NF-κB c-Rel Is Dispensable for the Development but Is Required for the Cytotoxic Function of NK Cells.

Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-κB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their expression. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c- Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

BAFF receptor antibody for mantle cell lymphoma therapy.

Mantle cell lymphoma (MCL) is an aggressive form of B cell non-Hodgkin's lymphoma and remains incurable under current treatment modalities. One of the main reasons for treatment failure is the development of drug resistance. Accumulating evidence suggests that B cell activating factor (BAFF) and BAFF receptor (BAFF-R) play an important role in the proliferation and survival of malignant B cells. High serum BAFF levels are often correlated with poor drug response and relapse in MCL patients. Our study shows that BAFF-R is expressed on both MCL patient cells and cell lines. BAFF-R knockdown leads to MCL cell death showing the importance of BAFF-R signaling in MCL survival. Moderate knockdown of BAFF-R in MCL cells did not affect its viability, but sensitized them to cytarabine treatment in vitro and in vivo, with prolonged mice survival. Anti-BAFF-R antibody treatment promoted drug-induced MCL cell death. Conversely, the addition of recombinant BAFF (rhBAFF) to MCL cells protected them from cytarabine-induced apoptosis. We tested the efficacy of a humanized defucosylated ADCC optimized anti-BAFF-R antibody in killing MCL. Our data show both in vitro and in vivo efficacy of this antibody for MCL therapy. To conclude, our data indicate that BAFF/BAFF-R signaling is crucial for survival and involved in drug resistance of MCL. Targeting BAFF-R using BAFF-R antibody might be a promising therapeutical strategy to treat MCL patients resistant to chemotherapy.

Combination Therapy for Treating Advanced Drug-Resistant Acute Lymphoblastic Leukemia.

Drug-resistant acute lymphoblastic leukemia (ALL) patients do not respond to standard chemotherapy, and an urgent need exists to develop new treatment strategies. Our study exploited the presence of B-cell activating factor receptor (BAFF-R) on the surface of drug-resistant B-ALL cells as a therapeutic target. We used anti-BAFF-R (VAY736), optimized for natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), to kill drug-resistant ALL cells. VAY736 antibody and NK cell treatments significantly decreased ALL disease burden and provided survival benefit in vivo However, if the disease was advanced, the ADCC efficacy of NK cells was inhibited by microenvironmental transforming growth factor-beta (TGFß). Inhibiting TGFß signaling in NK cells using the TGFß receptor 1 (R1) inhibitor (EW-7197) significantly enhanced VAY736-induced NK cell-mediated ALL killing. Our results highlight the potential of using a combination of VAY736 antibody with EW-7197 to treat advance-stage, drug-resistant B-ALL patients.

Wheat Germ Agglutinin as a Potential Therapeutic Agent for Leukemia.

Dietary lectins are carbohydrate-binding proteins found in food sources. We used a panel of seven dietary lectins to analyze cytotoxicity against hematological cancers. Wheat germ agglutinin (WGA), even at low doses, demonstrated maximum toxicity toward acute myeloid leukemia (AML) cells. Using AML cell lines, we show time- and dose-dependent killing by WGA. We also show that low doses of WGA kills primary patient AML cells, irrespective of subtype, with no significant toxicity to normal cells. WGA caused AML cell agglutination, but failed to agglutinate RBC's at this dose. WGA, primarily, binds to N-acetyl-D-glucosamine (GlcNAc) and is also reported to interact with sialic-acid-containing glycoconjugates and oligosaccharides. After neuraminidase pre-treatment, which catalyzes the hydrolysis of terminal sialic acid residues, AML cells were less sensitive to WGA-induced cell death. AML cells were also not sensitive to succinyl-WGA, which does not react with sialic acid. Incubation with LEL lectin, which recognizes GlcNAc or SNA, which binds preferentially to sialic acid attached to terminal galactose in α-2,6 and to a lesser degree α-2,3 linkage, did not alter AML cell viability. These data indicate that WGA-induced AML cell death is dependent on both GlcNAc binding and interaction with sialic acids. We did not observe any in vitro or in vivo toxicity of WGA toward normal cells at the concentrations tested. Finally, low doses of WGA injection demonstrated significant in vivo toxicity toward AML cells, using xenograft mouse model. Thus, WGA is a potential candidate for leukemia therapy.

Hexosamine Biosynthetic Pathway Inhibition Leads to AML Cell Differentiation and Cell Death.

Treatment for acute myeloid leukemia (AML) has remained unchanged for past 40 years. Targeting cell metabolism is a promising avenue for future cancer therapy. We found that enzymes involved in metabolic hexosamine biosynthetic pathway (HBP) are increased in patients with AML. Inhibiting GFAT (the rate-limiting enzyme of HBP) induced differentiation and apoptosis in AML cells, sparing normal cells. UDP-GlcNAc, the end product of HBP, is the substrate for O-GlcNAcylation, a posttranslational modification. O-GlcNAc transferase (OGT) is the enzyme which transfers GlcNAc from UDP-GlcNAc to target proteins. Inhibition of O-GlcNAcylation, using OGT inhibitors as well as genetic knockdown of OGT, also led to cell differentiation and apoptosis of AML cells. Finally, HBP inhibition in vivo reduced the tumor growth in a subcutaneous AML xenograft model and tumor cells showed signs of differentiation in vivo A circulating AML xenograft model also showed clearance of tumor load in bone marrow, spleen, and blood, after HBP inhibition, with no signs of general toxicity. This study reveals an important role of HBP/O-GlcNAcylation in keeping AML cells in an undifferentiated state and sheds light into a new area of potential AML therapy by HBP/O-GlcNAc inhibition. Mol Cancer Ther; 17(10); 2226-37. ©2018 AACR.

Activation of Hematopoietic Stem/Progenitor Cells Promotes Immunosuppression Within the Pre-metastatic Niche.

Metastatic tumors have been shown to establish microenvironments in distant tissues that are permissive to disseminated tumor cells. Hematopoietic cells contribute to this microenvironment, yet the precise initiating events responsible for establishing the pre-metastatic niche remain unclear. Here, we tracked the developmental fate of hematopoietic stem and progenitor cells (HSPC) in tumor-bearing mice. We show that a distant primary tumor drives the expansion of HSPCs within the bone marrow and their mobilization to the bloodstream. Treatment of purified HSPCs cultured ex vivo with tumor-conditioned media induced their proliferation as well as their differentiation into immunosuppressive myeloid cells. We furthered tracked purified HSPCs in vivo and found they differentiated into myeloid-derived suppressor cells in early metastatic sites of tumor-bearing mice. The number of CD11b(+)Ly6g(+) cells in metastatic sites was significantly increased by HSPC mobilization and decreased if tumor-mediated mobilization was inhibited. Moreover, pharmacologic mobilization of HSPCs increased metastasis, whereas depletion of Gr1(+) cells abrogated the metastasis-promoting effects of HSPC mobilization. Finally, we detected elevated levels of HSPCs in the circulation of newly diagnosed cancer patients, which correlated with increased risk for metastatic progression. Taken together, our results highlight bone marrow activation as one of the earliest steps of the metastatic process and identify circulating HSPCs as potential clinical indicators of metastatic niche formation.