Figure S3. Etomoxir-induced, pyroptotic DAMP release does not require GSDMD.

Figure S2. CPT1A ‘activity-signature’ score (GSVA) K-Means clustering and association with SCNA clusters in TCGA-UVM data.

Table S1. PScore and CPT1A ‘activity’ signature gene sets.

NRAS mutations occur in 10%-30% of cutaneous melanomas and are associated with high tumor mutational burden. Mutant NRAS signaling drives aberrant cell growth and proliferation, in part, through activation of the RAF-MEK-ERK1/2 kinase pathway; however, targeted therapies to this pathway have limited effectiveness in patients with NRAS mutant melanoma. The role of other targetable signaling pathways in NRAS mutant melanoma is poorly characterized. Here, we demonstrated that one isoform of diacylglycerol kinase, diacylglycerol kinase eta (DGKη), a lipid signaling regulator, was highly expressed in NRAS mutant melanoma patient samples. Knockdown of DGKH in NRAS mutant melanoma cell lines resulted in significant growth inhibition in vitro. Transcriptomic data indicated downregulation of the estrogen response late signature, including decreased CCND1 (cyclin D1) expression following DGKH knockdown. Cell growth inhibition and decreased cyclin D1 expression correlated to an inhibition of cell cycle after DGKH knockdown. These data suggest that DGKη mediates cell cycle progression in NRAS mutant melanoma cells and represents a potential therapeutic target for these patients.

Treatment of melanoma with BRAF inhibitors plus MEK inhibitors (BRAFi + MEKi) stimulates an intratumoral immune response, in part through pyroptosis mediated by the pore-forming protein gasdermin E (GSDME/Gsdme). How GSDME mediates effects on tumoral immunity is not well characterized. Using single-cell RNA sequencing and flow cytometry in BRAFi + MEKi-treated melanoma, we show herein that isogenic Gsdme knockout (KO) tumors show decreased infiltration with T cells, natural killer (NK) cells, and regulatory T cells (Treg) compared with control tumors. Infiltrated Tregs in Gsdme KO tumors displayed decreased expression of the IL2 receptor and phenotypic markers associated with suppressive function. Furthermore, intratumoral frequency of phenotypically suppressive Tregs was decreased after BRAFi + MEKi treatment in Gsdme KO tumors engineered to express a pyroptosis-defective mutant form of Gsdme (T6E) compared with Gsdme KO tumors engineered to reexpress wild-type Gsdme. Combining BRAFi + MEKi with a TLR9 agonist limited the regrowth of Gsdme-deficient tumors, and this was associated with a further reduction in intratumoral Tregs. Overall, we show a critical role of GSDME in the modulation of intratumoral immune cells in BRAFi + MEKi-treated melanoma.

Abstract Activating mutations in GNAQ and GNA11 (GNAQ oncogenes) are found in ∼93% of uveal melanoma (UVM) and 4% of skin cutaneous melanoma (SKCM), where they act as driver oncogenes. UVM is the most common primary cancer of the eye in adults, affecting more than 2,500 patients each year in the US alone, nearly 50% of whom will die from liver metastasis. To date, there are limited effective therapeutic options to prevent or treat UVM metastatic disease (mUVM), which typically also fails to respond to immunotherapies. By combining synthetic biology approaches, CRISPR/Cas9 genome-wide screens, and high-throughput chemogenetic drug screening, our team has revealed that classical and novel non-canonical GNAQ signaling circuits converge to promote UVM growth, survival, metastasis, and treatment resistance. Ultimately, elucidating GNAQ oncogenic signaling networks may reveal system vulnerabilities that can be exploited to develop new precision therapies for mUVM. In this regard, we have recently shown that darovasertib acts as a dual PKC-PKN inhibitor and exhibits the highest activity among thousands of drugs tested. Darovasertib has demonstrated encouraging activity in UVM patients, and clinical trials using darovasertib as a single agent in primary UVM lesions and in combination with crizotinib in mUVM are currently ongoing. However, few patients achieve complete responses, and tumors often progress due to the acquisition of resistance mechanisms. We aim to identify new targets that can overcome resistance to darovasertib. RNA-seq analysis revealed that long-term treatment with darovasertib increased the expression of YAP-target genes, and we hypothesized that YAP/TEAD activation may contribute to darovasertib resistance. Indeed, expression of an active YAP mutant (YAP2-5SA) or LATS1/2 inhibition was sufficient to induce darovasertib resistance in UVM cells. In turn, knockdown of YAP or TEAD, or the expression of a doxycycline-induced TEAD inhibitor (TEADi) peptide, increases darovasertib-induced apoptosis. Remarkably, co-targeting with small molecule TEADi decreases the expression of darovasertib-induced YAP targets and acts synergistically to reduce cell viability and increase UVM cell death. Ongoing studies are now exploring the preclinical benefit of combining darovasertib with small-molecule TEADi for the treatment of human UVM tumor xenografts in mice. Emerging evidence will be presented supporting that the Hippo YAP/TEAD pathway represents an adaptive mechanism of resistance to darovasertib treatment, and that the combination of TEADi with darovasertib may prevent the development of treatment resistance, thereby increasing the depth and duration of the anti-tumor response in UVM. Citation Format: Rodolfo Daniel Cervantes-Villagrana, Elena Sofia Cardenas Alcoser, Kuniaki Sato, Simone Lubrano, Tomohiko Ishikawa, Andrew E. Aplin, J. Silvio Gutkind. Targeting Hippo/YAP-TEAD increases the antitumor activity of darovasertib in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1837.

