Key Points• HMGB1 and DNA released from CLL cells induce nurse-like cell differentiation.• This differentiation appears TLR9/RAGE dependent.Chronic lymphocytic leukemia (CLL) is a disease of an accumulation of mature B cells that are highly dependent on the microenvironment for maintenance and expansion. However, little is known regarding the mechanisms whereby CLL cells create their favorable microenvironment for survival. High-mobility group protein B-1 (HMGB1) is a highly conserved nuclear protein that can be actively secreted by innate immune cells and passively released by injured or dying cells. We found significantly increased HMGB1 levels in the plasma of CLL patients compared with healthy controls, and HMGB1 concentration is associated with absolute lymphocyte count. We therefore sought to determine potential roles of HMGB1 in modulating the CLL microenvironment. CLL cells passively released HMGB1, and the timing and concentrations of HMGB1 in the medium were associated with differentiation of nurse-like cells (NLCs). Higher CD68 expression in CLL lymph nodes, one of the markers for NLCs, was associated with shorter overall survival of CLL patients. HMGB1-mediated NLC differentiation involved internalization of both receptor for advanced glycation end products (RAGE) and Toll-like receptor-9 (TLR9). Differentiation of NLCs can be prevented by blocking the HMGB1-RAGE-TLR9 pathway. In conclusion, this study demonstrates for the first time that CLL cells might modulate their microenvironment by releasing HMGB1. (Blood. 2014;123(11):1709-1719 IntroductionMany cancers arise from sites of infection, chronic irritation, and inflammation. An inflammatory microenvironment is an important participant in the neoplastic process, fostering proliferation, survival, and migration for cancers, including chronic lymphocytic leukemia (CLL).1-4 Stressed, injured, or dying cells release damage-associated molecular patterns (DAMPs), which initiate noninfectious inflammatory responses. [5][6][7] The DAMP high-mobility group protein B1 (HMGB1) is a major player associating inflammation and cancer. 8,9 HMGB1 is a nuclear protein that can be released passively by damaged or dead cells or actively by immune cells and stressed cancer cells. [10][11][12][13][14] HMGB1 regulates transcription factors but also behaves as a proinflammatory cytokine mediating inflammation. 13,[15][16][17][18] Nonprotein DAMPs, including DNA, RNA, and ATP, are also released by damaged or dying cells. 6,7,19 DAMPs are associated with acute inflammatory responses, chronic inflammation, and wound healing, but are also important components of the disordered tumor microenvironment. 8,20 HMGB1 is a DNA-binding protein, and increased serum concentrations of the HMGB1-DNA complex can activate the immune system and cause systemic autoimmune disease via the receptor for advanced glycation end products (RAGE) and toll-like receptor-9 (TLR9). 21 Interactions of HMGB1-RAGE-TLR9 constitute a tripod that triggers nuclear factor kB (NF-kB) activation 22 and promotes ...

Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2− lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2− Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.

1625 Diffuse large B-cell lymphoma (DLBCL) is characterised by overexpression of the anti-apoptotic protein Bcl-2. It has been recently observed that Bcl-2 also inhibits autophagy by binding and sequestering Beclin-1, an essential autophagy protein, but it is unclear whether Bcl-2 inhibits both apoptosis and autophagy in DLBCL cells. We aimed to determine the dual role of Bcl-2 in both apoptosis and autophagy in Bcl-2 positive cell lines (Su-DHL4 and CRL) and Bcl-2 negative cell lines (Su-DHL8 and Su-DHL10) using the BH3 mimetic compound ABT-737. The sensitivity of Bcl-2 positive and Bcl-2 negative cell lines to ABT-737-mediated mitochondrial depolarization (ΔΨmLOW) and cell death (DAPI positive) was assessed by flow cytometry. Treatment of the Bcl-2 positive cell lines Su-DHL4 and CRL with ABT-737 significantly increased (p<0.01) the percentage of both ΔΨmLOW cells, indicating mitochondrial damage as well as DAPI positive cells indicating cell death. Treatment with ABT-737 increased Bax activation and PARP cleavage in Bcl-2 positive cells, indicating that as expected, ABT-737-induced cell death is via apoptosis. ABT-737-induced cell death was not detected in Bcl-2 negative cell lines Su-DHL8 and Su-DHL10, demonstrating that, as expected, the sensitivity of DLBCL cell lines to ABT-737-induced apoptosis is Bcl-2 dependent. Treatment of Bcl-2 positive cells with ABT-737 also resulted in a decreased cellular co-localisation of Bcl-2 and Beclin-1 as detected by immunofluorescent staining. Degradation of p62 and LC3-II, selective substrates of autophagy, was detected by Western blotting in Bcl-2 positive but not in Bcl-2 negative cell lines after treatment with ABT-737 for 15 hours. LC3-I is a diffuse cytoplasmic protein which upon activation of autophagy becomes cleaved and lipidated to LC3-II which becomes punctate within cells. Punctuate LC3-II is a widely used marker of active autophagy. ABT-737-induced autophagosome formation was determined at an earlier time point (3 hours after ABT-737 treatment) using immune-fluorescent microscopy. ABT-737 induced increased numbers of larger punctate LC3-II in Bcl-2 positive Su-DHL4 and CRL cell lines but not in Bcl-2 negative cells, indicating that inhibition of Bcl-2 induces autophagy in Bcl-2 positive cells. We then determined whether autophagy affects ABT-737-induced apoptosis by blocking autophagy using an autophagy inhibitor chloroquine (CQ). Co-treatment with ABT-737 and CQ resulted in an increase in the percentage of ΔΨmLOW cells, DAPI positive cells and PARP cleavage compared to cells treated with ABT-737 alone in Bcl-2 positive cell lines. Combined, these results indicate that inhibition of autophagy by chloroquine further sensitises Bcl-2 positive cells to ABT-737-induced apoptosis. In summary, our results indicate that Bcl-2 inhibits autophagy in lymphoma cells by sequestering Beclin-1. Disruption of this interaction by ABT-737 induces autophagy which in turn inhibits apoptosis. Inhibition of autophagy results in increased sensitivity of Bcl-2 positive cells to ABT-737-induced apoptosis, suggesting a role for autophagy inhibitors in lymphoma treatment. Disclosures: No relevant conflicts of interest to declare.

