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

The methyl CpG-binding protein 2 (MECP2) gene has copy-number gain in a number of human cancers and functions as an oncogene through an unusual epigenetic mechanism. We explored the possibility that MECP2 might be a therapeutic target and whether its epigenetic mode of action is amenable to specific therapy. Constitutively expressed or inducible lentiviral short hairpin RNA directed at MECP2 in human triple-negative breast cancer (TNBC) cell lines with high or low level of MECP2 protein were grown as xenografts to assess oncogene addiction. We next evaluated the effect of DNA methylation inhibitors or histone deacetylase inhibitors on monolayer or soft agar growth of isogenic human mammary epithelial cells with or without MECP2 overexpression and xenograft growth of MECP2-dependent or MECP2-independent human TNBC or lung cancer cell lines. We then investigated the mechanism of MECP2-induced activation of the MAPK pathway and assessed the effect of drug treatment. Human cancer cell lines with MECP2 overexpression show MECP2 dependence, and epigenetic drugs are effective in these models. Activated RAS and other activators of the MAPK pathway caused resistance to these therapies, giving insight into their novel mode of action and demonstrating specificity. The kinase PAK3 is important for MECP2-mediated induction of the MAPK pathway, is modulated by epigenetic drugs affecting MECP2 action as expected, and may itself be a therapeutic target for MECP2-driven cancers. These preclinical studies show that tumors overexpressing MECP2 might benefit from epigenetic therapy targeting MECP2 function and demonstrate a novel mechanism of action for these drugs.

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

Table S2. UM patient ICb treatment groups in figure 7.

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

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.

Supplementary Figure S2 shows the effect of MECP2 overexpression on genes that may mediate the oncogenic activity of MECP2 and their properties

Supplementary Figure S1 shows the effect of HDAC inhibitors on MECP2 protein levels

Figure S1. GSDMD cleavage fragments induced by Etomoxir.

Supplementary Figure Legends and References

of the study: MECP2 amplification plays a role similar to activated oncogenic RAS in tumorigenesis, activating the MAPK and PI3K signaling pathways. MECP2 amplification and RAS mutations are mutually exclusive across human cancers; solid tumors such as high-grade serous ovarian cancer (HGSOC), triple-negative breast cancer (TNBC) and lung adenocarcinoma have 38%, 33% and 29% rates of MECP2 amplification respectively. The MECP2 protein has DNA- binding activity which is required for oncogenic transformation and binding is dependent on cytosine methylation. Therefore, MECP2 DNA binding may be targeted by inhibiting cytosine methylation with DNMTi. Additionally, since MECP2 protein requires HDAC activity for its stability, treatment with either HDACi and/orDNMTi could be therapeutic strategies in MECP2- dependent tumors. Experimental Procedures and Results: The MDAMB468 (TNBC) cell line was modified stably with a doxycycline-inducible shRNA construct targeting the 3’UTR of MECP2 (shMECP2-3’UTR) and growth was dependent on MECP2 in culture. The cells were then implanted in nude mice subcutaneously and tumors allowed to reach approximately 150 mm3 before shRNA was induced. Tumor growth in doxycycline-treated mice immediately ceased whereas tumors in vehicle-treated mice continued to grow, showing dependence on MECP2 activity in vivo. To verify that this was an on-target effect, the modified cell line was further infected with lentiviruses carrying both splicing isoforms of MECP2 without the 3’UTR shRNA target. This reconstitution allowed growth as a xenograft at a similar rate regardless of shRNA induction, confirming inhibition was on-target. We then assessed the therapeutic efficacy of HDACi and DNMTi alone and in combination in xenograft (for TNBC and lung) or PDX (HGSOC) models of MECP2-dependent and independent tumors. To determine if growth inhibition was an on-target effect of these drugs, MDAMB468 was stably transduced with activated KRAS or GFP as a control and was injected in nude mice. Activated KRAS rescued the epigenetic drug sensitivity of xenografts, demonstrating that these drugs inhibit tumor growth by preventing the ability of MECP2 to induce RAS-activated growth factor pathways. The antitumor effects of the commonly used epigenetic drugs are thought to result from cell reprogramming through re-expression of tumor suppressors that had been silenced by hypermethylation or by repressive chromatin marks. However, in this context, we show that these epigenetic drugs can act directly by interfering with the oncogenic activities of MECP2, culminating in inhibition of key growth factor pathways for transformation. Targeting MECP2 function with epigenetic drugs provides an unanticipated therapeutic opportunity for treating the subset of tumors that overexpress MECP2. Manish Neupane, Mukesh Kumar, Malela M. Werner, Colleen M. Donnelly, Alsu Ibragimova, Brian Curcio, Inna Chervoneva, Irina Vasilevskaya, Daniel P. Silver. Epigenetic drugs inhibit the oncogenic activity of methyl CpG binding protein 2 (MECP2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7187.

Motivation The intra-tumor heterogeneity of protein expression is well recognized and may provide important information for cancer prognosis and predicting treatment responses. Analytic methods that account for spatial heterogeneity remain methodologically complex and computationally demanding for single-cell protein expression. For many functional proteins, single-cell expressions vary independently of spatial localization in a substantial proportion of the tumor tissues, and incorporation of spatial information may not affect the prognostic value of such protein biomarkers.Results We developed a new framework for using the distributions of functional single-cell protein expression levels as cancer biomarkers. The quantile functions of single-cell expressions are used to fully capture the heterogeneity of protein expression across all cancer cells. The quantile index (QI) biomarker is defined as an integral of an unspecified function which may depend linearly or nonlinearly on a tissue-specific quantile function. Linear and nonlinear versions of QI biomarkers based on single-cell expressions of ER, Ki67, TS, and CyclinD3 were derived and evaluated as predictors of progression-free survival or high mitotic index in a large breast cancer dataset. We evaluated performance and demonstrated the advantages of nonlinear QI biomarkers through simulation studies.Availability and implementation The associated R package Qindex is available at https://CRAN.R-project.org/package=Qindex and R package hyper.gam is available at https://github.com/tingtingzhan/hyper.gam. Examples of R code and detailed instructions could be found in vignette quantile-index-predictor (https://CRAN.R-project.org/package=hyper.gam/vignettes/applications.html#quantile-index-predictor).

