What if an MCL1 inhibitor fights melanoma not mainly by killing tumor cells, but by removing the immune-suppressing cells that help tumors evade treatment? This study tests that idea and combines the inhibitor with anti-PD-1. Melanoma remains a significant and rising health burden in the United States, and tumors in a significant proportion of patients are or will become resistant to immune checkpoint inhibitors.

The study focuses on myeloid-derived suppressor cells, or MDSCs, because they pose a significant obstacle to successful antitumor immunity and effective immune checkpoint inhibitor treatment. Its aim is to explore the therapeutic potential of MCL1 inhibitors to target MDSCs and improve the efficacy of immune checkpoint inhibitors for melanoma.

MCL1 expression in melanoma patient tumors has been linked with worse responses to targeted or chemotherapies, while interest in MCL1 inhibitors for solid tumors, including melanoma, has grown. However, prior studies had not examined the immunomodulatory properties of the MCL1 inhibitor S64315, which is the focus of this study.

MDSCs are a heterogeneous population of immature myeloid cells that accumulate in cancer and are a significant source of treatment resistance in melanoma and other cancers. In tumors and peripheral tissues, MDSCs promote tumor growth and immune escape through immunosuppressive molecules such as interleukin-ten, reactive oxygen species, and vascular endothelial growth factor.

They also express cell-surface receptors that suppress T-cell responses, while immune checkpoint inhibitors rely on proper immune function to be effective. The study investigates S64315 as an immunomodulatory agent in mouse melanoma models and human immune cells.

In mouse models, the effects examined included tumor growth, tumor-infiltrating MDSC frequency, CD8-positive T-cell function, and the efficacy of anti-PD-1 therapy. In human cells, the study examined the effects of S64315 on MDSCs and T cells, with the broader goal of testing next-generation MCL1 inhibitors in combination with immune checkpoint inhibitor therapy.

The animal experiments used eight- to ten-week-old female C57BL6J mice or NCRNU nude mice, and all studies had five mice per treatment group. Mice received melanoma cells by implantation, and S64315 was used at a dose of twenty-five milligrams per kilogram through the intraperitoneal route unless otherwise mentioned.

For flow-cytometry studies, S64315 was administered on days four, six, and nine, and tumors were harvested on day ten. All in vivo treatment began four days after tumor inoculation. Figure one tests whether the MCL1 inhibitor S64315 acts directly on melanoma cells or depends on the host immune system.

In nude, immunocompromised mice, S64315 does not clearly separate tumor growth from vehicle, while in immunocompetent C57BL/6J mice both dosing regimens are associated with reduced B16.F10 tumor volume, with significance marked at the final measurements. Consistent with this, S64315 leaves B16.F10 and YUMM1.7 cell viability near control levels in vitro, and MCL1 knockdown mainly affects B16.F10 tumor growth, supporting an immune-mediated mechanism.

In immunocompromised nude mice, there was no statistically significant change in B16.F10 melanoma tumor growth after S64315 treatment. In immunocompetent C57BL6J mice, tumor growth was significantly reduced by S64315, suggesting an immunomodulatory role for the inhibitor.

Both tested regimens inhibited tumor growth in C57BL6J mice, while neither regimen affected tumor growth in nude mice. S64315 had little to no direct effect on cell viability, and MCL1 knockdown in tumor cells was not sufficient to inhibit tumor growth. Together, these results indicate that S64315 functions in vivo through modulation of immune cells.

The study used single-cell RNA sequencing on fluorescence-activated cell sorting-purified myeloid cells from tumors of patients who had relapsed after anti-PD-1 treatment. The analysis identified heterogeneous tumor-infiltrating myeloid populations, including macrophages, monocytes, dendritic cells, and an MDSC-like cluster with low antigen-processing machinery and high expression of immunosuppressive genes.

The MDSC-like cells in cluster four expressed high levels of MCL1 compared with other cells, and MDSCs also expressed high levels of BCL2A1. Figure two profiles MDSCs from an anti-PD-1-resistant melanoma metastasis.

UMAP and gene-expression dot plots identify clusters with an MDSC transcriptional signature, while the BCL2-family analysis and feature plots show MCL1, BCL2, and BCL2L1 across those clusters. Flow-cytometry gating and representative histograms extend this observation to MDSCs from ten melanoma patients, supporting MCL1 as a candidate vulnerability for targeting these immunosuppressive cells.

Figure three tests how the MCL1 inhibitor S64315 reshapes the immune environment in B16.F10 tumors. After treatment, flow cytometry quantified myeloid and T-cell populations: the figure marks significant differences for PMN-MDSCs, MO-MDSCs, CD8-positive T cells, regulatory T cells, and activated PD-1-positive, granzyme B-positive CD8-positive T cells, while dendritic cells and CD4-positive T cells are marked not significant.

These findings support the authors’ proposal that MCL1 inhibition can reduce immunosuppressive myeloid cells and alter antitumor immunity. The measured T-cell populations included CD8-positive T cells and highly activated CD8-positive PD-1-positive Gzmb-positive T cells. Overall, the antitumor immunomodulatory effects of S64315 were related to a significant reduction in immunosuppressive MDSCs.

Human MDSCs were isolated from five healthy human subjects, and the study also used HL-60 as an immature MDSC model. After twenty-four hours of S64315 treatment, viability of human MDSCs and HL-60 was significantly reduced in a dose-dependent manner, while the effect on A375 and B16.F10 melanoma cell viability was negligible.

The data indicate that S64315 can kill human MDSCs at nanomolar ranges, and that MDSCs were ten times more sensitive to MCL1 inhibition than melanoma cell lines. Figure four tests whether the MCL1 inhibitor S64315 preferentially affects myeloid-derived suppressor cells, or MDSCs, while preserving T-cell activation.

In panel A, S64315 reduces the viability of human MDSCs and HL-60 cells, with reported IC50 values of zero point five nine and zero point zero seven micromolar, while melanoma cell sensitivities are also shown. Panels B through E measure CD8 and CD4 proliferation, plus CD8 interferon gamma and granzyme B expression after anti-CD3/CD28 activation, providing evidence that the inhibitor does not negatively impact these T-cell responses.

Figure five connects patient data with a mouse experiment. In panel A, the Kaplan–Meier plot shows that MCL1 expression is associated with survival among patients treated with anti-PD-1, with a hazard ratio of one point seven seven, a log-rank p-value of zero point zero zero zero one, and an FDR of three percent.

Panels B and C then show the treatment schedule and that combining the MCL1 inhibitor S six four three one five with anti-PD-1 significantly inhibited B sixteen.F ten tumor growth compared with vehicle or either single treatment. In the C57BL6J syngeneic model, S64315 at twenty-five milligrams per kilogram combined with anti-PD-1 at ten milligrams per kilogram significantly inhibited B16.F10 tumor growth compared with vehicle or a single agent.

The combination was tolerable at these doses and did not significantly reduce mouse weight. These results indicate that the MCL1 inhibitor S64315 can enhance the efficacy of anti-PD-1. Although immune checkpoint inhibitors have revolutionized cancer treatment, many melanoma patients do not respond to immune checkpoint inhibitor therapy, making alternative therapeutic approaches necessary.

Targeting BCL2 family proteins has been explored to sensitize cancer cells to apoptosis and reduce the development of therapy resistance. Previous studies showed the feasibility of using MCL1 inhibitors to target melanoma, but had not investigated their effects on immune cells.

S64315 reduced melanoma growth through immune-cell modulation, reduced immunosuppressive MDSCs, and enhanced anti-PD-1 treatment in mice. The results support developing next-generation MCL1 inhibitors for combination immunotherapy.