A Schematic presentation of approach for CRSPR/Cas9 based kinome knockout screening to discover the potential kinase responsible for 5FU resistance in OSCC. B, C Primary screening of 840 kinases was performed in H357 5FUR line with sublethal dose of 5FU (8 μM). The kinases (n = 506 nos) whose knockout alone induced significantly higher cell death (>30%) depicted in red were excluded. From the rest of the kinases (n = 334 nos) depicted in green, the survival fraction (5FU treated/Vehicle Control) was determined and top 60 candidates having lowest survival fraction were considered for secondary screening described in panel (C). D For secondary screening with top 60 kinases, four cell lines were considered i.e., H357 5FUR, SCC4 5FUR, SCC9 5FUR and H357CisR. After overlapping all three 5FUR cell lines, MINK1, SBK1, FKBP1A were found to be the common kinases among them. MINK1 was selected as a potential kinase target purely based on having the lowest survival fraction among all common candidates. E Heat map based on % of cell viability with or without treatment of 5FU or cisplatin followed by knocking out 60 individual kinases in H357 5FUR, SCC4 5FUR, SCC9 5FUR and H357 CisR cells. F The fluorescent images acquired from high content analyzer with indicated treated group in H357 5FUR lines during kinome screening. G Lysates were collected from indicated cells and immunoblotting (n = 3) was performed with indicated antibodies. H Protein expression of MINK1 was analyzed by IHC in chemotherapy- responder and chemotherapy-non-responder OSCC tumors. Scale bars: 50 μm. I IHC scoring for MINK1 from panel (H) (Q Score = Staining Intensity × % of Staining), (Median, n = 11 for chemotherapy-responder and n = 23 for chemotherapy-non-responder) *P < 0.05 by 2-tailed Student’s t test. J Protein expression of MINK1 was analyzed by immunohistochemistry (IHC) in pre- and post-TPF treated paired tumor samples from chemotherapy-non-responder patients. Scale bars: 50 μm. K IHC scoring for MINK1 from panel (J) (Q Score = Staining Intensity × % of IHC Staining). *P < 0.05 by 2-tailed Student’s t test.

A 5FU resistant cells stably expressing MINK1sgRNA (#1 and #2) and NTsgRNA were treated with 5FU for 48 h and cell viability was determined by MTT assay (n = 3 and 2-way ANOVA, ****P < 0.0001). B Indicated cells were treated with 5FU for 48 h, after which cell death was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death (early and late apoptotic) with respective treated groups (Mean ± SEM, n = 3, Two-way ANOVA, ****P < 0.0001). C Left panel: Indicated cells were treated with 10 μM of 5FU for 48 h, after which immunostaining was performed for γ-H2AX. Right panel: The number of foci from panel C are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. D Indicated cells were treated with 5FU for 48 h and immunoblotting (n = 3) was performed with indicated antibodies. E PDC2 MINK1WT cells were implanted in right upper flank of athymic male nude mice and PDC2 MINK1KO cells were implanted in left upper flank, after which they were treated with 5FU at indicated concentration. At the end of the experiment mice were euthanized, tumors were isolated and photographed (n = 5). F Tumor growth was measured in indicated time points using digital slide caliper and plotted as a graph (mean ± SEM, n = 5, Two-way ANOVA, *P < 0.05). G Bar diagram indicates the tumor weight measured at the end of the experiment (mean ± SEM, n = 5, Two-way ANOVA, ****P < 0.0001). H After completion of treatment, tumors were isolated and paraffin-embedded sections were prepared as described in materials and methods to perform immunohistochemistry with indicated antibodies. Scale bars: 50 μm. I Lateral view of fluorescent transgenic [Tg(fli1:EGFP)] zebrafish embryos at Day 0 and Day 5 injected with Dil-Red stained PDC2 control and MINK1 KO cells with and without treatment of 5FU. J The tumor growth and migration were assessed by an increase in fluorescence intensity on the 5th day compared to the day of injection. The quantitation of fluorescence intensity (I) was performed using ImageJ software and represented as fold change of fluorescence intensity where day 0 reading was taken as baseline (mean ± SEM, n = 6, Two-way ANOVA, ****P < 0.0001). K Zoomed image of distal part of embryo (5days post injection) to monitor migration of tumor cells.