SOX10-knockout RPPA data (log2-transformed median-centered average protein expression), related to Fig. 1A

Pavlidis Template Matching R-values and p-values for significantly upregulated and downregulated proteins in SOX10-knockout cells compared to parental cells using RPPA data, related to Fig. 1A and Supp. Figure 1B

TGM2 expression in melanoma patient samples

Few treatment options are available for patients with metastatic uveal melanoma. Although the bispecific tebentafusp is FDA approved, immunotherapy has largely failed, likely given the poorly immunogenic nature of uveal melanoma. Treatment options that improve the recognition of uveal melanoma by the immune system may be key to reducing disease burden. We investigated whether uveal melanoma has the ability to undergo pyroptosis, a form of immunogenic cell death. Publicly available patient data and cell line analysis showed that uveal melanoma expressed the machinery needed for pyroptosis, including gasdermins D and E (GSDMD and E), caspases 1, 3, 4, and 8, and ninjurin-1. We induced cleavage of GSDMs in uveal melanoma cell lines treated with metabolic inhibitors. In particular, the carnitine palmitoyltransferase 1 (CPT1) inhibitor, etomoxir, induced propidium iodide uptake, caspase 3 cleavage, and the release of HMGB1 and IL-1β, indicating that the observed cleavage of GSDMs led to pyroptosis. Importantly, a gene signature reflecting CPT1A activity correlated with poor prognosis in patients with uveal melanoma and knockdown of CPT1A also induced pyroptosis. Etomoxir-induced pyroptosis was dependent on GSDME but not on GSDMD, and a pyroptosis gene signature correlated with immune infiltration and improved response to immune checkpoint blockade in a set of patients with uveal melanoma. Together, these data show that metabolic inhibitors can induce pyroptosis in uveal melanoma cell lines, potentially offering an approach to enhance inflammation-mediated immune targeting in patients with metastatic uveal melanoma. Implications: Induction of pyroptosis by metabolic inhibition may alter the tumor immune microenvironment and improve the efficacy of immunotherapy in uveal melanoma.

Melanoma heterogeneity contributes to therapy resistance and immune evasion. The loss of SOX10, a neural crest lineage-specific transcription factor, leads to phenotypic switching from a proliferative cell state to an invasive, drug-tolerant cell state. SOX10-deficient cells are able to persist during immunotherapy treatment, highlighting the need to characterize the factors that regulate immune evasion downstream of SOX10 loss. In this study, we found that SOX10-deficient melanoma cell lines and patient samples express elevated levels of TGM2, a transglutaminase family member. TGM2 upregulation in SOX10 knockout cells was reversed by inhibition of epigenetic reader BET proteins. Knockdown of TGM2 did not affect the SOX10-deficient invasive cell state; however, overexpression of TGM2 in syngeneic melanomas promoted tumor onset in immunocompetent mice, but not in immunodeficient mice, suggesting an immune-mediated effect. TGM2 overexpression in melanoma was associated with decreased intratumoral CD4+ T cells, and depletion of CD4+ T cells abolished the tumor-promoting effect of TGM2. These data indicate that TGM2 is negatively regulated by SOX10 in melanoma and can promote an immunosuppressive tumor microenvironment.Significance: The transglutaminase TGM2 is negatively associated with the neural crest lineage-specific transcription factor SOX10 and is an immunomodulatory protein in cutaneous melanoma.

Representative gating strategy for YUMMER1.7-EV and TGM2-overexpressing tumors analyzed by flow cytometry

Despite the success of targeted inhibitors in cutaneous melanoma, therapeutic responses are limited by the aged tumor microenvironment and drug-tolerant residual cells. Given the similarities between drug tolerance and cellular dormancy, we studied the dormancy marker, nuclear receptor subfamily 2 group F member 1 (NR2F1), in response to BRAF-V600E inhibitors (BRAFi) plus MEK inhibitors (MEKi) in BRAF-mutant melanoma models. Transcriptomic analysis of melanoma patient samples treated with BRAFi + MEKi showed increased NR2F1. NR2F1 was highly expressed in the drug-tolerant invasive cell state of minimal residual disease in patient-derived and mouse-derived xenografts on BRAFi + MEKi. NR2F1 over-expression was sufficient to reduce BRAFi + MEKi effects on tumor growth in vivo, and cell proliferation, death, and invasion in vitro. Effects were linked to genes involved in mTORC1 signaling. These cells were sensitive to the combination of BRAFi, MEKi plus rapamycin. Melanomas from aged mice, known to exhibit decreased responses to BRAFi + MEKi, displayed higher levels of NR2F1 compared to tumors from young mice. Depleting NR2F1 in an aged mouse melanomas improved the response to targeted therapy. These findings show high NR2F1 expression in 'invasive-state' residual cells and that targeting NR2F1-high cells with mTORC1 inhibitors may improve outcomes in patients with melanoma.

TGM2 overexpression modulates tumor onset in immunocompetent mice, but does not affect 3D spheroid growth