Loss of TET2 Increases MHC Class I Expression in Acute Myeloid Leukemia

Background:

One of the major challenges in the treatment of acute myeloid leukemia (AML) is the elimination of undifferentiated immature blood cells, often referred to as leukemia stem and progenitor cells (LSPC). LSPCs persist after treatment and are considered a major cause of relapse and refractory disease. Despite initially high remission rates with recent advances in treatment strategies, relapse occurs in a large proportion of patients, leading to high mortality in AML. Myeloid neoplasia (MN) is driven by somatic mutations, with loss of function TET2 mutations (TET2^MT) being one of the most common somatic lesions in MN. TET2, a member of the TET-family of DNA dioxygenases, is the major methyl-cytosine oxidase in hematopoietic cells, including T-cells. TET2 (along with TET1/3) are Fe2+ and αKG-dependent dioxygenases that utilize molecular oxygen to progressively oxidize 5-methyl cytosine (mC) in promoters, enhancers and silencer-associated mCpG segments of the genome, leading to their demethylation. This process is essential for mounting an accurate and efficient gene transcription profile that determines cell lineage fate, differentiation and proliferation. Here we report, while loss of TET2 creates a proliferative advantage to the LSPCs, it may also make them immunogenic and susceptible to immune surveillance due to higher expression of MHC class I/II molecules.

Methods:

We generated isogenic TET2 knockout (TET2^KO) leukemic cell models in THP1 and K562 cell lines using CRISPR-Cas9 gene editing. We also utilized natural TET2-deficient cell models, including OCI-AML5 (TET2^+/-) and SIMG5 (TET2^-/-). Western blot (WB) analysis, bicolor immunofluorescent (IF) imaging and confocal microscope were employed to assess protein expression and protein subcellular localization in these models. To validate our findings, we performed flow cytometry. In addition, we analyzed the HLA expression in TET2^MT mutant associated MN patients and compared it with WT.

Results:

Our study revealed that HLA- A/-B/-C (pan-HLA) expression in THP1 cells (MHC Class I positive) increased by more than 2-fold as a consequence of TET2 deletion compared to their isogenic wildtype counterparts, as observed in both WB and IF analyses. Additionally, pan-HLA protein levels were also significantly higher in cells with naturally TET2 deficiency (OCI-AML5 and SIGM5) compared to TET2 wildtype cells (THP1). The increased HLA protein levels was further confirmed by treatment with small molecule inhibitor of TET2 (TETi). Our data demonstrated that TETi treatment significantly increased pan-HLA expression in TET2^WT cells but not in TET2^KO cells. Furthermore, treatment with interferon gamma (INFg), a known inducer for HLA expression, significantly increased pan-HLA protein levels in TET2^WT but not in TET2^KO, suggesting that HLA expression is epigenetically regulated by TET2 in interferon mechanism of action. Interestingly, K562, an erythroid leukemia cell line known to be negative for surface expression of HLA- A/-B/-C had a significant pan-HLA upregulation upon genetic deletion or small molecule inhibition of TET2 dioxygenase activity. This observation was further confirmed in our analysis of human myeloid neoplasia (MDS and AML) patient cohort. Analysis of the RNAseq data from a cohort of 157 MN patients, including 38 with TET2^MT and 64 healthy bone marrow samples, demonstrated that both MHC class I and II were upregulated in TET2 mutant MN patients.

Conclusion:

Our findings demonstrate that the loss of TET2 function significantly increased the surface expression of MHC class I/II proteins in both isogenic and natural TET2-deficient leukemic cell models, as well as in MDS and AML patients, compared to TET2^WT and healthy control. The use of TET family inhibitors to chemically mimic TET2 loss further substantiated the increased pan-HLA expression, specifically in TET2 wildtype cells. Moreover, the differential response to INFγ treatment between TET2 wildtype and TET2 knockout cells highlights the critical role of TET2 in regulating HLA protein expression. These results suggest that targeting TET2 could enhance anti-cancer immune responses by increasing MHC class I/II expression, thereby improving the efficacy of immunotherapy therapies. This study provides a promising avenue for advancing cancer immunotherapy through epigenetic modulation.