Bortezomib augments lymphocyte stimulatory cytokine signaling in the tumor microenvironment to sustain CD8+T cell antitumor function.

Tumor-induced immune tolerance poses a major challenge for therapeutic interventions aimed to manage cancer. We explored approaches to overcome T-cell suppression in murine breast and kidney adenocarcinomas, and lung fibrosarcoma expressing immunogenic antigens. We observed that treatment with a reversible proteasome inhibitor bortezomib (1 mg/kg body weight) in tumor-bearing mice significantly enhanced the expression of lymphocyte-stimulatory cytokines IL-2, IL-12, and IL-15. Notably, bortezomib administration reduced pulmonary nodules of mammary adenocarcinoma 4T1.2 expressing hemagglutinin (HA) model antigen (4T1HA) in mice. Neutralization of IL-12 and IL-15 cytokines with a regimen of blocking antibodies pre- and post-adoptive transfer of low-avidity HA518-526-specific CD8+T-cells following intravenous injection of 4T1HA cells increased the number of pulmonary tumor nodules. This neutralization effect was counteracted by the tumor metastasis-suppressing action of bortezomib treatments. In bortezomib-treated 4T1HA tumor-bearing mice, CD4+T-cells showed increased IL-2 production, CD11c+ dendritic cells showed increased IL-12 and IL-15 production, and HA-specific activated CD8+T-cells showed enhanced expression of IFNγ, granzyme-B and transcription factor eomesodermin. We also noted a trend of increased expression of IL-2, IL-12 and IL-15 receptors as well as increased phosphorylation of STAT5 in tumor-infiltrating CD8+T-cells following bortezomib treatment. Furthermore, bortezomib-treated CD8+T-cells showed increased phosphorylation of mitogen-activated protein kinase p38, and Akt, which was abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitor. These data support the therapeutic potential of bortezomib in conjunction with other immunotherapies to augment the strength of convergent signals from CD8+T-cell signaling molecules including TCR, cytokine receptors and downstream PI3K/Akt/STAT5 pathways to sustain CD8+T-cell effector function in the tumor microenvironment.

Bortezomib enhances expression of effector molecules in anti-tumor CD8+ T lymphocytes by promoting Notch-nuclear factor-κB crosstalk.

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFß-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NFκB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions.

Multivalent Forms of the Notch Ligand DLL-1 Enhance Antitumor T-cell Immunity in Lung Cancer and Improve Efficacy of EGFR-Targeted Therapy.

Activation of Notch signaling in hematopoietic cells by tumors contributes to immune escape. T-cell defects in tumors can be reversed by treating tumor-bearing mice with multivalent forms of the Notch receptor ligand DLL-1, but the immunologic correlates of this effect have not been elucidated. Here, we report mechanistic insights along with the efficacy of combinational treatments of multivalent DLL-1 with oncoprotein targeting drugs in preclinical mouse models of lung cancer. Systemic DLL-1 administration increased T-cell infiltration into tumors and elevated numbers of CD44(+)CD62L(+)CD8(+) memory T cells while decreasing the number of regulatory T cells and limiting tumor vascularization. This treatment was associated with upregulation of Notch and its ligands in tumor-infiltrating T cells enhanced expression of T-bet and phosphorylation of Stat1/2. Adoptive transfer of T cells from DLL1-treated tumor-bearing immunocompetent hosts into tumor-bearing SCID-NOD immunocompromised mice attenuated tumor growth and extended tumor-free survival in the recipients. When combined with the EGFR-targeted drug erlotinib, DLL-1 significantly improved progression-free survival by inducing robust tumor-specific T-cell immunity. In tissue culture, DLL1 induced proliferation of human peripheral T cells, but lacked proliferative or clonogenic effects on lung cancer cells. Our findings offer preclinical mechanistic support for the development of multivalent DLL1 to stimulate antitumor immunity.

Bortezomib Improves Adoptive T-cell Therapy by Sensitizing Cancer Cells to FasL Cytotoxicity.

Cancer immunotherapy shows great promise but many patients fail to show objective responses, including in cancers that can respond well, such as melanoma and renal adenocarcinoma. The proteasome inhibitor bortezomib sensitizes solid tumors to apoptosis in response to TNF-family death ligands. Because T cells provide multiple death ligands at the tumor site, we investigated the effects of bortezomib on T-cell responses in immunotherapy models involving low-avidity antigens. Bortezomib did not affect lymphocyte or tissue-resident CD11c(+)CD8(+) dendritic cell counts in tumor-bearing mice, did not inhibit dendritic cell expression of costimulatory molecules, and did not decrease MHC class I/II-associated antigen presentation to cognate T cells. Rather, bortezomib activated NF-κB p65 in CD8(+) T cells, stabilizing expression of T-cell receptor CD3ζ and IL2 receptor-α, while maintaining IFNγ secretion to improve FasL-mediated tumor lysis. Notably, bortezomib increased tumor cell surface expression of Fas in mice as well as human melanoma tissue from a responsive patient. In renal tumor-bearing immunodeficient Rag2(-/-) mice, bortezomib treatment after adoptive T-cell immunotherapy reduced lung metastases and enhanced host survival. Our findings highlight the potential of proteasome inhibitors to enhance antitumor T-cell function in the context of cancer immunotherapy.