene-marked cells were infused by IM or IV administration after myeloablative irradiation. RESULTS: Peripheral blood granulocyte marking peaked at 2 to 3 weeks after transplantation and decreased thereafter before stabilizing. In all animals, marking levels of IM-injected cells (GFP) were lower than those of IV-injected cells (YFP) early after transplantation. However, in two of the four monkeys, GFP marking steadily increased after 2 months resulting in higher marking levels from IM-injected cells. In one animal, this trend sustained up to the last follow-up at 1 year after transplantation, with marking levels of 63.4% and 9.7% from IM- and IV-injected cells, respectively. Transplantation of both IM- and IV-injected CD34+ cells resulted in polyclonal multilineage engraftment. CONCLUSION: Our data show efficient gene marking after IM injection and suggest a different engraftment profile for IM- vs IV-injected repopulating cells.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Animais , Marcadores Genéticos , Injeções Intravenosas , Camundongos , Camundongos SCID , Papio , Retroviridae/genética , Integração ViralRESUMO
Genetic lesions affecting polycomb repressive complex 2 (PRC2) have been found in more than 40% of pediatric cases of early T-cell precursor acute lymphoblastic leukemia. The functional role of these PRC2 alterations has been obscure. Our recent data suggest that compromise of PRC2 blocks differentiation and accentuates growth and survival signaling.
RESUMO
Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autorrenovação Celular , Leucemia Mieloide Aguda/patologia , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia , Células-Tronco Neoplásicas/patologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Células Cultivadas , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Mitofagia/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K. All three dogs had stable gene marking and donor chimerism before receiving a course of O(6) -benzylguanine (O(6) BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) between days 210 and 589 after transplantation. One to four doses led to a marked increase in gene marking in all dogs. Furthermore, the transduced cells conferred chemoprotection and prevented severe neutropenia. Our results suggest that drug resistance gene therapy is feasible and safe in the nonmyeloablative transplantation setting.
Assuntos
Resistência a Medicamentos/genética , Terapia Genética/métodos , Transplante de Células-Tronco , Animais , Sequência de Bases , Carmustina/uso terapêutico , Primers do DNA/genética , Cães , Vetores Genéticos , Facilitação Imunológica de Enxerto , Lentivirus/genética , Quimeras de Transplante , Transplante HomólogoRESUMO
Clinical application of gene therapy for genetic and malignant diseases has been limited by inefficient stem cell gene transfer. Here we studied in a clinically relevant canine model whether genetic chemoprotection mediated by a mutant of the DNA-repair enzyme methylguanine methyltransferase could circumvent this limitation. We hypothesized that genetic chemoprotection might also be used to enhance allogeneic stem cell transplantation, and thus we evaluated methylguanine methyltransferase-mediated chemoprotection in an allogeneic setting. We demonstrate that gene-modified allogeneic canine CD34+ cells can engraft even after low-dose total body irradiation conditioning. We also show that cytotoxic drug treatment produced a significant and sustained multilineage increase in gene-modified repopulating cells. Marking in granulocytes rose to levels of up to 98%, the highest in vivo marking reported to date to our knowledge in any large-animal or human study. Increases in transgene-expressing cells after in vivo selection provided protection from chemotherapy-induced myelosuppression, and proviral integration site analysis demonstrated the protection of multiple repopulating clones. Drug treatment also resulted in an increase in donor chimerism. These data demonstrate that durable, therapeutically relevant in vivo selection and chemoprotection of gene-modified cells can be achieved in a large-animal model and suggest that chemoprotection can also be used to enhance allogeneic stem cell transplantation.
Assuntos
Antígenos CD34/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Células-Tronco Hematopoéticas/fisiologia , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Animais , Antígenos CD34/imunologia , Antineoplásicos/metabolismo , Carmustina/metabolismo , Linhagem da Célula , Cães , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Modelos Animais , Quimeras de Transplante , Condicionamento Pré-TransplanteRESUMO
The MLL1 histone methyltransferase gene undergoes many distinct chromosomal rearrangements to yield poor-prognosis leukemia. The remaining wild-type allele is most commonly, but not always, retained. To what extent the wild-type allele contributes to leukemogenesis is unclear. Here we show, using rigorous, independent animal models, that endogenous MLL1 is dispensable for MLL-rearranged leukemia. Potential redundancy was addressed by co-deleting the closest paralog, Mll2. Surprisingly, Mll2 deletion alone had a significant impact on survival of MLL-AF9-transformed cells, and additional Mll1 loss further reduced viability and proliferation. We show that MLL1/MLL2 collaboration is not through redundancy, but regulation of distinct pathways. These findings highlight the relevance of MLL2 as a drug target in MLL-rearranged leukemia and suggest its broader significance in AML.
Assuntos
Proteínas de Ligação a DNA/genética , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Animais , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/fisiologia , Humanos , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
Early T-Cell precursor acute lymphoblastic leukemia (ETP-ALL) is a relatively newly identified subset of T-lineage ALL. There are conflicting results regarding prognosis, and the genetic basis of this condition is variable. Here, we summarize the current status of the field and discuss the role of mutations in the Polycomb Repressive Complex 2 frequently identified in ETP-ALL patients.
RESUMO
Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/genética , Regulação Leucêmica da Expressão Gênica , Genes ras , Complexo Repressor Polycomb 2/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Fator de Transcrição STAT3/genética , Animais , Modelos Animais de Doenças , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Células Precursoras de Linfócitos T/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Leucemia Mieloide Aguda/metabolismo , Metiltransferases/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Feminino , Histona-Lisina N-Metiltransferase/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Metiltransferases/genética , Camundongos , Camundongos Knockout , Proteína Meis1 , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogênicas/genética , Transativadores , Proteínas Supressoras de Tumor/genéticaRESUMO
While DNA abnormalities have long been recognized as the cause of cancer, the contribution of chromatin is a relatively recent discovery. Excitement in the field of cancer epigenetics is driven by 3 key elements: 1. Chromatin may play an active and often critical role in controlling gene expression, DNA stability and cell identity. 2. Chromatin modifiers are frequent targets of DNA aberrations, in some cancers reaching near 100%. Particularly in cancers with low rates of DNA mutations, the key "driver" of malignancy is often a chromatin modifier. 3. Cancer-associated aberrant chromatin is amenable to pharmacologic modulation. This has sparked the rapidly expanding development of small molecules targeting chromatin modifiers or reader domains, several of which have shown promise in clinical trials. In parallel, technical advances have greatly enhanced our ability to perform comprehensive chromatin/histone profiling. Despite the discovery that distinct histone profiles are associated with prognostic subgroups, and in some instances may point towards an underlying aberration that can be targeted, histone profiling has not entered clinical diagnostics. Even eligibility for clinical trials targeting chromatin hinges on traditional histologic or DNA-based molecular criteria rather than chromatin profiles. This review will give an overview of the philosophical debate around the role of histones in controlling or modulating gene expression and discuss the most common techniques for histone profiling. In addition, we will provide prominent examples of aberrantly expressed or mutated chromatin modifiers that result in either globally or locally aberrant histone profiles, and that may be promising therapeutic targets.