Biology subjects

Onken, M. D.

Publications and source records attributed to Onken, M. D..

5 recordsLinked to original sources

bioRxiv2026

The Alzheimer's Disease-associated gene CD2AP regulates permeability of endothelial cells and transendothelial migration of immune cells

Variants at the CD2AP locus, which encodes a branched-actin regulator, are associated with late-onset Alzheimer's disease (AD), and CD2AP-deficient mice show increased blood-brain barrier (BBB) permeability. CD2AP is enriched in endothelial cells, but its function there is poorly understood. In endothelium, branched actin networks maintain cell-cell junctions and are also required for immune cells to cross through individual endothelial cells (transcellular transendothelial migration, TEM). We previously showed that the Arp2/3 activators N-WASP and WAVE2 contribute to these processes, but the capping protein (CP)-interacting proteins involved are unknown. Here we tested whether CD2AP, a CP-interacting protein recruited to cell-cell contacts, regulates both junction integrity and TEM. In primary human microvascular endothelial cells, shRNA knockdown of CD2AP increased TNF-induced monolayer permeability, measured by transendothelial electrical resistance, and altered VE-cadherin junction morphology. Unexpectedly, on soft hydrogels the same knockdown reduced TEM of NK-92 MI cells, lowering the proportion of transcellular events. Thus, loss of CD2AP weakens the endothelial barrier yet impairs a distinct, actin-dependent route of immune-cell passage. These findings suggest that CD2AP risk variants may affect the BBB through more than permeability, and they identify endothelial immune-cell trafficking as a testable link between CD2AP and AD.

cell biology↗

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↗

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↗

bioRxiv2020

Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium

Uveal melanomas (UM) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells, notably transendothelial migration (TEM), is a key step in formation of metastases. UM cells execute TEM via a stepwise process of intercalation into the endothelial monolayer involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations in Gq/11, which activate TRIO, a guanine nucleotide exchange factor (GEF) for RhoA and Rac1. Pharmacologic inhibition of Gq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 also inhibited mesenchymal motility, but invasion was less affected; in this case, the amoeboid blebbing behavior of the cells led to transmigration without intercalation, a direct mechanism similar to that of immune cells. BAP1-deficient (+/-) UM subclones displayed motility behavior and increased levels of TEM, similar to effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.

cell biology↗