Stoffwechselveränderungen in T Zellen im Verlauf der Chronisch Lymphatischen Leukämie (CLL)

Chronic lymphocytic leukemia (CLL), leukemia with the highest incidence amongst adults in Western countries, is characterized by the expansion and accumulation of malignant B cell clones in the peripheral blood (PB) and lymphoid tissues. Nowadays, the front-line treatment is the use of targeted therapies, including Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) inhibitors. In addition, CLL was one of the first malignancies where chimeric antigen receptor (CAR) T cells were used. Despite the wide variety of treatment regimens, CLL remains incurable. Among the underlying reasons are the protective tumor microenvironment (TME), where CLL cells establish interactions with other cell types to support their growth, and the immunocompromised T cell compartment. One of the most studied characteristics of the T cell compartment is the enrichment of highly differentiated T cells and the “pseudo-exhaustion” phenomenon, where T cells display increased expression of exhaustion markers and impaired cytotoxic activity but retain their ability to secrete cytokines. Given the well- established link between T cell differentiation and metabolism, and the lack of studies in this regard in the context of CLL, we sought out to study potential metabolic alterations that T cells might undergo during disease onset and progression. Using human CLL samples and two CLL murine models, we observed that T cells undergo metabolic activation during early disease, which can most likely be attributed to (hyper-)activation of the PI3K/AKT/mTOR pathway. However, at advanced disease stages, defined as a frequency of >80% CLL cells among circulating mononuclear cells, T cells failed to maintain the previously observed enhanced metabolic activity while entering a rather exhausted state. At this time point, metabolic dysfunction was mostly evidenced by impaired mitochondrial fitness. We hypothesized that this metabolic exhaustion is driven by the constant antigen presentation caused by the high CLL burden and, at least in part, by the high uptake of lipids, which have the potential to induce mitochondrial dysfunction and lipotoxicity. Given these premises, we propose a mechanism to prevent T cell hyperactivation and to enhance mitochondrial fitness during disease progression. To do so, we treated T cells with short-chain fatty acids (SCFAs), known to activate the AMPK/PGC1/PPAR pathway. In contrast to the PI3K/AKT/mTOR pathway, which supports effector T cell differentiation by inducing aerobic glycolysis, AMPK rather supports oxidative phosphorylation and memory-like properties. In fact, we observed that SCFAs restored mitochondrial fitness even in presence of high CLL burden. Many advances still need to be made regarding SCFAs and their clinical application. However, we believe in their potential to restore T cell mitochondrial fitness in the context of CLL and likely other diseases where T cells undergo similar metabolic alterations.Die chronisch lymphatische Leukämie (CLL) ist die am häufigsten auftretende Leukämie im Erwachsenenalter in westlichen Ländern. Sie ist gekennzeichnet durch Expansion und Akkumulation maligner B Zell Klone sowohl im peripheren Blut als auch in lymphatischen Organen. Heutzutage werden in erster Linie zielgerichtete Therapien eingesetzt. Darüber hinaus gehört die CLL zu den ersten malignen Erkrankungen, die mit CAR T Zellen behandelt wurden. Trotz des breiten Spektrums an Behandlungsmöglichkeiten, ist die CLL nach wie vor unheilbar. Dies ist unter anderem auf die schützende Tumormikroumgebung (TME) zurückzuführen, in der das CLL- Wachstum durch andere Zelltypen unterstützt wird, sowie auf das immungeschwächte T Zell-Kompartiment. Eines der am besten untersuchten Merkmale des T Zell- Kompartiments ist die Anreicherung hoch differenzierter T Zellen und das Phänomen der sogenannten Pseudo-Erschöpfung, bei dem T Zellen eine erhöhte Expression von Erschöpfungsmarkern und eine verminderte zytotoxische Aktivität aufweisen, aber weiterhin in der Lage sind, Zytokine zu freizusetzen. Angesichts der Tatsache, dass T Zell-Differenzierung und T Zell-Metabolismus eng miteinander verknüpft sind, es aber wenige Daten im Kontext der CLL gibt, haben wir versucht, die metabolischen Veränderungen der T Zellen während des Krankheitsverlaufs zu charakterisieren. Anhand von humanen CLL Proben und zwei CLL Mausmodellen konnten wir zeigen, dass T Zellen in den ersten CLL Stadien eine Stoffwechselaktivierung erfahren, die wahrscheinlich durch eine Hyperaktivierung des PI3K/AKT/mTOR-Stoffwechselwegs vermittelt wird. Erreicht die Erkrankung ein fortgeschrittnes Stadium mit mehr als 80% CLL Zellen im peripheren Blut, konnten die T Zellen ihre erhöhte metabolische Aktivität nicht weiter aufrechterhalten und gingen in einen Erschöpfungzustand über. Zu diesem Zeitpunkt zeigt sich die metabolische Dysfunktion vor allem in einer Beeinträchtigung der mitochondrialen Fitness. Wir stellten die Hypothese auf, dass diese metabolische Erschöpfung durch die chronische T Zell-Aktivierung und, zumindest teilweise, durch die signifikante Aufnahme von Lipiden verursacht wird, die das Potenzial haben, eine mitochondriale Dysfunktion der T Zellen und Zelltod zu verursachen. Entsprechend verfolgen wir eine Möglichkeit die Hyperaktivierung der T Zellen zu verzögern und die mitochondriale Fitness im fortgeschrittenen Krankheitsstadium zu verbessern. Zu diesem Zweck behandelten wir T Zellen mit kurzkettigen Fettsäuren (SCFAs), von denen bekannt ist, dass sie sich positiv auf die Fitness und Funktion von T-Zellen auswirken. Interessanterweise stellten wir fest, dass SCFAs die mitochondriale Fitness selbst bei hoher CLL Belastung wiederherstellten, was wahrscheinlich durch die Aktivierung der AMPK/PGC1/PPAR-Achse erreicht wurde. Im Gegensatz zum PI3K/AKT/mTOR- 2 Signalweg, der die Differenzierung von Effektor T Zellen durch Induktion der aeroben Glykolyse unterstützt, ist dieser molekulare Signalweg dafür bekannt, dass er die oxidative Phosphorylation fördert und somit gedächtnisähnliche Eigenschaften bewahrt. Im Hinblick auf SCFAs und ihre klinische Anwendung müssen noch viele Untersuchungen unternommen werden, insbesondere bei ihres Wirkmechanismus und ihrer Verabreichung

Mecanismo de la muerte celular inducida por inhibición del metabolismo energético en células de mieloma múltiple

La oncogénesis es un proceso mediante el cual una célula normal se convierte en célula tumoral, con capacidad de proliferación descontrolada y posibilidad de formar un tumor maligno. Entre las múltiples alteraciones que sufre una célula tumoral, cabe destacar las variaciones en el metabolismo energético, ya que las células tumorales utilizan la glucólisis para obtener energía incluso en presencia de oxígeno, lo que se conoce como “Efecto Warburg”. Esta característica resulta interesante para generar estrategias terapéuticas contra el cáncer y por ello, en este trabajo se pretende evaluar el potencial que tiene el bloqueo del metabolismo energético para la terapia antitumoral. Para ello, las estrategias seguidas consisten en la búsqueda de efectos sinérgicos entre fármacos metabólicos (ritonavir, metformina y 2-deoxiglucosa) y fármacos quimioterápicos dirigidos contra las células del mieloma (carfilzomib y Byl-719). Los resultados obtenidos muestran que ritonavir, un fármaco inhibidor de la proteasa del VIH y bloqueante del transportador de glucosa GLUT4, sinergiza con carfilzomib, un inhibidor del proteasoma, potenciando el efecto del mismo en las líneas celulares ensayadas. Por otro lado, se aprecia un ligero efecto sumatorio entre la acción de 2-deoxiglucosa, un fármaco inhibidor de la glucólisis, y Byl-719, fármaco emergente para el tratamiento del mieloma que inhibe selectivamente la ruta de la fosfoinositol 3-quinasa (PI3K). De forma que, en ambos casos se obtienen mayores niveles de muerte celular en las células tratadas con la combinación de fármacos que con los fármacos por separado. Asimismo, al analizar la expresión de proteínas anti-apoptóticas en diferentes condiciones, se observa un aumento de Bcl-2 y Mcl-1, lo que podría estar relacionado con un mecanismo de resistencia de las células ante a los cambios inducidos en el metabolismo energético

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell–mediated elimination. Here, we introduce sialic acid–binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6–specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy

Role of CAR T Cell Metabolism for Therapeutic Efficacy

Chimeric antigen receptor (CAR) T cells hold enormous potential. However, a substantial proportion of patients receiving CAR T cells will not reach long-term full remission. One of the causes lies in their premature exhaustion, which also includes a metabolic anergy of adoptively transferred CAR T cells. T cell phenotypes that have been shown to be particularly well suited for CAR T cell therapy display certain metabolic characteristics; whereas T-stem cell memory (TSCM) cells, characterized by self-renewal and persistence, preferentially meet their energetic demands through oxidative phosphorylation (OXPHOS), effector T cells (TEFF) rely on glycolysis to support their cytotoxic function. Various parameters of CAR T cell design and manufacture co-determine the metabolic profile of the final cell product. A co-stimulatory 4-1BB domain promotes OXPHOS and formation of central memory T cells (TCM), while T cells expressing CARs with CD28 domains predominantly utilize aerobic glycolysis and differentiate into effector memory T cells (TEM). Therefore, modification of CAR co-stimulation represents one of the many strategies currently being investigated for improving CAR T cells’ metabolic fitness and survivability within a hostile tumor microenvironment (TME). In this review, we will focus on the role of CAR T cell metabolism in therapeutic efficacy together with potential targets of intervention

Lipotoxicity as a Barrier for T Cell-Based Therapies

Nowadays, T-cell-based approaches play an increasing role in cancer treatment. In particular, the use of (genetically engineered) T-cells has heralded a novel era for various diseases with previously poor outcomes. Concurrently, the relationship between the functional behavior of immune cells and their metabolic state, known as immunometabolism, has been found to be an important determinant for the success of immunotherapy. In this context, immune cell metabolism is not only controlled by the expression of transcription factors, enzymes and transport proteins but also by nutrient availability and the presence of intermediate metabolites. The lack of as well as an oversupply of nutrients can be detrimental and lead to cellular dysfunction and damage, potentially resulting in reduced metabolic fitness and/or cell death. This review focusses on the detrimental effects of excessive exposure of T cells to fatty acids, known as lipotoxicity, in the context of an altered lipid tumor microenvironment. Furthermore, implications of T cell-related lipotoxicity for immunotherapy will be discussed, as well as potential therapeutic approaches

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic acid-binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6-specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CART cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naive) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy