The Contribution of Toll-like Receptor (TLR) Pathway Hyper-Reactivity to Clonal Selection in Secondary MDS and AML

Background: Stem cell (HSC) hypersensitivity to inflammatory cytokines and exaggerated TLR-dependent production of such cytokines contribute to bone marrow failure and clonal selection in Fanconi anemia (FA). Clonal neoplasms in FA patients and FA-deficient mice exhibit either resistance or paradoxical proliferative responses to tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN). Because FA MDS/AML shares cytogenetic and clinical features in common with secondary MDS/AML developing after prior MDS or exposure to cytotoxic chemotherapy (sMDS/sAML), we tested the idea that an FA-like TLR/cytokine hypersensitive phenotype might underlie clonal selection in sMDS/sAML. A pilot study of 4 hematologically normal individuals with histories of prior cytotoxic chemotherapy (including alkylating agents) revealed 2 whose committed progenitor cells were hypersensitive to TNF and IFN. Three patients with sMDS/sAML exhibited resistance to IFN and paradoxical growth responses to TNF. To determine the true prevalence of these FA-like phenotypes, we conducted a larger study of patients with sMDS/sAML, quantifying a) progenitor cell growth in response to inflammatory cytokines and b) TLR7/8 or TLR4-dependent cytokine production in peripheral blood monocytes.

Methods: Patients with newly diagnosed sMDS or sAML were eligible. All potential subjects with active infections were excluded. Research bone marrow aspirates and peripheral blood (PB) were obtained at the same time. TNF was quantified (ELISA) in the supernatants of PB CD14⁺ cells cultured in the presence of 1 ng/ml lipopolysaccharide (LPS, a TLR-4 agonist) or 3-5 μM R848 (TLR-7/8 agonist) for 24-hours. BFU-E and CFU-GM were quantified in methylcellulose cultures of low density bone marrow mononuclear cells (LDBMCs) (SCF, IL-3, EPO +/- TNF or IFN). Progenitor responses were classified as resistant/paradoxical if either CFU-GM or BFU-E colonies were >120% of control (normal volunteer bone marrow cells) at TNF 1 ng/ml, or >100% at IFN 0.1 ng/ml; all other responses were categorized as sensitive. TNF production was classified as exaggerated if levels exceeded normal mean values by more than two standard deviations.

Results: A total of 22 patients with sMDS/sAML had bone marrow and PB analysis, including 13 patients with complex karyotypes and 9 patients with non-complex karyotypes. An additional 18 patients had PB samples drawn without providing bone marrow samples. Progenitor responses: In colony assays, 18/22 patients demonstrated resistant/paradoxical growth responses to TNF/IFN, a phenomenon seen in patients with both complex and non-complex karyotypes. Interestingly, TNF resistance was observed only in samples that were also IFN resistant. Among patients with a complex karyotype, only 3 demonstrated normal progenitor sensitivity. None of these 3 had received prior chemotherapy (2 with radiation only). Macrophage responses: CD14⁺ cells from 63% of patients with resistant/paradoxical colony assay responses overproduced TNF in response to either R848 or LPS. Mean TNF levels from CD14⁺ cells of these patients were higher compared to patients with sensitive colony responses [1756 pg/ml (R848) and 1699 pg/ml (LPS), versus 783 pg/ml (R848) and 959 pg/ml (LPS), for resistant/paradoxical versus sensitive patients respectively]. Among all patients with PB samples (including those with/without marrow results), CD14⁺ cells from 68% of these patients overproduced TNF. Mean TNF levels in response to R848 or LPS stimulation of patient cells were elevated compared to normal donor controls [1730 pg/ml (R848) and 1795 pg/ml (LPS), versus 719 pg/ml (R848) and 555 pg/ml (LPS), from patient and normal donor CD14⁺ cells, respectively].

Conclusions: In patients with sMDS/sAML there is a high prevalence of TNF resistance in committed progenitors and TLR-hyper-reactivity in CD14⁺ cells. In FA the MDS/AML clonal resistance phenotype is selected because it possesses a selective advantage over the unfit pool of non-clonal FA HSC. We suggest that prior chemotherapy can create a fixed phenotype of HSC unfitness (e.g. hypersensitivity to inflammatory cytokines) and that the desultory fitness landscape in that pool permits the selection of cytokine resistant clones. Mechanistic studies should provide opportunities to identify patients at risk of sMDS/sAML and to develop pharmacological approaches to leukemia prevention.