Immunogenic cell death (ICD) is a type of cell death that can enhance anti-tumour immune responses of chemotherapies and targeted therapies by releasing DAMPs and cytokines that activate dendritic cells and T cells, thereby engaging the patient’s immune system to combat the cancer. Pyroptosis and necroptosis are strongly immunogenic because they release DAMPs and inflammatory signals through pore-forming proteins, whereas apoptosis can be tolerogenic. This immunogenic response is contingent on a functional immune system. Unfortunately, most conventional chemotherapies and many targeted therapies also impair the immune system. Here, we investigated the mechanism by which the tumour-selective treatment of Checkpoint kinase 1 inhibitor (CHK1i) combined with low-dose hydroxyurea (LDHU) promotes ICD and anti-tumour immunity. We show that CHK1i+LDHU induces S-phase arrest and caspase-dependent lytic cell death with features of pyroptosis, including gasdermin E cleavage, but cell death was not dependent solely on gasdermin cleavage. Inhibiting caspases was sufficient to block both tumour cell killing and treatment immunogenicity. The mechanism does not rely on any single caspase or gasdermin, consistent with the contributions from multiple caspase-dependent processes. By contrast, doxorubicin that predominantly triggers apoptosis was less effective at stimulating anti-tumour immune responses despite triggering similar levels of cell death. These findings demonstrate that caspase-dependent lytic cell death with pyroptotic features promotes a more effective stimulus for anti-tumour immunity.

Figure S1. GSDMD cleavage fragments induced by Etomoxir.

The 2025 Cure Ocular Melanoma (CURE OM) Global Science Meeting took place in Amsterdam, The Netherlands on October 25th, 2025. Several promising drug candidates are in development; however, there is still an urgent need for better prevention, detection, and treatment of uveal melanoma. The purpose of this meeting was to promote international collaboration and idea exchange between scientists, industry, and patient advocates.

Background: Treatment options are limited, and outcomes remain poor for patients with metastatic uveal melanoma (MUM). We conducted an investigator-initiated, prospective, single-arm, single-institution, phase II study evaluating the combination of an FAK inhibitor (defactinib) with a RAF/MEK inhibitor (avutometinib) for the treatment of MUM. Methods: From February 2021 through January 2023, 12 patients with MUM were treated with the combination of defactinib and avutometinib. Defactinib was given 200 mg twice daily, and avutometinib was given 3.2 mg twice a week. Both drugs were given for 3 weeks on and 1 week off (28-day cycle). Disease control rate was the primary endpoint of this study. Results: Median lines of prior therapies for the patients were two. After two cycles, six patients achieved stable disease while six patients developed progressive disease (disease control rate of 50%). With a median follow-up of 20.0 months, the median progression-free survival was 3.0 months and the median overall survival was 20.0 months. The combination was quite tolerable for patients, with no patients requiring dose reduction or discontinuation. Trial enrollment was stopped early by study sponsors before the anticipated accrual of 18 patients due to no patients having a significant reduction in disease. Conclusions: This is the first study reporting on the use of an FAK inhibitor combination in MUM. Further research should seek to elucidate an optimal combination treatment strategy for MUM, such as FAK and PKC inhibitors, for improved blockade of the signaling pathways downstream of GNAQ/GNA11 driver mutations.

Supplementary Figure Legends and References

Widespread BRAF mutations result in persistent RAS-RAF-MEK-ERK (MAPK) signaling in melanoma. BRAF (BRAFi) and MEK (MEKi) inhibitors are approved for BRAF V600E melanomas, including those progressing on immunotherapy; however, rapid resistance to these agents highlights the need for novel strategies. Here, transcriptome analysis of BRAF V600E melanomas from patients resistant to BRAFi and MEKi shows activation of focal adhesion signaling. Consistently, BRAFi, MEKi, and the RAF-MEK clamp avutometinib activate focal adhesion kinase (FAK) in melanoma cells. Mechanistically, inhibition of an MAPK-RhoE (RND3) feedback loop results in the adaptive activation of RhoA-FAK-AKT. In turn, FAK inhibitors (FAKi) exert potent pro-apoptotic activity when combined with MAPK pathway inhibition. FAKi plus avutometinib overcomes resistance in multiple models derived from BRAFi plus MEKi-resistant melanoma patients and immunotherapy-resistant syngeneic mouse models. These findings provide a rationale for the development of avutometinib in combination with FAKi for patients with BRAF V600E melanoma progressing on BRAFi plus MEKi or immunotherapy.

Ocular malignancies, though relatively rare, pose significant risks to patients' vision, quality of life, and potential for developing metastatic disease. Due to the eye's complicated and delicate anatomy, diagnosing ocular malignancy and monitoring its progression can be technically challenging. Current approaches include direct tumor biopsy, vitreous humor sampling, and imaging. However, these techniques are associated with rare, yet potentially severe, side effects and can be invasive or limited in detecting microscopic disease. Recent studies have introduced aqueous humor sampling as a promising, minimally invasive alternative to diagnose ocular malignancy, guide therapeutic management, monitor response to treatment, and track early recurrence. As oncologic biomarker research continues to progress, incorporating aqueous humor sampling into clinical practice may refine diagnosis, risk stratification, and therapeutic approaches to enhance patient outcomes. This article reviews the latest advances in aqueous humor biomarkers in common ocular malignancies, including uveal melanoma, intraocular lymphoma, and retinoblastoma.

Figure S1: Representative CT images used to calculate tumor volume. Tumor volume was calculated using the following formula: 4π/3 x (a/2) x (b/2) x (c/2) and 3-dimensional images of tumor in the liver. The longest tumor diameter (a) was scaled and then the shortest diameter (b) of the tumor was measured in the Axial view. Z-axis of the tumor (c) was scaled with Coronal view or Sagittal view depending on tumor shape in the liver.

Figure S7: Quantification of UM001 cell western blots from Fig. 7. Bands were normalized to GAPDH quantification. Error bars represent the mean + SEM from 3 independent experiments. P < 0.05 (*), P < 0.005 (**), P < 0.0005 (***), P < 0.0001 (****), Two-way ANOVA.

Supplementary Figure from A Genome-Wide Screen Identifies PDPK1 as a Target to Enhance the Efficacy of MEK1/2 Inhibitors in NRAS Mutant Melanoma

Uveal melanoma (UVM) is the most common eye cancer in adults, with 50% of patients developing overt metastasis that often proves fatal. The majority of UVM harbor mutations in GNAQ or GNA11, encoding constitutively active Gαq proteins. Combined inhibition of MEK and FAK downstream of Gαq has shown promising effects in UVM cells by inducing apoptotic cell death, but resistance to this strategy can occur in the clinic. Here, we aimed to identify new targets to overcome resistance to MEK + FAK inhibition (FAKi + MEKi). Reverse-phase protein array (RPPA) analysis in UVM cells treated with FAKi + MEKi showed increased levels of pro-apoptotic proteins, such as PUMA and BIM, which promoted cell death. However, we observed an adaptive increase in anti-apoptotic proteins, including BCL2, upon FAK + MEK blockade. We generated UVM cells resistant to FAKi + MEKi by prolonged exposure. Whole-exome sequencing did not reveal relevant acquired mutations; instead, resistant cells exhibit increased BCL2 levels. Moreover, expression of a stable BCL2 mutant confers resistance to both FAKi + MEKi and FAKi+"RAF-MEK clamp" (avutometinib) treatment. Of direct translational relevance, we found that an approved BCL2 inhibitor (venetoclax) displays synergistic efficacy with FAK + MEK blockade and overcomes acquired resistance, including when combined with darovasertib, a dual PKC/PKN inhibitor limiting MEK and FAK signaling that is under clinical evaluation. Our findings suggest that resistance to FAKi + MEKi in UVM cells can be driven by an adaptive upregulation of the anti-apoptotic protein BCL2, and that, in turn, BCL2 inhibitors represent a promising precision-targeted strategy to overcome FAKi + MEKi treatment resistance and improve therapeutic outcomes.

Figure S6: Quantification of OMM1.3 cell western blots from Fig. 7. Bands were normalized to GAPDH quantification. Error bars represent the mean + SEM from 3 independent experiments. P < 0.05 (*), P < 0.005 (**), P < 0.0005 (***), P < 0.0001 (****), Two-way ANOVA.