Discovery and characterization of a novel highly potent and selective type II native and drug-resistant V299L mutant BCR-ABL inhibitor (CHMFL-ABL-039) for Chronic Myeloid Leukemia (CML)

ABSTRACT

BCR fused ABL kinase is the critical driving oncogene for chronic myeloid leukemia (CML) and has been extensively studied as the drug discovery target in the past decade. The successful introduction of tyrosine kinase inhibitors (TKI) such as Imatinib, Dasatinib and Bosutinib has greatly improved the CML patient survival rate. However, upon the chronic treatment, a variety of TKI resistant mutants, such as the V299L mutant which has been found in more and more patients with the high-throughput sequencing technology, are observed, although the incidence is still considered rare compared to the more prevalent gatekeeper T315I mutant. However, with the progress of the precision medicine concept, the rare mutation (or the orphan drug target) has attracted more and more attention. Here we report a novel type II BCR-ABL kinase inhibitor, CHMFL-ABL-039, which not only displayed great potency (IC₅₀: 7.9 nM) and selectivity (S score (1) = 0.02) against native ABL kinase among other kinases in the kinome, but also exhibited great potency (IC₅₀: 27.9 nM) and selectivity against Imatinib-resistant V299L mutant among other frequently observed ABL kinase mutants. CHMFL-ABL-039 has demonstrated greater efficacies than Imatinib regarding to the anti-proliferation, inhibition of the signaling pathway, arrest of cell cycle progression, induction of apoptosis in vitro and suppression of the tumor progression in vivo in the native and V299L mutated BCR-ABL kinase-driven cells/xenograft models. It would be a useful pharmacological tool to study the TKI resistant ABL V299L mutant-mediated pathology and provide a potential precise treatment approach for this orphan CML subtype in the precision medicine era.

Introduction

Chronic myeloid leukemia (CML), which accounts for about 15% of the adult leukemias, is a white blood cell disorder and affects about 1–2 person per 100,000 every year¹. The BCR gene and ABL kinase gene fusion, resulting from the reciprocal translocation of t(9;22) chromosome (Ph+ chromosome) has been identified as the critical driving force of the pathogenesis of CML². The majority of CML patients express a 210 kD BCR-ABL (p210 BCR-ABL) fusion protein³. Fusion of the BCR gene and ABL kinase gene results in the constitutive activation of ABL kinase, which is essential for the subsequent activation of the mitogenic signaling pathways⁴, suppression of the apoptosis⁵ as well as the enhanced adhesive capability to stromal cells for the proliferation⁶. Thus, inhibition of the BCR-ABL kinase activity has been considered as an effective approach to control the CML. The first BCR-ABL kinase inhibitor Imatinib, which adopts a special type II binding mode, featured by a DFG-out inactive conformation, has achieved great success in the clinic and validated this approach¹. However, upon the chronic clinic treatment with Imatinib, a variety of drug resistance was observed, among them the most prevalent resistant mechanism was drug-acquired resistance induced by the point mutations in the kinase domain⁷. Besides the gatekeeper T315I resistant mutant, about 30 other mutants have been detected from the patients such as F317L, H369P, Y253H, and V299L, etc.. Although V299L has been considered as the rare mutations, it is resistant to Imatinib, Bosutinib⁸, and Dasatinib⁹ but still sensitive to the secondary generation BCR-ABL inhibitor Nilotinib¹⁰. However, Nilotinib also bears strong activities against other important kinases such as KIT kinase, LCK kinase and ZAK kinase, etc., which induced some contraindications¹¹. With the progress of the precision medicine for the personalized treatment, a highly selective BCR-ABL kinase which could selectively overcome some special drug-resistant mutant is highly desired from the clinic. Here we report a novel type II BCR-ABL kinase inhibitor which displayed better potencies and selectivity than Imatinib and could selectively overcome V299L mutant among other drug-resistant mutants.

Materials and methods

Chemicals

Imatinib and Nilotinib were purchased from Haoyuan Chemexpress Inc. (Shanghai, China). CHMFL-ABL-039 (N-(4-(2-((4-(cyclopropanecarboxamido)phenyl)amino)-2-oxoethyl)phenyl)-4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide) was synthesized in the lab, and the synthetic procedure is described in the supplementary information.

