Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC 50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg null mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

SFKs have been implicated as intermediates in signaling pathways that regulate cellular functions such as proliferation, differentiation, survival, and migration in various cell lineages ( 13 ). Of these, Lyn, Fgr, and HCK are expressed in myeloid lineages and are involved in normal myelopoiesis ( 14 ). The importance of HCK, Lyn, and Fgr in myeloid proliferation and differentiation has been demonstrated in knockout mice ( 15 , 16 ). The myeloid-specific SFKs participate in wild-type and mutant KIT and FLT3 signaling and in the activation of the signal transducer and activator of transcription 5 (STAT5) and extracellular signal–regulated kinase (ERK) pathways downstream of BCR-ABL, and are involved in leukemogenesis in mouse model of BCR-ABL + B-ALL ( 17 – 23 ).

Over the last decade, leukemia stem cells (LSCs) have become recognized as key players in human AML pathogenesis as well as chemotherapy resistance and relapse ( 6 , 7 ). In two recent studies, the expression of a LSC gene signature was independently associated with poor clinical outcome ( 8 , 9 ). We have previously demonstrated chemotherapy resistance of human LSCs in an in vivo mouse model for human primary AML ( 10 ). Forced cell cycle entry increases the chemotherapy responsiveness of human primary LSCs and leads to decreased capacity for in vivo leukemia initiation ( 11 ). Recently, we determined that mRNA for hematopoietic cell kinase (HCK), a member of the Src family of nonreceptor tyrosine-protein kinases (SFKs), is overrepresented in primary human AML LSCs in comparison to normal hematopoietic stem cells (HSCs) ( 12 ).

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults, with ~28,000 new cases reported in the United States, Europe, and Japan in 2010 ( 1 ). The prognoses for AML other than acute promyelocytic leukemia remain poor. Although 70 to 80% of patients younger than 60 years and 40 to 50% of patients 60 years and older initially achieve complete remission after a combination chemotherapy regimen that includes cytarabine (AraC) and an anthracycline, the majority of patients ultimately experience disease relapse, with 5-year overall survival of 40 to 45% for patients below 60 years of age and <10% in patients 60 years and older ( 2 – 5 ). Therefore, prevention of relapse is crucial for improving long-term outcomes in AML.

Results

HCK expression and function in chemotherapy-resistant human AML LSCs Nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rgnull (NSG) mice lack multiple immune subsets, making them highly useful recipients for xenotransplantation models of human normal and malignant hematopoiesis (24). Consistent with our previous report that HCK transcript is overrepresented in human AML LSCs, HCK protein was expressed in human (h)CD45+ AML cells in the endosteal region of the bone marrow (BM) of human AML–engrafted NSG recipients, where Ki-67–negative, cell cycle–quiescent, chemotherapy-resistant human LSCs reside (Fig. 1A, left panel) (10–12). HCK expression persisted in hCD45+ AML cells that remained in the BM endosteal region after in vivo treatment with the chemotherapy agent cytarabine (AraC) (Fig. 1A, right panel). We therefore hypothesized that HCK is a promising molecular target for anti–human AML LSC therapy development. Fig. 1 HCK expression in human AML–engrafted BM and inhibition of HCK expression by shRNA. (A) Femurs from NSG recipients engrafted with human primary AML at baseline and after in vivo AraC administration were labeled with anti-CD45 (green) and anti-hHCK (red) antibodies. Nuclei were labeled by 4′,6-diamidino-2-phenylindole (DAPI; blue). (B) After lentiviral transduction, GFP+ hCD34+CD38− TF1a and GFP+ hCD45+ K562 cells were cultured in triplicate (1 × 104 cells per well). Photomicrographs of TF1a (upper left panels) and K562 (lower left panels) cells transduced with either control or HCK shRNA at day 7. Cells were harvested, and the numbers of viable cells were determined by flow cytometric beads enumeration (upper right and upper left panels). Dotted lines indicate starting number of sorted cells. *P = 0.0048 by two-tailed t test. To assess the functional role of HCK, we examined the effect of HCK down-regulation by short hairpin RNA (shRNA). We transduced the human AML cell line TF1a and the human chronic myeloid leukemia (CML) cell line K562 with HCK shRNA and control shRNA. Green fluorescent protein (GFP)–positive hCD34+CD38− cells were fluorescence-activated cell sorting (FACS)–purified and cultured in vitro for 7 days to determine the effect of HCK down-regulation on proliferation and survival. Reduced HCK expression led to significant inhibition of proliferation and reduction in the survival of TF1a cells (which expressed HCK before transduction), whereas the cell growth and viability of K562 cells [which do not express HCK (25)] were not affected (Fig. 1B and fig. S1, A and B). HCK shRNA did not affect the transcript levels of the SFKs Lyn and Src, reportedly involved in human AML pathogenesis (fig. S1C). These findings suggested that HCK expression is required for proliferation and survival of human AML cells and that pharmacologic inhibition of HCK may eliminate human AML LSCs.