Background: Human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the most common forms of indolent and aggressive NHL, respectively. The t(14;18) translocation characterizes approximately 85% of FL and 20% of DLBCL and results in constitutive overexpression of the anti-apoptotic protein BCL-2. It was previously reported that BCL-2 plays dual roles in preventing apoptosis and autophagy. Autophagy is a physical and pathological process whereby cells sequester portions of cytoplasm including organelles to form autophagosomes where they are degraded and recycled. Growing evidence demonstrates that autophagy plays important roles in tumorigenesis, tumor progression, and resistance to chemotherapy. Aims: The autophagy status in human B-cell lymphomas is unknown. We hypothesized that overexpression of BCL-2 could change autophagy status and aimed to determined expression of autophagy-related genes and proteins in FL and DLBCL primary samples by PCR array and tissue microarray. We aimed to evaluate whether expression of the autophagy-related proteins p62, Beclin-1 and LC3 individually and in combination with BCL-2 protein expression could risk-stratify FL and DLBCL patients at diagnosis. Patients and methods: Using PCR array, the autophagy-related gene expression profiles were determined in purified and unpurified reactive and malignant human lymph node tissue biopsies. Diagnostic tissues from FL (n=117) and DLBCL (n=109) patients were microarrayed and autophagy protein expression was evaluated using immunohistochemistry. Univariate and multivariate analyses on both continuous and categorical variables were conducted to measure overall survival (OS), disease specific survival (DSS), and progression-free survival (PFS). Results: Seven autophagy machinery genes were up-regulated in purified FL B-cells, namely ATG9A, ATG16L1, MAP1LC3A, GABARAPL1, ULK1, LAMP1 and HDAC6 compared with reactive B-cells. Two autophagy machinery genes, MAP1LC3A and DRAM1, were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. CTSD (cathepsin D) and TGM2 (transglutaminase 2) genes and proteins were mainly up-regulated in DLBCL tumor-infiltrating macrophages. These results demonstrate that FL and DLBCL showed increased expression of autophagy-related genes, regardless of the heterogeneity of these diseases. p62, a selective autophagy substrate; LC3, an autophagosome membrane protein; and Beclin-1, an essential autophagy effector, are often used to evaluate autophagy activity in the cell. 91% FL samples were BCL-2 positive but significantly decreased expression of p62, LC3 and Beclin-1 in FL samples was observed in both intra-follicular and non-malignant inter-follicular areas. This suggests that increased basal autophagy activity in both malignant FL cells and surrounding tumor infiltrating cells, indicating that BCL-2 does not inhibit basal autophagy activity. DLBCL samples displayed heterogeneous expression patterns of BCL-2, p62, LC3 and Beclin-1. We found that decreased p62 expression confers worse OS (continuous P=0.015; and categorical P=0.003), DSS (continuous P=0.037; categorical P=0.014) and PFS (categorical P=0.002) in DLBCL patients. Decreased expression of Beclin-1 was also confers poor prognosis in both FL and DLBCL as conducted by categorical analysis, OS (DLBCL, P=0.015; FL, P=0.004), DSS (FL, P=0.006), and PFS (DLBCL, P=0.029). p62 retains prognostic significance after adjustment for the International Prognostic Index (IPI) score and levels of BCL-2, Beclin-1 and LC3 in multivariate analysis. Beclin-1 retains its prognostic significance in FL after adjusting for FLIPI scores. Low p62 plus high BCL-2 expression in DLBCL confers the worst OS (P<0.0001) and DSS (P=0.001) compared with other combinations. Conclusions: These results demonstrate that FL has increased basal autophagy activity, while it varies in DLBCL. p62 is a novel, independent prognostic biomarker for DLBCL but not for FL. Combining p62 with BCL-2 provides a more robust and reliable method to risk-stratify DLBCL patients at diagnosis. Importantly, we report for the first time that overexpression of BCL-2 in human NHL does not inhibit basal autophagy activity. We propose that increased autophagy activity could be a therapeutic target for treatment of NHL. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.