RESUMO
The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.
Assuntos
Engenharia Celular/métodos , Transplante de Células/métodos , Células Matadoras Induzidas por Citocinas/imunologia , Resistencia a Medicamentos Antineoplásicos , Terapia Genética/métodos , Xenoenxertos , Imunoterapia Adotiva/métodos , Leucemia Experimental/terapia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos Quiméricos/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Animais , Estudos de Viabilidade , Técnicas de Transferência de Genes , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células THP-1 , Transposases/genética , Transposases/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Eradicating the malignant stem cell is the ultimate challenge in the treatment of leukaemia. Leukaemic stem cells (LSC) hijack the normal haemopoietic niche, where they are mainly protected from cytotoxic drugs. The anti-leukaemic effect of L-asparaginase (ASNase) has been extensively investigated in acute lymphoblastic leukaemia, but only partially in acute myeloid leukaemia (AML). We explored the susceptibility of AML-LSC to ASNase as well as the role of the two major cell types that constitute the bone marrow (BM) microenvironment, i.e., mesenchymal stromal cells (MSC) and monocytes/macrophages. Whilst ASNase was effective on both CD34+ CD38+ and CD34+ CD38- LSC fractions, MSC and monocytes/macrophages partially counteracted the effect of the drug. Indeed, the production of cathepsin B, a lysosomal cysteine protease, by BM monocytic cells and by AML cells classified as French-American-British M5 is related to the inactivation of ASNase. Our work demonstrates that, while MSC and monocytes/macrophages may provide a protective niche for AML cells, ASNase has a cytotoxic effect on AML blasts and, importantly, LSC subpopulations. Thus, these features should be considered in the design of future clinical studies aimed at testing ASNase efficacy in AML patients.
Assuntos
Asparaginase/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Asparaginase/farmacologia , Linhagem Celular , HumanosRESUMO
Bone marrow-mesenchymal stromal cells (BM-MSCs) are key components of the BM niche, where they regulate hematopoietic stem progenitor cell (HSPC) homeostasis by direct contact and secreting soluble factors. BM-MSCs also protect the BM niche from excessive inflammation by releasing anti-inflammatory factors and modulating immune cell activity. Thanks to these properties, BM-MSCs were successfully employed in pre-clinical HSPC transplantation models, increasing the rate of HSPC engraftment, accelerating the hematological reconstitution, and reducing the risk of graft failure. However, their clinical use requires extensive in vitro expansion, potentially altering their biological and functional properties. In this work, we analyzed the transcriptomic profile of human BM-MSCs sorted as CD45-, CD105+, CD73+, and CD90+ cells from the BM aspirates of heathy-donors and corresponding ex-vivo expanded BM-MSCs. We found the expression of immune and inflammatory genes downregulated upon cell culture and selected the transcription factor EGR1 to restore the MSC properties. We overexpressed EGR1 in BM-MSCs and performed in vitro tests to study the functional properties of EGR1-overexpressing BM-MSCs. We concluded that EGR1 increased the MSC response to inflammatory stimuli and immune cell control and potentiated the MSC hematopoietic supportive activity in co-culture assay, suggesting that the EGR1-based reprogramming may improve the BM-MSC clinical use.
RESUMO
In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.
Assuntos
Células Matadoras Induzidas por Citocinas , Leucemia Mieloide Aguda , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Leucemia Mieloide Aguda/terapia , Imunoterapia Adotiva , Interferon gama , Microambiente TumoralRESUMO
Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T-cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual-CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-induced killer (CIK) cells, coexpressing a first-generation low affinity anti-CD123 interleukin-3-zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.
Assuntos
Interleucina-3 , Leucemia Mieloide Aguda , Humanos , Criança , Idoso , Interleucina-3/metabolismo , Células Endoteliais/metabolismo , Linfócitos T , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/patologiaRESUMO
Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.
Assuntos
Elementos de DNA Transponíveis/genética , Leucemia/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Doença Aguda , Ensaios Clínicos como Assunto , Humanos , Leucemia/genéticaRESUMO
BACKGROUND: Chondrosarcomas are malignant cartilage-producing tumors showing mutations and changes in gene expression in metabolism related genes. In this study, we aimed to explore the metabolome and identify targetable metabolic vulnerabilities in chondrosarcoma. METHODS: A custom-designed metabolic compound screen containing 39 compounds targeting different metabolic pathways was performed in chondrosarcoma cell lines JJ012, SW1353 and CH2879. Based on the anti-proliferative activity, six compounds were selected for validation using real-time metabolic profiling. Two selected compounds (rapamycin and sapanisertib) were further explored for their effect on viability, apoptosis and metabolic dependency, in normoxia and hypoxia. In vivo efficacy of sapanisertib was tested in a chondrosarcoma orthotopic xenograft mouse model. RESULTS: Inhibitors of glutamine, glutathione, NAD synthesis and mTOR were effective in chondrosarcoma cells. Of the six compounds that were validated on the metabolic level, mTOR inhibitors rapamycin and sapanisertib showed the most consistent decrease in oxidative and glycolytic parameters. Chondrosarcoma cells were sensitive to mTORC1 inhibition using rapamycin. Inhibition of mTORC1 and mTORC2 using sapanisertib resulted in a dose-dependent decrease in viability in all chondrosarcoma cell lines. In addition, induction of apoptosis was observed in CH2879 after 24â¯h. Treatment of chondrosarcoma xenografts with sapanisertib slowed down tumor growth compared to control mice. CONCLUSIONS: mTOR inhibition leads to a reduction of oxidative and glycolytic metabolism and decreased proliferation in chondrosarcoma cell lines. Although further research is needed, these findings suggest that mTOR inhibition might be a potential therapeutic option for patients with chondrosarcoma.
RESUMO
Chondrosarcomas are malignant cartilage tumors that are relatively resistant towards conventional therapeutic approaches. Kinase inhibitors have been investigated and shown successful for several different cancer types. In this study we aimed at identifying kinase inhibitors that inhibit the survival of chondrosarcoma cells and thereby serve as new potential therapeutic strategies to treat chondrosarcoma patients. An siRNA screen targeting 779 different kinases was conducted in JJ012 chondrosarcoma cells in parallel with a compound screen consisting of 273 kinase inhibitors in JJ012, SW1353 and CH2879 chondrosarcoma cell lines. AURKA, CHK1 and PLK1 were identified as most promising targets and validated further in a more comprehensive panel of chondrosarcoma cell lines. Dose response curves were performed using tyrosine kinase inhibitors: MK-5108 (AURKA), LY2603618 (CHK1) and Volasertib (PLK1) using viability assays and cell cycle analysis. Apoptosis was measured at 24 h after treatment using a caspase 3/7 assay. Finally, chondrosarcoma patient samples (N = =34) were used to examine the correlation between AURKA, CHK1 and PLK1 RNA expression and documented patient survival. Dose dependent decreases in viability were observed in chondrosarcoma cell lines after treatment with MK-5108, LY2603618 and volasertib, with cell lines showing highest sensitivity to PLK1 inhibition. In addition increased sensitivity to conventional chemotherapy was observed after CHK1 inhibition in a subset of the cell lines. Interestingly, whereas AURKA and CHK1 were both expressed in chondrosarcoma patient samples, PLK1 expression was found to be low compared to normal cartilage. Analysis of patient samples revealed that high CHK1 RNA expression correlated with a worse overall survival. AURKA, CHK1 and PLK1 are identified as important survival genes in chondrosarcoma cell lines. Although further research is needed to validate these findings, inhibiting CHK1 seems to be the most promising potential therapeutic target for patients with chondrosarcoma.