Abstract

Pancreatic ductal adenocarcinoma (PDA) was responsible for ~ 44,000 deaths in the United States in 2018 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS1,2,3,4. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis5. However, paradoxically, inhibition of this pathway has provided no clinical benefit to patients with PDA6. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling elicits autophagy, a process of cellular recycling that protects PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, inhibition of MEK1/2 leads to activation of the LKB1→AMPK→ULK1 signaling axis, a key regulator of autophagy. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic anti-proliferative effects against PDA cell lines in vitro and promotes regression of xenografted patient-derived PDA tumors in mice. The observed effect of combination trametinib plus chloroquine was not restricted to PDA as other tumors, including patient-derived xenografts (PDX) of NRAS-mutated melanoma and BRAF-mutated colorectal cancer displayed similar responses. Finally, treatment of a patient with PDA with the combination of trametinib plus hydroxychloroquine resulted in a partial, but nonetheless striking disease response. These data suggest that this combination therapy may represent a novel strategy to target RAS-driven cancers.

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The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We dedicate this work to the memory of our patient and his family, whose courage continues to inspire us to further improve the diagnosis and treatment of pancreatic cancer. We thank the members of the McMahon Lab for their support, advice, guidance, comments and discussions during the course of this work. Additionally we thank I. Garrido-Laguna (HCI/Univ. of Utah), M. Tempero, E. Collisson and F. McCormick (U.C. San Francisco), E. White (Rutgers University), D. Tuveson (Cold Spring Harbor), and K. Olive (Columbia) for advice and guidance, J. Mulcahy-Levy and A. Thorburn (U.C. Denver) for inspiration, advice, guidance and reagents, H. Land (University of Rochester Medical Center) and K. Shokat (UCSF) for providing reagents, NCI Patient-Derived Models Repository for supplying the NCI-516677 NRAS-mutated melanoma PDX, K. Owings, D. Lum and the HCI Preclinical Research Resource for assistance with tumor xenografts and drug treatments and M. Silvis for assistance with drug dosing. C.K. & M.M. wish to acknowledge the collegiality of K. Bryant and C. Der (University of North Carolina, Chapel Hill) for ongoing discussions and for sharing data in advance of publication. M.M. acknowledges financial support from the National Cancer Institute (R01-CA176839, R01-CA131261 & P30-CA042014), the Pancreatic Cancer Collective, Melanoma Research Alliance, Five for the Fight, and the Huntsman Cancer Foundation. E.L.S. was supported in part by a Career Award for Medical Scientists from the Burroughs Wellcome Fund, a V Scholar Award, the Huntsman Cancer Foundation, and the NIH (R01CA212415). B.E.W. acknowledges support from the National Cancer Institute and DoD (U54CA224076 and W81XWH1410417). A.L.W. acknowledges support from the Huntsman Cancer Foundation. C.G.K acknowledges support from the Huntsman Cancer Foundation. A.M.B. acknowledges support from Fondation pour la Recherché Medicale (FDM20150633361) and Societe Francaise de Dermatologie.

Author information

Affiliations

  1. Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA

    • Conan G. Kinsey
    • , Soledad A. Camolotto
    • , Amelie M. Boespflug
    • , Katrin P. Gullien
    • , Mona Foth
    • , Amanda Truong
    • , Sophia S. Schuman
    • , Jeffrey T. Yap
    • , Lance D. Burrell
    • , David H. Lum
    • , Jonathan R. Whisenant
    • , G. Weldon Gilcrease III
    • , Courtney C. Cavalieri
    • , Kaitrin M. Rehbein
    • , Stephanie L. Cutler
    • , Kajsa E. Affolter
    • , Alana L. Welm
    • , Bryan E. Welm
    • , Courtney L. Scaife
    • , Eric L. Snyder
    •  & Martin McMahon
  2. Department of Internal Medicine, Division of Oncology, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Conan G. Kinsey
    • , Jonathan R. Whisenant
    •  & G. Weldon Gilcrease III
  3. Department of Dermatology, Centre Hospitalier Lyon-Sud, Pierre Benite, Cedex, France

