RESUMO
BACKGROUND: New therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells. METHODS: The pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL's immunomodulatory effects in vitro and in vivo. RESULTS: 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts' immune system, specifically CD8+ T cells. 5-NL's pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL's induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment. CONCLUSIONS: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.
Assuntos
Linfócitos T CD8-Positivos , Melanoma , Camundongos , Animais , Regulação para Cima , Camundongos Endogâmicos C57BL , Vírus da Coriomeningite Linfocítica/genética , Melanoma/tratamento farmacológicoRESUMO
The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese , Transformação Celular Neoplásica , Proteínas com Domínio LIM/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/fisiologia , Histocitoquímica , Camundongos , Timo/patologiaRESUMO
The majority of childhood leukemias are precursor B-cell acute lymphoblastic leukemias (pB-ALLs) caused by a combination of prenatal genetic predispositions and oncogenic events occurring after birth. Although genetic predispositions are frequent in children (>1% to 5%), fewer than 1% of genetically predisposed carriers will develop pB-ALL. Although infectious stimuli are believed to play a major role in leukemogenesis, the critical determinants are not well defined. Here, by using murine models of pB-ALL, we show that microbiome disturbances incurred by antibiotic treatment early in life were sufficient to induce leukemia in genetically predisposed mice, even in the absence of infectious stimuli and independent of T cells. By using V4 and full-length 16S ribosomal RNA sequencing of a series of fecal samples, we found that genetic predisposition to pB-ALL (Pax5 heterozygosity or ETV6-RUNX1 fusion) shaped a distinct gut microbiome. Machine learning accurately (96.8%) predicted genetic predisposition using 40 of 3983 amplicon sequence variants as proxies for bacterial species. Transplantation of either wild-type (WT) or Pax5+/- hematopoietic bone marrow cells into WT recipient mice revealed that the microbiome is shaped and determined in a donor genotype-specific manner. Gas chromatography-mass spectrometry (GC-MS) analyses of sera from WT and Pax5+/- mice demonstrated the presence of a genotype-specific distinct metabolomic profile. Taken together, our data indicate that it is a lack of commensal microbiota rather than the presence of specific bacteria that promotes leukemia in genetically predisposed mice. Future large-scale longitudinal studies are required to determine whether targeted microbiome modification in children predisposed to pB-ALL could become a successful prevention strategy.
Assuntos
Suscetibilidade a Doenças , Disbiose/complicações , Fezes/microbiologia , Microbioma Gastrointestinal , Leucemia Experimental/prevenção & controle , Fator de Transcrição PAX5/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/prevenção & controle , Animais , Feminino , Leucemia Experimental/genética , Leucemia Experimental/microbiologia , Leucemia Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/microbiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
Somatic loss of function mutations in cohesin genes are frequently associated with various cancer types, while cohesin disruption in the germline causes cohesinopathies such as Cornelia-de-Lange syndrome (CdLS). Here, we present the discovery of a recurrent heterozygous RAD21 germline aberration at amino acid position 298 (p.P298S/A) identified in three children with lymphoblastic leukemia or lymphoma in a total dataset of 482 pediatric cancer patients. While RAD21 p.P298S/A did not disrupt the formation of the cohesin complex, it altered RAD21 gene expression, DNA damage response and primary patient fibroblasts showed increased G2/M arrest after irradiation and Mitomycin-C treatment. Subsequent single-cell RNA-sequencing analysis of healthy human bone marrow confirmed the upregulation of distinct cohesin gene patterns during hematopoiesis, highlighting the importance of RAD21 expression within proliferating B- and T-cells. Our clinical and functional data therefore suggest that RAD21 germline variants can predispose to childhood lymphoblastic leukemia or lymphoma without displaying a CdLS phenotype.
Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Linfoma , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptose , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Pontos de Checagem da Fase G2 do Ciclo Celular , Células Germinativas/metabolismo , Humanos , Linfoma/genética , Mutação , Fenótipo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Similar to pediatric acute myeloid leukemia (AML) the subgroup of biphenotypic acute lymphoblastic leukemia (ALL) is a rare complex entity with adverse outcome, characterized by the surface expression of CD33. Despite novel and promising anti-CD19 targeted immunotherapies such as chimeric antigen receptor T cells and bispecific anti-CD19/CD3 antibodies, relapse and resistance remain a major challenge in about 30% to 60% of patients. To investigate the potential role of the fully humanized bispecific antibody CD16 × CD33 (BiKE) in children with CD33+ acute leukemia, we tested whether the reagent was able to boost NK cell effector functions against CD33+ AML and biphenotypic ALL blasts. Stimulation of primary NK cells from healthy volunteers with 16 × 33 BiKE led to increased cytotoxicity, degranulation and cytokine production against CD33+ cell lines. Moreover, BiKE treatment significantly increased degranulation, IFN-γ and TNF-α production against primary ALL and AML targets. Importantly, also NK cells from leukemic patients profited from restoration of effector functions by BiKE treatment, albeit to a lesser extent than NK cells from healthy donors. In particular, those patients with low perforin and granzyme expression showed compromised cytotoxic function even in the presence of BiKE. In patients with intrinsic NK cell deficiency, combination therapy of CD16xCD33 BiKE and allogeneic NK cells might thus be a promising therapeutic approach. Taken together, CD16xCD33 BiKE successfully increased NK cell effector functions against pediatric AML and biphenotypic ALL blasts and constitutes a promising new option for supporting maintenance therapy or "bridging" consolidation chemotherapy before hematopoietic stem cell transplantation.
Assuntos
Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Receptores de IgG/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Anticorpos Biespecíficos/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Citotoxicidade Imunológica/imunologia , Proteínas Ligadas por GPI/imunologia , Células HL-60 , Humanos , Imunoterapia/métodos , Ativação Linfocitária/imunologiaRESUMO
Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Resposta ao Choque Térmico/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Animais , Antineoplásicos/química , Sítios de Ligação , Biomarcadores Tumorais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Mesilato de Imatinib/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Multimerização Proteica/efeitos dos fármacos , Análise Espectral , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of more than one cancer-related pathway by multi-target agents is an emerging approach in modern anticancer drug discovery. Here, based on the well-established synergy between histone deacetylase inhibitors (HDACi) and alkylating agents, we present the discovery of a series of alkylating HDACi using a pharmacophore-linking strategy. For the parallel synthesis of the target compounds, we developed an efficient solid-phase-supported protocol using hydroxamic acids immobilized on resins (HAIRs) as stable and versatile building blocks for the preparation of functionalized HDACi. The most promising compound, 3 n, was significantly more active in apoptosis induction, activation of caspase 3/7, and formation of DNA damage (γ-H2AX) than the sum of the activities of either active principle alone. Furthermore, to demonstrate the utility of our preloaded resins, the HAIR approach was successfully extended to the synthesis of a proof-of-concept proteolysis-targeting chimera (PROTAC), which efficiently degrades histone deacetylases.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Resinas Sintéticas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Estrutura MolecularRESUMO
The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53-p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated ß-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.
Assuntos
Ciclo Celular , Diferenciação Celular , Senescência Celular , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/biossíntese , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/biossíntese , Animais , Eritropoese/genética , Células-Tronco Hematopoéticas/patologia , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Translocação GenéticaAssuntos
Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Fusão Oncogênica/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteogenômica/métodos , Translocação GenéticaRESUMO
Combining multiple drugs broadens the window of therapeutic opportunities and is crucial for diseases that are currently lacking fully curative treatments. A powerful emerging tool for selecting effective drugs and combinations is the high-throughput drug screening (HTP). The histone deacetylase inhibitor (HDACi) givinostat (ITF2357) has been shown to act effectively against CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a subtype characterized by poor outcome and enriched in children with Down Syndrome, very fragile patients with a high susceptibility to treatment-related toxicity. The aim of this study is to investigate possible synergies with givinostat for these difficult-to-treat patients by performing HTP screening with a library of 174 drugs, either approved or in preclinical studies. By applying this approach to the CRLF2-r MHH-CALL-4 cell line, we identified 19 compounds with higher sensitivity in combination with givinostat compared to the single treatments. Next, the synergy between givinostat and the promising candidates was further validated in CRLF2r cell lines with a broad matrix of concentrations. The combinations with trametinib (MEKi) or venetoclax (BCL2i) were found to be the most effective and with the greatest synergy across three metrics (ZIP, HAS, Bliss). Their efficacy was confirmed in primary blasts treated ex vivo at concentration ranges with a safe profile on healthy cells. Finally, we described givinostat-induced modifications in gene expression of MAPK and BCL-2 family members, supporting the observed synergistic interactions. Overall, our study represents a model of drug repurposing strategy using HTP screening for identifying synergistic, efficient, and safe drug combinations.
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A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles' heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.