A Using a lentiviral approach, 5FU resistant OSCC lines and PDC2 were stably transfected with ShRNA which targets 3′UTR of MINK1 mRNA (MINK1 UTRKD). For ectopic overexpression, pLenti CMV/TO Puro DEST MINK1 and control vector were transiently transfected to indicated MINK1 UTRKD cells and immunoblotting (n = 3) was performed with indicated antibodies. B MINK1 was ectopically overexpressed in 5FUR cells stably expressing MINK1ShRNA targeting 3′UTR and treated with 5FU at indicated concentration for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001. C Cells were treated as indicated in (B) panel and cell death (early and late apoptotic) was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death with respective treated groups (Mean ± SEM, n = 3), 2-way ANOVA, ****P < 0.0001. D Left panel: MINK1 was overexpressed in 5FUR cells stably expressing MINK1ShRNA targeting 3′UTR and treated with 5FU for 48 h, after which immunostaining was performed for γ-H2AX as described in materials and methods. Right panel: The number of foci from panel (D) are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. E MINK1 was overexpressed in chemoresistant cells stably expressing MINK1ShRNA targeting 3′ UTR, followed by 5FU treatment for 48 h, and immunoblotting (n = 3) was performed with indicated antibodies. F 5FU sensitive cells were stably transfected with either pSilencer™ 4.1-CMV puro-MINK1WT or pSilencer™ 4.1-CMV puro-MINK1 K54R (kinase dead) followed by puromycin selection (up to 5 µg/ml). Two clones (C1 and C2) from each line were selected and immunoblotting was performed with indicated antibodies. G 5FU sensitive OSCC lines stably expressing MINK1 WT or MINK1 K54R (two clones C1 and C2 from each line) were treated with 5FU at indicated concentrations for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001.

A High-throughput phosphorylation profiling with 1318 site-specific antibodies from over 30 signaling pathways was performed in the lysates of MINK1KO and MINK1WT clones of H357 5FUR cells as described in methods. The top 20 upregulated phosphoproteins (MINK1 KO/MINK1WT) are represented in left panel, whereas top 20 downregulated phosphorylated proteins are represented in right panel. The upregulated targets considered in the study is marked in red box, whereas downregulated targets in green box. B Lysates were collected from indicated cells and immunoblotting (n = 3) was performed with indicated antibodies. C pLenti CMV/TO Puro DEST MINK1 (ectopic overexpression of MINK1) was transiently transfected in 5FUR lines stably expressing MINK1 ShRNA (targeting 3′UTR) and immunoblotting (n = 3) was performed with indicated antibodies. D Lysates were collected from indicated cells and immunoblotting (n = 3) was performed with indicated antibodies. E MINK1 was ectopically overexpressed in 5FUR lines stably expressing MINK1 ShRNA (targeting 3′UTR) and immunoblotting (n = 3) was performed with indicated antibodies. F pLNCX myr HA Akt1 (ectopic overexpression of myr AKT) was transiently transfected in indicated MINK1KO cells and immunoblotting (n = 3) was performed with indicated antibodies. G MINK1 was ectopically overexpressed in 5FUR lines stably expressing MINK1ShRNA (UTRKD) as described in panel C and treated with AKT inhibitor (Akti-1/2) for 24 h and immunoblotting (n = 3) was performed with indicated antibodies. H Lysates were collected from indicated cells and immunoblotting (n = 3) was performed with indicated antibodies.

