Fig 2: Therapeutic inhibition of HCK reproduces antitumor immunity observed in HckKO mice.(A) Mass of individual subcutaneous MC38 tumors from WT hosts treated with RK20449 (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) for a total of 10 days. Vehicle control mice were treated with 12% Captisol and an isotype-matched IgG control. Representative tumors are depicted above graphs. Scale bar, 1 cm. Each symbol represents an individual mouse. (B) Tumor volume of subcutaneous MC38 tumors established in HckKO hosts treated with dasatinib (30 mg/kg, twice daily) and/or αPD1 (200 μg, once every 3 days) until the experimental end point when tumors reached ≥600 mm3. n ≥ 9 mice per group. (C) t-distributed stochastic neighbor embedding (tSNE) plot depicting Csf1r, Hck, and Pik3cg gene expression in subcutaneous MC38 tumors analyzed from Zhang et al. (18). (D) Heatmap showing the relative expression of CSF1R, PIK3CG, HCK, and other SFK family members in human colon cancer samples analyzed from Zhang et al. (18). (E) Mass of individual subcutaneous MC38 tumors from HckKO hosts treated with IPI549 (15 mg/kg, once daily) or BLZ945 (200 mg/kg, once daily) in the presence or absence of αPD1 (200 μg, once every 3 days) for 20 days. Each symbol represents an individual mouse. Data represent mean ± SEM; ***P < 0.001; n.s., not significant, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig 3: Genetic ablation of HCK in myeloid cells promotes CD8+ T cell recruitment and activation.(A) Flow cytometry quantification of CD4+ T cells, CD8+ T cells, and NK cells from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. Each symbol represents an individual mouse. (B) Representative immunohistochemical staining for CD8+ T cells in subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. Scale bar, 100 μm. Quantification of staining per square micrometer tumor area is also shown. Each symbol represents an individual mouse. (C) qPCR analysis on FACS-purified tumor-associated CD45+TCRβ+CD8+ T cells for markers associated with immune cell activation. Cells were isolated from subcutaneous MC38 tumors of WT and HckKO hosts treated as described in Fig. 1A. n = 4 mice per group. (D and E) Subcutaneous MC38 tumor volume of WT and HckKO hosts following treatment with αPD1 and/or depleting antibodies against CD8 or NK1.1. Depletion antibodies were administered once every 3 days before subcutaneous MC38 tumor cell injection (total of three treatments) and continued until the experimental end point when tumors reached ≥600 mm3. n ≥ 10 mice per group. Data represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, with statistical significance determined by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig 4: Analysis of Flt3-ITD phosphotyrosine content in TF-1 cells following A-419259 treatment.Each TF-1 cell population from Fig 3 was treated with A-419259 at the concentrations shown or with the DMSO carrier solvent (0.1%) alone as control. Following overnight incubation, Flt3 was immunoprecipitated and analyzed for phosphotyrosine (pTyr) content and Flt3 protein recovery by immunoblotting. Flt3 and pTyr immunoreactivity were quantified using the Odyssey infrared imaging system. Each experiment was performed in triplicate, and the resulting Flt3 pTyr:Flt3 protein ratios were fit by non-linear regression analysis to determine the IC50 value. (A) Individual concentration-response curves for TF-1 cells expressing Flt3-ITD (ITD), as well as the inhibitor-resistant mutant, D835Y, either alone or in the presence of Hck or Fgr. Each replicate is represented by a different color (red, blue, green). (B) Summary of IC50 values for all experiments (Flt3-ITD cells, blue; ITD D835Y cells, red; ITD F691L cells, green). Each point represents a single IC50 determination, with the vertical bar representing the mean value ± SE. Student’s t-tests did not show a significant difference between the IC50 values obtained with TF-1 cells expressing Flt3-ITD alone or in the presence of Hck or Fgr. A-419259 treatment did not inhibit the Flt3 pTyr:Flt3 ratios for cell populations expressing the D835Y mutant alone or the F691L mutant either alone or in the presence of Hck or Fgr (highest concentration tested was 1,000 nM).

Fig 5: Development of selective HCK inhibitors and identification of BT424 as a specific HCK inhibitor.A SAR study of dasatinib to design selective HCK inhibitors. B Screen HCK specific inhibitors for all our designed compounds. Five inhibitors (BT294, 331, 332, 342 and 424) with high efficiency of HCK inhibitions are highlighted in red arrows. C HCK inhibition profiling for BT424 with 10 dose 1:3 serial dilution. D Inhibition of all SFKs for 6 candidate HCK inhibitors at 50uM. SFKs inhabitation assay was performed at Reaction Biology Corp. Values were shown for % of enzyme activity compared to DMSO control. Cell proliferation (E) and cytotoxicity (F) were measured by MTT assay and release LDH with various concentration of BT424 for 24 h in Raw264.7 cells. Source data are provided as a Source Data file.