Several genetic and clinical markers are established as prognostic factors in chronic lymphocytic leukemia (CLL). However, additional markers are needed for risk stratification. Flow cytometric analysis is a mainstay of CLL diagnostics, thus identification of novel prognostic surface markers can improve risk assessment without increasing burden for patients and physicians. Furthermore, surface molecules preferentially expressed in high-risk cases could serve as therapeutic targets for immunotherapy. CD105 (endoglin) is a TGF-beta coreceptor and activates endothelial cells in healthy tissues and cancer. In addition, it is expressed on healthy hematopoietic precursors as well as lymphoid and myeloid leukemias. In acute myeloid leukemia (AML), a CD105 antibody is successfully applied in clinical studies. In CLL, mRNA expression of the CD105 gene ENG reportedly correlates with other risk factors but failed to show significant correlation with overall survival. However, CD105 protein expression in CLL has never been studied. We here analyzed CD105 surface expression on CLL cells from 71 patients by flow cytometry and report for the first time that substantial levels of CD105 are detectable on CLL cells in 70.4% of patients. Using receiver operating characteristics, we established a cutoff of 5.99% positive cells to distinguish between low and high CD105 levels, the latter correlating with decreased time to first treatment and overall survival. High CD105 expression further correlates with CD38 expression. Our study identified membrane expression of CD105 as a potential risk marker and therapeutic target in high-risk CLL. However, multivariant analyses of large cohorts should be performed in confirmatory studies.
Recent success of novel therapies has improved treatment of chronic lymphocytic leukemia (CLL) patients, but most of them still require several treatment regimes. To improve treatment choice, prognostic markers suitable for prediction of disease outcome are required. Several molecular/genetic markers have been established, but accessibility for the entirety of all patients is limited. We here evaluated the relevance of GITR/4-1BB as well as their ligands for the prognosis of CLL patients. Surface expression of GITR/GITRL and 4-1BB/4-1BBL was correlated with established prognostic markers. Next, we separated our patient population according to GITR/GITRL and 4-1BB/4-1BBL expression in groups with high/low expression levels and performed Kaplan-Meier analyses. Interestingly, no correlation was observed with the defined prognostic markers. Whereas no significant difference between high and low expression of GITR, GITRL and 4-1BBL was observed, high 4-1BB levels on leukemic cells were associated with significantly shorter survival. Thereby we identify 4-1BB as prognostic marker for CLL.
Genetic loss of the transcription factor NFAT2 (NFATc1), known as important player in lymphocyte development, causes an aggressive course of CLL, as it enhances BCR signaling and leads to highly malignant and proliferative disease (Märklin et al. Nat Commun 2017). To investigate whether and how NFAT2 affects treatment resistance in CLL, we generated a CRISPR/Cas9 based NFAT2 knockout (KO) in MEC-1 CLL cells to model aggressive CLL. In immunodeficient NSG-mice, the NFAT2 KO cells showed a ten-fold higher engraftment in the bone marrow after three weeks compared to scrambled (SCR) controls. Cytotoxicity assays revealed a profoundly higher resistance of NFAT2 KO cells to NK cell killing without detectable differences at the level of recognition as revealed by analyses of NK cell activation, degranulation and IFNy release, which excluded resistance to NK cells was due to altered immunogenicity. As CLL patients are routinely treated with anti-CD20 antibody Rituximab, which mediates its therapeutic efficacy in large part by inducing antibody dependent cellular cytotoxicity (ADCC) of NK cells via secretion of perforin and granzymes. When we exposed the target cells to the cytotoxic content isolated out of NK cells, MEC-1 NFAT2 KO cells showed higher resistance for membrane permeabilization compared to SCR controls. In line, perforin blockade equalized susceptibility to membrane permeabilization of both, NFAT2 KO cells and controls, confirming the involvement of NFAT2 in maintaining membrane integrity. In addition, caspase 3/7 activation was reduced in NFAT2 KO cells upon exposure to NK cell granule content as well as upon treatment with staurosporine. This shows an important role of NFAT2 for resistance of the leukemic cells at early stages of apoptosis. The results on the role of NFAT2 in resistance to NK effector function were confirmed with primary CLL patient cells, which were grouped according to high and low NFAT2 expression followed by treatment with NK cell content. Our results demonstrate that loss of NFAT2 allows CLLs cells to evade NK cell effector function. This holds true for both, constitutive cytotoxicity and therapeutically induced ADCC and is due to lowered susceptibility of the leukemic cells to perforin mediated membrane permeabilization and intrinsic resistance to granzyme induced apoptosis. Thus, NFAT2 loss, which correlates with aggressive clinical course in patients, facilitates resistance of CLL cells to CD20 antibody-based treatment. Citation Format: Samuel J. Holzmayer, Sarah M. Greiner, Kuebra Kaban, Jonas S. Heitmann, Helmut R. Salih, Melanie Maerklin. NFAT2 determines susceptibility of chronic lymphocytic leukemia cells to constitutive and Rituximab-induced NK cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2102.
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