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Abstract The standard treatment for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), which target the aberrant MAPK signaling pathway. Although patients initially respond well to this therapy, many develop acquired resistance over time. This resistance can be attributed to various altered signaling pathways, including the reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. Despite extensive omics studies aimed at deciphering the mechanisms behind acquired resistance, the specific alterations within these pathways remain poorly understood. To gain further insights into acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patient-derived cell lines and in vitro studies using PDX models. We investigated differential transcript usage (DTU) to detect specific splice variants altered during the development of resistance. Our analysis revealed significant transcript alterations during on-treatment that reverted to baseline states once resistance was established, underscoring the dynamic and adaptive nature of these changes. Genes with DTU were enriched in pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, and signaling by RTKs. Furthermore, the DTU-centered analysis provided better insights into MAPKi resistance mechanisms compared to standard differential gene expression (DEG) analysis, with DTUs showing higher enrichment scores in MAPKi resistance-related pathways. Further, identifying developmental splicing signatures highlights the complexity of MAPKi resistance, as it reveals significant upregulation of transcripts associated with embryonic melanoblast stem cells (MSCs) among the altered transcripts in on-treatment cell lines. This reprogramming through RNA splicing may confer an adaptive advantage, enabling melanoma cells to revert to a more plastic, stem-like state, with developmental splicing events playing a crucial role in the adaptive response to MAPKi therapy. Next, using a regulatory model based on the expression of splicing factors (SFs), we accurately predicted transcript alterations in on-treatment samples and identified key SFs. Our study highlights the crucial role of RNA splicing in the adaptive response of melanoma cells to MAPKi treatment. These insights pave the way for future research and therapeutic strategies focusing on RNA splicing to combat drug resistance in cancer. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A Ronai, Eytan Ruppin, Sridhar Hannenhalli. RNA splicing alterations in the development of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A004.

Abstract The primary therapy for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), targeting the dysregulated MAPK signaling pathway. Despite the initial response, many patients often develop acquired resistance. Resistance to MAPKi could be linked to various altered signaling pathways, including reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. While numerous omics studies have aimed to understand the mechanism of acquired resistance, how these pathways are altered remains elusive. To gain further insights into the acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events in this context by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patients and in vitro studies using cell lines. We investigated the differential transcript usage (DTU) to detect the specific splice variants that are altered during the development of resistance. We find that the genes involving DTU are enriched in various pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, signaling by RTKs, etc. Furthermore, the DTU-centered analysis provides better insights into MAPKi resistance mechanisms compared to standard differential gene expression analysis. Next, we analyzed the developmental splicing signatures during MAPKi resistance development, revealing developmental reprogramming as a key factor in MAPKi resistance. Further, we identified the top 10 transcription factors as potential upstream regulators of the perturbed transcripts, among which five have been previously reported to be involved in various stages of embryonic development. This finding suggests a link between the reactivation of developmental pathways and MAPKi resistance development. Further, by analyzing MAPKi-treated melanoma cell-line models at different time points, such as no-treatment, on-treatment, and at the resistant stage, we observed significant alterations in transcript isoforms during the development of resistance. These changes involve lipid metabolic reprogramming and PI3K/Akt/mTOR signaling, providing further insights into the mechanisms of MAPKi resistance. Our study also uncovers a global shift in transcript profiles towards shorter 3’ and 5’ UTRs in response to MAPKi treatment, suggesting the existence of a master regulatory mechanism influencing global splicing changes during therapy resistance. In summary, our study deciphered the post-transcriptional mechanism coordinating the signaling changes, underscoring the complexity of MAPKi resistance in melanoma, as revealed by the joint analysis of both transcript alterations and splicing events. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A. Ronai, Eytan Ruppin, Sridhar Hannenhalli. Dynamics of transcript alternations and differential splicing in the evolution of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB250.

