Ellen Brenner

Abstract Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16 Ink4a /p19 Arf ( Cdkn2a ) or p21 Cip1 ( Cdkn1a ) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication.

T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated β-galactosidase (SA-β-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-β-gal activity, and-except palbociclib-increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.

BACKGROUND: Although antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors. METHODS: Growth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry. RESULTS: On immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF. CONCLUSIONS: Tumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence.