Study on the Immune Escape Mechanism of Acute Myeloid Leukemia With DNMT3A Mutation

Huang

Clinical & Translational Med

et al. 2022

Self Cite

Background Chimeric antigen receptor T‐cell (CAR‐T) therapy for acute myeloid leukaemia (AML) has thus far been elusive, in part due to target restriction and phenotypic heterogeneity of AML cells. Mutations of the FMS‐like tyrosine kinase 3 (FLT3) and DNA methyltransferase 3A (DNMT3A) genes are common driver mutations that present with a poor prognosis in AML patients. We found that AML patients with FLT3 or DNMT3A mutations had higher expression of CD44 isoform 6 (CD44v6) compared to normal specimens. Therefore, we intended to demonstrate CD44v6 could be a specific option for AML with FLT3 or DNMT3A mutations. Methods Internal tandem duplication (ITD) mutations of FLT3 (FLT3/ITD) knock‐in clone and DNMT3A‐R882H mutant clones of SKM‐1 cells were generated using CRISPR/Cas9 and lentiviral transfection, respectively. CD44v6 CAR‐T cells were constructed by transfecting T cells with lentivirus containing CD44v6 CAR. CD44v6 expression in AML cell lines, AML patients and healthy donors was evaluated by flow cytometry. DNA methylation assays were used to analyse the mechanisms of FLT3 and DNMT3A mutations affecting CD44v6 expression. Results Aberrant overexpression of CD44v6 was observed in AML cell lines with FLT3 or DNMT3A mutations compared to the wild‐type SKM‐1 or K562 cells. AML patients with FLT3 or DNMT3A mutations had higher expression of CD44v6 compared to normal specimens. Then we constructed CD44v6 CAR‐T cells and found that CD44v6 CAR‐T specifically lysed CD44v6+ cells, accompanied by cytokines release. No significant killing effect was observed from CD44v6‐ AML cells and normal cells after co‐culture with CD44v6 CAR‐T. These results were also observed in vivo. Furthermore, we found that FLT3 or DNMT3A mutations induced CD44v6 overexpression by downregulating the CpG methylation of CD44 promoter. Conclusions Collectively, CD44v6 is a promising target of CAR‐T for AML patients with FLT3 or DNMT3A mutations.

Section: Methodsmentioning

confidence: 99%

“…For detailed construction methods and related data, please refer to previous research. 22 The brief process was as follows: a lentiviral vector pCDH-EF1-MCS-T2A-copGFP was used to clone the full-length human DNMT3A cDNA. Mutant strain of DNMT3A-R882H (CGC > CAC) was identified by DNA sequence.…”

Section: Construction Of Dnmt3a-r882h Mutant Cell Linementioning

confidence: 99%

CD44v6 chimeric antigen receptor T cell specificity towards AML with FLT3 or DNMT3A mutations

Huang

Clinical & Translational Med

et al. 2022

Self Cite

“…Due to the growing evidence that leukemic cells interact with bone marrow (BM) reactive cells, including macrophages, the concept of leukemia-associated macrophages has been formulated [ 39 , 40 ]. A possible correlation between an immunosuppressive microenvironment and leukemia progression has been hypothesized for acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) [ 41 ].…”

Section: Tam Role In Leukemiamentioning

confidence: 99%

“…A possible correlation between an immunosuppressive microenvironment and leukemia progression has been hypothesized for acute lymphoblastic leukemia (ALL), adult T-cell leukemia/lymphoma (ATLL), acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) [ 41 ]. The pathophysiology could be linked at least in part to an immune escape, due to a reduced ability of macrophages to recognize antigens and to perform their phagocytosis [ 39 , 40 , 41 , 42 , 43 ].…”

