FDG-PET is a good biomarker of both early response and acquired resistance in BRAFV600 mutant melanomas treated with vemurafenib and the MEK inhibitor GDC-0973

Andreas R Baudy, Taner Dogan, Judith E Flores-Mercado, Klaus P Hoeflich, Fei Su, Nicholas van Bruggen, Simon-Peter Williams, Andreas R Baudy, Taner Dogan, Judith E Flores-Mercado, Klaus P Hoeflich, Fei Su, Nicholas van Bruggen, Simon-Peter Williams

Abstract

Background: The BRAF inhibitor, vemurafenib, has recently been approved for the treatment of metastatic melanoma in patients harboring BRAFV600 mutations. Currently, dual BRAF and MEK inhibition are ongoing in clinical trials with the goal of overcoming the acquired resistance that has unfortunately developed in some vemurafenib patients. FDG-PET measures of metabolic activity are increasingly employed as a pharmacodynamic biomarker for guiding single-agent or combination therapies by gauging initial drug response and monitoring disease progression. However, since tumors are inherently heterogeneous, investigating the effects of BRAF and MEK inhibition on FDG uptake in a panel of different melanomas could help interpret imaging outcomes.

Methods: 18 F-FDG uptake was measured in vitro in cells with wild-type and mutant (V600) BRAF, and in melanoma cells with an acquired resistance to vemurafenib. We treated the cells with vemurafenib alone or in combination with MEK inhibitor GDC-0973. PET imaging was used in mice to measure FDG uptake in A375 melanoma xenografts and in A375 R1, a vemurafenib-resistant derivative. Histological and biochemical studies of glucose transporters, the MAPK and glycolytic pathways were also undertaken.

Results: We demonstrate that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous BRAFV600 but ineffective in cells with acquired resistance or having WT BRAF status. However, combination with GDC-0973 results in a highly significant increase of efficacy and inhibition of FDG uptake across all twenty lines. Drug-induced changes in FDG uptake were associated with altered levels of membrane GLUT-1, and cell lines harboring RAS mutations displayed enhanced FDG uptake upon exposure to vemurafenib. Interestingly, we found that vemurafenib treatment in mice bearing drug-resistant A375 xenografts also induced increased FDG tumor uptake, accompanied by increases in Hif-1α, Sp1 and Ksr protein levels. Vemurafenib and GDC-0973 combination efficacy was associated with decreased levels of hexokinase II, c-RAF, Ksr and p-MEK protein.

Conclusions: We have demonstrated that 18 F-FDG-PET imaging reflects vemurafenib and GDC-0973 action across a wide range of metastatic melanomas. A delayed post-treatment increase in tumor FDG uptake should be considered carefully as it may well be an indication of acquired drug resistance.

Trial registration: ClinicalTrials.gov NCT01271803.

Figures

Figure 1
Figure 1
BRAF and MEK inhibition of FDG uptake. BRAF inhibition reduces FDG uptake associated with presence of BRAFV600E mutations, while coadministration of MEK inhibitor broadly increases the effect. (A) A panel of melanoma cell lines that were homozygous null; heterozygous or homozygous positive for BRAFV600 mutations were treated with drug for 3 days, and FDG uptake was assessed (Asterisk, lines with increased FDG uptake from vemurafenib treatment). (B) Cell lines with both BRAFV600E and RAS mutation were most susceptible to increased FDG uptake. Standard error of the mean shown, n = 4/group.
Figure 2
Figure 2
BRAF and MEK modulation of GLUT-1. BRAF and MEK inhibition results in changes in the amount of GLUT-1 at the cellular membrane associated with levels of FDG uptake. Immunofluorescent staining was performed for GLUT-1 (green) and nuclei (blue) on all panels of cells from Figure 1, which had been treated with drug for 3 days. (A) A375s, (B) resistant clone A375R1, (C) SK-Mel-30 melanomas and (D) HCT 116 colorectal cells.
Figure 3
Figure 3
FDG-PET imaging. FDG-PET imaging is effective for monitoring vemurafenib and GDC-0973 combination drug action in BRAFV600E mutant and resistant xenografts. (A) A375- and (B) A375 R1-resistant KRAS mutant melanomas were implanted in athymic nude mice and were administered vehicle, vemurafenib (50 mg/kg BID) or vemurafenib (50 mg/kg BID) and GDC-0973 (7.5 mg/kg QD). Dynamic FDG-PET imaging was performed at baseline, day 3 and day 6 after treatment. A reduction in Ki and MRGlucMAX was induced on day 6 of imaging by both vemurafenib and 0973 combination treatment (Student's t test showing standard error of the mean A375: MRGlucMax − vemuraf; *p = 0.02, combination; **p = 0.01, Ki − vemuraf; *p = 0.02, combination; **p = 0.001, tumor volume; ***p = 0.001. A375R1: MRGlucMax − vemuraf; *p = 0.04, tumor volume; ***p = 0.001). White arrow points at the tumor.
Figure 4
Figure 4
Immunohistochemical staining of GLUT-1. Vemurafenib treatment induces decreases in both total and membrane GLUT-1 staining in A375 tumors but not in the resistant A375R1 line. Immunohistochemical staining for GLUT-1 in (A) A375 tumor xenografts and (B) A375R1-resistant xenografts.
Figure 5
Figure 5
Effects of BRAF and MEK on pathway proteins. Vemurafenib treatment induced reductions of the MAPK pathway in A375s but caused increases in MAPK and glucose metabolism/signaling resistant A375 R1s. Western blot analysis of PET-imaged tumors that had been treated with drug for 6 days is shown.

