Fig 2: Therapeutic inhibition of HCK reduces PDX tumor burden in humanized mice.(A and B) Tumor volume and mass of individual subcutaneous PDXs from humanized NSG-SGM3 hosts following treatment with vehicle (12% Captisol) or RK20449 (30 mg/kg, twice daily) for 25 days. Each symbol represents an individual mouse. (C) qPCR analysis on whole PDX tumors from humanized NSG-SGM3 hosts treated as described in (A) for genes associated with human immune cells. n = 4 mice per group. (D) qPCR analysis on whole PDX tumors from humanized NSG-SGM3 hosts treated as described in (A) for genes associated with immune cell activation (i.e., IL12, IFNG, IL1B, TNF, CXCL9, and CXCL10) and immune suppression (i.e., CD163, MRC1, TGFB, IL10, and CXCL12). n = 4 mice per group. Data represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, with statistical significance determined by an unpaired Student’s t test.

Fig 3: Hck and Fgr gatekeeper mutants confer resistance to A-419259 in vitro.(A) Close-up view of the A-419259 binding pocket in the crystal structure of Hck bound to A-419259 (PDB: 4LUE). The carbon backbone of A-419259 is shown in yellow. The side chain of the Hck gatekeeper residue (Thr338; cyan) and forms a hydrogen bond with the primary amine on the pyrrolopyrimidine core of A-419259 (dotted line). Using PyMOL, T338 was substituted with methionine, leucine, and phenylalanine as shown, resulting in loss of the H-bond and steric clash predicted to interfere with inhibitor action. (B) Near-full-length Hck, Fgr and Lyn (wild-type and gatekeeper mutants) were expressed in E. coli, purified and assessed for A-419259 sensitivity in vitro using the Z’-Lyte kinase assay. The Km value for ATP for each kinase was determined first, and subsequent concentration-response experiments with A-419259 were performed with the ATP concentration set to the Km for each kinase. IC50 values for A-419259 were determined by non-linear regression analysis of the concentration-response curves, and are shown as the mean ± SE from three independent determinations. Additional kinetic data for all three recombinant kinases is provided in the Supporting Information, S4 Fig.

Fig 4: Flt3-ITD N676S mutation confers resistance to A-419259 in TF-1 cells.(A) TF-1 cells expressing Flt3-ITD or the Flt3-ITD N676S mutant were incubated over a range of A-419259 concentrations or the DMSO carrier solvent (0.1%) alone as control. Cell viability was determined 72 hours later using the CellTiter Blue cell viability assay. Results are shown relative to the DMSO control values, and IC50 values were determined by non-linear regression analysis of the resulting concentration-response curves. (B) Cell populations from part A were incubated overnight in the presence of the A-419259 concentrations shown. Flt3 was then immunoprecipitated and analyzed for phosphotyrosine (pTyr) content and Flt3 protein recovery by immunoblotting followed by Odyssey infrared imaging. The mean pTyr:Flt3 protein ratios ± SE for three independent experiments were fit by non-linear regression analysis. (C) The IC50 values for growth inhibition (Cells) and Flt3 phosphotyrosine context (pTyr) are shown, and were compared for statistical significance by Student’s t test. (D) Model of A-419259 bound to the Flt3 kinase domain. The crystal structure of the Flt3 kinase domain bound to quizartinib (PDB: 4XUF; green) was aligned with the crystal structure of the Hck kinase domain bound to A-419259 (PDB: 4LUE; blue) using PyMol. The overall alignment is shown at left, and a close-up of the inhibitor binding site is shown at right. Significant overlap was observed in the positions of quizartinib (yellow) and A-419259 (orange) in the ATP-binding site. In the Flt3 structure, residue Asn676 forms a local network of polar contacts with the side chains of Glu692, Lys826 and the main chain of His671. Acquired mutations in this residue were associated with resistance to A-419259 in AML cell lines. Whole exome sequencing also identified a substitution of Flt3-ITD Asp839 (shown) in MV4-11 population R3; this mutation was not verified in subsequent Sanger sequencing of individual clones (Table 1).

Fig 5: Transformation by Flt3-ITD sensitizes TF-1 myeloid cells to growth suppression by A-419259.TF-1 myeloid cells were transformed to cytokine independence by expression of Flt3-ITD or the inhibitor-resistant mutants D835Y or F691L. These mutations are associated with clinical resistance to quizartinib and other Flt3 kinase inhibitors (see main text). (A) The parent TF-1 cell line, as well as the Flt3-ITD+ AML cell line MV4-11, were incubated in the presence of a range of A-419259 concentrations or the DMSO carrier solvent (0.1%) alone as control. Cell viability was determined 72 hours later using the CellTiter Blue cell viability assay. Results were normalized to DMSO control values, and are presented as mean percent control ± SD for triplicate determinations. TF-1 cells expressing Flt3-ITD (black curves), Flt3-ITD D835Y (red) or Flt3-ITD F691L (blue) either alone (B) or in combination with Hck (C) or Fgr (D) were assayed for sensitivity to growth arrest by A-419259 as per part A. IC50 values for each experiment were determined by non-linear regression analysis and are summarized in the table.