Targeting High Mobility Group Box-1 (HMGB1) Promotes Cell Death in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous and preleukemic clonal stem cell disorder characterized by aberrant hematopoiesis and bone marrow failure (1). Current therapies include hypomethylating chemotherapies and immunomodulatory drugs (1). Patients with MDS are often advanced in age and may have comorbidities that exclude these treatment options because of excess toxicities (2). For these reasons, novel therapies are needed for the treatment of MDS that cause less toxicity than either standard chemotherapies or hematopoietic stem cell transplantation.

A newer approach to the treatment of MDS is to target the innate immune system, which may be dysregulated in MDS (3). Within the innate immune system, Toll-like receptor (TLR) signaling regulates the inflammatory response by activating transcription factors for IFNs and inflammatory cytokines (4). TLR4 was first shown to be overexpressed in both mononuclear cells and CD34⁺ cells of patients with MDS compared with healthy controls, to mediate inflammatory cytokine production in MDS (5), and that normalization of inflammatory pathways could impair MDS survival (6). In addition, TLR4 activation on bone marrow macrophages can increase proinflammatory cytokines like TNFα (7). Subsequently, TLR2, along with its TLR partners for heterodimerization, TLR1 and TLR6, were also found to be overexpressed in MDS compared with healthy controls (4). These data suggest that TLR signaling could be altered in MDS and could represent a therapeutic target in this disease.

We report that high mobility group box 1 (HMGB1) can modulate MDS cell expansion and long-term engraftment in human-mouse studies. HMGB1 is a nonhistone chromatin-binding protein that bears two DNA-binding domains (8). It is a member of the damage-associated molecular patterns and functions as a mediator of inflammatory processes, binds to a subset of TLRs, and is a regulator of cytokine storm (9). Here, we identify HMGB1 as a previously undescribed target to modulate the innate immune system in MDS.

MDS-L cells were a gift from Kaoru Tohyama, MD, PhD (Department of Laboratory Medicine, Kawasaki Medical School, Kurashiki, Okayama, Japan; refs. 10, 11). MDS-L cells were cultured as described previously (11). Mycoplasma testing was most recently performed by PCR (Sigma Aldrich) in January 2019 and was negative.

Studies with human samples and bone marrow from 13 patients with biopsy-proven MDS were performed in accordance with the Declaration of Helsinki and approved by the Duke Institutional Review Board. Written, informed consent was obtained from all patients. Samples were selected at random based on sample availability. Details of patient demographics are outlined in Supplementary Table S1. Marrow mononuclear cells were isolated with Lymphoprep (STEMCELL Technologies). CD34⁺ cells were isolated and cultured as described previously (12) with addition of 20 ng/mL rhIL-3 to culture media (R&D Systems).

RNA was extracted with RNeasy Mini or MicroKit (Qiagen). Gene expression analyses were performed according to the manufacturer's specifications (Thermo Fisher Scientific). Data were normalized to GAPDH and are shown following ΔΔC_t analysis (13). SiRNAs for HMGB1 (pool of 4 siRNAs) and nontargeting control from Dharmacon (GE Healthcare) were used according to the manufacturer's instructions.

HMGB1, RAGE, and TLRs analyses: cells were stained with anti-CD34 (BD Biosciences), fixed with 4% paraformaldehyde, permeabilized with Perm Buffer III (BD Biosciences), and labeled with anti-HMGB1 antibody (Abcam). HMGB1 was detected with an Alexa-Fluor 488 goat anti-rabbit IgG (Thermo Fisher Scientific). For RAGE, cells were labeled with anti-RAGE antibody (Abcam) and PE-conjugated goat anti-rabbit IgG (Abcam). For TLRs, cells were surface-labeled with FITC anti-TLR2 or PE anti-TLR4 antibodies (BioLegend). Isotype controls were included for all analyses.

