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Discovery of a selective inhibitor of doublecortin like kinase 1

Abstract

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

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Fig. 1: DCLK1-IN-1 is a potent DCLK1/2 inhibitor.
Fig. 2: DCLK1-IN-1 engages DCLK1 potently and selectively in cells.
Fig. 3: DCLK1 is a target of aberrant KRAS-ERK signaling and is dispensable in DCLK1+ PDAC cell lines.
Fig. 4: DCLK1 is a vulnerability in DCLK1+ patient-derived organoids.

Data availability

KINOMEscan and KiNativ data are provided in Supplementary Datasets 1 and 2. Cell line RNA-sequencing data has been deposited to the NCBI GEO (accession number GSE140490). Cell line and deidentified patient-derived organoid RNA-sequencing analyzed data files are provided in Supplementary Dataset 3. Cell line and deidentified patient-derived organoid mass spectrometry-based proteomics and phosphoproteomics analyzed data files are provided in Supplementary Datasets 4 and 5.

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Acknowledgements

We thank M. Kostic for critical reading of the manuscript and S. Nabet and members of the Gray laboratory for helpful discussions. We gratefully acknowledge S. Gygi for use of CORE for mass spectrometry data analysis software. This work was supported by an American Cancer Society Postdoctoral Fellowship PF-17-010-01-CDD (B.N.), Claudia Adams Barr Program in Innovative Basic Cancer Research Award (B.N.), Katherine L. and Steven C. Pinard Research Fund (N.S.G. and B.N.), Hope Funds for Cancer Research Postdoctoral Fellowship (S.R.), Harvard Catalyst KL2/CMeRIT Fellowship (S.R.), Perry Levy Fellowship (S.R.), the Lustgarten Foundation (S.R., B.M.W., A.J.A. and W.C.H.), NCI HCMI program (A.J.A. and S.R.), American Cancer Society 129089-PF-16-088-01-TBG (E.J.P.), KU-KIST Graduate School of Converging Science and Technology Program (T.S.), Spanish Ministerio de Economia y Competitividad grant no. SAF2015-60268R, cofunded by Fondo Europeo de Desarrollo Regional funds (J.M.L.), Pancreatic Cancer Action Network Catalyst Award (A.J.A.), Doris Duke Charitable Foundation Clinician Scientist Development Award (A.J.A.), NCI K08 CA218420 (A.J.A.), NCI U01 CA176058 (W.C.H.), NCI U01 CA199253 (W.C.H.) and NCI U01 CA224146 (W.C.H.), American Cancer Society Award 132205-RSG-18-039-01-DMC (K.D.W.), Welch Foundation grant no. I1829 (K.D.W.), 2017 AACR-Bayer Innovation and Discovery grant no. 17-80-44-GRAY (N.S.G.), DF/HCC GI SPORE Developmental Research Project Award P50CA127003 (N.S.G. and K.M.H.) and Hale Center for Pancreatic Research (J.D.M., B.M.W., A.J.A., W.C.H. and N.S.G.).

Author information

Affiliations

Authors

Contributions

F.M.F., B.N. and N.S.G. conceived and led the study. F.M.F. performed medicinal chemistry optimization, with input from J.W. B.N. developed and characterized dTAG model systems, performed cellular PDAC studies and performed DCLK1 nanoBRET assays. B.N., S.R., R.L.K., R.W.S.N. and R.S. performed patient-derived organoid culture assays and prepared organoid samples for RNA-sequencing, proteomics and phosphoproteomics. B.N. and A.L.L. performed pulldown experiments and prepared cellular samples for KiNativ, RNA-sequencing, proteomics and phosphoproteomics. Y.L., W.H. and L.L. performed DCLK1 protein purification, ITC and molecular shift assay. M.K. performed proteomics and phosphoproteomics experiments. A.Y. performed the maximum tolerated dose studies. S.H. performed zebrafish toxicity assays. E.J.P. performed western blot and immunohistochemistry analysis of mouse model tissues. L.H. performed validation of organoid sensitivity. J.K. performed RNA-sequencing data analysis. N.D.-M. and S.E performed ERK5 biochemical and cellular assays. Z.Z., C.R.C., J.D.V. and M.B.R. developed the DCLK1 nanoBRET assay reagents. R.O. performed viability and morphology testing on rat cortical neurons. T.S. and N.D.K. performed molecular modeling. J.M.L., S.M., C.Y.L., A.T.L., K.M.H., J.D.M., B.M.W., A.J.A., W.C.H., K.D.W. and N.S.G. supervised the study. F.M.F., B.N. and S.R. wrote the manuscript, with edits from N.S.G. All authors read and approved the manuscript.

Corresponding author

Correspondence to Nathanael S. Gray.

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Competing interests

F.M.F. and N.S.G. are inventors on a patent application related to the DCLK1 inhibitors described in this manuscript (WO/2018/075608). B.N. is an inventor on patent applications related to the dTAG system described in this manuscript (WO/2017/024318, WO/2017/024319, WO/2018/148443, WO/2018/148440). Z.Z., C.R.C., J.D.V. and M.B.R. are employees of Promega Corporation. B.M.W. receives research funding from Celgene, Inc, and is a consultant for G1 Therapeutics, BioLineRx and GRAIL. A.J.A. has consulted for Oncorus, Inc. W.C.H. is a consultant for Thermo Fisher, AjuIB, MPM Capital, iTeos and Paraxel and is a Scientific Founder and serves on the Scientific Advisory Board (SAB) for KSQ Therapeutics. K.D.W. is a member of the SAB for Vibliome Therapeutics. N.S.G. is a Scientific Founder, member of the SAB and equity holder in C4 Therapeutics, Syros, Soltego, B2S, Gatekeeper and Petra Pharmaceuticals. The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voroni, Her2llc, Deerfield and Sanofi.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2, Figs. 1–22 and Note.

Reporting Summary

Supplementary Dataset 1. KinomeSCAN.

Full KINOMEscan dataset.

Supplementary Dataset 2. KiNativ.

Full KiNativ dataset from lysate and live cell format.

Supplementary Dataset 3. RNA-sequencing.

RNA-sequencing dataset for PANFR0172_T2, PANFR0172_T3 and PATU-8988T on comparison of DMSO and 2.5 µM DCLK1-IN-1 treatments for 24 h.

Supplementary Dataset 4. Proteomics.

Mass spectrometry-based proteomics dataset for PANFR0172_T2, PANFR0172_T3 and PATU-8988T on comparison of DMSO and 2.5 µM DCLK1-IN-1 treatments for 24 h.

Supplementary Dataset 5. Phosphoproteomics.

Mass spectrometry-based phosphoproteomics dataset for PANFR0172_T2, PANFR0172_T3 and PATU-8988T on comparison of DMSO and 2.5 µM DCLK1-IN-1 treatments for 24 h.

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Ferguson, F.M., Nabet, B., Raghavan, S. et al. Discovery of a selective inhibitor of doublecortin like kinase 1. Nat Chem Biol 16, 635–643 (2020). https://doi.org/10.1038/s41589-020-0506-0

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