Search

Cancer immunotherapy using chimeric antigen receptor–armed T (CAR T) cells have been shown to improve outcomes significantly in patients with hematological malignancies. However, cytokine release syndrome (CRS) remains a risk. CRS is characterized by the excessive activation of CAR T cells and macrophages. Signs and symptoms of CRS are usually resolved after steroid administration, but steroids abrogate the expansion and persistence of CAR T cell populations. Tocilizumab is a humanized monoclonal antibody (mAb) that attenuates CRS without significant loss of CAR T cell activity. However, interleukin-6 (IL-6)/IL-6 receptor (IL-6R) blockade alone cannot relieve CRS symptoms fully, and novel treatments are needed to prevent or cure CRS. TO-207 is an N-benzoyl-L-phenylalanine derivative that significantly inhibits inflammatory cytokine production in human monocyte and macrophage-specific manner. We investigated whether TO-207 could inhibit cytokine production without impairing CAR T cell function in a CRS-simulating co-culture system.

Myofibroblast accumulation is a pathological feature of lung diseases requiring oxygen therapy. One possible source for myofibroblasts is through the epithelial-to-mesenchymal transition (EMT) of alveolar epithelial cells (AEC). To study the effects of oxygen on alveolar EMT, we used RLE-6TN and ex vivo lung slices and found that hyperoxia (85% O2, H85) decreased epithelial proteins, presurfactant protein B (pre-SpB), pro-SpC, and lamellar protein by 50% and increased myofibroblast proteins, α-smooth muscle actin (α-SMA), and vimentin by over 200% ( P < 0.05). In AEC freshly isolated from H85-treated rats, mRNA for pre-SpB and pro-SpC was diminished by ∼50% and α-SMA was increased by 100% ( P < 0.05). Additionally, H85 increased H2O2 content, and H2O2 (25–50 μM) activated endogenous transforming growth factor-β1 (TGF-β1), as evident by H2DCFDA immunofluorescence and ELISA ( P < 0.05). Both hyperoxia and H2O2 increased SMAD3 phosphorylation (260% of control, P < 0.05). Treating cultured cells with TGF-β1 inhibitors did not prevent H85-induced H2O2 production but did prevent H85-mediated α-SMA increases and E-cadherin downregulation. Finally, to determine the role of TGF-β1 in hyperoxia-induced EMT in vivo, we evaluated AEC from H85-treated rats and found that vimentin increased ∼10-fold ( P < 0.05) and that this effect was prevented by intraperitoneal TGF-β1 inhibitor SB-431542. Additionally, SB-431542 treatment attenuated changes in alveolar histology caused by hyperoxia. Our studies indicate that hyperoxia promotes alveolar EMT through a mechanism that is dependent on activation of TGF-β1 signaling.

Cancer immunotherapy using chimeric antigen receptor-armed T cells (CAR-T cells) have shown excellent outcomes in hematological malignancies. However, cytokine release syndrome (CRS), characterized by excessive activation of CAR-T cells and macrophages remains to be overcome. Steroid administration usually resolves signs and symptoms of CRS but abrogates CAR-T cell expansion and persistence. Tocilizumab, a humanized monoclonal antibody against interleukin-6 receptor (IL-6R), attenuates CRS without significant loss of CAR-T cell activities, while perfect rescue of CRS symptoms cannot be achieved by IL-6/IL-6R blockade. There is actual need for novel strategies to prevent or cure CRS. TO-207, an N-benzoyl-L-phenylalanine derivative compound, significantly inhibits inflammatory cytokine production in a human monocyte/macrophage-specific manner. Here we tested TO-207 for its ability to inhibit cytokine production without impaired CAR-T cell function in a CRS-simulating co-culture system consisting of CAR-T cells, target leukemic cells and monocytes. To observe a precise pattern of cytokine release from CAR-T cells and monocytes, we first established a co-culture system that mimics CRS using K562/CD19 cells, 19-28z CAR-T cells, and peripheral blood CD14+ cells. IFN-γ was produced exclusively from CAR-T cells, and TNF-α, MIP-1α, M-CSF, and IL-6 were produced from both CAR-T cells and monocytes, but monocytes were the major source of these cytokine production. MCP-1, IL-1β, IL-8, and IL-10 were released exclusively from monocytes. To observe the effect of drugs on cytokine production, prednisolone (PSL), TO-207, tocilizumab, and anakinra (an IL-1R antagonist) were added to the co-culture. PSL exhibited suppressive effects on TNF-α and MCP-1 production. Tocilizumab did not suppress these cytokines. Anakinra up-regulated IL-6 and IL-1β production, probably due to activation of negative feedback loops. Interestingly, TO-207 widely suppressed all of these monocyte-derived cytokines including TNF-α, IL-6, IL-1β, MCP-1, IL-8, and GM-CSF. Next, we observed whether the cytokine inhibition by TO-207 attenuates killing effect of CAR-T cells. PSL attenuated killing effect of CD4+ CAR-T cells and CD8+ CAR-T cells toward K562/CD19 cells. In contrast, TO-207 did not exhibit any change in cytotoxicity of CD4+ CAR-T cells and CD8+ CAR-T cells. To determine whether the effect of PSL and TO-207 on cytotoxicity changes in the presence of CD14+ monocytes, CD14+ cells were added to the co-culture. In the absence of CAR-T cells, PSL induced a modest attenuation of cytotoxicity, whereas to the CAR-T cells, PSL exhibited a significant attenuation of cytotoxicity. TO-207 exhibited a minimal effect on cytotoxicity in the absence or presence of CAR-T cells. These results suggested that CAR-T cells play a major role in the cytotoxicity toward leukemia cells, and drugs that do not affect CAR-T cell functions, such as TO-207, maintain their cytotoxic effects on leukemia cells. In conclusion, our present co-culture model with K562/CD19 cells, 19-28z CAR-T cells, and CD14+ monocytes accurately recapitulate killing effect and cytokine release profiles. IFN-γ was produced exclusively by CAR-T cells, but majority of other cytokines such as TNF-α, MIP-1α, M-CSF, IL-6, MCP-1, IL-1β, IL-8, and IL-10 were from CD14+ monocytes/macrophages. Because killing effect was largely dependent on CAR-T cells while cytokine production was dependent on monocytes/macrophages, selective inhibition of pro-inflammatory cytokines from monocytes by TO-207 would be ideal for treatment of CAR-T-related CRS. These results encourage us to consider a clinical application for CRS. Figure Disclosures Futami: Torii Pharmaceutical: Research Funding. Suzuki:Torii Pharmaceutical: Employment. Kato:Torii Pharmmaceutical: Research Funding. Tahara:Torii Pharmaceutical: Employment. Imai:Celgene: Honoraria, Research Funding; Janssen Pharmaceutical K.K: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Mimura:Torii Pharmaceutical: Employment. Watanabe:Torii Pharmaceutical: Employment. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