Cell Death, PUMA, activated caspase 3, γ-H2AX, and phosphorylated ERK1/2 analysis: cell death was performed with Annexin V Apoptosis Detection Kit and 7-AAD (BD Biosciences). MDS-L cells were fixed and permeabilized as above, labeled with AlexaFluor 488 mouse anti–γ-H2AX antibody (BD Biosciences), anti-PUMA (Abcam), anti-activated caspase 3 (Cell Signaling Technology), or anti-phospho ERK1/2 (Cell Signaling Technology), and then labeled with AlexaFluor 488-goat anti-rabbit IgG (Thermo Fisher Scientific). Data were analyzed with FlowJo software (v10.5.0).

Cord blood (CB), MDS-L, or primary MDS cells were treated with sivelestat (ONO-5046; Selleckchem), azacitidine (Mylan Institutional), or decitabine (Selleckchem). Cells were quantified with a hemacytometer using trypan blue (Lonza). For cultures of colony-forming cells (CFC), cells were plated in specified doses in MethoCult H4434 (STEMCELL Technologies) and scored between days 10–14 by two independent investigators.

Whole-cell lysates were prepared in RIPA buffer with protease inhibitors according to the manufacturer's specifications (Thermo Fisher Scientific). To prepare conditioned media, media from 10⁶ cultured MDS-L cells was concentrated with Amicon Ultra-4 (Millipore). For analysis of conditioned media from primary MDS cells, media from 3 × 10⁵ CD34⁺ MDS marrow cells were incubated with 2 μg of anti-HMGB1 antibody overnight at 4ºC. HMGB1 protein in media was precipitated with Dynabeads Protein A (Thermo Fisher Scientific). After protein quantification, samples either from cell lysates or media were resolved on SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with appropriate antibodies. These include anti-HMGB1, anti-actin, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-RelA, and anti-IκBα antibodies (all from Cell Signaling Technology). Secondary antibodies are conjugated with horseradish peroxidase, IRdye 700, or IRdye 800. Blots were stained with either Ponceau S or REVERT^TM Stain (LI-COR Biosciences) to visualize total loaded protein. Quantification of signals were performed as described previously (14).

Animal studies were approved by the Duke Institutional Animal Care and Use Committee. Male and female mice, ages 8–12 weeks, were used in studies. At 24 hours following 250 cGy total body irradiation, NOD-scidIL2Rg^null (NSG) mice (Jackson Laboratory) were injected intraperitoneally with 250–300 mg/kg 2,2,2-Tribromoethanol (Sigma-Aldrich). MDS-L cells were transplanted by intrafemoral injection. For in vitro studies, 10⁶ MDS-L cells and progeny were transplanted following 72-hour culture with 300 μg/mL sivelestat or DMSO. For in vivo analysis, mice were treated with either 5 mg/kg sivelestat or DMSO in 200 μL of 1 × PBS by intraperitoneal injection once daily for 7 days.

Wright stains were performed on an Aerospray Hematology Pro Series 2 (EliTech Group). Hematoxylin and eosin staining was performed by the Duke Research Immunohistology Lab. Microscopic evaluation was performed by a hematopathologist. Between 80–150 cells were counted for each replicate. MDS-L cell engraftment was analyzed by FACS on red blood cell–depleted bone marrow. Bone marrow and spleen aspirates were captured on Zeiss Axio Imager Z2 with Zen 2 Software (Oberkochen,). Images of femur and spleen sections were captured on an Olympus BX43 microscope with a Spot Idea camera 5MP and software Spot PathSuite v2.0 (Sterling Heights).

Cells were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton-X-100, and treated with 3% goat serum. Cells were stained with rabbit anti-HMGB1 and goat anti-rabbit secondary antibody Alexa Fluor 488 or Alexa Fluor 488 mouse anti–γ-H2AX antibody (BD) and DAPI. The mean fluorescent intensity of HMGB1 and γ-H2AX per nucleus was quantified with background subtraction from DAPI-positive cells. Immunofluorescence images were captured on Zeiss Axio Imager Z2.

