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RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging

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Abstract

Purpose

Macropinocytosis serves as a highly conserved endocytotic process that has recently been shown as a critical mechanism by which RAS-transformed cells transport extracellular protein into intracellular amino acid pathways to support their unique metabolic needs. We developed NIR fluorescently labeled molecular imaging probes to monitor macropinocytosis-mediated uptake of albumin in a K-RAS-dependent manner.

Procedures

Using western blot analysis, immunofluorescence, and flow cytometry, albumin retention was characterized in vitro across several RAS-activated lung and pancreatic cancer cell lines. AF790-albumin was synthesized and administered to mice bearing K-RAS mutant xenograft tumors of H460 (K-RAS p.Q61H) and H358 (K-RAS p.G12C) non-small cell lung cancers on each flank. Mice were treated daily with 2 mg/kg of ARS-1620, a targeted RAS p.G12C inhibitor, for 2 days and imaged following each treatment. Subsequently, the mice were then treated daily with 10 mg/kg of amiloride, a general inhibitor of macropinocytosis, for 2 days and imaged. Intratumoral distribution of AF790-albumin was assessed in vivo using near-infrared (NIR) fluorescence imaging.

Results

Albumin retention was observed as a function of K-RAS activity and macropinocytosis across several lung and pancreatic cancer cell lines. We documented that ARS-1620-induced inhibition of K-RAS activity or amiloride-mediated inhibition of macropinocytosis significantly reduced albumin uptake. Tumor retention in vivo of AF790-albumin was both RAS inhibition-dependent as well as abrogated by inhibition of macropinocytosis.

Conclusions

These data provide a novel approach using NIR-labeled human serum albumin to identify and monitor RAS-driven tumors as well as evaluate the on-target efficacy in vivo of inhibitors, such as ARS-1620.

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Acknowledgements

The authors would like to thank members of the D. Piwnica-Worms laboratory for discussion and suggestions and the Small Animal Imaging Facility of MD Anderson Cancer Center for animal imaging assistance, especially Charles Kingsley and Jorge Delacerda. The Biostatistics Resource, Flow Cytometry Core Facility, and Small Animal Imaging Facility obtain support from NCI Cancer Center Support Grant P30 CA016672.

This work was supported by NIH Grant P50 CA94056 to the MD Anderson Cancer Center–Washington University Inter-Institutional Molecular Imaging Center; the Gerald Dewey Dodd, Jr., Endowed Distinguished Chair of the University of Texas MD Anderson Cancer Center; and a generous gift from the Estate of Barbara Cox Anthony/Koch Foundation. M.N.S. was supported by a Ruth L. Kirchstein Postdoctoral Individual Research Service Award (F32) NCI F32CA250323, the Harold C. and Mary L. Dailey Endowed Fellowship Recognition of Research Excellence (M.D. Anderson Cancer Center), and the Diane Denson Tobola Endowed Fellowship in Ovarian Cancer Research (M.D. Anderson Cancer Center).

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Authors and Affiliations

  1. Department of Cancer Systems Imaging, The University of Texas, M. D. Anderson Cancer Center, Unit 1907, 1515 Holcombe Blvd., Houston, TX, 77030, USA

    Margie N. Sutton, Seth T. Gammon, Riccardo Muzzioli, Federica Pisaneschi, Bhasker Radaram, Ping Yang & David Piwnica-Worms

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  1. Margie N. Sutton
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  2. Seth T. Gammon
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  3. Riccardo Muzzioli
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  4. Federica Pisaneschi
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  5. Bhasker Radaram
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  6. Ping Yang
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  7. David Piwnica-Worms
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Contributions

M.N.S. and D.P.W. designed the study. M.N.S., S.T.G., R.M., F.P., B.R., and P.Y. carried out experiments. D.P.W. provided resources and reagents. M.N.S., S.T.G., and D.P.W. analyzed the data, and M.N.S. and D.P.W. wrote the manuscript. All authors edited and approved the manuscript.

Corresponding author

Correspondence to David Piwnica-Worms.

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Sutton, M.N., Gammon, S.T., Muzzioli, R. et al. RAS-Driven Macropinocytosis of Albumin or Dextran Reveals Mutation-Specific Target Engagement of RAS p.G12C Inhibitor ARS-1620 by NIR-Fluorescence Imaging. Mol Imaging Biol 24, 498–509 (2022). https://doi.org/10.1007/s11307-021-01689-8

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  • DOI: https://doi.org/10.1007/s11307-021-01689-8

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