Biology subjects

Blumer, K. J.

Publications and source records attributed to Blumer, K. J..

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↗

bioRxiv2024

Examination of INPP5A in uveal melanoma uncovers novel calcium transients

Uveal melanoma is a highly aggressive intraocular cancer that metastasizes in about half of patients whereupon it is inexorably fatal. Uveal melanomas (UM) are distinct from other melanomas because they are driven by constitutively activating mutation in the heterotrimeric G protein alpha subunits Gq (GNAQ) and G11 (GNA11). This results in constitutive production of inositol trisphosphate (IP3) by phospholipase C-beta downstream of Gq/11. In normal cells, increased IP3 causes calcium release from the endoplasmic reticulum, which would be cytotoxic if maintained chronically, but UM cells are able to survive constitutive IP3 production. INPP5A, which dephosphorylates and thus inactivates IP3, is highly upregulated in UM cells compared to other melanomas and another study has shown that INPP5A is necessary for UM cell survival. To understand the mechanism of calcium regulation in response to IP3, we collected single-cell calcium measurements and found that UM cells driven by constitutively active Gq/11 produce spontaneous calcium transients. These calcium oscillations are not seen in any other melanoma cell lines unless induced by an agonist, but they are present in patient UM tumor samples. Moreover, these calcium oscillations are lost in UM cells treated with the Gq/11 inhibitor FR900359, demonstrating their dependence on constitutive Gq/11 activity. We found that the INPP5A inhibitor YU144369 causes significant changes in calcium oscillations in UM cells, demonstrating a role for INPP5A in this system. INPP5A is tethered to membranes by C-terminal prenylation and palmitoylation, suggesting that localization may play a role in INPP5A regulation of IP3 levels. GFP-tagged INPP5A was localized to plasma membrane, nuclear envelop, endoplasmic reticulum, and lysosomes. Mutation of the palmitoylation site significantly reduced localization to the plasma membrane, while mutation of the prenylation site resulted in purely nucleoplasmic localization of INPP5A. These results demonstrate a role of palmitoylation in the regulation of INPP5A localization and mobilization in UM cells.

cancer biology↗

bioRxiv2023

G protein activation occurs via a largely universal mechanism

Understanding how signaling proteins like G proteins are allosterically activated is a long-standing challenge with significant biological and medical implications. Because it is difficult to directly observe such dynamic processes, much of our understanding is based on inferences from a limited number of static snapshots of relevant protein structures, mutagenesis data, and patterns of sequence conservation. Here, we use computer simulations to directly interrogate allosteric coupling in six G protein -subunit isoforms covering all four G protein families. To analyze this data, we introduce automated methods for inferring allosteric networks from simulation data and assessing how allostery is conserved or diverged among related protein isoforms. We find that the allosteric networks in these six G protein -subunits are largely conserved and consist of two pathways, which we call pathway-I and pathway-II. This analysis predicts that pathway-I is generally dominant over the second pathway, which we experimentally confirmed by showing that mutations to pathway-I perturb nucleotide exchange more than mutations to pathway-II. In the future, insights into unique elements of each G protein family could inform the design of isoform-specific drugs. More broadly, our tools should also be useful for studying allostery in other proteins and assessing the extent to which this allostery is conserved in related proteins.

biophysics↗

bioRxiv2021

Targeting primary and metastatic uveal melanoma with a G protein inhibitor

Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models, and reduced blood pressure transiently. FR did not regress xenografted UM tumors, or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM, and potentially other diseases caused by constitutively active Gq/11.

cancer biology↗

bioRxiv2018

Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding

Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ~30 [A] apart, remains elusive. We construct an atomically-detailed model of G protein activation by combining three powerful computational methods; metadynamics, Markov state models (MSMs), and CARDS analysis of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochemical data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. {beta}-Strands 1-3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examining unbinding processes with slow off-rates.

biophysics↗