References

    1. Bastiaannet E, Hoekstra HJ, Hoekstra OS. Melanoma. Methods Mol Biol. 2011;727:123–139. doi: 10.1007/978-1-61779-062-1_8.
    1. Gogas HJ, Kirkwood JM, Sondak VK. Chemotherapy for metastatic melanoma: time for a change? Cancer. 2007;109:455–464. doi: 10.1002/cncr.22427.
    1. Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, Spevak W, Zhang C, Zhang Y, Habets G, Burton EA, Wong B, Tsang G, West BL, Powell B, Shellooe R, Marimuthu A, Nguyen H, Zhang KYJ, Artis DR, Schlessinger J, Su F, Higgins B, Iyer R, D'Andrea K, Koehler A, Stumm M, Lin PS, Lee RJ, Grippo J. et al.Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature. 2010;467:596–599. doi: 10.1038/nature09454.
    1. Ibrahim N, Haluska FG. Molecular pathogenesis of cutaneous melanocytic neoplasms. Annu Rev Pathol. 2009;4:551–579. doi: 10.1146/annurev.pathol.3.121806.151541.
    1. El-Osta H, Falchook G, Tsimberidou A, Hong D, Naing A, Kim K, Wen S, Janku F, Kurzrock R. BRAF mutations in advanced cancers: clinical characteristics and outcomes. PLoS One. 2011;6:e25806. doi: 10.1371/journal.pone.0025806.
    1. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF, Nolop K, Chapman PB. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809–819. doi: 10.1056/NEJMoa1002011.
    1. Oberholzer PA, Kee D, Dziunycz P, Sucker A, Kamsukom N, Jones R, Roden C, Chalk CJ, Ardlie K, Palescandolo E, Piris A, MacConaill LE, Robert C, Hofbauer GFL, McArthur GA, Schadendorf D, Garraway LA. RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors. J Clin Oncol. 2011;30:316–321.
    1. Hatzivassiliou G, Song K, Yen I, Brandhuber BJ, Anderson DJ, Alvarado R, Ludlam M, Stokoe D, Gloor SL, Vigers G, Morales T, Aliagas I, Liu B, Sideris S, Hoeflich KP, Jaiswal BS, Seshagiri S, Koeppen H, Belvin M, Friedman LS, Malek S. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature. 2010;464:431–435. doi: 10.1038/nature08833.
    1. Emery CM, Vijayendran KG, Zipser MC, Sawyer AM, Niu L, Kim JJ, Hatton C, Chopra R, Oberholzer PA, Karpova MB, MacConaill LE, Zhang J, Gray NS, Sellers WR, Dummer R, Garraway LA. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc Natl Acad Sci USA. 2009;106:20411–20416. doi: 10.1073/pnas.0905833106.
    1. Su F, Bradley WD, Wang Q, Yang H, Xu L, Higgins B, Kolinsky K, Packman K, Kim MJ, Trunzer K, Lee RJ, Schostack K, Carter J, Albert T, Germer S, Rosinski J, Martin M, Simcox ME, Lestini B, Heimbrook D, Bollag G. Resistance to selective BRAF inhibition can be mediated by modest upstream pathway activation. Cancer Res. 2011;72:969–978.
    1. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, Chen Z, Lee MK, Attar N, Sazegar H, Chodon T, Nelson SF, McArthur G, Sosman JA, Ribas A, Lo RS. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010;468:973–977. doi: 10.1038/nature09626.
    1. Aplin AE, Kaplan FM, Shao Y. Mechanisms of resistance to RAF inhibitors in melanoma. J Invest Dermatol. 2011;131:1817–1820. doi: 10.1038/jid.2011.147.
