Biology subjects

Riordan, J. D.

Publications and source records attributed to Riordan, J. D..

5 recordsLinked to original sources

bioRxiv2026

A functional genomics screen identifies novel drivers of FR900359 resistance in uveal melanoma cells

Uveal melanoma (UM) is the most common form of intraocular cancer in adults and has a median survival rate of [~]1 year after metastasis occurs. Metastatic UM is largely refractory to treatment and there are no effective pharmacological therapies, resulting in poor overall survival. Activating mutations in GNAQ and GNA11 proteins (GNAQ/11) are the oncogenic initiators in >90% UM cases. While there are no targeted therapies yet identified for the GNAQ/11 oncoproteins, a natural compound called FR900359 (FR) is a selective inhibitor for both oncogenic and wild type GNAQ/11. We performed a functional genomics screen to identify drivers of FR resistance in two UM cell lines (92.1 and MEL202). The screen identified eleven genes as candidate FR resistance drivers in both cell lines. Over-expression of five of these genes (ABCB1, PLCB4, GRM1, PLCE1, PDGFRB) was predicted to provide resistance to FR treatment. Enforced expression of ABCB1 or PLCB4 did not provide immediate resistance to FR, although over-expression of either transgene led to the emergence of resistant colonies at a much higher rate than occurs spontaneously in parental cells. We show that a relatively small fraction of UM cells can tolerate the initial over-expression of PLCB4 and ABCB1, but FR treatment leads to expansion of this cell population. Expression of an ABCB1-tGFP fusion protein was used to isolate drug naive UM cells. We show that these cells are uniformly resistant to FR, unlike the bulk tumor cell population. Finally, additional experiment of the drug naive ABCB1-tGFP+ UM cells led to the observation that these cells exhibit a significantly lower rate of protein translation, like BAP1-deficient UM cells. These findings suggest that resistance to targeted GNAQ/11 inhibitors is dictated by interaction between acquired genetic alterations and epigenetic states within heterogenous UM cell populations.

cancer biology↗

bioRxiv2025

Co-targeting the PI3K-Akt pathway improves response to MEK inhibition in low-grade serous ovarian cancer cell lines

Low grade serous ovarian cancer (LGSOC) accounts for [~]5% of all ovarian cancers, with >1,000 new cases diagnosed each year in the United States. Currently, there is no established standard-of-care treatment for this subtype, and chemotherapy response rates are only [~]4%. One defining characteristic of LGSOC is a high prevalence of mutations within the MAPK pathway, which has motivated studies using MEK inhibitors to treat this disease. A recent phase II/III clinical trial using trametinib to treat patients with recurrent LGSOC improved the response rate to 26%; however, all patients eventually progressed due to the emergence of therapeutic resistance. Ongoing clinical trials are investigating combined inhibition of MEK and FAK with avutometinib plus defactinib for LGSOC patients, with preliminary results demonstrating response rates near 50%. While these results are highly encouraging, a significant subset of patients still fails to respond to existing targeted treatments. Additionally, development of therapeutic resistance in initial responders remains a major obstacle. Here, we sought to characterize molecular responses of LGSOC to MEK inhibition, both acutely and during the development of spontaneous drug resistance, with a goal of identifying adaptive mechanisms LGSOC cells utilize to withstand MAPK inhibition. Our research identified significant upregulation of the PI3K-Akt pathway in response to MEK inhibition. While targeted inhibition of AKT had minimal impact on its own, combination with MEK inhibitors produced strong synergistic suppression of proliferation in LGSOC cells. This combination strategy could potentially be used to prevent or reverse the emergence of MEK inhibitor resistance in LGSOC patients.

cancer biology↗

bioRxiv2024

A critical role of FAK signaling in Rac1-driven melanoma cell resistance to MAPK pathway inhibition

The Rac1 P29S hotspot mutation in cutaneous melanoma is associated with resistance to MAPK pathway inhibitors (MAPKi) and worse clinical outcomes. Moreover, activation of Rac1 guanine exchange factors (GEFs) also promotes MAPKi-resistance, particularly in undifferentiated melanoma cells. Here we delineate mechanisms of Rac1-driven MAPKi-resistance and identify strategies to inhibit the growth of this class of cutaneous melanomas. We find that Rac1-driven melanomas manifest pleiotropic resistance mechanisms including (i) reduced dependence on BRAF/MEK, (ii) activation of alternative MAPK pathways utilizing Jun kinase and p38 MAP kinase, and (iii) a partial reliance on YAP/TAZ signaling. Importantly, although Rac1-driven melanoma cells display reduced dependence on BRAF/MEK, they are not completely ERK-independent. Additionally, the presence of activated Rac1 appears to create a dependency on focal adhesion kinase (FAK) signaling in undifferentiated melanoma cells. Therefore, despite the pleiotropic mechanisms of Rac1-driven MAPKi resistance, we find that combined inhibition of RAF and MEK with the RAF/MEK clamp auvotometinib and FAK with the FAK inhibitor defactinib is a promising approach for suppressing the growth of Rac1-driven melanoma cells. Thus, the avutometinib plus defactinib combination, which is currently being investigated for brain metastatic cutaneous melanoma may also have utility against Rac1-driven MAPKi-resistance in heavily pre-treated, advanced disease.

cancer biology↗

bioRxiv2019

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the Sleeping Beauty transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

genomics↗

bioRxiv2019

Src-dependent DBL family members drive resistance to vemurafenib in human melanoma

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/Rac1/Pak signaling axis capable of driving resistance to both current and next-generation BRAF inhibitors. However, we show that the Src inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance.

cancer biology↗