    • Amelie M. Boespflug
  4. Cancer Research Center of Lyon, Claude Bernard Lyon-1 University, INSERM 1052, CNRS 5286, Villeurbanne, France

    • Amelie M. Boespflug
  5. Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Jill E. Shea
    • , Michael T. Seipp
    • , Bryan E. Welm
    •  & Courtney L. Scaife
  6. Department of Radiology and Imaging Services, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Jeffrey T. Yap
  7. Department of Pharmacy Services, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA

    • Courtney C. Cavalieri
  8. Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Kajsa E. Affolter
    •  & Eric L. Snyder
  9. Department of Oncological Sciences, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Alana L. Welm
  10. Department of Dermatology, University of Utah School of Medicine, Salt Lake City, UT, USA

    • Martin McMahon

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Contributions

C.G.K. and M.M designed all experiments. C.G.K., S.A.C., A.M.B., K.P.G., M.F., A.T. and S.S.S. performed in vitro and in vivo experiments and collected data. J.T.Y. and L.D.B. designed, performed and analyzed in vivo FDG-PET/MRI/CT imaging studies. J.E.S., M.T.S., D.H.L, A.L.W., and C.L.S established, maintained and provided PDX mouse models. B.E.W. and E.L.S supervised experiments performed by K.P.G. and S.A.C., respectively. C.G.K., J.R.W., G.W.G., C.C.C., K.M.R., and S.L.C. provided patient care. K.E.A. assisted with pathologic analysis of samples. C.G.K. and M.M. analyzed data, prepared the manuscript and guided the manuscript through review.

Competing interests

M.M. served on external advisory boards for Novartis (June 2017), Genentech (December 2017) and Merck (June 2018). M.M. is the recipient of research funding from Pfizer, through a grant peer-reviewed, co-funded and awarded by the Melanoma Research Alliance.

Corresponding author

Correspondence to Martin McMahon.

Extended data

  1. Extended Data Fig. 1 Flow cytometry analysis of autophagic flux reporter with autophagy inhibitors and inducers.

    a-e: Autophagic flux was assessed by flow cytometry in Mia-PaCa2AFR cells following 48 h treatment with control, chloroquine (CQ), SAR-405, temsirolimus, or trametinib. Experiments were repeated three times with similar results. f: Autophagic flux was assessed by fluorescent imaging in Mia-PaCa2AFR cells following 48 h treatment with control, chloroquine (CQ), VPS34i (SAR-405), or trametinib. Experiments were repeated three times with similar results.

  2. Extended Data Fig. 2 Inhibition of RAS→RAF→MEK→ERK signaling pathway induces autophagic flux (AF) as seen by p62 degradation and LC3 conversion in pancreatic cancer cells.

    a & b: Cell lysates prepared from Mia-PaCa2 (a) or BxPC3 (b) cells treated with 0.1–100 nM of trametinib for 48 h were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. c: Cell lysates prepared from Mia-PaCa2 cells treated with ARS-853 (KRASG12Ci), SCH772984 (ERKi), or cobimetinib (MEKi) for 48 h were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. d: Cell lysates prepared from Mia-PaCa2AFR cells transiently expressing exogenous ULK1WT, ULKM92A (dominant negative), AMPKWT, or AMPKK45R (dominant negative) were analyzed by immunoblotting for ULK1, AMPK, or actin as indicated. Experiments were repeated three times with similar results. e: Cell lysates prepared from Mia-PaCa2AFR cells lentivirally transduced with shRNAs targeting LKB1 or scrambled control were analyzed by immunoblotting for LKB1 or actin as indicated. Experiments were repeated three times with similar results. Source Data

  3. Extended Data Fig. 3 Trametinib and chloroquine are synergistically cytotoxic in vitro.

    Mia-PaCa2 cells, BxPC3 and PDX220 cells were treated for 48–96 as indicated with trametinib and chloroquine and analyzed for cell viability by ATPlite assay. Synergy scores were generated utilizing Combenefit Software. Experiments were repeated four times with similar results.

