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Investigating Epigenetic Mechanisms of Response to Allogenic CAR T Cell Therapy

Institution: 
St. Jude Children’s Research Hospital
Researcher(s): 
Caitlin Zebley, MD/PhD
Grant Type: 
Young Investigator Grants
Year Awarded: 
2021
Type of Childhood Cancer: 
General Pediatric Cancer
Project Description: 

While autologous chimeric antigen receptor (CAR) T cell therapy has proven efficacious for many patients with acute lymphoblastic leukemia, CD19-positive relapse often occurs after CD19-CAR T cell therapy indicating that the CAR T cells either lack persistence or functionality. Based on our longitudinal assessment of patient samples, we have now shown that autologous CD19 CAR T cells undergo exhaustion-associated epigenetic programming during an in vivo tumor response. We hypothesize that acquisition of the exhaustion-associated epigenetic programs is hastened in autologous-derived CAR T cells due to underlying leukemia-induced T cell dysfunction. Therefore, I will explore whether CAR T cell therapy can be improved by the use of allogeneic CAR T cells generated from healthy donors. In this application, I propose 1) To determine if leukemia-induced T cell dysfunction promotes acquisition of exhaustion-associated epigenetic programs. 2) To determine the kinetics of exhaustion-associated epigenetic programs in allogeneic CAR T cells during a clinical response. Identifying mechanisms that contribute to the decline in CAR T cell survival and effector function (defining features of T cell exhaustion) is critical for addressing this challenge of limited therapeutic response. We have previously reported that DNA methyltransferase 3A (DNMT3A) programming is causal in establishing T cell exhaustion in murine systems and have confirmed this mechanism in humans using a variety of CAR T cell models. Building upon our insights into the epigenetic basis of T cell exhaustion, we have recently generated a bioinformatic tool termed the Multipotency Index (MPI) which is based on the DNA methylation programs of T cells and can predict the T cell’s differentiation potential. To determine the impact of leukemia-induced T cell dysfunction on the efficacy of CAR T cell therapy, I will compare autologous- (leukemia patient) and allogeneic- (healthy donor) derived CAR T cells by performing whole genome bisulfite sequencing methylation profiling to apply our MPI as well using our established ex vivo chronic antigen stimulation assays to examine proliferation and anti-tumor function. Furthermore, having identified the epigenetic signature of T cell exhaustion acquired in autologous CAR T cells, we will determine the kinetics of allogeneic CAR T cell dysfunction and highlight epigenetic differences between autologous and allogeneic CAR T cells during an in vivo tumor response.

Project Goal:

The research proposed here will identify DNA methylation programs that limit expansion and persistence of CAR T cells generated from a healthy donor. In pediatrics, a parent can serve as a T cell donor and our study will provide further insight into the epigenetic programs that restrict allogeneic CAR T cell survival after infusion into patients. Furthermore, our study will provide insight into leukemia-associated dysfunction of autologous T cells used for CAR T cell therapy in pediatric patients. Lastly, these studies will identify targetable molecular mechanisms that can be exploited to prolong CAR T cell anti-tumor responses.

Project Update 2024:

Chimeric antigen receptor (CAR) T cells are a promising immunotherapy for patients with relapsed/refractory leukemias. CD19 CAR T cells function by targeting the expression of CD19 which is on the surface of B-cells. We have shown that many patients who have received CD19-directed CAR T cell therapy relapse with CD19-positive cancer indicating that the CAR T cells lack functional persistence. Indeed, autologous CD19 CAR T cells (CAR T cells generated using T cells from the patient) acquire exhaustion-associated epigenetic programming which limits their function against tumor. We hypothesize that autologous-derived CAR T cells are more susceptible to exhaustion due to underlying leukemia-induced T cell dysfunction. In other words, the T cells used to generate the therapeutic CAR T cells are not healthy and therefore may not perform optimally against cancer. This grant proposes to explore the role of leukemia-induced T cell dysfunction on cellular therapy and circumvent this issue by using allogeneic CAR T cells derived from a healthy donor. We have now published our findings on autologous CD19 CAR T cell exhaustion that occurs in patients during an in vivo tumor response (Zebley et al. Cell Reports 2021). My proposal discusses examining these dysfunctional programs in allogeneic T cells from patients who received CAR T cells from a healthy haploidentical donor. While awaiting patient accrual on the clinical trial, we have focused our efforts on the T cell dysfunction present in AML patients compared to ALL patients. Specifically, we are looking deeper into the factors in the tumor microenvironment that are contributing to the underlying T cell dysfunction. Inflammatory danger associated molecular patterns (DAMPs) have been shown to be elevated in the plasma of adults with myeloid hematologic malignancies prompting our investigation into the role of extracellular inflammatory molecules in pediatric leukemias. Our preliminary data shows that the presence of DAMPs changes the phenotypic properties of healthy donor-derived T cells. We will next explore the plasma of leukemia patients to quantify the level of DAMPs and further investigate whether this proinflammatory environment can limit T cell function in pediatric ALL and AML. Furthermore, we have identified that disruption of the epigenetic regulator ASXL1 allows T cells to resist the immunosuppressive tumor microenvironment and maintain anti-tumor function. Results from these studies will provide a rationale to develop allogeneic CAR T cell protocols from healthy donors as well as genetically modified ASXL1 KO CAR T cells with the goal of establishing a sustained anti-tumor response that can prevent disease relapse.

Final Project Update 2025:

Our investigation into leukemia-induced T cell dysfunction resulted in the discovery that damage associated molecular patterns (DAMPs) play a key role in shaping the acute myeloid leukemia (AML) tumor microenvironment. We show that DAMPs can induce T cell dysfunction which provides mechanistic insight into the suppressive myeloid tumor microenvironment limiting T cell-based immunotherapy. Further, we analyzed myelodysplastic syndrome (MDS) patient T cells with ASXL1 mutations given that patients with these mutations experience superior long-term survival after immune checkpoint blockade. The ASXL1 disrupted patient T cells underwent enhanced proliferation after anti-PD-L1 therapy. In mouse models, ASXL1 KO T cells were able to combat historically immunosuppressive tumors with enhanced anti-tumor ability in the presence of anti-PD-L1 therapy. These data collectively provide a mechanism to explain long-term survival of MDS patients after PD-L1 blockade and a rationale for future studies exploring ASXL1 disruption as a strategy to protect T cells from myeloid leukemia-induced immune suppression.
 

Co-funded by: 
Ava's Army

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