FLT3-ITD Driven CMTM6 Expression Contributes to Immune Escape of Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a highly diverse blood cancer caused by genetic mutations, including FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), which occurs in about 25% of AML cases and is linked to a high leukemic burden and poor prognosis. While allogeneic hematopoietic cell transplantation (allo-HCT) can be a curative treatment for high-risk AML, post-transplant relapse remains a common and challenging issue, frequently leading to death. Relapse after allo-HCT often results from the loss of the graft-versus-leukemia (GVL) effect due to immune escape mechanisms like programmed death-ligand 1 (PD-L1) expression. CKLF-like MARVEL transmembrane domain containing member 6 (CMTM6) was identified to bind and stabilize PD-L1, thereby maintaining its expression at the cell surface in solid tumor cells. In this study, we found that oncogenic FLT3-ITD drives CMTM6 and PD-L1 protein expression in leukemia cells, contributing to immune escape of AML cells. Methods: For in vitro studies, 32D and WEHI-3B cell lines transduced with pMig vectors carrying different oncogenic mutations and the human MV4-11 cell line were used to assess the effect of oncogenes on the PD-L1/CMTM6 axis via flow cytometry, qRT-PCR, co-immunoprecipitation (Co-IP) and immunofluorescence microscopy. FLT3 kinase inhibition assays were performed using the FLT3 inhibitors tandutinib, crenolanib, and quizartinib. To study in vivo effects, an allo-HCT mouse model with wild type (WT) or Cmtm6-deficient C57BL/6N bone marrow (BM) transduced with FLT3-ITD and BALB/c donor BM / T cells was analyzed for survival and T cell activation by flow cytometry and CyTOF. Additionally, a Rag2-/-γc-/- xenograft mouse model with pTRIPZ inducible CMTM6-knockdown or WT MV4-11 cells and human T cells was used to study the effect of CMTM6 on disease progression. Clinical relevance was assessed using peripheral blood samples from FLT3-ITD+ AML patients (n=23) and bone marrow aspirates from AML patients before and after relapse (n=17), provided by the Study Alliance Leukemia Biobank. Results: Transduction of murine AML cell lines and primary BM cells with different oncogenes revealed that FLT3-ITD mutation enhanced protein expression of CMTM6 and PD-L1, which was reversible when treating cells with FLT3 inhibitors. Mechanistically, we found FLT3 kinase co-localized with CMTM6 in the plasma membrane and endoplasmic reticulum in AML cell lines. Co-IP of FLT3 and CMTM6 in FLT3-ITD+ AML cells confirmed their interaction at the protein level, while FLT3-ITD did not increase Cmtm6 mRNA expression. In the FLT3-ITD+ leukemia allo-HCT mouse model, Cmtm6-deficient AML mice showed significantly prolonged survival (p=0.005) and reduced leukemia burden (p=0.002) compared to WT AML mice. FLT3-ITD+Cmtm6-deficient AML cells also showed decreased PD-L1 levels in peripheral blood and spleen ultimately leading to enhanced T cell activation with a significantly higher abundance of CD4 and CD8 effector T cells and reduced naïve T cell numbers in the spleen. In addition, effector T cells showed increased CD69 and IFNγ expression. In a second mouse model, knockdown of CMTM6 in MV4-11 cells reduced CMTM6/PD-L1 surface expression causing improved AML rejection (p=0.003) and increased T cell activation measured by CD69 expression on CD4 T cells. Similarly, tandutinib treatment reduced leukemia burden as well as CMTM6 protein levels in Rag2-/-γc-/- mice transplanted with WT MV4-11 cells. In CD34+ blasts derived from PBMCs and BM aspirates of 2 independent AML patient cohorts, we found elevated CMTM6 protein expression in FLT3-ITD+ patients, which was associated with reduced T cell effector function and activation. Conclusions: We found that oncogenic FLT3-ITD increased CMTM6 and PD-L1 expression in AML cells, while FLT3 inhibition reduced their expression. Transplantation of Cmtm6-deficient FLT3-ITD+ leukemia cells improved survival, reduced leukemia burden and enhanced T cell activation compared to Cmtm6-proficient FLT3-ITD+ leukemia cells in vivo. We confirmed enhanced CMTM6 expression on leukemia cells in 2 independent cohorts of FLT3-ITD+ AML patients. Taken together, our study identifies a new mechanism of oncogene-induced stabilization of CMTM6 in leukemia cells, resulting in tumor immune escape. Inhibiting the CMTM6/PD-L1 axis could enhance the GVL effect post allo-HCT in patients with FLT3-ITD+ AML.