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.

Fig. 1 |. DCLK1-IN-1 is a potent DCLK1/2 inhibitor.

(a) Chemical structure of DCLK1 inhibitor DCLK1-IN-1 and negative control DCLK1-NEG. (b) ITC thermograms showing titration of DCLK1-IN-1 and DCLK1-NEG into purified, recombinant DCLK1 kinase domain. Data are representative of n = 2 experiments per compound. Integrals shown with error bars depicting error estimates of peak integration, calculated according to ref. . (c-d) DMSO-normalized results of a ³³P-labeled ATP DCLK1 kinase assay (c) and DCLK2 kinase assay (d). Assays were performed at an ATP concentration of 50 μM (DCLK1) or 100 μM (DCLK2) (K_m). Data in c-d are presented as mean ± S.D. of n = 3 biologically independent samples. (e) Schematic of the DCLK1 NanoBRET assay. (f) NanoBRET evaluation of HCT116 cells expressing DCLK1-Nluc upon treatment with the indicated compounds. Data are presented as mean ± S.D. of n = 4 biologically independent samples and are representative of n = 3 independent experiments.

Fig. 2 |. DCLK1-IN-1 engages DCLK1 potently and selectively in cells.

(a-b) KiNativ assay profiling in PATU-8988T cell lysates treated with 2.5 μM DCLK1-IN-1 (a) or DCLK1-NEG (b). The % inhibition represented with circles are 68.5 (DCLK1, red) and 37.9 (ERK5, pink). Data in a-b are presented as the mean of n = 3 technical replicates and associated datasets are provided in Supplementary Dataset 2. (c) Docking model of DCLK1-IN-1 into the X-ray co-crystal structure of the DCLK1 kinase domain (PDB:5JZN). DCLK1 main chain shown grey carbons, DCLK1-IN-1 shown pink carbons. Hydrogen bonds shown as black dashed lines.

Fig. 3 |. DCLK1 is a target of aberrant KRAS-ERK signaling and is dispensable in DCLK1⁺ PDAC cell lines.

(a) Immunoblot analysis of the indicated cell lines. (b) Immunoblot analysis of PATU-8988T FKBP12^F36V-KRAS^G12V; KRAS^−/− clone treated with DMSO or dTAG-13 for the indicated time-course. Data in a-b are representative of n = 3 independent experiments. Uncropped immunoblots for a-b are displayed in Supplementary Fig. 18. (c) DMSO-normalized antiproliferation of PATU-8988T (top) or PATU-8902 (bottom) LACZ-FKBP12^F36V or FKBP12^F36V-KRAS^G12V; KRAS^−/− clones treated with DMSO or dTAG-13 for 120 h. Cells were cultured as 2D-adherent monolayers or as ultra-low adherent 3D-spheroid suspensions. Data are presented as mean ± S.D. of n = 4 biologically independent samples and are representative of n = 3 independent experiments. (d) Immunoblot analysis of PATU-8988T FKBP12^F36V-KRAS^G12V; KRAS^−/− clone treated with DMSO or dTAG-13 for the indicated time-course. (e) Immunoblot analysis of PATU-8988T cells treated with DMSO or trametinib for 48 h. Data in d-e are representative of n = 3 independent experiments. Uncropped immunoblots for d are displayed in Supplementary Fig. 18 and uncropped immunoblots for e are displayed in Supplementary Fig. 19. (f-g) DMSO-normalized antiproliferation of PATU-8988T cells treated with the indicated compounds for 120 h. Cells were cultured as 2D-adherent monolayers or as ultra-low adherent 3D-spheroid suspensions. Data in f-g are presented as mean ± S.D. of n = 4 biologically independent samples and are representative of n = 3 independent experiments.

Figure 4. |. DCLK1 is a vulnerability in DCLK1⁺ patient-derived organoids.

(a) DMSO-normalized antiproliferation of patient-derived organoid cultures treated with the indicated compounds for 7 d. Data are presented as mean ± S.D. of n = 3 biologically independent samples and are representative of n = 2 independent experiments. (b) GSEA signatures upon assessment of highly expressed (FPKM > 10) transcripts after treatment of PANFR0172_T2 organoids with 2.5 μM DCLK1-IN-1 for 24 h. (c-h) Transcript (c, f), protein (d, g) and phosphoprotein (e, h) abundance after treatment of PANFR0172_T2 (c-e) or PANFR0172_T3 (f-h) patient-derived organoid cultures with 2.5 μM DCLK1-IN-1 for 24 h compared to DMSO treatment. Volcano plots depict fold change abundance relative to DMSO versus P value. P value for c, f is derived from a two-tailed Welch’s t-test. P value for d, e, g and h is derived from a two-tailed Student’s t-test. Data in b-h are from n = 3 biologically independent samples and associated datasets are provided in Supplementary Datasets 3–5.