Identification of small-molecule HCK inhibitors To identify inhibitors for HCK, we performed a combination of a high-throughput enzyme inhibition assay, in silico screening, and x-ray crystallography (Fig. 2A). First, using a kinase mobility shift assay, which rapidly and directly determined the ability of a compound to inhibit HCK, we screened 47,574 commercially available small molecules with a mean molecular weight of 329.6 and an ALogP of 3.32. As a primary screen, each compound (final concentration of 2 μM) was tested for its effect on HCK activity by the mobility shift assay (26) with 0.5 mM adenosine 5′-triphosphate (ATP). The mean z′ value (27) was 0.61 across all plates, indicating a stable assay performance. Among 47,574 compounds, 146 hit compounds inhibited HCK activity by more than 50%, and among these, we identified 12 compounds with IC 50 (half maximal inhibitory concentration) less than 1 μM. In parallel with the enzyme inhibition assay, we performed in silico screening that allowed rapid screening of a much larger pool of compounds. We used a computational approach combining ligand-based two-dimensional (2D) and 3D similarity search against known inhibitors of HCK and other SFKs, docking simulation with published HCK crystal structures, and a machine-based learning algorithm for structural fingerprints of known HCK inhibitors. From 8,927,055 commercially available small molecules, we selected 3088 candidate compounds. HCK inhibition in enzymatic assays was determined for these compounds, yielding three compounds with IC 50 less than 1 μM. Fig. 2 Identification of small-molecule HCK inhibitors. (A) Screening strategy integrating high-throughput enzyme assay, in silico prediction, and x-ray crystallography. (B) Upper left: Chemical structures of PP2 and RK-24466. Upper right: Superimposed model of RK-24466 with PP2. Lower left and right: Docking models of RK-24466 and PP2 with HCK (PDB ID: 1QCF). The phenoxy moiety of HCK where hydrophobic interaction with RK-24466 may occur is indicated by squares. (C) RK-24466 bound to the ATP binding site of HCK. RK-24466 and AMPPNP [PDB ID: 1AD5 (61)] are represented in ball and stick model, and carbon atoms are colored yellow and cyan, respectively. The nitrogen, oxygen, and sulfur atoms are colored blue, red, and green, respectively. Hydrogen bonds are shown as broken lines. (D) RK-24466 binding with HCK based on crystal structure determination, highlighting strategies for designing compounds with higher affinity with HCK. Broken lines indicate hydrogen bonds. Meshed regions show contact preferences of ligand atoms by Molecular Operating Environment (MOE; Chemical Computing Group). Green regions favor hydrophobic ligand atoms, and magenta regions favor hydrophilic ligand atoms. Blue and pink shading indicate positively and negatively charged regions of HCK, respectively. The pyrrolo-pyrimidine nucleus of RK-24466 is circled, with the seventh position indicated. The areas targeted for modification strategies are indicated by a rectangle. Of the 15 compounds, we chose RK-24466 for further evaluation because it was the only compound with IC 50 <100 nM against HCK (IC 50 = 7.7 nM) and had the most favorable chemical structure (without reactive moieties or structures associated with nonspecific protein reactivity) (28). A 3D structural model of RK-24466 showed similarity (ROCS ComboScore) to a known HCK inhibitor, PP2 (Fig. 2B). An in silico docking model of RK-24466 with HCK [Protein Data Bank (PDB) ID: 2C0I] showed that RK-24466 inserts into the ATP-binding pocket of HCK and forms hydrogen bonds with Met341 and Glu339. This is predicted to stabilize RK-24466’s interaction with the hinge region of HCK, resulting in competitive interference with ATP binding (29) (fig. S2). The Glide docking score of RK-24466 binding with HCK predicted that it has a higher binding affinity than the known SFK inhibitor PP2 (IC 50 = 630 nM) (Fig. 2B) (30). The higher potency of HCK inhibition by RK-24466 than by PP2 may also be a result of additional protein–ligand surface contacts, for instance, at the phenoxy moiety where there is a hydrophobic interaction. The crystal structure of HCK complexed with RK-24466 (Fig. 2C and table S1) confirmed that the compound binds within the ATP-binding pocket and forms hydrogen bonds with the hinge region. The phenoxy moiety is buried in a hydrophobic pocket formed by Met314, Val323, Leu325, Ile336, Ala403, Phe405, and Leu407, which may contribute to the potency of this compound against HCK. We next proceeded to evaluate the activity of RK-24466 against LSCs derived from AML patients (patient information summarized in Table 1). In an in vitro assay in which FACS-purified CD34+CD38− AML cells are cultured in the presence of human stem cell factor (SCF), thrombopoietin (TPO), and FLT3 ligand, the phenotype and in vivo function of CD34+CD38− LSCs are maintained, as demonstrated by leukemia initiation and generation of CD34+CD38+ cells and CD34− cells in NSG recipients (fig. S3). After exposure of the cells to the test compounds for 3 days, we harvested the cells for flow cytometric determination of viability and proliferation. In vitro, RK-24466 potently inhibited CD34+CD38− human primary AML cell proliferation to a greater extent than did the known SFK inhibitor PP2 (fig. S4). However, pharmaceutical use of RK-24466 was expected to be difficult because of its poor aqueous solubility. Therefore, we went on to explore modifications of RK-24466 to obtain HCK inhibitors with superior chemical profile. Table 1 Patient information. FAB, French-American-British classification; NOS, not otherwise specified; MRC, myelodysplasia-related changes; Ida, idarubicin; AraC, cytarabine; MIT, mitoxantrone; HiDAC, high-dose cytarabine; MEC, mitoxantrone, etoposide, and cytarabine; CAG, cytarabine, aclarubicin, and granulocyte colony-stimulating factor; BHAC, behenoyl cytarabine; DMP, daunorubicin, 6-mercaptopurine, and prednisolone; DNR, daunorubicin; 6-MP, 6-mercaptopurine; CA, cytarabine and aclarubicin; FLAG, fludarabine, cytarabine, and granulocyte-colony stimulating factor; SCT, stem cell transplantation; GO, gemtuzumab ozogamicin; na, not available. View this table: There remained open spaces in the crystal structure of RK-24466–bound HCK, which may accommodate new substituents to improve the aqueous solubility as well as the potency of RK-24466 (Fig. 2D). One of these spaces was between the 7-substituent of the pyrrolo-pyrimidine nucleus and Asp348, which hydrogen-bonds with the ribose moiety of ATP (Fig. 2C). The corresponding space has been used to increase the potency and specificity of inhibitors against Lck, another SFK (31). Another available space extends toward Asp404 in the universally conserved Asp-Phe-Gly (DFG) motif in the activation segment of HCK. To use these two spaces, we synthesized seven compounds that are potentially better ligands for HCK than RK-24466 (table S2 and fig. S5). Of these, RK-20449 [compound 13 (31); Fig. 3A] demonstrated the most potent inhibition of HCK, with an IC 50 of 0.43 nM. Fig. 3 Crystal structure of HCK in complex with RK-20449, the leading candidate inhibitor. (A) Chemical structure of RK-20449. (B to D) Crystal structures of RK-24466 and RK-20449 bound to HCK, demonstrating an additional interaction that leads to the improved affinity of RK-20449 for HCK. Broken lines (yellow) indicate favorable interactions between each compound and HCK, with the interaction distances in angstrom units. (B) RK-24466 (green) forms hydrogen bonds with Met341, Glu339, and Thr338 in the hinge region of HCK (blue), enhancing its binding within the ATP-binding pocket, and the phenoxy moiety interacts with the hydrophobic pocket featuring Phe405. (C) In addition to the interactions with the hinge region and the hydrophobic pocket, the methylpiperazine group of RK-20449 (green) interacts with Asp348 of HCK (blue), leading to increased binding affinity. (D) An alternative view [45° rotation from (C)] showing how the acral tertiary amine nitrogen in the methylpiperazine group of RK-20449 interacts with Asp348 of HCK. (E) RK-20449 was screened against a panel of 106 human kinases, which are represented by circles on the kinase dendrogram. Kinases with IC 50 less than 1 μM are shown by red circles, with circle size indicating potency of inhibition. Kinases with IC 50 greater than 1 μM are indicated by small green circles. The complete data set is shown in table S3. The kinase dendrogram was adapted and is reproduced with permission from Cell Signaling Technology Inc. (http://www.cellsignal.com). To examine the mechanism of highly potent HCK inhibition by RK-20449, we determined the crystal structures and analyzed the binding modes of these seven compounds with HCK. RK-20444, RK-20445, and RK-20446 have phenyl groups connected through N-methylurea, amide, and urea linkers, respectively, which extend toward the DFG motif (fig. S6, A and B). In the structures of HCK complexed with these three compounds, the conformations of the Phe405 side chain vary. Correspondingly, the phenyl groups of the three compounds assume different orientations, but none of them reaches the Phe405 side chain. In addition, the side-chain conformation of Asp404 in the RK-20446–bound HCK structure is different from those in the RK-20444– and RK-20445–bound HCK structures, presumably to avoid a steric clash with the urea linker. These findings explain why these three compounds exhibit increased IC 50 values, as compared with the compounds with diphenylether, such as RK-24466 (table S2). Next, we determined the crystal structures of HCK bound with RK-20448, RK-20449, RK-20450, and RK-20451, four compounds with 7-substituents extending toward Asp348. Although all four compounds maintain hydrogen-bond interactions with the hinge region of HCK similar to RK-24466 (Fig. 3B), RK-20450 and RK-20451, with more hydrophilic oxane and methylpiperazine groups, respectively, do not interact with Asp348 (fig. S6C). In RK-20448, with the attachment of methylpiperazine to cyclohexane in the cis configuration, the side chain moves away from the negatively charged surface of HCK, precluding its interaction with Asp348 (fig. S6, C and D). In contrast, the trans configuration of methylpiperazine attached to cyclohexane in RK-20449 allows electrostatic interaction of the basic acral tertiary amine nitrogen atom with the acidic side chain of Asp348 (3.2 Å) (Fig. 3, C and D, and fig. S6, C to E). This interaction is expected to be stronger than the hydrogen bond between Asp348 and the ATP ribose moiety (3.4 Å) (fig. S6D), resulting in higher binding affinity and superior potency of inhibition of HCK by RK-20449. To examine the spectrum of inhibition by RK-20449, we determined the IC 50 against a panel of 106 human kinases. The findings are summarized in Fig. 3E and table S3. We found that RK-20449 showed an IC 50 <100 nM against seven kinases, all of which were SFKs (FYN, HCK, LCK, LYNa, LYNb, SRC, and YES), consistent with the high level of amino acid sequence homology among the SFKs. Four non-SFK kinases (EGFR, RET, PDGFRα, and FLT3) were inhibited at IC 50 of 100 nM to 1 μM. RK-20449 did not highly inhibit the other kinases tested (IC 50 >1 μM). Because constitutive activation of FLT3, most frequently in the form of FLT3−ITD+ mutations, is commonly associated with normal karyotype AML, we examined the significance of HCK expression in FLT3−ITD+ human AML by HCK shRNA. In two FLT3−ITD+ human AML cell lines, MV-4-11 and Molm13, we found that inhibition of HCK by shRNA reduced both viable cell number and viability in the remaining cells (fig. S7), suggesting that HCK inhibition by RK-20449 may be effective against AML with activating mutations of FLT3.

