Drugs selectively targeting replication stress have demonstrated significant preclinical activity, but this has not yet translated into an effective clinical treatment. Here we report that targeting increased replication stress with a combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea targets also promotes pro-inflammatory cytokine/chemokine expression that is independent of cGAS-STING pathway activation and immunogenic cell death in human and murine melanoma cells. In vivo, this drug combination induces tumour regression which is dependent on an adaptive immune response. It increases cytotoxic CD8+ T cell activity, but the major adaptive immune response is a pronounced NKT cell tumour infiltration. Treatment also promotes an immunosuppressive tumour microenvironment through CD4+ Treg and FoxP3+ NKT cells. The number of these accumulated during treatment, the increase in FoxP3+ NKT cells numbers correlates with the decrease in activated NKT cells, suggesting they are a consequence of the conversion of effector to suppressive NKT cells. Whereas tumour infiltrating CD8+ T cell PD-1 and tumour PD-L1 expression was increased with treatment, peripheral CD4+ and CD8+ T cells retained strong anti-tumour activity. Despite increased CD8+ T cell PD-1, combination with anti-PD-1 did not improve response, indicating that immunosuppression from Tregs and FoxP3+ NKT cells are major contributors to the immunosuppressive tumour microenvironment. This demonstrates that therapies targeting replication stress can be well tolerated, not adversely affect immune responses, and trigger an effective anti-tumour immune response.

Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(–)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.

Phenotypic and functional cancer cell heterogeneity limits the efficacy of targeted and immuno-therapies. The transcription factor MITF is known to regulate melanoma cell plasticity and, consequently, response to drugs. However, the underlying mechanisms of this phenomenon remain incompletely understood. Here, we show that MITF levels control functional melanoma cell heterogeneity by fine-tuning the ability to contract the extracellular matrix, the maturation of focal adhesions and ROCK-mediated melanoma cell contractility.Modulation of MITF expression alters extracellular matrix organization, melanoma cell morphology and solid stress in three-dimensional melanoma spheroids, thereby accounting for spatial differences in cell cycle dynamics. Together, our data identify MITF as a master regulator of the melanoma micro-architecture and point towards novel targeting strategies for cancer cell heterogeneity.Significance. Development of drug resistance is a major cause of melanoma therapy failure.The role of MITF in melanoma response to therapy has been discussed controversially, which can be explained, at least in part, through the rheostat model linking MITF activity to cell proliferation. Heterogeneity is widely associated with therapy resistance, however, whether cell phenotype switching, mediated by MITF, is responsible for treatment resistance is not known. Our findings provide an in-depth mechanistic understanding of the MITF-mediated regulation of cell cycle behavior and physical regulation of the tumor architecture. As MITF is not amenable to direct drug targeting, the identification of mediators of MITF-triggered functional heterogeneity reveals novel targets that can be deployed to control this phenomenon.

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