    1. Su F, Viros A, Milagre C, Trunzer K, Bollag G, Spleiss O, Reis-Filho JS, Kong X, Koya RC, Flaherty KT, Chapman PB, Kim MJ, Hayward R, Martin M, Yang H, Wang Q, Hilton H, Hang JS, Noe J, Lambros M, Geyer F, Dhomen N, Niculescu-Duvaz I, Zambon A, Niculescu-Duvaz D, Preece N, Robert L, Otte NJ, Mok S, Kee D, Ma Y. et al.RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors. N Engl J Med. 2012;366:207–215. doi: 10.1056/NEJMoa1105358.
    1. Lemech C, Infante J, Arkenau HT. The potential for BRAF V600 inhibitors in advanced cutaneous melanoma: rationale and latest evidence. Ther Adv Med Oncol. 2012;4:61–73. doi: 10.1177/1758834011432949.
    1. Hoeflich KP, Merchant M, Orr C, Chan J, Den Otter D, Berry L, Kasman I, Koeppen H, Rice K, Yang NY, Engst S, Johnston S, Friedman LS, Belvin M. Intermittent administration of MEK inhibitor GDC-0973 plus PI3K inhibitor GDC-0941 triggers robust apoptosis and tumor growth inhibition. Cancer Res. 2011;72:210–219.
    1. Almuhaideb A, Papathanasiou N, Bomanji J. 18 F-FDG PET/CT imaging in oncology. Ann Saudi Med. 2011;31:3–13. doi: 10.4103/0256-4947.75771.
    1. Krug B, Crott R, Lonneux M, Baurain JF, Pirson AS, Vander Borght T. Role of PET in the initial staging of cutaneous malignant melanoma: systematic review. Radiology. 2008;249:836–844. doi: 10.1148/radiol.2493080240.
    1. Kelloff GJ, Hoffman JM, Johnson B, Scher HI, Siegel BA, Cheng EY, Cheson BD, O'Shaughnessy J, Guyton KZ, Mankoff DA, Shankar L, Larson SM, Sigman CC, Schilsky RL, Sullivan DC. Progress and promise of FDG-PET imaging for cancer patient management and oncologic drug development. Clin Cancer Res. 2005;11:2785–27808. doi: 10.1158/1078-0432.CCR-04-2626.
    1. Havenga K, Cobben DC, Oyen WJ, Nienhuijs S, Hoekstra HJ, Ruers TJ, Wobbes T. Fluorodeoxyglucose-positron emission tomography and sentinel lymph node biopsy in staging primary cutaneous melanoma. Eur J Surg Oncol. 2003;29:662–664. doi: 10.1016/S0748-7983(03)00147-1.
    1. Eigtved A, Andersson AP, Dahlstrom K, Rabol A, Jensen M, Holm S, Sørensen SS, Drzewiecki KT, Højgaard L, Friberg L. Use of fluorine-18 fluorodeoxyglucose positron emission tomography in the detection of silent metastases from malignant melanoma. Eur J Nucl Med. 2000;27:70–75. doi: 10.1007/PL00006666.
    1. Morgan B. Opportunities and pitfalls of cancer imaging in clinical trials. Nat Rev Clin Oncol. 2011;8:517–527. doi: 10.1038/nrclinonc.2011.62.
    1. Li Q, Leahy RM. Statistical modeling and reconstruction of randoms precorrected PET data. IEEE Trans Med Imaging. 2006;25:1565–1572.
    1. Patlak CS, Blasberg RG. Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations. 1985;5:584–590. doi: 10.1038/jcbfm.1985.87.
    1. Green LA, Gambhir SS, Srinivasan A, Banerjee PK, Hoh CK, Cherry SR, Sharfstein S, Barrio JR, Herschman HR, Phelps ME. Noninvasive methods for quantitating blood time-activity curves from mouse PET images obtained with fluorine-18-fluorodeoxyglucose. J Nucl Med. 1998;39:729–734.
    1. Dean CB, Nielsen JD. Generalized linear mixed models: a review and some extensions. Lifetime Data Anal. 2007;13:497–512. doi: 10.1007/s10985-007-9065-x.
    1. Williams SP, Flores-Mercado JE, Port RE, Bengtsson T. Quantitation of glucose uptake in tumors by dynamic FDG-PET has less glucose bias and lower variability when adjusted for partial saturation of glucose transport. Eur J Nucl Med Mol Imaging. 2012;2:6.
