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3444 Dual Inhibition of Histone and DNA Methylation in AML Enhances the Effectiveness of TCR-Based Cellular Therapy through Upregulation of Surface MHC Class I/p53 Antigen Complex

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies other than CAR-T Cells: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Combination therapy, Translational Research, Diseases, Treatment Considerations, Myeloid Malignancies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Ulrike Regina Schmits1*, Edite Antunes Ferreira2*, Borhane Guezguez, PhD2,3*, David Gomez-Zepeda4,5*, Stefan Tenzer4,5,6*, Kevin Jan Legscha2*, Matthias Theobald, MD3,6,7 and Hakim Echchannaoui, PhD2,3*

1Department of Hematology and Medical Oncology, University Medical Center (UMC) Mainz, Mainz, AL, Germany
2Department of Hematology and Medical Oncology, University Medical Center (UMC) Mainz, Mainz, Germany
3German Cancer Consortium (DKTK), Partner site Frankfurt/Mainz, Mainz, Germany
4Helmholtz Institute for Translational Oncology Mainz (HI-TRON Mainz), Mainz, Germany
5DKFZ German Cancer Research Center, Heidelberg, Germany
6Institute of Immunology, Research Center for Immunotherapy (FZI), Mainz, Germany
7Department of Hematology and Oncology, Medical Center of the Johannes Gutenberg University, Mainz, Germany

Introduction: Despite recent innovations in the treatment of acute myeloid leukemia (AML), adverse risk AML continues to be associated with poor response rates and short-term survival. Mutations and epigenetic alterations are key drivers in the resistance to conventional treatment and progression of AML. In this landscape, alteration/mutation of TP53 is accompanied with poor prognosis and has been recently reported to confer resistance to CAR-T cell therapy. TP53 represents a promising target to overcome resistance to T cell-based therapies. Epigenetic modifiers, in particular hypomethylation drugs that target DNA methyltransferase (DNMT), have emerged as another promising approach, and have received FDA approval. CM-272 is a novel reversible and selective dual inhibitor of DNMT and Histone Methyltransferase (HMT) G9a with anti-tumor properties in AML cells in vitro. However, the potential role of this DNMT/HMT complex towards tumor outgrowth and immune evasion across the AML mutational spectrum remains to be fully determined. Here, we explored the therapeutic combination of CM-272 treatment with adoptive T cell immunotherapy as a synergistic approach to overcome immune resistance of AML.

Methods: The NPM1mut OCI-AML3 cell line (expressing Luciferase) was used as a model to study the effect of CM-272 in in vitro coculture with T cells. RNA sequencing (RNA-seq) and quantitative proteomics were used to assess the effect of CM-272 treatment in OCI-AML3. Changes in the heterogeneity of peptides bound to MHC class I (MHC-I) induced by CM-272 were analyzed by immunopeptidomics. Human T cells were isolated from healthy donor buffy coats and retrovirally transduced with an HLA-A2.1-restricted p53TCR. Luminescence-based cytotoxicity assays were used to evaluate the combination of antigen-specific T cell killing and CM-272. The effect of CM-272 on the effectiveness of antigen-specific T cells was assessed by flow cytometry focusing on the T cells viability, proliferation, changes in differentiation/exhaustion markers and induction of apoptosis.

Results: Gene expression (RNA-seq) analysis of the NPM1mut OCI-AML3 cell line demonstrated that CM-272 induces up-regulation of the TP53 gene signature. These transcriptional changes were confirmed by p21 induction, a downstream target of TP53. Combining CM-272 with p53 antigen-TCR-specific T cells resulted in improved leukemic cell elimination compared to CM-272 or T cell monotherapies. Furthermore, CM-272 treatment induced upregulation of HLA-A*02:01 and the co-stimulatory molecule CD86 on OCI-AML3 cells while inducing cell cycle arrest and apoptosis. CM-272 did not affect T cell viability and effector functions. Quantitative proteomics confirmed the upregulation of HLA-A, and immunopeptidomics detected an increase in the number of HLA-A*02:01-presented peptides on CM-272 treated OCI-AML3 cells. Importantly, this was associated with a significant increase of the HLA-A*02:01-bound p53-epitope. Globally, these results suggest that increased expression of HLA-A*02:01/p53 complex along with the co-stimulatory signal CD86 upon CM-272 treatment account for the increased antigen-specific T cell effectiveness.

Conclusion: Our study provides first evidence of increased human T cell effector functions upon combination with a reversible epigenetic drug and a proof of principle demonstration for this novel combinatorial treatment for AML. The combinatorial epigenetic/TCR therapeutic approach lays the groundwork for further research into overcoming immune resistance in AML therapy. The immunopeptidomics results revealed new targetable tumor-associated epitopes and already provide us with predicted binding affinities to evaluate the potential of new antigen-specific T cell therapies. Deep characterization of the MHC-I bound peptides will be extended to patient-derived AML samples and validated in xenograft mouse models.

Disclosures: Legscha: Takeda: Honoraria.

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