Research ArticleCancer

Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors

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Science Translational Medicine  14 Jun 2017:
Vol. 9, Issue 394, eaah6144
DOI: 10.1126/scitranslmed.aah6144
  • Fig. 1. Cellular profiling of RET inhibitors identifies AD80 and ponatinib as potent compounds.

    (A) Dose-response curves (72 hours) for AD80, cabozantinib (CAB), vandetanib (VAN), alectinib (ALE), regorafenib (REG), sorafenib (SOR), ponatinib (PON), crizotinib (CRI), ceritinib (CER), or PF06463922 (PF06) in KIF5B-RET–expressing Ba/F3 cells (n = 3 technical replicates). (B) Immunoblotting results of KIF5B-RET–rearranged Ba/F3 cells after treatment (4 hours). C, control. (C) Relative mean colony number of NIH-3T3 cells engineered with KIF5B-RET fusion by CRISPR/Cas9 was assessed in soft agar assays after 7 days under treatment. Representative images of colonies under AD80 treatment are displayed in the lower panel. Scale bars, 100 μm (n = 3). (D) Immunoblotting of CRISPR/Cas9-engineered, KIF5B-RET–rearranged NIH-3T3 cells treated with AD80, cabozantinib, or vandetanib (4 hours). KIF5B-RET expressing Ba/F3 cells (Ba/F3 ctrl.) serve as control for RET signaling (n = 3). (E) Dose-response curves (72 hours) for different inhibitors in LC-2/AD cells. (F) Immunoblotting was performed in LC-2/AD cells treated with AD80, cabozantinib, or vandetanib (4 hours).

  • Fig. 2. AD80 specifically targets RET and tightly binds to RET fusion kinase.

    (A) Scatterplot of log2-fold phosphorylation change for LC-2/AD cells treated (4 hours) with either 10 or 100 nM AD80. Each dot represents a single phosphosite; phospho-RET (Y900) is highlighted in red. (B) Difference in melting temperatures after AD80, sorafenib (SOR), vandetanib (VAN), or sunitinib (SUN) addition (ΔTm) and the respective SEM are shown for each construct. Thermal shift experiments were performed using independent preparations of each protein and were carried out in triplicates (left). Representative thermal melting curves for ΔKIF5B-KD incubated with either AD80 (1 μM) or the equivalent volume of dimethyl sulfoxide (DMSO) (ctrl.) are shown (right).

  • Fig. 3. AD80 is active against gatekeeper mutant RETV804M cells.

    (A) Optimized structures after extensive MD refinement followed by ALPB optimization. (i) RETwt/AD80 after 102 ns, (ii) RETwt/AD57 after 202 ns (92 ns from RETwt/AD80 simulation followed by 110 ns from TI-MD), and (iii) RETV804M/AD80 after 107 ns (side view). The DFG motif is shown in violet. Distances from the center of central phenyl to Val804-C(wt), Ile788-C(wt), and Met804-S(V804M) are 4.77, 3.90, and 4.29 Å, respectively. Dashed lines indicate the H-bond between the bound ligands and aspartate of the DFG motif. (B) Heat map of mean 50% growth inhibition (GI50) values (n ≥ 3) of Ba/F3 cells expressing CCDC6-RETV804M or KIF5B-RETV804M after 72 hours of treatment, as assessed for various inhibitors. (C) Immunoblotting of AD80-, cabozantinib-, or vandetanib-treated (4 hours) KIF5B-RETV804M Ba/F3 cells. (D) Immunoblotting of Ba/F3 cells expressing CCDC6-RET-RETwt or CCDC6-RETV804M under AD80 or vandetanib treatment (4 hours). wt, wild type. (E) Calculated Michaelis constant (Km) values of ATP binding to RETwt or RETV804M from three independent experiments. ***P < 0.001, n = 3.

  • Fig. 4. RETI788N mutations abrogate the activity of AD80 but not ponatinib.

