Skin cutaneous melanoma is characterized by high malignancy and prognostic heterogeneity. Immune cell networks are critical to the biological progression of melanoma through the tumor microenvironment. Thus, identifying effective biomarkers for skin cutaneous melanoma from the perspective of the tumor microenvironment may offer strategies for precise prognosis prediction and treatment selection. A total of 470 cases from The Cancer Genome Atlas and 214 from the Gene Expression Omnibus were systematically evaluated to construct an optimal independent immune cell risk model with predictive value using weighted gene co-expression network analysis, Cox regression, and least absolute shrinkage and selection operator assay. The predictive power of the developed model was estimated through receiver operating characteristic curves and Kaplan-Meier analysis. The association of the model with tumor microenvironment status, immune checkpoints, and mutation burden was assessed using multiple algorithms. Additionally, the sensitivity of immune and chemotherapeutics was evaluated using the ImmunophenScore and pRRophetic algorithm. Furthermore, the expression profiles of risk genes were validated using gene expression profiling interactive analysis and Human Protein Atlas resources. The risk model integrated seven immune-related genes: ARNTL, N4BP2L1, PARP11, NUB1, GSDMD, HAPLN3, and IRX3. The model demonstrated considerable predictive ability and was positively associated with clinical and molecular characteristics. It can be utilized as a prognostic factor for skin cutaneous melanoma, where a high-risk score was linked to a poor prognosis and indicated an immunosuppressive microenvironment. Furthermore, the model revealed several potential target checkpoints and predicted the therapeutic benefits of multiple clinically used drugs. Our findings provide a comprehensive landscape of the tumor immune microenvironment in skin cutaneous melanoma and identify prognostic markers that may serve as efficient clinical diagnosis and treatment selection tools.

Protons are conventionally regarded as a low-linear energy transfer (low-LET) radiation modality with a relative biological effectiveness (RBE) of 1.1, suggesting direct mechanistic similarity to X-rays in the underpinning biological effects. However, exposure to spread-out Bragg peak (SOBP) protons reveals instructive deviations from this assumption. Indeed, proton beams have a maximum LET of ~5 keV/µm but display reduced reliance on classical non-homologous end joining (c-NHEJ) as well as an increased dependence on homologous recombination (HR) and alternative end joining (alt-EJ). These features are well described in cells exposed to high-LET radiation and typically manifest between 100 and 150 keV/µm. We hypothesized that this apparent discrepancy reflects biological consequences of proton-beam properties that remain uncharacterized. In the present study, we outline exploratory experiments aiming at uncovering such mechanisms. We begin by investigating for both entrance and SOBP protons the dose-dependent engagement of HR we recently showed for X-rays. Consistent with our previous findings with X-rays, HR engagement after exposure to both types of proton beams declined with dose, from ~80% at 0.2 Gy to less than 20% at higher doses. RAD51/γH2AX foci ratios, reflecting HR engagement, were modestly higher following proton irradiation, in line with increased HR utilization. G₂-checkpoint activation, previously linked to HR, was also stronger after exposure to protons, as was DNA end resection. Moreover, the formation of structural chromosomal abnormalities (SCAs) was higher for SOBP than entrance protons and X-rays. Collectively, our results suggest quasi-high-LET characteristics for proton beams and raise the question as to the physical proton properties that underpin them. We discuss that the commonly employed definition of LET may be insufficient for this purpose.

Proton beam radiotherapy (PBT) offers superior accuracy of dose deposition, reducing the risk of adverse effects to surrounding healthy tissues. However, despite high medical need, molecular and cellular determinants of radiosensitivity to proton radiotherapy remain underexplored, and prognostic biomarkers and therapeutic targets informing precision medicine strategies for PBT are mostly missing. This study aimed to investigate the role of DNA double-strand break (DSB) repair pathways in shaping tumor response to proton versus photon radiation. The study employed genetic and pharmacological methods to impair DSB repair, including CRISPR-Cas9 gene editing to generate DSB-repair deficient (ATM, PARP1, and BRCA2 knockout) A549 and HCT116 cell lines, and pharmacological inhibitors of ATM and PARP using KU55933 and Olaparib, respectively. Cellular responses to photon (X-rays) and proton irradiation were evaluated through clonogenic survival assays, crystal violet proliferation, and Annexin V/7AAD apoptosis assays. To investigate DNA repair mechanisms, U2OS reporter systems were employed, complemented by chromosomal aberration analysis, and pulsed-field gel electrophoresis (PFGE). Finally, the translational relevance of the findings was validated using the chorioallantoic membrane (CAM) assay closer representing an in-vivo situation. PBT triggered a stronger activation of resection-dependent DNA repair pathways, primarily homologous recombination (HR) and alternative end-joining (alt-EJ), compared to photon irradiation. This increased activation was further supported by classical cytogenetics results. Tumor cells deficient in BRCA2, ATM, or PARP1 showed significantly increased sensitivity to proton radiotherapy, highlighting enhanced relative biological effectiveness in both, in-vitro and in the CAM model. Importantly, combining proton radiotherapy with Olaparib or KU55933 potentiated tumor cell killing, even in repair-proficient models, showing synergy not observed with photons. The observed genotype-specific or drug-induced increase in radiosensitivity towards proton beam radiotherapy highlights the promise of genetic profiling of DSB repair defects for biology-driven patient stratification and the use of PARP-inhibitors in guiding personalized proton radiotherapy strategies.

Small cell lung cancer (SCLC) is an aggressive malignancy, with most patients presenting with prognostically poor extensive-stage disease. Limited progress in standard care stresses the urgent need for novel therapies. Radiotherapy offers some survival benefit for selected patients with SCLC but could be enhanced with radiosensitizers. In this study, we identify HDAC3 as a novel radiosensitizing target in SCLC using a CRISPR knockout screen and demonstrate its efficacy and mechanism. SBC5 cells were transduced with a custom EpiDrug single-guide RNA library and treated with ionizing radiation (IR) to identify radiosensitizing genes. HDAC3 emerged as a candidate and was validated through genetic knockdown and pharmacologic inhibition (RGFP966) in multiple SCLC cell lines. Both approaches enhanced radiosensitivity, as shown by cell viability (dose modification factor10 = 1.14–1.69) and clonogenic assays (dose modification factor10 = 1.16–1.41). We assessed changes in chromatin accessibility by assay for transposase-accessible chromatin using sequencing and IR-induced DNA damage and repair using γH2AX foci detection, double-strand break (DSB) repair assays, and immunoblotting of repair proteins. HDAC3-deficient cells exhibited increased chromatin accessibility, greater IR-induced DSBs, and impaired repair capacity, resulting in persistent DNA damage. This repair defect sensitized cells to PARP inhibitors, for which combining RGFP966 with olaparib or talazoparib produced additive to synergistic effects. In SCLC xenograft models, HDAC3 knockdown or RGFP966, combined with IR, achieved significant tumor growth inhibition. Collectively, we identified HDAC3 as a novel radiosensitizing target in SCLC. Its functional loss increased the generation and persistence of IR-induced DNA DSBs, effectively sensitizing SCLC cell lines and xenografts to IR, providing a potential radiosensitization strategy to treat SCLC.

In vitro CRISPR dropout screen identifies HDAC3 among the top radiosensitizing candidate targets in SCLC.

HDAC3 functional loss radiosensitizes SCLC cell lines.