Multiple proteins of the NFκB pathway were assayed in parallel using a Proteome Profiler Array (R&D Systems). Briefly, MDS-L cells were incubated with sivelestat at indicated concentrations or DMSO for 24 hours. Cell lysates were prepared according to the manufacturer's instructions (Thermo Fischer Scientific). Each sample (700 μg) was loaded on membranes and incubated overnight at 4°C. Detection was determined with specific antibody cocktail, streptavidin-HRP, and Chemi Reagent Mix. The membranes were exposed to an X-ray film and the images of dot blots were scanned. The pixel densities of dots were analyzed using ImageJ with a gel analysis function as described previously (15).

All data are shown as means ± SEM. Student two-tailed, unpaired t test, Mann–Whitney two-tailed test, or ANOVA analyses were performed using GraphPad Prism (v8.0) as specified in figure legends.

To study HMGB1 in MDS, we utilized a cell line, MDS-L, derived from a patient with MDS with ring sideroblasts (10, 11). These cells display dysplastic features, including cytoplasmic vacuolation and prominent, irregular nucleoli (Fig. 1A). These cells do not demonstrate blast morphology following in vivo expansion, which distinguishes this cell line from typical acute leukemic cells (11). By both mRNA expression and immunofluorescence analysis, we found 2- to 3-fold higher levels of HMGB1 in MDS cells compared with either cord blood (CB) or healthy marrow (Fig. 1B and C; Supplementary Table S1). Receptors for HMGB1 include Toll-like receptors (TLR; ref. 16). We discovered that TLR2, TLR4, TLR6, and TLR9 displayed 7- to 24-fold greater mRNA expression in CD34⁺ primary MDS cells compared with CD34⁺ CB cells and CD34⁺ healthy marrow (Fig. 1D). In addition to TLRs, HMGB1 can also signal through another receptor, receptor for advanced glycation end products (RAGE; ref. 17). We found TLR2, TLR4, and RAGE were detected in primary CD34⁺ MDS cells (Fig. 1E), indicating that activation of these pathways could contribute to both inflammation and pathogenesis of MDS.

Treatment of HMGB1 siRNA (siHMGB1) in macrophages and dendritic cells can suppress the secretion of HMGB1 and diminish the production of inflammatory cytokines (18). Following culture of MDS-L cells with siHMGB1, the levels of HMGB1 were decreased by 85% by mRNA expression and 25% by protein expression compared with nontargeting siRNA control (Supplementary Fig. S1A and S1B). This decrease in HMGB1 resulted in a 30% reduction in MDS-L cell expansion following culture with siHMGB1 (Supplementary Fig. S1C). This corresponded to a nearly 40% reduction in the number of CFCs and increased apoptotic and necrotic cell-death compared with nontargeting RNA (Supplementary Fig. S1D and S1E). These data indicate that elevated levels of HMGB1 may be necessary for MDS-L cell expansion and survival.

Next, we sought to inhibit HMGB1 signaling with sivelestat, which is a small-molecule inhibitor for both HMGB1 and neutrophil elastase (NE) and can suppress TNFα and other inflammatory cytokines (19). Because NE is not detected in MDS-L cells (Supplementary Fig. S2), the target for sivelestat in these MDS-L studies is HMGB1. Whether sivelestat or other HMGB1 inhibitors could impact MDS is not yet defined. When MDS-L cells and primary CD34⁺ MDS cells were treated with 300 μg/mL sivelestat for 72 hours, HMGB1 protein levels were significantly decreased by up to 50% compared with cultures with vehicle alone (Fig. 2A and B). Likewise, sivelestat markedly reduced total cell expansion and CFCs in MDS-L and primary MDS cells (Fig. 2C and D). Cultures of nonmalignant CD34⁺ CB cells or healthy marrow with sivelestat displayed no differences compared with control cultures (Fig. 2C and D). Notably, sivelestat had no effect compared with control cultures for patient sample 0449, which was obtained from a patient with normal cytogenetics and high levels of HMGB1 (Supplementary Fig. S3A and S3B). These data indicate that sivelestat could inhibit MDS cell self-renewal and expansion in a subset of patients.

Because azacitidine and decitabine are standard therapies for the treatment of MDS (1), we sought to determine whether dual treatment of these chemotherapies with sivelestat would be additive to decrease MDS cell expansion. Following culture with 10 μmol/L azacitidine and 300 μg/mL sivelestat, MDS-L cells decreased total cell expansion compared with either azacitidine alone or vehicle alone (Fig. 2E). This decrease in cell expansion corresponded to a greater decrease in CFCs with both azacitidine and sivelestat (Fig. 2E). Similarly, when primary CD34⁺ MDS cells were cultured with both azacitidine and sivelestat, there was an additional 30% reduction in total cell expansion compared with monotherapy with azacitidine (Fig. 2F). High doses of azacitidine like 10 μmol/L used in these studies exert direct cytotoxic effects (20). Because high versus low doses azacitidine could result in differential mechanisms of anticancer effects, with low doses causing sustained alterations in gene expressions of crucial cancer signaling pathways (21), we also tested clinically relevant lower doses of azacitidine and sivelestat. Even with a lower dose of azacitidine (1 μmol/L, IC₂₅ compared with IC₅₀), combination therapy with sivelestat yielded an additive effect to decrease MDS-L cell expansion and promote cellular death (Supplementary Fig. S3C–S3E). Dual therapy with sivelestat and decitabine also displayed decreased cell expansion and increased Annexin V⁺ cells compared with decitabine alone (Fig. 2F; Supplementary Fig. S3F). Of note, sivelestat alone or in combination with azacitidine did not impact cell expansion or CFCs in CD34⁺ healthy marrow cells (Fig. 2G). These data demonstrate that dual treatment is more effective at blocking MDS cell expansion and promoting cellular apoptosis compared with chemotherapy alone, while sparing toxicity to normal hematopoietic stem/progenitor cell subsets.

NSG mice that were transplanted with MDS-L cells treated with sivelestat demonstrated decreased marrow engraftment compared with control cultures (Fig. 3A–C). Microscopic examination of bone marrow aspirates display an 8.3-fold decrease in MDS-L engraftment in recipients of sivelestat-treated cultures compared with control cultures (Fig. 3C). This decrease in MDS-L engraftment was consistent with lower marrow engraftment as measured by flow cytometric analysis for human CD45, CD13, CD33, and CD38 (Fig. 3D and E). As with prior reports of MDS-L–engrafted mice (11), we also observed engraftment of MDS-L cells within the spleen (Supplementary Fig. S4A–S4D). These data show that following treatment of MDS cells with sivelestat, inhibition of HMGB1 could decrease long-term MDS engraftment.

Because sivelestat could inhibit MDS self-renewal in vitro, we investigated whether sivelestat could decrease MDS engraftment in vivo. Following intrafemoral transplantation of MDS-L cells into NSG mice, mice were treated intraperitoneally with 5 mg/kg sivelestat or DMSO for consecutive 7 days (Fig. 4A). At 17-weeks posttransplantation, bone marrow displayed decreased MDS cells in sivelestat-treated mice compared with DMSO-treated mice (Fig. 4B–D). This corresponded to preserved splenic architecture in sivelestat-treated mice compared with DMSO-treated mice (Supplementary Fig. S4E and S4F). Using flow cytometric analysis, the percentage human CD45 of sivelestat-treated mice displayed a 2.5-fold reduction in MDS engraftment compared with DMSO-treated mice (Fig. 4D and E). This reduction in MDS engraftment showed a corresponding decrease in myeloid markers (3.6-, 2.3-, and 3.6-fold reduction for CD13, CD 33, and CD38, respectively), indicating that pharmacologic treatment with sivelestat could inhibit MDS engraftment in vivo.

Because MDS thrives in inflammatory microenvironments (3), we next investigated whether neutralization of this environment by reduction of HMGB1 could promote apoptosis. Both MDS-L cells and primary MDS cells displayed an increase in Annexin V⁺ cells following sivelestat treatment, while CD34⁺ healthy marrow cells did not (Fig. 5A), suggesting inhibition of HMGB1 facilitates apoptosis in MDS cells. Following cotreatment of azacitidine and sivelestat, MDS cells but not CD34⁺ healthy marrow cells displayed further increased Annexin V⁺ cells compared with azacitidine alone (Fig. 5B–D). These findings indicate that sivelestat spares healthy hematopoietic cells.

Because apoptosis is regulated, in part, through activation of p53-upregulated modulator of apoptosis (PUMA; ref. 22), culture with either siHMGB1 or sivelestat displayed increased PUMA mRNA and protein expression in MDS-L (Fig. 5E and F). Hematopoietic cell death is also regulated by caspase activity, in particular caspase 3, an effector protease that is functional in the late stages of apoptosis (23). Cultures of MDS-L cells and primary CD34⁺ MDS cells with sivelestat resulted in caspase 3 activation in a dose-dependent manner compared with vehicle alone (Fig. 5G and H). Taken together, these data demonstrate that sivelestat promotes apoptosis, at least in part, by increasing both PUMA signaling and activated caspase 3.

Because DNA double-strand breaks can be associated with apoptotic-mediated cell death, we sought to determine whether sivelestat promoted apoptotic cell death by generating DNA double-strand breaks (24). Cultures of MDS-L cells and primary CD34⁺ MDS cells with sivelestat increased γ-H2AX by flow cytometric analysis and immunofluorescence compared with control cultures (Fig. 5I–K). These data indicate that sivelestat increased double-strand DNA breaks and contributed to increased cellular death.

HMGB1 is an inflammatory cytokine when released into the extracellular space (9). To determine whether extracellular release of HMGB1 is modulated following treatment with sivelestat, we measured HMGB1 in conditioned media of MDS-L cultures and from cultures of primary CD34⁺ MDS cells. We found that sivelestat decreased the levels of HMGB1 by up to 70% compared with control cultures, and thus could decrease the inflammatory potential of extracellular HMGB1 (Fig. 6A; Supplementary Fig. S5A).

Because TLR expression is increased in MDS cells, we sought to determine whether inhibition of HMGB1 with sivelestat would normalize TLR expression. Treatment of MDS-L cells with sivelestat was sufficient to reduce mRNA expression of TLR2, TLR4, TLR6, and TLR9 (Fig. 6B; Supplementary Fig. S5B). Complementary to these mRNA analyses, both TLR2 and TLR4 protein expression in primary CD34⁺ MDS cells was reduced by up to 3-fold following sivelestat treatment compared with control cultures (Fig. 6C and D). Because phosphorylation of extracellular-signal regulated kinase (ERK1/2) is downstream of activated TLR signaling, we demonstrated that phospho-ERK1/2 was decreased by 2- to 3-fold 12 hours after sivelestat treatment by both flow cytometric analysis (Supplementary Fig. S5C) and by Western blot analysis (Supplementary Fig. S5D). These data indicate that sivelestat can modulate the innate immune system in MDS by decreasing extracellular levels of HMGB1, TLR signaling, and phospho-ERK1/2.

Along with TLR signaling, the regulation of both innate immune and inflammatory responses is largely associated with activation of the transcription factor NFκB (25). We sought to determine whether inhibition of HMGB1 could modulate the NFκB pathway. When MDS-L cells were treated with sivelestat for 12 hours, the level of NFκB-inhibitory protein, IκBα was increased compared with control cultures, suggesting that increased levels of IκBα could decrease activation of NFκB (Supplementary Fig. S5E; ref. 25). Consistent with these findings, after 24-hour culture with sivelestat, the RelA subunit of NFκB was decreased in both cultures with MDS-L and primary CD34⁺ MDS cells following sivelestat treatment compared with control cultures (Fig. 6E and F), indicating that sivelestat could negatively regulate NFκB activation.

Next, we further characterized whether inhibition of HMGB1 by sivelestat could alter NFκB-related pathways. Using a Proteome Profiler Array for NFκB signaling, we measured the levels of 45 targets in MDS-L cells following culture with sivelestat (300 μg/mL or 600 μg/mL) for 24 hours compared with control cultures (Fig. 6G). We discovered that sivelestat evoked a 50%–70% reduction in levels of c-Rel and phosphorylated RelA (pS529), which are core components of NFκB. Moreover, sivelestat downregulated the expression of receptor activators of NFκB, including TNFRSF10A and TNFRSF3, and their downstream adaptor protein FADD (Fas-associated protein with death domain; Fig. 6G). There was a 1.8-fold increase in TNFRII following sivelestat treatment (Fig. 6G). Notably, sivelestat induced a 3.2-fold increase in caspase recruitment domain 6 (CARD6) following sivelestat compared with control cultures (Fig. 6G). CARD6 has been shown to inhibit NFκB activation within pathogen-associated innate immune responses (26). Although interleukin receptor-associated kinase 1 (IRAK1) one key inflammatory mediator is overexpressed in MDS cells compared with healthy hematopoietic cells (Supplementary Fig. S7; ref. 6), sivelestat treatment did not affect its expression at the concentrations and time point tested (Fig. 6G). Other targets within these pathways are summarized in Supplementary Fig. S6. Taken together, these data demonstrate that inhibition of HMGB1 with sivelestat modulates NFκB signaling and could contribute to a reduction in the inflammatory response of the innate immune system in MDS.

Our findings that HMGB1 could regulate MDS cell survival are supported by reports that HMGB1 could be also functional in other cancer systems. A meta-analysis of 11 different solid cancers demonstrated that overexpression of HMGB1 was associated with shortened progression-free survival and overall survival (27). High expression of HMGB1 by tissue microarrays in primary ovarian cancers was associated with both shortened progression-free survival and overall survival (28). At the time of diagnosis, children with acute lymphoblastic leukemia demonstrate >70-fold increase in the serum levels of HMGB1 compared with healthy subjects (29). When in remission, the levels of HMGB1 decline and are comparable with healthy donors. A meta-analysis of 10 studies of non–small-cell lung cancer showed HMGB1 was increased in both serum and tissue of patients with cancer compared with samples from healthy lung samples (30). In models of bladder cancer (31), cutaneous squamous cell carcinoma (32), and gastric adenocarcinoma (33), inhibition of HMGB1 resulted in decreased cancer cell expansion. Overexpression of HMGB1 has been associated with increased resistance to chemotherapy, and suppression of HMGB1 results in increased chemosensitivity (34). These reports support our hypothesis that HMGB1 may have a functional role in MDS.

Our findings are buffeted by another report that demonstrated increased levels of HMGB1 in bone marrow supernatants and plasma of patients with MDS compared with healthy controls (7). Velegraki and colleagues show that increased levels of HMGB1 in cultures of primary MDS macrophages was at least partially due to impaired clearance of apoptotic cells (7). Here, we demonstrate that primary CD34⁺ MDS cells display higher levels of HMGB1 within hematopoietic stem/progenitor cell populations compared with healthy marrow or cord blood cells. Increased apoptosis or cell death by sivelestat corresponded to decreased protein levels of HMGB1 and did not increase extracellular HMGB1 in our culture systems.

To modulate HMGB1 signaling, we performed loss-of-function studies with both siHMGB1 and with sivelestat. In our murine studies in which transplanted cells were exposed to sivelestat or sivelestat was administered in vivo, sivelestat treatment resulted in increased percentage of murine cells compared with DMSO-treated mice, indicating there was no detected adverse impact on murine hematopoiesis. Consistent with these findings, sivelestat has been well tolerated in clinical trials and following postmarket studies without increased toxicity to hematopoietic systems compared with control groups (35, 36). When used in combination with azacitidine and decitabine, sivelestat was additive to promote MDS cell death in a dose-dependent manner. Because sivelestat could also alter TLR expression, combination therapy with TLR antagonists could offer another therapeutic approach. For example, combination chemotherapy and CX-01, a TLR2/TLR4 antagonist and heparin derivative, could reduce cancer burden in early-phase studies in relapsed acute myeloid leukemia (37) and MDS (NCT02995655). These data could provide a rationale for therapeutic combinations with standard chemotherapies, HMGB1 inhibitors, and TLR antagonists.