  4. Extended Data Fig. 4 Treatment of pancreatic tumors with trametinib and chloroquine results in decreased pERK and increased p62 abundance respectively.

    Representative images of immunohistochemical analysis of sections of PDX 227 tumors that were treated with 1. vehicle (Control), 2. trametinib; 3. chloroquine or; 4. the combination of both agents. Sections were stained with H&E or with antisera against pERK1/2 or p62 as indicated. Experiments were repeated four times with similar results. Scale bar is 500 μM located in the bottom right of the upper left panel and is consistent for all images.

  5. Extended Data Fig. 5 Treatment of orthopically xenografted pancreatic tumors with trametinib and hydroxychloroquine demonstrates regression consistent with subcutaneous xenografts.

    a: PDX220 tumors were orthotopically transplanted and after 3 weeks were imaged via FDG-PET/CT for baseline. They were then treated with trametinib, hydroxychloroquine or trametinib plus hydroxychloroquine for 2 weeks prior to re-imaging. n = 3 for control; n = 2 for HCQ; n = 3 for trametinib; n = 2 for trametinib + hydroxychloroquine. b & c: Quantification of total lesion glycolysis (b) and % change (c) for individual tumors within each treatment group.

  6. Extended Data Fig. 6 Regression of established NRAS driven melanoma tumors by combined inhibition of MEK1/2 plus chloroquine.

    a. The growth of an NRAS-mutated melanoma (NCI515677) PDX was assessed over 21 days in mice treated with: 1. vehicle (Control), 2. trametinib (1 mg/kg), 3. chloroquine (25 mg/kg) or; 4. the combination of both agents at the aforementioned doses as indicated. n = 4 for all treatment groups except combination of both agents n = 5. Center values are the mean; statistical testing was performed by two-sided t-test; ***p < 0.001 vs. control; tttp < 0.001 vs. trametinib. Error bars represent SD. b-e. The percentage weight change of HCI-Mel002 NRAS-mutated PDX was assessed over 21 days in mice treated with: b. vehicle (Control), c. trametinib (1 mg/kg), d. chloroquine (50 mg/kg) or; e. the combination of both agents at the aforementioned doses as indicated. However, side-effects of facial rash and hair loss were noted. Source Data

  7. Extended Data Fig. 7 Lack of autophagy induction by MEK1/2 inhibition results in resistance to combined trametinib and chloroquine treatment.

    a: Cell lysates prepared from two suitably manipulated KRASG12D/TP53Null mouse lung cancer-derived cell lines, SC196 or SC274 treated with 100 nM of trametinib were analyzed by immunoblotting for the phosphorylation (p) or total (t) abundance of ERK1/2, p62, LC3, or actin as indicated. Experiments were repeated three times with similar results. b: SC196 and SC274 KRASG12D/TP53Null mouse lung cancer cells were treated for 48 h respectively with trametinib and chloroquine and analyzed for cell viability by ATPlite assay. Synergy scores were generated utilizing Combenefit Software. Experiments were repeated four times with similar results. c & d: Autophagic flux was measured in SC196AFR (c) or SC274AFR (d) following treatment with 0.1–1000 nM trametinib for 48 h. n = 3; center values are the mean; statistical testing was performed by two-sided t-test of control high (red) versus experimental high; ***p < 0.001 vs. control. Error bars represent SD. e & f: The growth of SC196 (e) or SC274 (f) KRASG12D/TP53Null mouse lung cancer derived tumors in xenografted mice treated with: 1. vehicle (Control), 2. trametinib (1 mg/kg), 3. chloroquine (50 mg/kg) or; 4. the combination of both agents was assessed over ~ 15 days as indicated. n = 10 for all treatment groups. Center values are the mean; statistical testing was performed by two-sided t-test;***p < 0.001 vs. control; tttp < 0.001 vs. trametinib. Error bars represent SD. Source Data Source Data

Supplementary information

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https://doi.org/10.1038/s41591-019-0367-9