SAMHD1 dependence of cgas-sting-associated anti-tumor immune responses in classical Hodgkin lymphoma Free

Background: The tumor microenvironment (TME) plays a pivotal role in the pathogenesis of classical Hodgkin lymphoma (cHL) because of the multiple and complex interactions of Hodgkin and Reed Sternberg (HRS) cells with the inflammatory cells, through numerous cytokines and chemokines secreted by both HRS and TME cells. SAMHD1, a dNTP hydrolase, is involved in anti-tumor immune responses through its function in DNA damage mechanisms. In a previous study, we showed that SAMHD1 expression is an independent adverse prognostic factor in cHL (Xagoraris et al, Br J Haemat, 2021) and also demonstrated the therapeutic potential of SAMHD1 inhibitors in hematologic malignancies (Rudd et al, EMBO Mol Med, 2020). However, the biology underlying the clinical associations of SAMHD1 is unknown to date. Here, using an in vitro study model of SAMHD1-proficient and -deficient HRS cells, we investigated the biologic impact of SAMHD1 on anti-tumor immune responses, particularly focusing on the cGAS-STING pathway.

Methods: The in vitro system included 6 cHL cell lines, in 3 of which (L1236, L428, L540) SAMHD1-deficient clones (KO) were generated using CRISPR-Cas9 gene editing. To further explore the immunoregulatory role of SAMHD1, plasmid-mediated forced expression of wild-type (WT) SAMHD1 was performed in 2 cHL cell lines with low (L428) or undetectable (MDAV) endogenous SAMHD1 levels. The cHL cell lines were treated with increasing concentrations of a STING agonist, a STING inhibitor (C-176) or doxorubicin. Cell viability and growth were assessed by trypan blue exclusion assay in triplicate in all set of experiments. Expression and activation of proteins including critical components of the cGAS-STING pathway were analysed by Western blot. Immune responses were evaluated using the following methods: 1) RT-qPCR for gene expression (mRNA level) of critical immunomodulators (IFNβ, IFNγ, CXCL8, CXCL10); 2) Flow cytometry; 3) Cytokine arrays (105 cytokines, chemokines and growth factors) using cell culture supernates; 4) Targeted proteomics with Olink Target 48 Cytokine panel performed on cell culture supernates; 5) ⁵¹Chromium-based NK cell killing assays following STING stimulation.

Results: Gene expression of critical modulators of the innate immune responses, including IFNβ, IFNγ, CXCL8, CXCL10 and IL10, differed significantly between SAMHD1-deficient (KO) and SAMHD1-proficient (WT) cHL cells, although the differences were variable among the cHL cell lines tested. The effects of SAMHD1 deficiency were associated with certain changes in the activation (phosphorylation) status of cGAS-STING pathway proteins, including TBK1, IRF3 and IRF7. Using cytokine arrays, SAMHD1 deficiency led to increased secretion of Angiogenin and VCAM-1 in cell culture supernates. Targeted proteomics with Olink cytokine platform in cHL cell culture supernates showed substantial differences in the level of secreted IL6, IL7, IL18, IL27, TNFSF10/12, CXCL8, CCL4/7 as well as CCL11 between SAMHD1-proficient and -deficient cHL cells. STING stimulation with a STING agonist resulted in significantly increased IFNβ gene expression in SAMHD1-proficient but not in SAMHD1-deficient cHL cell lines strongly suggesting that SAMHD1 is required for induction of IFNβ upon STING stimulation. Notably, treatment of cHL cells with doxorubicin significantly upregulated IFNβ gene expression in SAMHD1-proficient but not in SAMHD1-deficient cells, again indicating the dependence of IFNβ induction on SAMHD1 gene in genotoxic states (chemotherapy). Also, transient depletion of SAMHD1 in cHL cells using VPX resulted in decreased NK cell-mediated killing of cultured cHL cells suggesting reduced immune response. Furthermore, forced expression of SAMHD1 resulted in upregulation of CXCL10 and -at a lesser degree- IFNβ gene expression (mRNA level) in both L428 and MDAV cells with low/undetectable endogenous SAMHD1 associated with changes in cGAS-STING pathway activation indicating enhanced pro-inflammatory response. Using cytokine arrays, forced expression of SAMHD1 also resulted in reduced secretion of IL6, IL10, IL34, CXCL8, CXCL9, CXCL10 and CD30 in cell culture supernates.

Conclusions: Taken together, the findings reveal the dependence of IFNβ gene expression on SAMHD1 upon STING stimulation or genotoxic effects in cHL. The cytokine and chemokine profile, which may shape the TME in cHL, is also modified by SAMHD1 further highlighting its critical immunomodulatory function in lymphoma TME.