    (A) Dose-response curves for AD80 against Ba/F3 cells expressing KIF5B-RETwt (black) or KIF5B-RETI788N (red) and CCDC6-RETwt (black dashed line) or CCDC6-RETI788N (red dashed line) (n = 3). (B) Bar graph of mean GI50 values + SD (from n = 3) for KIF5B-RETwt or KIF5B-RETI788N Ba/F3 cells treated (72 hours) with AD80, cabozantinib (CAB), vandetanib (VAN), or ponatinib (PON). ***P < 0.001; **P < 0.01; n.s., not significant. (C) Immunoblot of Ba/F3 cells expressing KIF5B-RETwt or KIF5B-RETI788N and CCDC6-RETwt or CCDC6-RETI788N treated (4 hours) with AD80. (D) Immunoblot of KIF5B-RETwt, KIF5B-RETV804M, or KIF5B-RETI788N expressing Ba/F3 cells treated (4 hours) with ponatinib. HSP90 is used as loading control. (E) Optimized structure after extensive MD refinement followed by ALPB optimization. RETwt/AD80 after 102 ns (side view). Distance from the center of central phenyl to Ile788-C(V804M) is 4.61 Å.

  • Fig. 5. MAPK pathway activation may be involved in the development of resistance against RET inhibition.

    (A) RNA-seq results of LC-2/AD cells treated (48 hours) with 100 nM AD80. Genes contained within the core enrichments of GSEA against the hallmark gene sets with genes up-regulated (KRAS up) or down-regulated (KRAS down) by active KRAS are highlighted by red and blue, respectively. The dashed line represents false discovery rate–adjusted Q value = 0.05. (B) Relevant genes from the top 50 genes with the strongest significant changes in RNA-seq after AD80 treatment (100 nM; 48 hours). (C) Predicted number of down-regulated phosphorylation sites for each kinase. All kinases with greater than or equal to six down-regulated phosphorylation sites are shown in hierarchical order. Kinases associated with MAPK pathway signaling are highlighted in red. (D) In immunoblotting assays, RET signaling was monitored in LC-2/AD and TPC-1 cells treated (48 hours) with AD80 (0.1 μM), trametinib (TRA) (0.1 μM), or a combination of both inhibitors. (E) LC-2/ADev or LC-2/ADKRAS G12V cells were treated (72 hours) with AD80. Results are shown as means + SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05. (F) Immunoblotting of LC-2/ADev or LC-2/ADKRAS G12V cells under AD80 treatment (100 nM; 4 hours).

  • Fig. 6. AD80 treatment effectively shrinks RET-rearranged tumors in PDX models.

    (A) Immunoblotting of tumor tissue from CRISPR/Cas9-induced NIH-3T3KIF5B-RET xenografts was performed. Mice were treated (4 hours) with vehicle control or 12.5 or 25 mg/kg AD80, CAB, or VAN and were sacrificed. (B) Median tumor volume was assessed using consecutive measurements of PDX tumors driven by CCDC6-RETwt or CCDC6-RETV804M rearrangements under treatment with either AD80 (25 mg/kg; 14 days) or vehicle control (14 days). Treatment started at day 0. (C) Waterfall plot for each CCDC6-RETwt fusion–positive PDX depicting best response (14 days) under AD80 or vehicle control treatment. ***P < 0.001. (D) Waterfall plot for each CCDC6-RETV804M–positive PDX depicting best response (7 days) under AD80 or vehicle control treatment. ***P < 0.001. (E) Representative immunohistochemistry (IHC) staining for hematoxylin and eosin (H&E) and Ki-67 of AD80- or vehicle control–treated CCDC6-RETwt PDX. Scale bars, 100 μm. (F) Quantification of Ki-67 IHC staining. ***P < 0.001; *P < 0.05.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/394/eaah6144/DC1

    Materials and Methods

    Fig. S1. Selective inhibition of signaling induced by rearranged RET and clinical activity in vivo.

    Fig. S2. Induction of KIF5B-RET rearrangements in NIH-3T3 cells via CRISPR/Cas9 and S6 kinase as an off-target of AD80.

    Fig. S3. Characterization of the activity profile of AD80.

    Fig. S4. Delineation of the cellular targets of AD80 using ligand screens and thermal shift experiments.

    Fig. S5. RMSD of RET and AD80 or cabozantinib over time and ALPB-optimized structures.

    Fig. S6. Inhibitory potential of AD80 derivatives and resistance mechanisms against RET inhibition.

    Fig. S7. Validation of PDX via fluorescent in situ hybridization (FISH) and in vivo effects induced by treatment with AD80.

    Table S1. IC50 values of AD80, cabozantinib, and vandetanib for phospho-RET in Ba/F3 cells expressing wild type or V804M KIF5B-RET.

    Table S2. Rates of clinical response to currently available anti-RET drugs and clinical information of patients used in retrospective analysis.

    Table S3. GI50 values of the panel of patient-derived cell lines.

    Table S4. Tabulated derivative melting temperatures (Tm) and differences in melting temperature (ΔTm) values.

    Table S5. In vitro kinase assay of RETwt, RETV804M, and RETV804L mutants with different inhibitors.

    Table S6. Experimental setup for saturated mutagenesis screening.

    References (4366)

  • Supplementary Material for:

    Drugging the catalytically inactive state of RET kinase in RET-rearranged tumors

    Dennis Plenker, Maximilian Riedel, Johannes Brägelmann, Marcel A. Dammert, Rakhee Chauhan, Phillip P. Knowles, Carina Lorenz, Marina Keul, Mike Bührmann, Oliver Pagel, Verena Tischler, Andreas H. Scheel, Daniel Schütte, Yanrui Song, Justina Stark, Florian Mrugalla, Yannic Alber, André Richters, Julian Engel, Frauke Leenders, Johannes M. Heuckmann, Jürgen Wolf, Joachim Diebold, Georg Pall, Martin Peifer, Maarten Aerts, Kris Gevaert, René P. Zahedi, Reinhard Buettner, Kevan M. Shokat, Neil Q. McDonald, Stefan M. Kast, Oliver Gautschi, Roman K. Thomas, Martin L. Sos*

    *Corresponding author. Email: martin.sos{at}uni-koeln.de

    Published 14 June 2017, Sci. Transl. Med. 9, eaah6144 (2017)
    DOI: 10.1126/scitranslmed.aah6144

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Selective inhibition of signaling induced by rearranged RET and clinical activity in vivo.
    • Fig. S2. Induction of KIF5B-RET rearrangements in NIH-3T3 cells via CRISPR/Cas9 and S6 kinase as an off-target of AD80.
    • Fig. S3. Characterization of the activity profile of AD80.
    • Fig. S4. Delineation of the cellular targets of AD80 using ligand screens and thermal shift experiments.
    • Fig. S5. RMSD of RET and AD80 or cabozantinib over time and ALPB-optimized structures.
    • Fig. S6. Inhibitory potential of AD80 derivatives and resistance mechanisms against RET inhibition.
    • Fig. S7. Validation of PDX via fluorescent in situ hybridization (FISH) and in vivo effects induced by treatment with AD80.
    • Table S1. IC50 values of AD80, cabozantinib, and vandetanib for phospho-RET in Ba/F3 cells expressing wild type or V804M KIF5B-RET.
    • Table S2. Rates of clinical response to currently available anti-RET drugs and clinical information of patients used in retrospective analysis.
    • Table S3. GI50 values of the panel of patient-derived cell lines.
    • Table S4. Tabulated derivative melting temperatures (Tm) and differences in melting temperature (ΔTm) values.
    • Table S5. In vitro kinase assay of RETwt, RETV804M, and RETV804L mutants with different inhibitors.
    • Table S6. Experimental setup for saturated mutagenesis screening.
    • References (4366)

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