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Histone deacetylase 6 (HDAC6) is an emerging drug target to treat oncological and non-oncological conditions. Since highly selective HDAC6 inhibitors display limited anticancer activity when used as single agent, they usually require combination therapies with other chemotherapeutics. In this work, we synthesized a mini library of analogues of the preferential HDAC6 inhibitor HPOB in only two steps via an Ugi four-component reaction as the key step. Biochemical HDAC inhibition and cell viability assays led to the identification of 1g (highest antileukemic activity) and 2b (highest HDAC6 inhibition) as hit compounds. In subsequent combination screens, both 1g and especially 2b showed synergy with DNA methyltransferase inhibitor decitabine in acute myeloid leukemia (AML). Our findings highlight the potential of combining HDAC6 inhibitors with DNA methyltransferase inhibitors as a strategy to improve AML treatment outcomes.
Assuntos
Antineoplásicos , Decitabina , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases , Leucemia Mieloide Aguda , Humanos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/síntese química , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Decitabina/farmacologia , Decitabina/química , Relação Estrutura-Atividade , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Peptoides/química , Peptoides/farmacologia , Peptoides/síntese química , Aminopiridinas , BenzamidasRESUMO
Central nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2D cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3D co-culture model combining human brain organoids and BCP-ALL-cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids as compared to non-ALL-cells. To validate translatability between organoid co-culture and in vivo murine models, we confirmed that targeting CNS leukemia relevant pathways like CD79a/Igα or CXCR4-SDF1 reduced the invasion of BCP-ALL-cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared to the non-invaded fraction revealed significant upregulation of AP-1 transcription factor-complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in ALL-PDX-cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX-cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1-gene JUN in patients initially diagnosed as CNS-positive compared to CNS-negative cases as well as CNS-relapse vs non-CNS-relapse cases in a cohort of 100 BCP-ALL-patients. Our results suggest CNS-organoids as a novel model to investigate CNS-involvement and identify the AP-1 pathway as a critical driver of CNS-disease in BCP-ALL.
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Meriolin derivatives represent a new class of kinase inhibitors with a pronounced cytotoxic potential. Here, we investigated a newly synthesized meriolin derivative (termed meriolin 16) that displayed a strong apoptotic potential in Jurkat leukemia and Ramos lymphoma cells. Meriolin 16 induced apoptosis in rapid kinetics (within 2-3 h) and more potently (IC50: 50 nM) than the previously described derivatives meriolin 31 and 36 [1]. Exposure of Ramos cells to meriolin 16, 31, or 36 for 5 min was sufficient to trigger severe and irreversible cytotoxicity. Apoptosis induction by all three meriolin derivatives was independent of death receptor signaling but required caspase-9 and Apaf-1 as central mediators of the mitochondrial death pathway. Meriolin-induced mitochondrial toxicity was demonstrated by disruption of the mitochondrial membrane potential (ΔΨm), mitochondrial release of proapoptotic Smac, processing of the dynamin-like GTPase OPA1, and subsequent fragmentation of mitochondria. Remarkably, all meriolin derivatives were able to activate the mitochondrial death pathway in Jurkat cells, even in the presence of the antiapoptotic Bcl-2 protein. In addition, meriolins were capable of inducing cell death in imatinib-resistant K562 and KCL22 chronic myeloid leukemia cells as well as in cisplatin-resistant J82 urothelial carcinoma and 2102EP germ cell tumor cells. Given the frequent inactivation of the mitochondrial apoptosis pathway by tumor cells, such as through overexpression of antiapoptotic Bcl-2, meriolin derivatives emerge as promising therapeutic agents for overcoming treatment resistance.
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Despite the early clinical promise, adverse events such as acquired resistance and dose-limiting toxicities have barred the widespread use of HSP90 inhibitors as anticancer drugs. A new approach involving proteolysis-targeting chimeras (PROTACs) to degrade the protein instead of inhibiting it may overcome these problems. In this work, we describe the design, synthesis, and evaluation of cereblon-recruiting geldanamycin-based HSP90 degraders based on the PROTAC technology. Our best degrader, 3a, effectively decreased HSP90α and HSP90ß levels in cells utilizing the ubiquitin-proteasome pathway.