    1. Hoeflich KP, Herter S, Tien J, Wong L, Berry L, Chan J, O'Brien C, Modrusan Z, Seshagiri S, Lackner M, Stern H, Choo E, Murray L, Friedman LS, Belvin M. Antitumor efficacy of the novel RAF inhibitor GDC-0879 is predicted by BRAFV600E mutational status and sustained extracellular signal-regulated kinase/mitogen-activated protein kinase pathway suppression. Cancer Res. 2009;69:3042–3051.
    1. Parente P, Coli A, Massi G, Mangoni A, Fabrizi MM, Bigotti G. Immunohistochemical expression of the glucose transporters Glut-1 and Glut-3 in human malignant melanomas and benign melanocytic lesions. J Exp Clin Cancer Res. 2008;27:34. doi: 10.1186/1756-9966-27-34.
    1. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–892. doi: 10.1056/NEJMoa1113205.
    1. Kostakoglu L, Agress H, Goldsmith SJ. Clinical role of FDG PET in evaluation of cancer patients. RadioGraphics. 2003;23:315–340. doi: 10.1148/rg.232025705.
    1. Nissan MH, Solit DB. The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both? Curr Oncol Rep. 2011;13:479–487. doi: 10.1007/s11912-011-0198-4.
    1. Shi H, Moriceau G, Kong X, Lee MK, Lee H, Koya RC, Ng C, Chodon T, Scolyer RA, Dahlman KB, Sosman JA, Kefford RF, Long GV, Nelson SF, Ribas A, Lo RS. Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat Commun. 2012;3:724.
    1. Solit DB, Garraway LA, Pratilas CA, Sawai A, Getz G, Basso A, Ye Q, Lobo JM, She Y, Osman I, Golub TR, Sebolt-Leopold J, Sellers WR, Rosen N. BRAF mutation predicts sensitivity to MEK inhibition. Nature. 2006;439:358–362. doi: 10.1038/nature04304.
    1. Sondergaard JN, Nazarian R, Wang Q, Guo D, Hsueh T, Mok S, Sazegar H, MacConaill LE, Barretina JG, Kehoe SM, Attar N, von Euw E, Zuckerman JE, Chmielowski B, Comin-Anduix B, Koya RC, Mischel PS, Lo RS, Ribas A. Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032. J Transl Med. 2010;8:39. doi: 10.1186/1479-5876-8-39.
    1. Young CD, Lewis AS, Rudolph MC, Ruehle MD, Jackman MR, Yun UJ, Ilkun O, Pereira R, Abel ED, Anderson SM. Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo. PLoS One. 2011;6:e23205. doi: 10.1371/journal.pone.0023205.
    1. Macheda ML, Rogers S, Best JD. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer. J Cell Physiol. 2005;202:654–662. doi: 10.1002/jcp.20166.
    1. Yun J, Rago C, Cheong I, Pagliarini R, Angenendt P, Rajagopalan H, Schmidt K, Willson JK, Markowitz S, Zhou S, Diaz LA, Velculescu VE, Lengauer C, Kinzler KW, Vogelstein B, Papadopoulos N. Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells. Science. 2009;325:1555–1559. doi: 10.1126/science.1174229.
    1. Calvo MB, Figueroa A, Pulido EG, Campelo RG, Aparicio LA. Potential role of sugar transporters in cancer and their relationship with anticancer therapy. Int J Endocrinol. 2010;2010:1–14.
    1. Doe MR, Ascano JM, Kaur M, Cole MD. Myc posttranscriptionally induces HIF1 protein and target gene expression in normal and cancer cells. Cancer Res. 2012;72:949–957. doi: 10.1158/0008-5472.CAN-11-2371.
    1. Behrooz A, Ismail-Beigi F. Stimulation of glucose transport by hypoxia: signals and mechanisms. News Physiol Sci. 1999;14:105–110.

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit