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Cutaneous melanoma is a highly invasive skin cancer. Mortality for melanoma is low if caught before metastasis occurs, but is poor if metastasis has occurred at diagnosis. Melanomas are among the most mutated cancer genomes and have a high neoantigen load, which is related to better response to immunotherapies. A subset of melanomas contains mutations in BRAF(V600E) and respond initially to BRAF inhibitors, though they become resistant through escape pathways. Focused development of BRAF and immunotherapies has resulted in the clinical use of targeted therapies against the oncogenic BRAF/MEK/ERK pathway and immune checkpoint inhibitors for the treatment of metastatic melanoma. Although some patients with metastatic melanoma benefit from these therapies, others either do not respond or become resistant. Because of this, new targets and drugs are needed for patients who are either intrinsically resistant or have acquired resistance to targeted therapies and immunotherapies. RSKs are serine/threonine kinases downstream of BRAF/ERK that activate transcription, proliferation and cell survival. Thus RSKs are potential new targets and the availability of a RSK inhibitor called PMD-026, now in Phase 1b clinical trials for breast cancer, provides a tool to explore this possibility in pre-clinical models of melanoma. PMD-026 is an ATP-competitive inhibitor of the N-terminal kinase domain. This inhibitor is highly stable and effective in cell-based assays with an average IC50 of 2nM and 200-400 nM in soft agar and inhibits tumor growth in multiple mouse models of breast cancer given 70-100 mg/kg given orally. It is currently the only RSK inhibitor that can be given to mice orally with demonstrated in vivo activity. To explore the opportunity for RSK inhibitors in melanoma, we screened 1,959 PDx models for RSK1-4 expression, which was evaluated based on RNAseq. In melanoma, RSK1 and RSK2 were highly expressed, unlike RSK3 and RSK4. In a study of 21 cases of melanoma, RSK2 was activated in 52% of the cases based on nuclear localization using our companion diagnostic. Using FRET based assays, PMD-026 inhibited RSK2 binding with an IC50=14 nM compared to BID1870 with IC50=94 nM demonstrating competitive in vitro potency. Importantly, PMD-026 inhibits viability of melanoma cells including those that are resistant to the BRAF inhibitor. Surprisingly, we also found that it significantly reduces PD-L1 expression at the cell surface of several melanoma lines. This is through down-regulation of PD-L1 transcription and subsequent reduced PD-L1 cell surface expression. As expected, in our melanoma models, PMD-026 inhibited RSK signaling through pYB-1, a transcription factor known to bind to the PD-L1 promoter. Testing in pre-clinical mouse models is in progress. We propose that PMD-026 may therefore potentiate melanoma immunotherapy approaches and also improve response of patients to BRAFV600E targeted therapies. Citation Format: Joe William Ramos, Stephanie Si, Takeo Fujii, Brien Haun, Won Seok Yang, Aarthi Jayanthan, Sandra E. Dunn. Targeting B-RAF and checkpoint inhibitor resistant melanoma with RSK inhibitor PMD-026 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1218.

Metastasis, the spreading of cancer cells from a primary tumor to secondary sites throughout the body, is the primary cause of death for patients with cancer. New therapies that prevent invasion and metastasis in combination with current treatments could therefore significantly reduce cancer recurrence and morbidity. Metastasis is driven by altered signaling pathways that induce changes in cell–cell adhesion, the cytoskeleton, integrin function, protease expression, epithelial-to-mesenchymal transition and cell survival. The ribosomal S6 kinase (RSK) family of kinases is a group of extracellular signal–regulated kinase/mitogen-activated protein kinase (ERK/MAPK) effectors that can regulate these steps of metastasis by phosphorylating both nuclear and cytoplasmic targets. However, our understanding of RSK function in metastasis remains incomplete and is complicated by the fact that the four RSK isoforms perform nonredundant, sometimes opposing functions. Although some isoforms promote cell motility and invasion by altering transcription and integrin activity, others impair cell motility and invasion through effects on the actin cytoskeleton. The mechanism of RSK action depends both on the isoform and the cancer type. However, despite the variance in RSK-mediated outcomes, chemical inhibition of this group of kinases has proven effective in blocking invasion and metastasis of several solid tumors in preclinical models. RSKs are therefore a promising drug target for antimetastatic cancer treatments that could supplement and improve current therapeutic approaches. This review highlights contradiction and agreement in the current data on the function of RSK isoforms in metastasis and suggests ways forward in developing RSK inhibitors as new antimetastasis drugs. Cancer Res; 73(20); 6099–105. ©2013 AACR.