Section: Tam Role In Leukemiamentioning

confidence: 99%

The Role of Tumor-Associated Macrophages in Hematologic Malignancies

Cencini

Fabbri

Sicuranza

et al. 2021

Cancers

The tumor microenvironment includes dendritic cells, T-cytotoxic, T-helper, reactive B-lymphoid cells and macrophages; these reactive cells could interplay with malignant cells and promote tumor growth and survival. Among its cellular components, tumor-associated macrophages (TAM) represent a component of the innate immune system and play an important role, especially in hematologic malignancies. Depending on the stimuli that trigger their activation, TAM are polarized towards form M1, contributing to antitumor responses, or M2, associated with tumor progression. Many studies demonstrated a correlation between TAM, disease progression and the patient’s outcome in lymphoproliferative neoplasms, such as Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL), even if with conflicting results. A critical hurdle to overcome is surely represented by the heterogeneity in the choice of the optimal markers and methods used for TAM analysis (gene-expression profile vs. immunohistochemistry, CD163vs. CD68vs. CD163/CD68 double-positive cells). TAM have been recently linked to the development and progression of multiple myeloma and leukemia, with a critical role in the homing of malignant cells, drug resistance, immune suppression and angiogenesis. As such, this review will summarize the role of TAM in different hematologic malignancies, focusing on the complex interplay between TAM and tumor cells, the prognostic value of TAM and the possible TAM-targeted therapeutic strategies.

“…DNMT3A mutation happened in a variety of cancers such as colorectal cancer, lung cancer, chronic myelocytic leukemia, but is mainly with AML [32][33][34] and usually occur with other types of mutations, such as FLT3, NPM1, C/EBP alpha, resulting in synergistic effects in AML [35][36][37]. Being the most common mutation, DNMT3A R882H mutation can not only lower methylation enzymatic activity [38,39] but can also co-act with other proteins to influence immune activity, cell apoptosis, proliferation, and DNA damage repair to regulate AML [40][41][42]. Researchers also found that Twist1, mTOR pathway, CDK1, and Dot1l [43][44][45][46] which might be the potential therapeutic targets of AML patients carry DNMT3A mutation.…”

Section: Discussionmentioning

confidence: 99%

DNMT3A R882H mutation drives daunorubicin resistance in acute myeloid leukemia via regulating NRF2/NQO1 pathway

et al. 2022

Background DNA methyltransferase 3A (DNMT3A) often mutate on arginine 882 (DNMT3AR882) in acute myeloid leukemia (AML). AML patients with DNMT3A R882 mutation are usually resistant to daunorubicin treatment; however, the associated mechanism is still unclear. Therefore, it is urgent to investigate daunorubicin resistance in AML patients with DNMT3A R882 mutant. Method AML cell lines with DNMT3A-wild type (DNMT3A-WT), and DNMT3A-Arg882His (DNMT3A-R882H) mutation were constructed to investigate the role of DNMT3A R882H mutation on cell proliferation, apoptosis and cells’ sensitivity to Danunorubin. Bioinformatics was used to analyze the role of nuclear factor-E2-related factor (NRF2) in AML patients with DNMT3A R882 mutation. The regulatory mechanism of DNMT3A R882H mutation on NRF2 was studied by Bisulfite Sequencing and CO-IP. NRF2 inhibitor Brusatol (Bru) was used to explore the role of NRF2 in AML cells carried DNMT3A R882H mutation. Results AML cells with a DNMT3A R882H mutation showed high proliferative and anti-apoptotic activities. In addition, mutant cells were less sensitive to daunorubicin and had a higher NRF2 expression compared with those in WT cells. Furthermore, the NRF2/NQO1 pathway was activated in mutant cells in response to daunorubicin treatment. DNMT3A R882H mutation regulated the expression of NRF2 via influencing protein stability rather than decreasing methylation of NRF2 promoter. Also, NRF2/NQO1 pathway inhibition improved mutant cells’ sensitivity to daunorubicin significantly. Conclusion Our findings identified NRF2 as an important player in the regulation of cell apoptosis through which helps mediate chemoresistance to daunorubicin in AML cells with DNMT3A R882H mutation. Targeting NRF2 might be a novel therapeutic approach to treat AML patients with a DNMT3A R882H mutation.