Cell lines

The human cancer cell lines MEC-1, MEG-01, MV4-11, HL-60, KU812, K562 and U937 cell lines were purchased from American Type Culture Collection (Manassas, VA). MOLM-14 cell line was provided by Dr. Scott Armstrong, Dana Farber Cancer Institute (DFCI), Boston, MA. KU812, HL-60, K562, MEG-01, MOLM-14, U937 and all the isogenic BaF3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% FBS and supplemented with 1% penicillin/streptomycin and 2% L-glutamine. MEC-1 and MV4-11 were cultured in IMDM supplemented with 10% FBS, 2% L-glutamine and 1% pen/strep.

Antibodies

β-Actin (Sigma–Aldrich) served as a loading control. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): Phospho-CrkL (Tyr207) antibody (3181), CrkL (32H4) Mouse mAb (3182), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) Rabbit mAb (4377) and p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (4695), Akt (pan)(C67E7) Rabbit mAb (4691), Phospho-Akt (Thr308) (244F9) Rabbit mAb (4056), Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (4060), STAT5 (3H7) Rabbit mAb (9358), Phospho-STAT5 (Tyr694)(C71E5) Rabbit mAb (9314), Phospho-c-Abl (Tyr245)(73E5) Rabbit mAb (2868), c-Abl antibody (2862).

ABL and ABL-V299L protein purification

A construct encoding c-ABL residues 229–500 with a His tag was cloned into baculovirus expression vector pFAST-HTA (Invitrogen). ABL with V299L mutation was obtained by site mutation based on wt vector. The proteins were expressed as described.¹²

Kinase biochemical assay

The ADP-Glo^TM kinase assay (Promega, Madison, WI) was used to screen CHMFL-ABL-039 and Imatinib for its ABL-WT and ABL-V299L inhibition effects. The kinase reaction system contains 4.95 μL ABL-WT (1.5 ng/μL), ABL-V299L (4 ng/μL), 0.55 μL of serially diluted drug and 5.5 μL substrate Abltide (0.2 μg/μL) (Promega, Madison, WI) with 50 μM ATP (Promega, Madison, WI). The reaction was performed as described.¹³

Binding Kd examination

The Kd was measured using the Monolith NT.115 from Nanotemper Technologies. Proteins were fluorescently labeled according to the manufacturer’s protocol, and the reaction was performed as described.¹⁴

Anti-proliferation assay

Cells were grown in 96-well culture plates (2500–3000/well). For adherent cell lines, compounds of various concentrations were added into the plates after cells were cultured for 12 h. Cell proliferation was determined after treatment with compounds for 72 h. GI₅₀ values were calculated as described.¹⁵

Colony formation assay

BaF3-BCR-ABL-V299L, KU812, MEG-01, and K562 cells were dispensed into individual wells of six-well tissue culture dishes with a density of 3000 cells per well. Cells were maintained 1.7 mL growth media was combined with 0.3 ml of 3% agarose solution and plated on top of the bottom layer which was composed of 1 mL of 3% agarose and 1 mL cell culture medium. Cells were maintained in a humidified 5% CO₂ incubator at 37°C for 15 days, and continuously treated with serially diluted CHMFL-ABL-039, Nilotinib or Imatinib in a soft agar medium. On the 15th day, the numbers of colonies in each well were counted and each measurement was performed in triplicate.

Signal transduction pathway study

BaF3-BCR-ABL-V299L, KU812, MEG-01, and K562 cells were treated with DMSO, CHMFL-ABL-039 and Imatinib for 2 h before immunoblotting. Cells were then collected and lysed. CrkL (32H4), Phospho-CrkL (Tyr207), Phospho-c-Abl (Tyr245), c-Abl, Akt, Phospho-Akt (Thr308), Phospho-Akt (Ser473), STAT5, Phospho-STAT5 (Tyr694), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2) were used for immunoblotting. β-Actin (Sigma–Aldrich) served as a loading control.

Cell cycle progression test

BaF3-BCR-ABL-V299L, MEG-01, KU812, and K562 cells were treated with DMSO, CHMFL-ABL-039, Nilotinib and Imatinib for 24 h. The cells were fixed in 70% cold ethanol and incubated at −20°C overnight, then stained with PI/RNase staining buffer (BD Pharmingen). Flow cytometry was performed using a FACS Calibur (BD), and results were analyzed by ModFit software.

Apoptosis test

MEG-01, BaF3-BCR-ABL-V299L, KU812, and K562 cells were treated with DMSO, CHMFL-ABL-039 and Imatinib for 24 h before cells were harvested. Cells were then washed in PBS and lysed in cell lysis buffer. PARP, Caspase-3, GAPDH antibodies were used for immunoblotting. FACS PI/Annexin V double staining analysis was performed using a FITC Annexin V Apotosis Detection Kit I (BD), and results were analyzed by Flowjo software.