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Although a great cure rate has been achieved for pediatric BCP-ALL, approximately 15% of patients do not respond to conventional chemotherapy and experience disease relapse. A major effort to improve the cure rates by treatment intensification would result in an undesirable increase in treatment-related toxicity and mortality, raising the need to identify novel therapeutic approaches. High-throughput (HTP) drug screening enables the profiling of patients' responses in vitro and allows the repurposing of compounds currently used for other diseases, which can be immediately available for clinical application. The aim of this study was to apply HTP drug screening to identify potentially effective compounds for the treatment of pediatric BCP-ALL patients with poor prognosis, such as patients with Down Syndrome (DS) or carrying rearrangements involving PAX5 or KMT2A/MLL genes. Patient-derived Xenografts (PDX) samples from 34 BCP-ALL patients (9 DS CRLF2r, 15 PAX5r, 10 MLLr), 7 human BCP-ALL cell lines and 14 hematopoietic healthy donor samples were screened on a semi-automated HTP drug screening platform using a 174 compound library (FDA/EMA-approved or in preclinical studies). We identified 9 compounds active against BCP-ALL (ABT-199/venetoclax, AUY922/luminespib, dexamethasone, EC144, JQ1, NVP-HSP990, paclitaxel, PF-04929113 and vincristine), but sparing normal cells. Ex vivo validations confirmed that the BCL2 inhibitor venetoclax exerts an anti-leukemic effect against all three ALL subgroups at nanomolar concentrations. Overall, this study points out the benefit of HTP screening application for drug repurposing to allow the identification of effective and clinically translatable therapeutic agents for difficult-to-treat childhood BCP-ALL subgroups.
Assuntos
Reposicionamento de Medicamentos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Criança , Ensaios de Triagem em Larga Escala , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêuticoRESUMO
Children with Down syndrome have an augmented risk for B-cell acute lymphoblastic leukemia (DS-ALL), which is associated with lower survival than in non-DS-ALL. It is known that cytogenetic abnormalities common in childhood ALL are less frequent in DS-ALL, while other genetic aberrancies (ie, CRLF2 overexpression and IKZF1 deletions) are increased. A possible cause for the lower survival of DS-ALL that we herewith evaluated for the first time was the incidence and prognostic value of the Philadelphia-like (Ph-like) profile and the IKZF1plus pattern. These features have been associated with poor outcome in non-DS ALL and therefore introduced in current therapeutic protocols. Forty-six out of 70 DS-ALL patients treated in Italy from 2000 to 2014 displayed Ph-like signature, mostly characterized by CRLF2 (n = 33) and IKZF1 (n = 16) alterations; only 2 cases were positive for ABL-class or PAX5-fusion genes. Moreover, in an Italian and German joint cohort of 134 DS-ALL patients, we observed 18% patients positive for IKZF1plus feature. Ph-like signature and IKZF1 deletion were associated with poor outcome (cumulative incidence of relapse: 27.7 ± 6.8% versus 13 ± 7%; P = 0.04 and 35.2 ± 8.6% versus 17 ± 3.9%; P = 0.007, respectively), which further worsens when IKZF1 deletion was co-occurring with P2RY8::CRLF2, qualifying for the IKZF1plus definition (13/15 patients had an event of relapse or treatment-related death). Notably, ex vivo drug screening revealed sensitivity of IKZF1plus blasts for drugs active against Ph-like ALL such as Birinapant and histone deacetylase inhibitors. We provided data in a large setting of a rare condition (DS-ALL) supporting that these patients, not associated with other high-risk features, need tailored therapeutic strategies.
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HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and ß) in sustaining malignant cells' growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90ß isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.
Assuntos
Antineoplásicos , Proteínas de Choque Térmico HSP90 , Leucemia , Neoplasias , Humanos , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Leucemia/tratamento farmacológico , Leucemia/genética , Mutação , Resistencia a Medicamentos AntineoplásicosRESUMO
In this work, we utilized the proteolysis targeting chimera (PROTAC) technology to achieve the chemical knock-down of histone deacetylase 6 (HDAC6). Two series of cereblon-recruiting PROTACs were synthesized via a solid-phase parallel synthesis approach, which allowed the rapid preparation of two HDAC6 degrader mini libraries. The PROTACs were either based on an unselective vorinostat-like HDAC ligand or derived from a selective HDAC6 inhibitor. Notably, both PROTAC series demonstrated selective degradation of HDAC6 in leukemia cell lines. The best degraders from each series (denoted A6 and B4) were capable of degrading HDAC6 via ternary complex formation and the ubiquitin-proteasome pathway, with DC50 values of 3.5 and 19.4 nM, respectively. PROTAC A6 demonstrated promising antiproliferative activity via inducing apoptosis in myeloid leukemia cell lines. These findings highlight the potential of this series of degraders as effective pharmacological tools for the targeted degradation of HDAC6.