Abstract RNF185 is a RING finger domain-containing ubiquitin ligase implicated in ER-associated degradation. Prostate tumor patient data analysis revealed a negative correlation between RNF185 expression and prostate cancer progression and metastasis. Likewise, several prostate cancer cell lines exhibited greater migration and invasion capabilities in culture upon RNF185 depletion. Subcutaneous inoculation of mouse prostate cancer MPC3 cells stably expressing short hairpin RNA against RNF185 into mice resulted in larger tumors and more frequent lung metastases. RNA-sequencing and Ingenuity Pathway Analysis identified wound-healing and cellular movement among the most significant pathways upregulated in RNF185-depleted lines, compared with control prostate cancer cells. Gene Set Enrichment Analyses performed in samples from patients harboring low RNF185 expression and in RNF185-depleted lines confirmed the deregulation of genes implicated in epithelial-to-mesenchymal transition. Among those, COL3A1 was identified as the primary mediator of RNF185’s ability to impact migration phenotypes. Correspondingly, enhanced migration and metastasis of RNF185 knockdown (KD) prostate cancer cells were attenuated upon co-inhibition of COL3A1. Our results identify RNF185 as a gatekeeper of prostate cancer metastasis, partly via its control of COL3A1 availability. Implications: RNF185 is identified as an important regulator of prostate cancer migration and metastasis, in part due to its regulation of COL3A1. Both RNF185 and COL3A1 may serve as novel markers for prostate tumors.

Abstract Rewiring of metabolic pathways often underlies the malignant state, including melanoma. In our previous studies we have demonstrated melanoma addiction to Gutaryl Co-dehydrogenase (GCDH), an enzyme in the lysine catabolism pathway. Our studies revealed that blocking GCDH activity in melanoma, but not colon, lung or breast cancer cells, led to cell death which abolished growth both in culture and in vivo. Important cellular component which was found to mediate melanoma addiction to GCDH was the transcription factor NRF2, which exhibited tumor suppressor function upon GCDH inhibition. Notably, coinciding with NRF2 tumor suppressor role seen upon GCDH inhibition was its glutarylation, a post translational modification which acquired NRF2 stability and ability to induce transcription of ATF4, ATF3 and CHOP which led to extensive cell death program. Knockdown of NRF2, ATF3 or DTHDK-1 effectively blocked the cell death phenotype seen upon GCDH knockdown. Analyses of patient data confirmed that low GCDH expression coincided with prolonged survival of melanoma but not colon, breast or prostate cancer patients. These findings led us to perform an unbiased screen to identify cellular components, which underlie melanoma addiction to GCDH. The results of this analysis will be discussed. Citation Format: Namratha Nadig, Sachin Verma, Ze'ev A. Ronai. Melanoma addiction to GCDH [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1802.

Abstract Growing evidence points to the importance of the tumor microenvironment (TME) in tumor growth control, progression, and therapy response. Among those is the greater understanding of immune system components within the TME as mediators of tumor growth control. NR2F6 is an orphan nuclear receptor, which was shown to serve as an immune intrinsic checkpoint component and has been suggested to elicit tumor-intrinsic suppression of anti-tumor immunity. Correspondingly, genetic depletion of NR2F6 in CD8 T cells abrogates the immune-activating cytokines (e.g., IL-2, IFNg). In addition, genetic ablation of NR2F6 in melanoma cells activates CD8 T cell-mediated anti-tumor immunity partly by suppressing the expression of tumor-intrinsic immune repressors NACC1 and FKBP10. Notably, combined genetic ablation of tumor cells (NR2F6 KO melanoma) and stroma cells (NR2F6 KO mouse) resulted in a more pronounced inhibition of tumor growth compared with ablation of either tumor cells or TME. The latter provides the foundation for screening NR2F6 inhibitors, which are expected to have a strong systemic (tumor inhibition and immune cell activation) effect on tumor growth. Interestingly, single-cell transcriptomic data from human tumors identified high expression of NR2F6 in stroma cells, cancer-associated fibroblasts (CAFs), and tumor-associated endothelial cells (TAEs). Consistent with these, scRNAseq analysis of mouse melanoma revealed higher expression of NR2F6 in SMA+ immune suppressive myo-CAFs than in immune activating CAFs. Studies that directly reveal the role of stromal NR2F6 in anti-tumor immunity and tumor growth and the state of NR2F6 inhibitors will be discussed. Citation Format: Hyungsoo Kim, Yongmei Feng, Marria Radaeva, Eduard Sergienko, Artem Cherkasov, Ze'ev A. Ronai. Anti-tumor immune function of NR2F6, an orphan nuclear receptor, in stromal cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6531.