Culture of primary human CML cells

The peripheral blood samples were obtained from newly diagnosed CML patients. Mononuclear cells were isolated by the Ficoll-Paque method from peripheral blood samples. Cells were tested in liquid culture (RPMI1640 medium, supplemented with 20% FBS, 1% penicillin/streptomycin) in the presence of different concentrations of CHMFL-ABL-039. All studies performed with human specimens were done with approval from the First Hospital of Anhui Medical University. Ethical approval and informed consent was obtained for the use of human samples.

CD34+ progenitor cells isolation

The peripheral blood were obtained from the First Hospital of Anhui Medical University. The mononuclear cells were isolated by Ficoll density gradient, and then we isolated human CD34+ cells from mononuclear cells using a CD34+ cell isolation kit (Miltenyi Biotec) according to the manufacturers protocol. The purity of CD34+ cells were tested in liquid culture (RPMI1640 medium, supplemented with 20% FBS, 1% penicillin/streptomycin, human SCF (100 ng/mL), human Flt-3 ligand (100 ng/mL), human TPO (20 ng/mL), human IL-3 (20 ng/mL), human IL-6 (20 ng/mL)) in the presence of different concentrations of CHMFL-ABL-039.

In vivo animal studies

Five weeks old female nu/nu mice were purchased from the Shanghai Experimental Center, Chinese Science Academy (Shanghai, China). All animals were housed in a specific pathogen-free facility and used according to the animal care regulations of Hefei Institutes of Physical Science Chinese Academy of Sciences. Prior to implantation, cells were harvested during exponential growth. For BaF3-BCR-ABL-V299L and K562 allograft, one million cells in PBS were formulated as a 1:1 mixture with Matrigel (BD Biosciences) and injected into the subcutaneous space on the right flank of nu/nu mice. Daily i.p. administration was initiated when tumors had reached a size of 200 to 400 mm³. Animals were then randomized into treatment groups for efficacy studies. CHMFL-ABL-039 was delivered daily in a HKI solution (0.5% Methocellulose/0.4% Tween 80 in dd H₂O) by i.p. injection. A range of doses of CHMFL-ABL-039 or its vehicle were administered, as indicated in figure legends, Imatinib was set as a control. Body weight and tumor growth were measured daily after CHMFL-ABL-039 treatment. Tumor volumes were calculated as follows: tumor volume (mm³) = [(W²× L)/2] in which width (W) is defined as the smaller of the two measurements and length (L) is defined as the larger of the two measurements.

Results

Characterization of CHMFL-ABL-039 as highly potent and selective ABL wt/V299L mutant inhibitor

During characterization of our in-house generated structure focused type II kinase inhibitor library we found that inhibitor CHMFL-ABL-039 (Structure shown in Figure 1a) exhibited potent anti-proliferative activities against BCR-ABL transformed BaF3 cells BaF3-BCR-ABL (GI₅₀: 0.059 μM) meanwhile exhibited good selectivity over parental BaF3 cells (GI₅₀: 8.3 μM) which was similar to Imatinib and Nilotinib and indicated that it might be a potent ABL kinase inhibitor. (Table 1) Further testing in a panel of ABL mutants transfected BaF3 cells showed that it was more potent against V299L mutant (GI₅₀: 0.009 μM) than Imatinib (GI₅₀: 0.25 μM) and Nilotinib (GI₅₀: 0.036 μM)). It also exhibited moderated potencies to other mutants such as F317I/L, H369P, M351T and Y253H which was either better or equally potent as Imatinib but less potent to Nilotinib. However, the most prevalent drug-resistant T315I mutant was resistant to both the Imatinib, Nilotinib and CHMFL-ABL-039. In order to further confirm CHMFL-ABL-039’s on-target effect, we then tested it against the purified ABL kinase proteins. It could potently inhibit ABL wt kinase’s activity (IC₅₀: 7.9 ± 2.5 nM) as well as ABL V299L mutant protein (IC₅₀: 27.9 ± 11.8 nM) in the ADP-glo biochemical Assay. (Figure 1b) In addition, it exhibited a binding Kd of 54.7 nM to ABL wt and 228 nM to ABL V299L mutant, respectively. (Figure 1c) Give the fact that most of the type II ABL kinase inhibitors could also potently inhibit KIT kinase, we then examined CHMFL-ABL-039 against a panel of KIT wt/mutants transfected BaF3 cells. The results showed that unlike Imatinib and Nilotinib, it showed great selectivity over KIT kinase comparing to ABL kinase and did not display any apparent activity to KIT kinase and mutants. (Table 1) In order to further understand its selectivity profile against other protein kinases in the kinome, we then subjected it to the KinomeScan^TM profiling platform which examined the inhibitors relative binding capability against 472 kinases/mutants.¹⁶ At the concentration of 1 μM it showed a selectivity score (1) of 0.02, which indicated a good selectivity profile. (Figure 1d and Supplemental Table 1) Besides the ABL kinase, it also displayed the strong binding to BRAF V600E, CSF1R, DDR1/2, KIT, PDGFRα/β which showed percent control scores less than 1 (indicating a binding Kd might less than 1000 nM). Given the fact the KinomeScan is a binding assay and sometimes may not really reflect the inhibitory activity such as in the KIT kinase case aforementioned, we then tested these targets in the BaF3 transfected assays. The results showed that CHMFL-ABL-039 was also potent against PDGFRα (GI₅₀: 0.003 μM) and PDGFRβ (GI₅₀: 0.083 μM) but not BRAF V600E, DDR1/2. (Table 1)

