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Epigenetic and transcriptional regulation of CD4 T-cell dysfunction in chronic lymphocytic leukemia (Master thesis)

Τριανταφύλλου, Χαρίσιος/ Triantafyllou, Charisios

Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in the Western world, characterized by the clonal proliferation and accumulation of mature CD5+ B cells. It predominantly affects the elderly with an average diagnosis age of 70. The disease is characterized by a slow progression over many years and patients can be on a watch and wait regimen for long time until symptoms like fever and fatigue start to develop and treatment is necessary. CLL is not considered curable and while the scientific community is focused on targeted therapeutic strategies such as inhibitors of key players of the BCR-signaling such as Bruton tyrosine kinase (BTK), BCL2 and PI3K, these treatments ultimately lead to resistance and are therefore considered insufficient. This led to trials utilizing alternative strategies such as transfer of autologous chimeric antigen receptor (CAR) T cells focused on cancer-specific antigens, but this type of therapy faces significant limitations. For instance, its ability to induce complete response its limited to only a minor subset of CLL patients. The effect of this type of treatment is often compromised by T-cell dysfunction which is commonly observed in T cells of CLL patients. For CAR-T therapy to be successful T-cells need to effectively expand and persist after being injected to the patient. This is hindered by conditions such as terminal T-cell exhaustion or skewness of the T-cell population towards short-lived effector cells. While dysfunctional T cells share a lot of similarities with Exhausted and Effector cells, they are characterized by distinct metabolic and transcriptional profiles that are indicative of a distinct epigenetic profile and distinct functions of the transcription factor (TF). While our knowledge in this field is still lacking, it is known that T-cell dysfunction can be induced through the interaction with CLL cells. As CD4+ T cells have a central role on immunity and our knowledge about their dysfunction in CLL is limited, we decided to focus on CLL-induced changes in their regulatory landscape on the epigenetic and transcriptomic level, particularly regarding transcription factors. Thus, we employed a multi-omics approach to elucidate the CLL-induced regulatory changes in CD4+ T cells, focusing on epigenetic and transcriptional alterations. Utilizing data from inhouse ATAC-seq and RNA-seq experiments, we identified significant differences in chromatin accessibility and gene expression between CD4+ T cells co-cultured with CLL cells and those cultured in isolation. Using diffTF, we detected differential activation of 28 TFs, with notable alterations observed in members of the Jun/Fos and ATF/CREB families. Specifically, Jun/Fos TFs were consistently inactivated under CLL conditions, while ATF/CREB TFs were activated, suggesting a systematic reprogramming of the epigenetic landscape induced by the interaction with CLL cells. Complementary analyses using decoupleR on RNA-seq data, alongside an independent single-cell RNA-seq dataset from CLL patients treated with ibrutinib showed both similarities and differences compared to diffTF results. The consistent activation of androgen receptor (AR) across different data types and its role in T-cell differentiation suggest an involvement in CLL-induced dysfunction. In addition, the inactivation of numerous members of the Jun/Fos family of TFs in diffTF analysis underscores their potential role as key regulators of CLL-induced T cell dysfunction, especially when considering the anti-exhaustion role of members of Jun family such as c-Jun. Therapeutically, enhancing CAR-T cell therapies through c-Jun overexpression could mitigate T cell dysfunction, thereby improving treatment outcomes. Additionally, targeting androgen receptor (AR) signaling, represents another promising target. Integrating AR-targeted therapies with CAR-T cell approaches could yield more durable responses in CLL patients. In conclusion, this study provides a multilayered analysis of the epigenetic and transcriptional alterations underlying CLL-induced alteration that could lead to T cell dysfunction. By underscoring the significance of TFs that yield therapeutic potential this work contributes to the international endeavor for research aiming at the development of effective and potentially curable treatments against CLL.