In vitro inhibition of human AML LSC by RK-20449 We then evaluated the activity of RK-20449 in the in vitro LSC culture assay. Compared with SFK inhibitors PP2, PP20, and RK-24466; multikinase inhibitors PP121 and PI-103; and a mammalian target of rapamycin (mTOR) inhibitor, RK-20449 demonstrated the highest activity against human AML CD34+CD38− cells (Fig. 4, A and B) (32). RK-20449 showed dose-dependent inhibition of LSC proliferation with IC 50 <1 μM in 17 of 25 AML cases that we examined, exhibiting higher in vitro activity against human AML CD34+CD38− cells than PP2 (IC 50 <1 μM in 0 of 18 cases) and RK-24466 (IC 50 <1 μM in 7 of 21 cases) (Fig. 4C, table S4, and Table 1). Fig. 4 Potent in vitro activity of RK-20449 against human AML LSCs. (A and B) Potency of various SFK inhibitors, RK-24466, and RK-20449 against hCD34+CD38− cells in vitro. Representative (A) photomicrographs and (B) flow cytometry plots of sorted hCD34+CD38− cells exposed to 300 nM PP2, RK-24466, RK-20449, PP121, PP20, PI-103, or rapamycin. (C) Inhibition of cell proliferation by (left) PP2 (n = 18), (middle) RK-24466 (n = 21), and (right) RK-20449 (n = 25). For each dose, the numbers of viable cells, determined by flow cytometry bead enumeration, are represented as fraction of viable cells in the DMSO alone–treated group. Broken line indicates 50% reduction in the number of viable cells compared with the DMSO-alone group. (D) RK-20449 inhibition of phosphorylation of SFK-activating tyrosine and downstream intermediates. P-flow scores are obtained by log 2 transformation of the fold change of the mean fluorescent intensity (MFI). Fold change was calculated by dividing the MFI of RK-20449– or rapamycin-treated cells by the MFI of DMSO only–treated cells. Case #1 RK-20449 n = 3, rapamycin n = 2; case #2 RK-20449 n = 1, rapamycin n = 1; case #3 RK-20449 n = 6, rapamycin n = 2; case #4 RK-20449 n = 3, rapamycin n = 3. Red, green, purple, and blue bars show P-flow scores (mean ± SEM) after 1 μM RK-20449 for cases #1, 2, 3, and 4, respectively. Gray bars, P-flow scores (mean ± SEM) after 1 μM rapamycin for cases #1 to 4 combined. Next, we examined the effect of RK-20449 binding to HCK on the phosphorylation of the activating tyrosine residue in the homologous SFK protein kinase domain (pY418) and further downstream mediators in four AML patient cases (representative flow cytometry plots are shown in fig. S8). P-flow score, representing the change in phosphorylation status compared with dimethyl sulfoxide (DMSO) alone–treated cells, showed consistent suppression of SFK, S6, p4E-BP1, and STAT5 phosphorylation in hCD45+ total AML cells and in hCD34+CD38− LSCs with in vitro exposure to RK-20449 (Fig. 4D). In contrast, rapamycin suppressed phosphorylation of the TORC1 substrate S6 but not of pSRC and pSTAT5, consistent with previous reports (33–35). We then compared the effect of 100 and 300 nM RK-20449 at 24 and 48 hours on human AML LSCs and human cord blood (CB) CD34+CD38− HSCs in vitro. Although there was some reduction in CB HSC cluster size with higher concentration of RK-20449, the effect was more pronounced in AML LSCs, suggesting relative sparing of normal HSCs (fig. S9A). To determine the effect of RK-20449 treatment on LSC function, we evaluated the in vivo leukemia initiation capacity of in vitro RK-20449–treated LSCs through NSG transplantation. After 3 days in culture with RK-20449, viable hCD45+ cells were FACS-purified and transplanted into irradiated newborn NSG mice. We found that recipients of LSCs that had been treated in vitro with RK-20449 showed attenuated AML engraftment [representative flow cytometry plots of peripheral blood (PB) engraftment are shown in fig. S9B]. In addition, recipients of RK-20449–treated LSCs showed significantly improved survival compared with recipients of DMSO only–treated LSCs, consistent with reduced PB engraftment of hCD45+ AML cells (fig. S9, C and D). These data indicated reduced leukemia initiation capacity with in vitro exposure to RK-20449. When CB HSCs were similarly exposed to RK-20449 and transplanted into newborn NSG mice, we observed long-term, multilineage human hematopoietic reconstitution in the recipient BM, showing that HSCs retained in vivo hematopoietic repopulation capacity after exposure to RK-20449 (fig. S9, E to H).