Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance

Directly probing the mechanical properties of the spindle and its matrix

The spindle matrix does not make a significant mechanical contribution to metaphase spindle length.Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

The product of the von Hippel-Lindau tumor suppressor gene stabilizes microtubules by inhibiting GTPase activity

Directly probing the mechanical properties of the spindle and its matrix

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 188 (2010): 481-489, doi:10.1083/jcb.200907110.Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.This work was supported by National Institute of General Medicine grants to J.C. Gatlin (F32GM080049) and E.D. Salmon (GM24364). T.J. Mitchison was funded by a grant from the National Cancer Institute (CA078048-09)

GSK3β phosphorylation modulates CLASP–microtubule association and lamella microtubule attachment

Polarity of the microtubule (MT) cytoskeleton is essential for many cell functions. Cytoplasmic linker-associated proteins (CLASPs) are MT-associated proteins thought to organize intracellular MTs and display a unique spatiotemporal regulation. In migrating epithelial cells, CLASPs track MT plus ends in the cell body but bind along MTs in the lamella. In this study, we demonstrate that glycogen synthase kinase 3beta (GSK3beta) directly phosphorylates CLASPs at multiple sites in the domain required for MT plus end tracking. Although complete phosphorylation disrupts both plus end tracking and association along lamella MTs, we show that partial phosphorylation of the identified GSK3beta motifs determines whether CLASPs track plus ends or associate along MTs. In addition, we find that expression of constitutively active GSK3beta destabilizes lamella MTs by disrupting lateral MT interactions with the cell cortex. GSK3beta-induced lamella MT destabilization was partially rescued by expression of CLASP2 with mutated phosphorylation sites. This indicates that CLASP-mediated stabilization of peripheral MTs, which likely occurs in the vicinity of focal adhesions, may be regulated by local GSK3beta inactivation

Abstract 3673: High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes

Abstract Exosomes play a crucial role in the progression of cancer through the transport of a variety molecular cargo, including proteins, lipids, and nucleic acids, to and from cells as a means of intercellular communication. Unraveling mechanisms of exosome-cell interactions may open avenues for studying cellular communication and lead to new therapies. Cellular exosome uptake depends on cholesterol-rich membrane microdomains called lipid rafts. Non-specific depletion of lipid raft cholesterol reduces cellular exosome uptake; however, to our knowledge, no targeted mechanism of inhibiting cellular exosome uptake has been reported. Scavenger receptor type B-1 (SR-B1) localizes to lipid rafts, and is a high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL). SR-B1 is an intriguing therapeutic target because it is upregulated in many different cancers due to the high need for cholesterol of rapidly dividing cancer cells. Therefore, we hypothesized that specific targeting of SR-B1 and modulation of cholesterol flux through this receptor with biomimetic HDL-like nanoparticles (HDL NPs) would disrupt cellular exosome uptake. As a model, we explored exosomes derived from melanoma cells as they have been shown to promote angiogenesis and immunosuppression both crucial events in melanoma progression. Melanoma exosomes have also been shown to actively prepare metastatic sites, creating a suitable microenvironment allowing for the development of metastasis. Because of this, targeting exosomes and intercellular signaling could be beneficial for the treatment of metastatic melanoma. Using a variety of techniques including confocal microscopy, flow cytometry and automated image analysis, data demonstrate that HDL NPs specifically target SR-B1 in lipid rafts in melanoma cells and modulate cholesterol flux through this receptor. This leads to a clustering of SR-B1 at the cell membrane and potent inhibition of the cellular uptake of melanoma cell-derived exosomes. Citation Format: Michael P. Plebanek, Alexandre Matov, Kannan Mautharasan, Jesse Gatlin, C. Shad Thaxton. High-density lipoprotein-like nanoparticles target SR-B1 and inhibit the cellular uptake of melanoma-cell derived exosomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3673. doi:10.1158/1538-7445.AM2015-3673</jats:p