Inhibition of High Mobility Group Box-1 (HMGB1) Eradicates Human Myelodysplastic Syndrome

Abstract

Myelodysplastic syndrome (MDS) is a heterogeneous and pre-leukemic clonal stem cell disorder characterized by aberrant hematopoiesis and bone marrow failure. Since MDS is associated with a dysregulated innate immune system, we performed a genetic screen of the human toll-like receptor (TLR) pathway and found that high mobility group box-1 (HMGB1) was abundantly expressed in MDS. HMGB1 is a ubiquitously expressed, non-histone chromatin-binding protein. In solid cancers, high expression of HMGB1 was associated with both shortened progression-free and overall survival, but its role in hematologic malignancies is not well defined. We hypothesized that targeting HMGB1 could be therapeutic in MDS. To study HMGB1 in human MDS, we utilized a cell line, MDS-L, derived from a patient with myelodysplastic syndrome with ring sideroblasts. Morphologically, the cells display dysplastic features, including cytoplasmic vacuolation and prominent, irregular nucleoli. We found that HMGB1 was detected at high levels in both MDS-L and in primary CD34+ MDS compared to CD34+ cord blood and healthy human marrow (*p<0.0001 for CD34+ cord blood compared to untreated MDS and treated MDS or CD34+ healthy marrow cells. n=3-10 biologic replicates/group). HMGB1 can activate several immune receptors, including Toll-like Receptors (TLRs). Since TLRs have been implicated in the pathogenesis of MDS, we screened several TLRs in primary MDS cells. We discovered that TLR2, TLR4, TLR6, and TLR9 were more highly expressed in primary MDS cells by 11 to 32-fold greater compared to CD34+ cord blood cells (*p<0.03, n=3-7 biologic replicates/group). These data indicate that the HMGB1-TLR axes exist in MDS and could contribute to inflammation and the pathogenesis of MDS.Next, we sought to pharmacologically inhibit HMGB1 signaling with sivelestat, which is a small molecule inhibitor for neutrophil elastase and HMGB1. Since sivelestat has not been studied in either normal or malignant hematopoiesis, whether it or other HMGB1 inhibitors could impact MDS is not yet defined. Following 72 h culture, HMGB1 protein levels were decreased in both MDS-L cells and in primary CD34+ MDS cells treated with 300 mg/ml sivelestat compared to cultures with vehicle alone (*p<0.01). Cultures with sivelestat had a marked reduction in total cell expansion and the capacity to generate colony forming cells in MDS-L cells and a subset of primary MDS cells (*p=0.02). Importantly, cultures of non-malignant CD34+ cord blood cells or healthy human marrow with sivelestat displayed no differences compared to control cultures. Following culture with azacitidine and sivelestat for 7 days, MDS-L cells displayed decreased total cell expansion and colony forming units compared to either azacitidine alone or vehicle alone (*p<0.0001). These data demonstrate that dual treatment with sivelestat and hypomethylating chemotherapies are more effective at blocking MDS cell expansion compared to chemotherapy alone, while sparing toxicity to normal hematopoietic cells.Since treatment with sivelestat could decrease both MDS cell expansion and impair the ability to generate colonies in methylcellulose assays, we sought to determine whether blocking HMGB1 could decrease MDS engraftment in NOD-scid ILR2gnull (NSG) mice. At 17 weeks, irradiated NSG mice that were transplanted with MDS-L cells treated with sivelestat demonstrated a 3.2-fold decrease in human CD45 marrow engraftment with MDS-L compared to control cultures (*p<0.006). Similarly, sivelestat treated mice displayed a 2.5-fold reduction in MDS engraftment compared to DMSO-treated mice at 17 weeks post-transplantation.Mechanistically, sivelestat increased cellular apoptosis in cultures of MDS cells compared to control cultures (*p=0.001). This was done in part by increasing levels of p53 upregulated modulator of apoptosis (PUMA, *p=0.0003), activated caspase 3 (*p<0.04), and gamma-H2AX (*p=0.0001). Inhibition of HMGB1 reduced expression of TLR2, TLR4, TLR6, and TLR9 (*p<0.006) and had a corresponding reduction of downstream phosphorylation of ERK1/2 compared to control cultures (*p<0.0004). Our findings demonstrate that inhibition of HMGB1 could normalize a dysregulated immune system and that HMGB1 could be a therapeutic target in MDS, and possibly for other hematologic diseases. DisclosuresNo relevant conflicts of interest to declare.