Malignant gliomas are one of the most aggressive and deadly forms of cancer and can affect any age group. In glioblastoma multiforme (GBM), infiltration of primary tumor cells into the normal tissue and dispersal throughout the brain is a central challenge to successful treatment that remains unmet. Patients with malignant gliomas respond poorly to the standard therapeutic regimen of radiotherapy and chemotherapy that follow tumor resection and have only a 16-month median survival. It is therefore imperative to identify new approaches to specifically attack GBM cell survival, proliferation and invasion. The cellular mechanisms driving GBM-mediated migration and invasion are not fully understood. RSK2 (p90 ribosomal S6 kinase 2) is a kinase that regulates proliferation and adhesion and can promote metastasis. We find that RSK2 is significantly upregulated in vivo in human GBM patient tumors, and that high RSK2 expression significantly correlates with advanced tumor stage and poor patient survival. We demonstrate that active RSK2 regulates GBM adhesion and is essential for cell motility and invasion of patient-derived GBM neurospheres. Importantly, inhibition of RSK2 by either RSK inhibitors or shRNA silencing impairs invasion and combining RSK2 inhibitors with temozolomide improves efficacy in vitro. We further show that the effects of RSK2 on GBM invasion are mediated in part through activation of Rho GTPases. Rho family of GTPases are key regulators of cell polarity, migration and invasion. Here, we identify Rho A, B and C as downstream effectors of (RSK2) in GBM cell migration and invasion. Here, we identified a unique signaling pathway in which RSK2 confers invasiveness through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 and Cdc42. Our data support a fundamental importance of residue of Thr577 to the invasive signaling of RSK2. The invasive signaling of RSK2 appears to depend on RhoA and B, but not RhoC. RSK2 activates RhoA through activation of LARG by phosphorylation at Ser1288. Intriguingly, LARG activity is only required for RSK2-T577E, but not RSK2-Y707A-mediated RhoA activation and cell invasion. This further supports a pivotal role of Thr577 in invasive RSK2 signaling. These results establish a unique RSK2-dependent LARG-Rho signaling cascade as an uncompensated factor key to GBM cell migration and invasion. Together, our data provide strong evidence that RSK inhibitors could enhance the effectiveness of existing GBM treatment, and support RSK2 targeting as a promising approach for novel GBM therapy. Citation Format: Geng-Xian Shi, Ling Jin, Michelle L. Matter, Santosh Kesari, Joe W. Ramos. RSK2 provokes invasive signaling in glioblastoma through LARG-dependent activation of Rho GTPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1363. doi:10.1158/1538-7445.AM2017-1363

Glioblastoma (GBM) is associated with high mortality in patients due in large part to the highly migratory and invasive nature of glioma cells. The 90kDa ribosomal S6 Kinases (RSKs) are a family of serine/theronine kinases that are an integral component of the extracellular signal regulated kinase (ERK)/ mitogen activated protein kinase (MAPK) pathway, which is highly active in many types of cancer. The RSK family contains four human isoforms (RSK1-4) that regulate a variety of cellular processes including cell proliferation, survival, and motility, however a role for individual RSK isoforms in GBM cell invasion has not clearly been defined. Here, we analyze the roles of RSK1-4 in glioblastoma cell invasion. Using transwell assays and a novel three-dimensional tumor spheroid assay, we found that the highly specific RSK inhibitor, BI-D1870, decreases glioblastoma cell motility and invasion. To identify which RSK isoform was associated with this decrease in invasion, we knocked down each RSK isoform using lentiviral mediated shRNA transduction and found that different RSK isoforms could decrease invasion depending on the cell line, without significantly affecting proliferation. Additionally, RSK2 knockdown in primary patient-derived glioblastoma cells decreased cell migration, and Kaplan-Meier analysis revealed that patients overexpressing RSK2 had a poorer prognosis compared to those expressing normal levels of RSK2. Our studies support a differential role for individual RSKs in glioblastoma invasion, suggesting that targeting RSKs may provide an effective means for the treatment of glioblastoma. Citation Format: Amanda Prechtl, Florian Sulzmaier, Pengfei Jiang, Santosh Kesari, Joe Ramos. RSK isoform regulation of migration and invasion in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5007. doi:10.1158/1538-7445.AM2014-5007