The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8 + T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8 + T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Abstract This study is aimed to investigate the effect of ATR inhibitor (ATRi) ceralasertib on tumor microenvironment (TME). Ceralasertib has demonstrated encouraging clinical benefits in patients with non-small cell lung cancer who were resistant to PD1/PDL1 treatment. Using different pre-clinical mouse tumor models, we found that antitumor effect of ceralasertib was dependent on CD8 T cells. In fact, depletion of CD8 cells abrogated therapeutic effect of the ATRi. Ceralasertib treatment caused pleotropic effect in TME. It improved the function of T cells, depleted tumor associated macrophages and monocytic myeloid-derived-suppressor cells (MDSC) and inactivated suppressive activity of PMN-MDSC. Ceralasertib treatment resulted in activation of dendritic cells (DC) in tumor-bearing mice. DCs demonstrated increased ability to stimulate T cells, that resulted in enhanced antigen-specific T cell responses in mice treated with ceralasertib in combination with anti-PDL1. Gene expression profile showed significant up-regulation of type I interferon (IFN1) pathway in mice and patients treated with ceralasertib. Using BM chimera mice reconstituted with IFANR1KO for or IFNAR1SA bone marrow we demonstrated a cancelation of antitumor effect, or an enhancement of antitumor effect respectively induced by ceralasertib or combination therapy. DC cells isolated from IFNAR1KO mice treated with Ceralasertib failed to show an activated phenotype and to increase T cell activation demonstrating the pivotal role of type I IFN in ceralasertib-mediated immune activation. In conclusion, our finding showed that ATR inhibitor ceralasertib has a second mechanism of action necessary for its efficacy: modulating the tumor immune microenvironment and increasing sensitivity to anti-PDL1 treatment. Citation Format: Emilio Sanseviero, Devon Taylor, Sehmus Tohumeken, Mimi Mai, Ali Mostafa, Marta Milo, Kathy Mulgrew, Serge Fuchs, Alan Lau, Simon Barry, Mark Cobbold, Dmitry Gabrilovich. Ceralasertib enhance efficacy of anti-PDL1 treatment by modulating the tumor microenvironment in a IFNAR I dependent manner [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B006.

Background Ceralasertib is a specific ATR inhibitor (ATRi) that hinders the DNA damage response in tumor cells, making them more susceptible to death in situations of high replication stress. Ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. The mechanism of this effect remained unclear. Methods In this study, we employed different mouse tumor models treated with ceralasertib to investigate the effect of the ATRi on tumor microenvironment. Results Antitumor effect of ceralasertib was dependent on the presence of CD8 cells, since in vivo depletion of CD8 cells abrogated therapeutic effect of the ATRi. Analysis of the gene expression profile using RNAseq demonstrated significant up-regulation of type I interferon (IFNI) pathway in tumors of mice treated with ceralasertib. Neutralizing anti IFNI receptor (IFNAR1) antibody abrogated antitumor effect of ceralasertib. Antitumor effect of ceralasertib in combination with anti-PD-L1 was eliminated in mice reconstituted with bone marrow from IFNAR1-KO mice. Reconstitution with bone marrow from mice with constitutively active IFNAR1 markedly enhanced antitumor effect of ceralasertib. Treatment of tumor-bearing mice with ceralasertib caused accumulation of DCs with activated phenotype (up-regulation of CD40, CD86, MHC class II). DCs isolated from tumor of ceralasertib-treated mice demonstrated an enhanced ability to stimulate T cells in a mixed lymphocyte reaction assay. Notably, this effect was reversed when DCs were isolated from IFNAR1 KO mice. Moreover, in vitro-generated DCs treated with ceralasertib exhibited activation similar to that induced by lipopolysaccharide (LPS), but this effect was not observed in DCs generated from IFNAR1 KO mice. Treatment with ceralasertib led to the depletion in tumor of M-MDSC and TAMs, but not PMN-MDSC. However, PMN-MDSCs suppressive activity was abrogated after the treatment. This was associated with increased IFNI signature observed in tumor PMN-MDSC. Treatment with ceralasertib enhanced tumor antigen-specific response of T cells. Conclusions Our findings demonstrate IFNI mediated modulation of TME by ceralasertib resulting in enhanced antitumor activity of T cells and potentiated effect of PD-L1 antibody.

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

Abstract Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

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Cancer immunotherapy often depends on recognition of peptide epitopes by cytotoxic T lymphocytes (CTLs). The tumor microenvironment (TME) is enriched for peroxynitrite (PNT), a potent oxidant produced by infiltrating myeloid cells and some tumor cells. We demonstrate that PNT alters the profile of MHC class I bound peptides presented on tumor cells. Only CTLs specific for PNT-resistant peptides have a strong anti-tumor effect in vivo, whereas CTLs specific for PNT-sensitive peptides are not effective. Therapeutic target-ing of PNT in mice reduces resistance of tumor cells to CTLs. Melanoma patients with low PNT activity in their tumors demonstrate a better clinical response to immunotherapy than patients with high PNT activity. Our data suggest that intratumoral PNT activity should be considered for the design of neoantigen-based therapy and also may be an important immunotherapeutic target.