A In vitro MINK1 kinase assay was performed using three compounds potentially binding to MINK1 (based on IUPHAR database). All compounds (10 µM) were incubated with recombinant human MINK1 along with substrate MBP and ATP and further subjected to ADP-Glo™ Kinase Assay as described in materials and methods section (n = 3), one-way ANOVA, *P < 0.05. B Determination of EC50 value for kinase activity of top two MINK1 inhibitors selected from panel (A) (n = 3), one-way ANOVA, ****P < 0.0001. C, D The indicated cells were treated with indicated concentration of MINK1 inhibitors and cell viability was determined by MTT assay. Selection of highest dose of Lestaurtinib and Pexmetinib that does not affect cell viability when treated alone (viability > 80%) in 5FU resistant OSCC lines (n = 3). E 5FU resistant cells were treated with indicated doses of MINK1 inhibitors (50 nM Lestaurtinib, 500 nM Pexmetinib) in combination with increasing concentrations of 5FU for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001. F 5FU resistant OSCC lines and PDC2 cells were treated with indicated doses of MINK1 inhibitors (50 nM Lestaurtinib, 500 nM Pexmetinib) in combination with increasing concentrations of 5FU for 48 h, after which cell death (early and late apoptotic) was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death with respective treated groups (Mean ± SEM, n = 3), Two-way ANOVA, ****P < 0.0001. G Left panel: Indicated 5FU resistant OSCC lines and PDC2 cells were treated with 5 μM of 5FU and/or 50 nM of Lestaurtinib for 48 h, after which immunostaining was performed for γ-H2AX as described in materials and methods. Right panel: The number of foci from panel (G) are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. H Indicated 5FU resistant OSCC lines and PDC2 cells were treated with Lestaurtinib for 48 h, after which immunoblotting (n = 3) was performed with indicated antibodies. I Effect on 5FU IC50 upon Lestaurtinib treatment in cells with or without MINK1 knockout in indicated 5FU resistant OSCC lines and PDC2 cells (n = 3), *P < 0.05 by 2-way ANOVA.

A Patient-derived cells (PDC2) were earlier established from tumor of chemotherapy (TPF) non-responder patient. PDC2 were implanted in the right upper flank of athymic male nude mice, after which they were treated (i.p) with 5FU and/or Lestaurtinib at indicated concentrations. At the end of the experiment mice were euthanized, and tumors were isolated and photographed (n = 5). B Bar diagram indicates the tumor weight measured at the end of the experiment (mean ± SEM, n = 5). Two-way ANOVA, ****P < 0.0001. C Tumor growth was measured at the indicated time points using digital slide caliper and plotted as a graph (mean ± SEM, n = 5). Two-way ANOVA, ****P < 0.0001. D After completion of treatment, tumors were isolated, and paraffin-embedded sections were prepared as described in Methods to perform IHC with indicated antibodies. Scale bars: 50 μm. E Schematic presentation of the mechanism by which MINK1 regulates p53 expression through AKT/MDM2 axis.

A, B PDC2 cells were treated with indicated concentrations of cisplatin, 5FU and lestaurtinib for 48 h and cell viability was measured by MTT assay (n = 3 and, ****P < 0.0001 by 2-way ANOVA). C PDC2 cells were treated with indicated concentrations of cisplatin, 5FU and lestaurtinib for 48 h after which cell death was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death (early and late apoptotic) with respective treated groups (Mean ± SEM, n = 3 by Two-way ANOVA, ****P < 0.0001). D PDC2 cells were treated with indicated concentrations of cisplatin, 5FU and lestaurtinib for 48 h and immunoblotting (n = 3) was performed with indicated antibodies. E PDC2 cells were treated with indicated concentrations of cisplatin, 5FU, lestaurtinib for 12 days and colony forming assays were performed as described in method section. Left panel: Bar diagram indicate the relative colony number (n = 3 and, ****P < 0.0001 by 2-way ANOVA). Right panel: representative photographs of colony forming assay in each group.