Biology subjects

Siruvallur Murali, V.

Publications and source records attributed to Siruvallur Murali, V..

5 recordsLinked to original sources

bioRxiv2026

Modulation of Oncogenic KRAS Signaling by Branched Actin-driven Cell Membrane Protrusions

For over three decades, we have known that oncogenic RAS alters the actin cytoskeleton organization and cell surface morphology1,2. RAS activates the GTPase RAC1, which triggers the growth of branched actin networks to promote cell membrane protrusions3,4. In melanoma, the hyperactive RAC1 mutant, Rac1P29S, was recently shown to drive extended lamellipodia, which then empower cell proliferation through sequestration and localized inhibition of the merlin tumor suppressor5. This discovery illustrates cell morphological programs not only as outputs but also as regulators of human oncogenic signals. Hence, we wondered whether the pronounced branched actin-driven membrane protrusions (BAMPs) downstream of oncogenic RAS are not mere outputs of RAS signaling but rather an active component in mediating the oncogenic penetrance of RAS mutants. We used volumetric light sheet microscopy and biochemical approaches to investigate the role of BAMPs in regulating the molecular signaling of oncogenic KRAS in pancreatic and lung cancer models. We found that elevated BAMP formation regulated the interaction of oncogenic KRAS with downstream effectors, specifically with the RAC1 GEF TIAM1. This implies that BAMPs amplify their own upstream regulators in a positive feedback. This meritorious cycle upregulates cyclin D1 expression by inactivating the merlin tumor suppressor, independently of the mitogen activated protein kinase pathway (MAPK). In the absence of BAMPs, cells carrying oncogenic KRAS mutations are unable to attain their full penetrance in proliferation. Overall, this work unveils the long-overlooked role of branched actin-driven cell morphology in the functionalization of KRAS mutants as potent oncogenes.

cancer biology↗

bioRxiv2024

RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells.

Cutaneous melanomas harboring a B-RafV600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeletons role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.

cancer biology↗

bioRxiv2021

Cancer drug discovery as a low rank tensor completion problem

The heterogeneity of cancer necessitates developing a multitude of targeted therapies. We propose the view that cancer drug discovery is a low rank tensor completion problem. We implement this vision by using heterogeneous public data to construct a tensor of drug-target-disease associations. We show the validity of this approach computationally by simulations, and experimentally by testing drug candidates. Specifically, we show that a novel drug candidate, SU11652, controls melanoma tumor growth, including BRAFWT melanoma. Independently, we show that another molecule, TC-E 5008, controls tumor proliferation on ex vivo ER+ human breast cancer. Most importantly, we identify these chemicals with only a few computationally selected experiments as opposed to brute-force screens. The efficiency of our approach enables use of ex vivo human tumor assays as a primary screening tool. We provide a web server, the Cancer Vulnerability Explorer (accessible at https://cavu.biohpc.swmed.edu), to facilitate the use of our methodology.

cancer biology↗

bioRxiv2018

Hyperactive Rac1 drives MAPK-independent proliferation in melanoma by assembly of a mechanosensitive dendritic actin network

AbstractCancer cells use a variety of mechanisms to subvert growth regulation and overcome environmental challenges. Often, these same mechanisms enable cancer cells to also develop resistance to targeted therapies. Here, we describe how a hyperactivating mutation of the Rac1 GTPase (Rac1P29S) harnesses Rac1s role as a regulator of actin polymer assembly to sustain cell cycle progression in growth limiting conditions. This proliferative advantage supports metastatic colonization of melanoma cells and confers insensitivity to inhibitors of the mitogen-activated protein kinase (MAPK) pathway, a frequent target for melanoma treatment. Rac1P29S bypasses the MAPK axis through a mechanism that necessitates cell-matrix attachment, however, does not depend on integrin-mediated focal adhesion assembly and focal adhesion kinase signaling. Even without involvement of canonical adhesion signaling, cells carrying the Rac1P29S mutation show elevated traction upon drug treatment and require mechanical resistance from their surrounding matrix to gain a proliferative advantage. We describe an alternative arm for cell mechanosensing, whereby actin polymerization against a matrix of minimal rigidity organizes biochemical cues to drive proliferative signals. Hyperactivation of Rac1 by the P29S mutation channels this pathway in melanoma through Arp 2/3-dependent formation of a constrained actin brush network that results in the inactivation of tumor suppressor NF2/Merlin. These data suggest an alternative mechanism for mechanosensitive growth regulation that can be hijacked by cancer cells to circumvent the adverse conditions of foreign microenvironments or drug treatment.

cell biology↗

bioRxiv2018

Robust high content assessment of heterogeneous drug effects in 3D microenvironments

BackgroundEvery biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drugs screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput. MethodsHere we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput. ResultsWe demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. ConclusionIn summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.

cancer biology↗