BACKGROUND: Chronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL.
METHODS: We generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay.
RESULTS: GIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1β, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314(+) cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγ(null) immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL.
CONCLUSION: GIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.
Carfilzomib (CFZ) is a second generation proteasome inhibitor approved for the treatment of patients with multiple myeloma. It induces apoptosis in human cancer cells; but the underlying mechanisms remain undefined. In the present study, we show that CFZ decreases the survival of several human cancer cell lines and induces apoptosis. Induction of apoptosis by CFZ occurs, at least in part, due to activation of the extrinsic apoptotic pathway, since FADD deficiency protected cancer cells from undergoing apoptosis. CFZ increased total and cell surface levels of DR5 in different cancer cell lines; accordingly it enhanced TRAIL-induced apoptosis. DR5 deficiency protected cancer cells from induction of apoptosis by CFZ either alone or in combination with TRAIL. These data together convincingly demonstrate that DR5 upregulation is a critical mechanism accounting for CFZ-induced apoptosis and enhancement of TRAIL-induced apoptosis. CFZ inhibited the degradation of DR5, suggesting that DR5 stabilization contributes to CFZ-induced DR5 upregulation. In summary, the present study highlights the important role of DR5 upregulation in CFZ-induced apoptosis and enhancement of TRAIL-induced apoptosis in human cancer cells.
B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Rasmutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAILinduced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.
Fusokines are chimeric proteins generated by the physical coupling of cytokines in a single polypeptide, resulting in proteins with highly pleiotropic activity and the potential to treat cancer and autoimmune ailments. For instance, the fusokine GIFT15 (GM-CSF and Interleukin 15 Fusion Transgene) has been shown to be a powerful immunosuppressive protein able to convert naì ve B cells into IL-10-producing B cells. To date, the mammalian cell systems used for the expression of GIFT15 allow for secretion of the protein in the culturing media, an inefficient system for producing GMPcompliant fusokines. In this study we report the bacterial expression of bioactive recombinant GIFT15 (rGIFT15). Indeed, there is a constant demand to improve the expression systems for therapeutic proteins. Expression of a maltose-binding protein (MBP) fusion protein efficiently allowed the accumulation of soluble protein in the intracellular milieu. Optimizing the bacterial culture significantly increased the yield of recombinant protein. The biological activity of rGIFT15 was comparable to that of fusokine derived from a mammalian source. This approach led to the production of soluble, endotoxin-free functional protein, averaging 5 mg of rGIFT15 per liter of culture. This process is amenable to scale up for the development of Food and Drug Administration (FDA)-compliant immune-modulatory rGIFT15.
This study aimed to explore the interactions among long non-coding RNA H19, transcriptional factor CCCTC-binding factor (CTCF) and polycystic kidney disease 1 (PKD1), and to investigate its potentially regulatory effect on vulnerable plaque formation and angiogenesis of atherosclerosis. We established an atherosclerosis mouse model in ApoE knockout mice, followed by gain-and loss-of-function approaches. H19 was upregulated in aortic tissues of atherosclerosis mice, but silencing of H19 significantly inhibited atherosclerotic vulnerable plaque formation and intraplaque angiogenesis, accompanied by a downregulated expression of MMP-2, VEGF, and p53 and an upregulated expression of TIMP-1. Moreover, opposite results were found in the aortic tissues of atherosclerosis mice treated with H19 or CTCF overexpression. H19 was capable of recruiting CTCF to suppress PKD1, thus promoting atherosclerotic vulnerable plaque formation and intraplaque angiogenesis in atherosclerosis mice. The present study provides evidence that H19 recruits CTCF to downregulate the expression of PKD1, thereby promoting vulnerable plaque formation and intraplaque angiogenesis in mice with atherosclerosis.
Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the unde rlying mechanism. We found that several MEK inhibitors including MEK162, AZD6244, and PD0325901 effectively decreased DR4 protein levels including cell surface DR4 in different cancer cell lines. Accordingly, pre-treatment of TRAIL-sensitive cancer cell lines with a MEK inhibitor desensitized them to TRAIL-induced apoptosis. These results indicate that MEK inhibition negatively regulates DR4 expression and cell response to TRAIL-induced apoptosis. MEK inhibitors did not alter DR4 protein stability, rather decreased its mRNA levels, suggesting a transcriptional regulation. In contrast, enforced activation of MEK/ERK signaling by expressing ectopic B-Raf (V600E) or constitutively activated MEK1 (MEK1-CA) or MEK2 (MEK2-CA) activated ERK and increased DR4 expression; these effects were inhibited when a MEK inhibitor was present. Promoter analysis through deletion and mutation identified the AP-1 binding site as an essential response element for enhancing DR4 transactivation by MEK1-CA. Furthermore, inhibition of AP-1 by c-Jun knockdown abrogated the ability of MEK1-CA to increase DR4 promoter activity and DR4 expression. These results suggest an essential role of AP-1 in mediating MEK/ERK activation-induced DR4 expression. Our findings together highlight a previously undiscovered mechanism that positively regulates DR4 expression through activation of the MEK/ERK/AP-1 signaling pathway.
The clinical efficacy of immune cytokines used for cancer therapy is hampered by elements of the immunosuppressive tumor microenvironment such as TGFβ. Here we demonstrate that FIST15, a recombinant chimeric protein composed of the T-cell-stimulatory cytokine IL15, the sushi domain of IL15Rα and a TGFβ ligand trap, can overcome immunosuppressive TGFβ to effectively stimulate the proliferation and activation of natural killer (NK) and CD8 + T cells with potent antitumor properties. FIST15-treated NK and CD8 + T cells produced more IFNγ and TNFα compared with treatment with IL15 and a commercially available TGFβ receptor-Fc fusion protein (sTβRII) in the presence of TGFβ. Murine B16 melanoma cells, which overproduce TGFβ, were lysed by FIST15-treated NK cells in vitro at doses approximately 10-fold lower than NK cells treated with IL15 and sTβRII. Melanoma cells transduced to express FIST15 failed to establish tumors in vivo in immunocompetent murine hosts and could only form tumors in beige mice lacking NK cells. Mice injected with the same cells were also protected from subsequent challenge by unmodified B16 melanoma cells. Finally, mice with pre-established B16 melanoma tumors responded to FIST15 treatment more strongly compared with tumors treated with control cytokines. Taken together, our results offer a preclinical proof of concept for the use of FIST15 as a new class of biological therapeutics that can coordinately neutralize the effects of immunosuppressive TGFβ in the tumor microenvironment while empowering tumor immunity.
A major challenge in vaccinology is to prospectively determine vaccine efficacy. Here we have used a systems biology approach to identify early gene ‘signatures’ that predicted immune responses in humans vaccinated with yellow fever vaccine YF-17D. Vaccination induced genes that regulate virus innate sensing and type I interferon production. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4—an orchestrator of the integrated stress response—that correlated with and predicted YF-17D CD8+ T cell responses with up to 90% accuracy in an independent, blinded trial. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy. These data highlight the utility of systems biology approaches in predicting vaccine efficacy.
Inhibition of B-Raf/MEK/ERK signaling is an effective therapeutic strategy against certain types of cancers such as melanoma and thyroid cancer. While demonstrated to be effective anticancer agents, B-Raf or MEK inhibitors have also been associated with early tumor progression and development of secondary neoplasms. The ligation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with its receptor, death receptor 5 (DR5), leading to induction of apoptosis, offers a promising anticancer strategy. Importantly, this is also a natural immunosurveillance mechanism against cancer development. We previously demonstrated that activated B-Raf/MEK/ERK signaling positively regulates DR5 expression. Hence, our current work sought to address whether B-Raf/MEK/ERK inhibition and the consequent suppression of DR5 expression impede cancer cell response to DR5 activation-induced apoptosis and activated immune cell-induced killing. We found that both B-Raf (for example, PLX4032) and MEK inhibitors (for example, AZD6244 and PD0325901) effectively inhibited ERK1/2 phosphorylation and reduced DR5 levels in both human thyroid cancer and melanoma cells. Similar to the observed effect of genetic knockdown of the B-Raf gene, pre-treatment of cancer cell lines with either B-Raf or MEK inhibitors attenuated or abolished cellular apoptotic response induced by TRAIL or the DR5 agonistic antibody AMG655 or cell killing by activated T cells. Our findings clearly show that inhibition of B-Raf/MEK/ERK signaling suppresses DR5 expression and impairs DR5 activation-induced apoptosis and T cell-mediated killing of cancer cells. These findings suggest a potential negative impact of B-Raf or MEK inhibition on TRAIL- or DR5-mediated anticancer therapy and on TRAIL/DR5-mediated immune-clearance of cancer cells.