Abstract Growing evidence supports the importance of the gut microbiota in controlling tumor development, often via activation of anti-tumor immunity. Earlier studies from our laboratory identified 11 bacterial strains that were sufficient to inhibit melanoma development by inducing anti-tumor immunity in germ-free mice. Of these, three were predominant at the time of tumor collection. We thus set out to address whether fewer bacterial strains may be sufficient for melanoma growth inhibition. Here, we demonstrate that the administration of one bacterial strain, B. rodentium, was sufficient to attenuate melanoma growth in gnotobiotic mice. In all cases, gnotobiotic mice were also administered the altered Schadler flora (ASF), a mixture of 8 bacterial strains that are provided to establish minimal flora in these mice. RNAseq-based gene expression studies identified enrichment of immune-related genes, suggesting enhanced anti-tumor immunity. Metabolomic analysis identified reduced levels of tryptophane and isoleucine, which were implicated in control of immune system function. These findings point to a mechanism which may provide novel means to alter anti-tumor immunity and limit melanoma growth. Citation Format: Ximena Diaz Olea, Kristin Beede, Andrei Osterman, David Scott, Christopher Petucci, Daniel Kelly, Amanda Ramer-Tait, Ze’ev A. Ronai. Control of melanoma development by B. rodentium in germ free mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6685.

Abstract Growing evidence supports the important role of myeloid-derived suppressor cells (MDSC) in melanoma progression and response to therapy. MDSC contributes to a pro-tumorigenic phenotype, yet the mechanistic basis for the immune suppressor properties acquired by MDSC remains largely unknown. Mammals have two homologous Siah genes, Siah1 (Siah1a and Siah1b in mice) and Siah2. In the context of melanoma, the ubiquitin ligases Siah1a and Siah2 have melanoma-intrinsic and -extrinsic (immune) functions. Correspondingly, ablation of Siah1a/2 in melanoma or its microenvironment attenuates melanoma development in a number of mouse melanoma models. Given that number of macrophage clusters infiltrated into melanoma when grown in Siah2 KO mice, we set to assess macrophage function in melanoma. Selective ablation of Siah1a or Siah2 in macrophages, using Lyz2Cre, revealed that Siah1a but not Siah2 elicits a tumor suppressor function in melanoma. Inoculation of mouse melanoma Yummer1.7 cells (Braf mutated and Pten deleted) in mice lacking Siah1a in macrophages, resulted in bigger melanoma tumors compared to melanoma grown in either WT or mice in which macrophages were ablated of Siah2. FACS analysis of melanoma tumors grown in mice harboring Siah1a mutant macrophages revealed a significant increase in MDSC, which coincided with a decrease in CD4+ and CD8+ cells, compared with WT mice. These observations suggest that a lack of Siah1a in macrophages leads to an expansion of MDSC that affects surrounding immune cells, including T cells. RNA-seq analysis of MDSC that were differentiated in vitro from bone marrow cells of WT or Siah1a ablated macrophage mice revealed increased expression of genes involved in proliferation and alternative macrophage activation in MDSC lacking Siah1a when compared to the WT genotype. Mapping mechanisms underlying Siah1a tumor suppressor function is expected to reveal novel regulatory cues in melanoma control by the MDSC population while mapping novel means for stratifications of melanoma to therapy, and possibly new means for therapy of this tumor type. Citation Format: Marzia Scortegagna, Yuanning Du, Yongmei Feng, Ze'ev Ronai. Tumor suppressor function of the ubiquitin ligase Siah1a in melanoma is mediated by myeloid derived suppressor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6864.

[This corrects the article DOI: 10.3389/fphar.2022.1029093.].

Abstract The translation initiation factor 4F (eIF4F) complex assembly is a rate-limiting step in mRNA translation. eIF4F subunits including eIF4A, eIF4E, and eIF4G, are often upregulated in cancer and neurodegeneration diseases. Elevated eIF4F level/activity has been correlated with poor prognosis and drug resistance. Leveraging our findings with the small molecule SBI-756, which interacts with eIF4G1 and impairs eIF4F complex assembly, we set to map domains that are required for SBI-756 activity. A CRISPR screen using sgRNAs that target different sequences on eIF4G1 led to the identification of the MA3 domain, as a putative binding site for SBI-756. Deletion/mutation of the eIF4G1 MA3 domain attenuated melanoma cells and spheroids growth. Polysome profiling assays confirmed attenuated translation activity, which resembled those seen with SBI-756. In silico virtual screen identified 64 small molecules (out of >10 million) that interact with the MA3 domain. Of these, we have selected four that effectively attenuated melanoma growth in culture. Analogs developed for these four compounds were more potent in impairing the assembly of the eIF4F complex, inhibition of protein translation and the 2D and 3D growth of melanoma cells. RNA-sequencing analysis highlighted altered expression of genes implicated in apoptosis, UPR, cell cycle, and ROS pathways, leading us to test possible combination with pathways that may complement the above. Among those, autophagy inhibitors synergized with our lead compound, M19-6, resulting in efficient melanoma cell death, using notably lower concentrations of these inhibitors. Our findings identify the eIF4G1 MA3 domain as an important player in eIF4F assembly and a potential target for cancer therapy. Citation Format: Yongmei Feng, Mariia Radaeva, Hyungsoo Kim, Anagha Deshpande, Ani Deshpande, Predrag Jovanovic, Rabi Murad, Ivan Topisirovic, Steven Olson, Artem Cherkasov, Ze'ev Ronai. Targeting the translation initiation complex component eIF4G1 in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2085.

List of genes driving the enrichment score for HALLMARL_EMT in MPC3.shRNF185#3 compared to MPC3.pLKO in GSEA analysis.

Proteolysis-targeting chimera (PROTAC) and other targeted protein degradation (TPD) molecules that induce degradation by the ubiquitin-proteasome system (UPS) offer new opportunities to engage targets that remain challenging to be inhibited by conventional small molecules. One fundamental element in the degradation process is the E3 ligase. However, less than 2% amongst hundreds of E3 ligases in the human genome have been engaged in current studies in the TPD field, calling for the recruiting of additional ones to further enhance the therapeutic potential of TPD. To accelerate the development of PROTACs utilizing under-explored E3 ligases, we systematically characterize E3 ligases from seven different aspects, including chemical ligandability, expression patterns, protein-protein interactions (PPI), structure availability, functional essentiality, cellular location, and PPI interface by analyzing 30 large-scale data sets. Our analysis uncovers several E3 ligases as promising extant PROTACs. In total, combining confidence score, ligandability, expression pattern, and PPI, we identified 76 E3 ligases as PROTAC-interacting candidates. We develop a user-friendly and flexible web portal ( https://hanlaboratory.com/E3Atlas/ ) aimed at assisting researchers to rapidly identify E3 ligases with promising TPD activities against specifically desired targets, facilitating the development of these therapies in cancer and beyond.

Supplementary Figure 1-7 from Increased Expression of the E3 Ubiquitin Ligase RNF5 Is Associated with Decreased Survival in Breast Cancer

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Supplementary Figures S1-S11 from Preclinical Studies of Celastrol and Acetyl Isogambogic Acid in Melanoma

Supplementary Table S2: List of SPANX-interacting proteins identified by LC-MS/MS

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