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
Authors: Schmits Ulrike Regina , Antunes Ferreira Edite , Guezguez Borhane , Gomez-Zepeda David , Tenzer Stefan , Legscha Kevin Jan , Theobald Matthias , Echchannaoui Hakim 
Published: December 2024

Introduction: Despite recent innovations in thetreatment ofacute 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.