COP1 is a Ring-Finger E3 ubiquitin ligase that is involved in plant development, mammalian cell survival, growth, and metabolism. Here we report that COP1, whose expression is enhanced by insulin, regulates FoxO1 protein stability. We found that in Fao hepatoma cells, ectopic expression of COP1 decreased, whereas knockdown of COP1 expression increased the level of endogenous FoxO1 protein without impacting other factors such as C/EBPα and CREB (cAMP-response element-binding protein). We further showed that COP1 binds FoxO1, enhances its ubiquitination, and promotes its degradation via the ubiquitin-proteasome pathway. To determine the biological significance of COP1-mediated FoxO1 protein degradation, we have examined the impact of COP1 on FoxO1-mediated gene expression and found that COP1 suppressed FoxO1 reporter gene as well as FoxO1 target genes such as glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two key targets for FoxO1 in the regulation of gluconeogenesis, with corresponding changes of hepatic glucose production in Fao cells. We suggest that by functioning as a FoxO1 E3 ligase, COP1 may play a role in the regulation of hepatic glucose metabolism.

TRB3 is a pseudokinase whose expression is regulated during stress response and changing of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3 expression and Akt activation. We find that insulin induces TRB3 expression in cell type dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3 expression whereas inhibition of PKCzeta expression impairs TRB3 expression induced by insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3 expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced.

Members of Akt family are highly conserved protein kinase and yet, they show clearly distinct in vivo functions. Here, we have examined the abilities of Akt1 and Akt2 to activate CREB. We found that, in contrast to Akt1 that induces CREB phosphorylation at Ser-133 and CREB target gene expression, Akt2 was unable to induce CREB phosphorylation at Ser-133 in vivo and CREB target gene expression. This difference is specific to CREB as both Akt1 and Akt2 similarly inhibits FoxO1 mediated gene expression. We further showed that the regulatory domain of Akt plays a critical role to confer Akt substrate specificity as substitution of regulatory domain of Akt1 with that of Akt2 abolished the ability of Akt1 to activate CREB. We suggest that the regulatory domain of Akts contributes to the functional difference between Akt1 and Akt2.

TRB3 is a member of TRB protein family characterized by containing a variant kinase domain without enzymatic activity. Interacting with Ser/Thr protein kinases Akt, TRB3 impairs Akt activation induced by growth factors and insulin. In this study we have examined the potential role of TRB3 in muscle differentiation. Our data indicated that the expression of TRB3 is downregulated during C2C12 cells undergoing muscle differentiation and that overexpression of TRB3 inhibits Akt activation during differentiation. Correspondingly, overexpression of TRB3 inhibits, while knockdown TRB3 enhances C2C12 differentiation. Thus, our studies indicated that TRB3 plays a critical role in muscle differentiation.