American Association for Cancer Research
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Supplementary Figure 2 from RANKL Expression, Function, and Therapeutic Targeting in Multiple Myeloma and Chronic Lymphocytic Leukemia

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journal contribution
posted on 2023-03-30, 21:48 authored by Benjamin Joachim Schmiedel, Carolin Andrea Scheible, Tina Nuebling, Hans-Georg Kopp, Stefan Wirths, Miyuki Azuma, Pascal Schneider, Gundram Jung, Ludger Grosse-Hovest, Helmut Rainer Salih

PDF file - 264K, (A) The ability of MM cells to release sRANKL does not influence NK reactivity induced by RANK-Fc-ADCC. The increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells using RANKL surface-positive MM cells that do (+sRANKL) or do not (-sRANKL) release sRANKL as targets upon treatment with RANK-Fc-ADCC was calculated. To this end, specific lysis (E:T ratio 40:1) or cytokine release (E:T ratio 1:1) of NK cells in the presence of isotyp control-treated target cells was set to 1 in each individual data set. No statistically significant difference (p=0.39 and p=1, respectively; Mann-Whitney U-Test) was observed upon analysis of results from 8 (cytotoxicity) and 6 (cytokine release) independent experiments. (B) Surface levels of RANKL do not correlate with induction of NK reactivity by RANK-Fc-ADCC. Expression of RANKL on patient MM (circles) and CLL cells (triangles) was correlated with the increase in cytotoxicity (left) and cytokine production (right) of allogeneic NK cells upon treatment with RANK-Fc-ADCC calculated by as described in A. Each symbol represents the result of one independent experiment. The number of independent experiments, correlation coefficients (Pearson correlation) and p values (T-Test) are indicated. (C) NK reactivity against RANKL surface-negative MM cells is not affected by the fusion proteins. RPMI 8226 MM cells (left) and primary MM cells (right) that lack RANKL surface expression were incubated with allogeneic NK cells in the presence or absence of 10mug/ml of the indicated RANK fusion proteins or isotype control. Cytotoxicity was determined by 2h Europium release assays (upper panels) and IFN-gamma levels in supernatants were analyzed after 24h by ELISA (lower panels). (D) RANKL transfectants were cultured with allogeneic NK cells in the presence or absence of RANK-Fc-ADCC or isotype control (10�g/ml each) and the indicated concentrations of rRANKL. Cytotoxicity was determined by 2h Europium release assays (left) and IFN-� levels in supernatants were analyzed after 24h by ELISA (right). One repesentative experiment each of a total of three with similar results is shown in C and D.



Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK–RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity. Cancer Res; 73(2); 683–94. ©2012 AACR.

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