Figure 1.

Biochemical characterization of CHMFL-ABL-039. (a) Chemical structure of CHMFL-ABL-039. (b) ADP-glo enzymatic IC₅₀ examination of CHMFL-ABL-039. (c) Binding Kd determination of CHMFL-ABL-039 with MST. (d) Kinome-wide selectivity profiling of CHMFL-ABL-039 with KinomeScan^TM technology.

Table 1.

Anti-proliferative effect of CHMFL-ABL-039 against a panel of kinase transformed BaF3 cells^a.

Cell line (GI50: μM)	CHMFL-ABL-039	Imatinib	Nilotinib
BaF3	8.3 ± 0.2	>10	4.9 ± 0.074
BaF3-BCR-ABL	0.059 ± 0.003	0.24 ± 0.031	0.013 ± 0.008
BaF3-BCR-ABL-F317I	0.97 ± 0.023	0.91 ± 0.044	0.032 ± 0.002
BaF3-BCR-ABL-F317L	0.31 ± 0.011	0.85 ± 0.057	0.074 ± 0.006
BaF3-BCR-ABL-H369P	0.32 ± 0.015	1.1 ± 0.058	0.033 ± 0.004
BaF3-BCR-ABL-M351T	0.14 ± 0.011	0.5 ± 0.026	0.005 ± 0.001
BaF3-BCR-ABL-T315I	5.2 ± 0.45	7.6 ± 0.32	>10
BaF3-BCR-ABL-V299L	0.009 ± 0.0005	0.25 ± 0.01	0.036 ± 0.002
BaF3-BCR-ABL-Y253H	0.12 ± 0.01	1.5 ± 0.058	0.098 ± 0.001
BaF3-tel-KIT	9.1 ± 0.18	0.44 ± 0.003	0.19 ± 0.012
BaF3-tel-KIT-D816H	9 ± 0.073	1.5 ± 0.031	0.57 ± 0.006
BaF3-tel-KIT-D816V	4.6 ± 0.02	3.6 ± 0.05	>10
BaF3-tel-KIT-L567P	1.9 ± 0.063	0.068 ± 0.004	0.041 ± 0.003
BaF3-tel-KIT-N822K	8 ± 0.05	0.92 ± 0.017	0.024 ± 0.001
BaF3-tel-KIT-T670I	>10	>10	>10
BaF3-tel-KIT-V559D	2.3 ± 0.019	0.076 ± 0.002	0.014 ± 0.003
BaF3-tel-KIT-V559D-V654A	4 ± 0.005	1.1 ± 0.006	0.37 ± 0.005
BaF3-tel-KIT-V654A	1.1 ± 0.047	0.88 ± 0.009	0.002 ± 0.0002
BaF3-BRAF-V600E	>10	>10	0.89 ± 0.004
BaF3-tel-CSF1R	2.3 ± 0.17	0.16 ± 0.058	0.44 ± 0.005
BaF3-tel-DDR1	4.2 ± 0.036	8.7 ± 0.041	3.6 ± 0.017
BaF3-tel-DDR2	>10	7.7 ± 0.13	>10
BaF3-tel-PDGFRα	0.003 ± 0.0008	0.046 ± 0.003	0.024 ± 0.002
BaF3-tel-PDGFRβ	0.083 ± 0.001	0.021 ± 0.002	0.033 ± 0.004