Although sivelestat could inhibit MDS cell expansion in MDS-L cells and in many primary MDS cells, it was not universally effective. The levels of HMGB1 within CD34⁺ MDS cells did not correspond with prognostic scoring indices or severity of disease in our cohort of samples. One study limitation is that we were not able to measure the extracellular levels of HMGB1 in primary MDS patient samples. Measurements of both extracellular and intracellular HMGB1 could more clearly represent disease status and might predict disease response.

In addition to promoting cancer cell death directly, inhibiting HMGB1 with sivelestat could also have additional paracrine effects in cancer. HMGB1 released from dying cells following injury like radiation or chemotherapy could stimulate proliferation of living cells via an apoptosis-stimulated tumor repopulation mechanism named the “Phoenix Rising” pathway (38, 39). Inhibition of HMGB1, either with the small-molecule inhibitor glycyrrhizin or by deletion of HMGB1 expression and function with CRISPR/Cas9 technology, abrogated proliferation of living cancer cells through reduction of phospho-ERK activation (39). We show that sivelestat markedly reduced extracellular release of HMGB1 into culture media and reduced phospho-ERK activity by up to 3-fold as quantified using flow cytometric analysis and Western blot analysis. These data indicate that sivelestat could alter the paracrine microenvironment and possibly limit apoptosis-stimulated tumor repopulation in MDS.

We sought to associate HMGB1 with the innate immune system and inflammatory responses via NFκB signaling. We identified several targets in this pathway that were downregulated or upregulated following sivelestat culture compared with controls. That proteins RelA, c-Rel, TNFRSF10A, TNFRSF3, and FADD were downregulated following sivelestat demonstrate inhibition of NFκB and inflammatory pathways (40). Likewise, upregulation of CARD6 and TNFRII also block NFκB activation (26). In our system, there were several proteins that were not altered with sivelestat therapy. For example, the levels of IRAK1 did not appear be altered following 24-hour cultures with sivelestat with the concentrations tested. In an elegant study by Rhyasen and colleagues, treatment with an IRAK1 inhibitor of MDS cells in vitro resulted in decreased activation of NFκB, promoted apoptosis, and normalization of the hematopoietic system compared with control mice (6). It is possible that timing of the assay (i.e., at 24 hours in culture with sivelestat) could determine whether targets in the NFκB pathway are modulated with sivelestat. We do not exclude the possibility that variable activation of NFκB could be measured in primary in MDS cells compared with MDS-L cells.

Our findings indicate that HMGB1 is a therapeutic target in MDS. Reduction of HMGB1 levels was sufficient to impair MDS cell self-renewal and promote apoptotic cell death. Inhibitors of HMGB1 signaling could provide a first-in-class therapeutic option for patients with MDS. These inhibitors of HMGB1 could be used as monotherapy or in combination with chemotherapies to improve sensitization of MDS cells or other hematologic malignancies.

D.A. Rizzieri reports receiving speakers bureau honoraria from Celgene and is a consultant/advisory board member for Abbvie and Pfizer. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A.Y.F. Kam, P.L. Doan

Development of methodology: A.Y.F. Kam, P.L. Doan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.Y.F. Kam, S.O. Piryani, H.S. Park, D.A. Rizzieri, P.L. Doan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.Y.F. Kam, S.O. Piryani, C.M. McCall, D.A. Rizzieri, P.L. Doan

Writing, review, and/or revision of the manuscript: A.Y.F. Kam, S.O. Piryani, C.M. McCall, D.A. Rizzieri, P.L. Doan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.Y.F. Kam, D.A. Rizzieri, P.L. Doan

Study supervision: P.L. Doan

We thank Christopher Holley, MD, PhD, for scientific discussion and Julia Lloyd-Cowden for extracting clinical patient information. This independent research was supported by the American Association for Cancer Research Judah Folkman Career Development Award for Angiogenesis Research, Grant Number 14–20-18-DOAN (to P.L. Doan), Gilead Sciences Research Scholars Program in Hematology/Oncology (to P.L. Doan), NCI of the NIH under Award Number K08CA184552 (to P.L. Doan), and the Duke Cancer Institute (to P.L. Doan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.