Genotoxicity of cytokines at chemotherapy-induced ‘storm’ concentrations in a model of the human bone marrow

Abstract

Donor cell leukaemia (DCL) is a complication of haematopoietic stem cell transplantation where donated cells become malignant within the patient’s bone marrow. As DCL predominates as acute myeloid leukaemia, we hypothesised that the cytokine storm following chemotherapy played a role in promoting and supporting leukaemogenesis. Cytokines have also been implicated in genotoxicity, thus we explored a cell line model of the human BM to secrete myeloid cytokines following drug treatment, and their potential to induce micronuclei. HS-5 human stromal cells were exposed to mitoxantrone (MTX) and chlorambucil (CHL) and for the first time, were profiled for 80 cytokines using an array. Fifty-four cytokines were detected in untreated cells, of which 24 were upregulated, and 10 were downregulated by both drugs. FGF-7 was the lowest cytokine to be detected in both untreated and treated cells. Eleven cytokines not detected at baseline were detected following drug exposure. TNFα, IL6, GM-CSF, G-CSF and TGFβ1 were selected for micronuclei induction. TK6 cells were exposed to these cytokines in isolation and in paired combinations. Only TNFα and TGFβ1 induced micronuclei at healthy concentrations, but all five cytokines induced micronuclei at storm levels, which was further increased when combined in pairs. Of particular concern was that some combinations induced micronuclei at levels above the mitomycin C positive control, however most combinations were less than the sum of micronuclei induced following exposure to each cytokine in isolation. These data infer a possible role for cytokines through chemotherapy-induced cytokine storm, in the instigation and support of leukaemogenesis in the BM, and implicate the need to evaluate individuals for variability in cytokine secretion as a potential risk factor for complications such as DCL.

IL-6 knockdown in a model of the human bone marrow, abrogates DNA damage induction in bystander cells post-chemotherapy induced cytokine release syndrome

“…Reagents quoted in this research were sourced from Sigma-Aldrich (UK) except where otherwise stated. All drugs, positive controls and cell lines were as described in Asurappulige [ 18 ]. Briefly, chlorambucil (CHL) was used at 4 µM and mitoxantrone (MTX) was used at 1.12 µM (500 ng/ml), considered clinically relevant doses [ [19] , [20] , [21] ].…”

“…HS-5 cells (CRL-11882; ATCC; from LGC, UK), represented the BM microenvironment. Cells were cultured as described in Asurappulige [ 18 ]. For consistency within assays, cells within passages 3–9 (TK6) and 6–10 (HS-5) were selected.…”

“…TK6 cells were exposed to recombinant IL-6 at ‘healthy’ doses (50–1000 pg/ml) [ 23 , 24 ] or CS/CRS levels (2000–4000 pg/ml) [ 25 , 26 ] as described in Asurappulige [ 18 ] and collected to assess viability and genotoxicity (MN).…”

“…In a cell line model of the human BM [ 18 ] we previously confirmed increased cytokine release following chemotherapy and demonstrated that direct exposure to CS concentrations of selected myeloid cytokines (IL-6, TNFα, TGF-β1, GM-CSF and G-CSF) could induce genotoxicity relative to untreated cells or cells exposed to healthy cytokine levels. Furthermore, when these cytokines were paired, the genotoxicity statistically increased above untreated, and in some cases exceeded our positive genotoxic control, mitomycin C (MMC) [ 18 ].…”

“…Here we follow our previous publication [ 18 ] by exploring the role of IL-6 secretion following exposure to the alkylating agent chlorambucil, and the topoisomerase II inhibitor mitoxantrone. We show that siRNA reduction of IL-6 to healthy levels, reduced genotoxicity in bystander cells supporting the observations and recommendations of Turnquist [ 11 ] of anti-IL-6 therapy for patients suffering from CS/CRS.…”