Background & Aim: Replication stress is common in solid cancers, including melanoma, and can be selectively targeted using Checkpoint kinase 1 inhibitors (CHK1i). However, CHK1i as single agents have shown limited clinical efficacy. We previously demonstrated that combining CHK1i with low-dose hydroxyurea (CHK1i+LDHU) selectively targets replication stress in tumours with minimal toxicity, inducing pro-inflammatory responses and immunogenic cell death. Notably, CHK1i+LDHU is effective in BRAFi- and immune checkpoint inhibitor (ICI)- resistant melanomas. This study aims to further elucidate the mechanisms of anti-tumour immune responses triggered by CHK1i+LDHU and to explore strategies to enhance these responses by targeting tumour-associated macrophages (TAMs).
Methods: (1) immune cell depletion studies to identify key mediators of response; (2) multiple mouse melanoma models to evaluate efficacy of CHK1i+LDHU combined with immunotherapy; (3) tumour microenvironment profiling using multiparameter flow cytometry, imaging flow cytometry, and CITE-seq with scRNA-seq and TCR sequencing.
Results: CHK1i+LDHU induced robust anti-tumour immune responses independent of baseline tumour immune status, including in immunologically “cold” tumours. The responses were dependent on CD8 T cells and involved NK cell activation. Treatment increased CD8 T cell cytotoxicity (granzyme B) and PD-1 expression, but anti-PD-1 did not improve outcomes. In contrast, PD-L1 expression was upregulated on tumour and tumour-associated myeloid cells, and anti-PD-L1 significantly enhanced therapeutic efficacy. CITE-seq and scRNA-seq data demonstrated that PD-L1 blockade reprograms PD-L1⁺ myeloid cells, indirectly boosting CD8 T cell activity. CHK1i+LDHU shifted TAMs toward a more inflammatory phenotype, although immunosuppressive features persisted, while combination with anti-PD-L1 strengthened the inflammatory reprogramming. A proliferating effector CD8 T cell cluster emerged specifically in tumours treated with the CHK1i+LDHU and anti-PD-L1 combination. TCR sequencing revealed increased repertoire diversity following CHK1i+LDHU.
Conclusions: CHK1i+LDHU selectively targets melanoma with minimal toxicity while inducing potent CD8 T cell-dependent anti-tumour immunity. Its enhanced efficacy with PD-L1, but not PD-1, blockade suggests that PD-L1 directed therapy reprograms TAMs to augment anti-tumour immune responses. Also, these findings support further investigation on TAM-targeting approaches to optimise treatment responses. Given the activity of CHK1i+LDHU in BRAFi- and ICI-resistant disease, together with the clinical availability of anti-PD-L1 agents, this combination represents a readily translatable treatment strategy.