Fn14•Trail effectively inhibits hepatocellular carcinoma growth.

BACKGROUND: New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL\u27s capacity to inhibit HCC growth was tested. RESULTS: Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL\u27s molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice. CONCLUSIONS: In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein\u27s potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies

UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in Skeletal Muscle?

Hereditary inclusion body myopathy (HIBM) is a rare neuromuscular disorder caused by mutations in GNE, the key enzyme in the biosynthetic pathway of sialic acid. While the mechanism leading from GNE mutations to the HIBM phenotype is not yet understood, we searched for proteins potentially interacting with GNE, which could give some insights about novel putative biological functions of GNE in muscle. We used a Surface Plasmon Resonance (SPR)-Biosensor based assay to search for potential GNE interactors in anion exchanged fractions of human skeletal muscle primary culture cell lysate. Analysis of the positive fractions by in vitro binding assay revealed alpha-actinin 1 as a potential interactor of GNE. The direct interaction of the two proteins was assessed in vitro by SPR-Biosensor based kinetics analysis and in a cellular environment by a co-immunoprecipitation assay in GNE overexpressing 293T cells. Furthermore, immunohistochemistry on stretched mouse muscle suggest that both GNE and alpha-actinin 1 localize to an overlapping but not identical region of the myofibrillar apparatus centered on the Z line. The interaction of GNE with alpha-actinin 1 might point to its involvement in alpha-actinin mediated processes. In addition these studies illustrate for the first time the expression of the non-muscle form of alpha-actinin, alpha-actinin 1, in mature skeletal muscle tissue, opening novel avenues for its specific function in the sarcomere. Although no significant difference could be detected in the binding kinetics of alpha-actinin 1 with either wild type or mutant GNE in our SPR biosensor based analysis, further investigation is needed to determine whether and how the interaction of GNE with alpha-actinin 1 in skeletal muscle is relevant to the putative muscle-specific function of alpha-actinin 1, and to the muscle-restricted pathology of HIBM

DSP107 combines inhibition of CD47/SIRPα axis with activation of 4-1BB to trigger anticancer immunity

BACKGROUND: Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα ‘don’t eat me’ signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. METHODS: Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. RESULTS: Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. CONCLUSIONS: DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13046-022-02256-x

Fn14•TRAIL induces death of HCC cell lines in a dose dependent manner.

<p>SK-HEP-1 (<b>A</b>) HCC cells were incubated with indicated concentrations of Fn14•TRAIL for 72 [left panel], 48 and 24 hours [right panel]. (<b>B</b>,<b>C</b>) HepG2 [B, left panel] and Huh7 [B, right panel] HCC cell lines, as well as NKNT3 [C, left panel] and FHB [C, right panel] hepatocyte cell lines, were incubated with indicated concentrations of Fn14•TRAIL for 48 and 24 hours. Viable cells were stained with trypan blue and counted. Results represent the mean +/- SE of 3 independent experiments (* <i>p</i> < 0.05 ** <i>p</i> < 0.01 vs no Fn14•TRAIL). </p

Fn14•TRAIL is more potent than soluble forms of Trail and Fn14 alone or in combination.

<p>(<b>A</b>) SK-HEP-1 [A, left panel], HepG2 [A, middle panel] and Huh7 [A, right panel] HCC cell lines, as well as NKNT3 [B, left panel] and FHB [B, right panel] hepatocyte cell lines, were incubated with 0, 3, 30 or 300 ng/ml of Fn14•TRAIL, TRAIL, Fn14-Fc or combination of the later two for 48 hours. Viable cells were stained with trypan blue and counted. The results represent the mean +/- SD of three independent experiments (* <i>p</i> ≤ 0.05). (<b>B</b>) HepG2 HCC cells were incubated with 30ng/ml Fn14•TRAIL for 24 hours, in the presence or absence of anti Fn14 or anti TRAIL blocking antibodies. Treated cells were stained by Annexin V-FITC and Propidium Iodide, and counted by flow cytometer (2x10⁴ cells per sample). The results represent the mean +/- SD of two independent experiments (* p ≤ 0.05).</p

HCC and hepatocyte cell lines express TRAIL, TRAIL receptors, Fn14 and TWEAK.

<p>(<b>A</b>) The mRNA expression level of TRAIL, TRAIL receptors (DR4, DR5, DCR-1, DCR-2, OPG), Fn14 and TWEAK was determined by quantitive real-time PCR analysis. A representative experiment of three independent experiments is shown. Data are shown as average of triplicates (SD < 0.3), normalized against two endogenous control human genes, TBP and Actin-B, as calculated by Dataassist v2.0 software. (<b>B</b>,<b>C</b>) Protein expression of TRAIL, TRAIL receptors (DR4, DR5, DcR1, DcR2), Fn14 and TWEAK was determined by flow cytometeric analysis. (<b>D</b>) Fn14•TRAIL binds to HCC cells – HepG2 cells were incubated with Fn14•TRAIL, soluble TRAIL, Fn14 or the combination of the latter for 30 min at 4°c, immune-stained with PE-conjugated anti-Fn14, and analyzed by flow-cytometry. The results represent the mean +/- SD of triplicates (* <i>p</i> ≤ 0.05). </p

LDH-B does not show BIAcore binding signal with GNE.

<p>[A] Sensorgram of the interaction of GNE with 5 µM LDH-B, compared to 5 µM α-actinin 1. [B] Sensorgram showing the effect of 2 µM LDH-B on the interaction of GNE with 0.5 µM α-actinin 1 [Resp. Diff., response difference; RU, response units].</p

No hepatotoxicty is observed in Fn14•TRAIL treated mice.

<p>HepG2 Nude mice were treated daily with subcutaneous injections of 200μg of Fn14•TRAIL (n=4) or vehicle (n=4) for 8 days. One hour after last injection blood counts (<b>A</b>) liver enzymes and urea (<b>B</b>) were measured. Livers were harvested and H&E stained sections were tested (<b>C</b>). Data are presented as mean +/- S.E. </p