^aall GI50s were obtained from the the triple test

Binding mechanism illustration of CHMFL-ABL-039 for ABL wt/V299L

In order to better understand CHMFL-ABL-039’s binding mechanism, we then docked it to ABL wt (PDB ID: 2HYY). (Figure 2a) Unsurprisingly, it adopted a typical type II binding mode to the ABL kinase and formed a hydrogen bond between Met318 and aminopyridine in the hinge binding area¹⁷. Two canonical hydrogen bonds of the type II inhibitors was formed between the amide bond in the inhibitor and Glu286 located in the cHelix as well as the Asp381 in the DFG motif. The trifluromethylbenzyl piperazine moiety occupied the hydrophobic pocket generated by the DFG motif out shifting¹⁸. Comparably, besides the similar two hydrogen bonds formed by the Glu286 and Asp381 as well as the newly generated hydrophobic pocket occupation, Imatinib formed one hydrogen bond with its pyridine moiety in the hinge binding area. (Figure 2b) In addition, Imatinib formed another hydrogen bond with gatekeeper residue of ABL The315, which lacks in the CHMFL-ABL-039 due to the acylamide replacement of the NH linkage in the Imatinib. Furthermore, this acylamdie together with the benzene ring and amide bond for the typical hydrogen bond formation formed a U shape, which generated larger space enough to accommodate the relatively large size Leu299 mutant residue. (Figure 2c) While for Imatinib the NH linkage and the benzene ring as well as the amide bond formed a linear shape which generated the steric hindrance for the Leu299 mutant. (Figure 2d) This explained why ABL V299L mutant was more sensitive to CHMFL-ABL-039 than Imatinib.

Figure 2.

Structural binding mechanism of CHMFL-ABL-039 against ABL wt and V299L mutant. (a) Docking of CHMFL-ABL-039 to ABL wt (PDB ID 2HYY). (b) Docking of Imatinib to ABL wt (PDB ID: 2HYY). (c) Docking of CHMFL-ABL-039 to ABL V299L (homology model made from PDB ID: 2HYY). (d) Docking of Imatinib to ABL V299L (homology model generated from PDB ID: 2HYY).

Characterization of CHMFL-ABL-039 in ABL wt and V299L mutant driven cells

We then tested anti-proliferative effects of CHMFL-ABL-039 against a panel of BCR-ABL dependent and independent established cell lines. (Table 2) Interestingly, it was as potent as Nilotinib and was 6–10 fold more sensitive than Imatinib to BCR-ABL driven cancer cell lines including K562, KU812, and MEG-01. While for the BCR-ABL independent cell lines such as HL-60, MOLM-14, MV4-11 and U937, etc., it displayed a great selectivity window comparing to the BCR-ABL driven cell lines. In addition, neither CHMFL-ABL-039 nor Imatinib/Nilotinib showed any apparent inhibitory activity against normal cells such as CHL and CHO. CHMFL-ABL-039 also displayed anti-proliferative effect against BCR-ABL wt positive CML patients derived primary cells although it was slightly less potent than Imatinib and Nilotinib. This might be due to the heterogeneous genetic backgrounds of the patients derived cells. Furthermore, it also exhibited a a similar range of anti-proliferative effect against CD34+ progenitor cells isolated from normal PBMC cells, which indicated that there is no general cytotoxicity of these compounds. (Table 2 and Supplemental Figure 2) In the anti-colony formation assay, CHMFL-ABL-039 could almost completely block the colon formation of K562 and BaF3-BCR-ABL-V299L cells at the concentration of 0.1 μM, which was much better than Imatinib at the similar concentration. In the KU812 and MEG-01 cells, it displayed a similar effect as Imatinib. (Figure 3a and Supplemental Figure 3)

Table 2.

Anti-proliferative effect of CHMFL-ABL-039 against a panel of established cancer cell lines^a.

Cell line	Cell type	CHMFL-ABL-039 (GI50: μM)	Imatinib (GI50: μM)	Nilotinib (GI50: μM)
K562	CML(BCR-ABL)	0.010 ± 0.001	0.32 ± 0.007	0.045 ± 0.002
KU812	CML(BCR-ABL)	0.007 ± 0.0004	0.087 ± 0.004	0.008 ± 0.0005
MEG-01	CML(BCR-ABL)	0.006 ± 0.0002	0.049 ± 0.005	0.007 ± 0.0003
HL-60	AML(PML-RARα)	9.3 ± 0.2	>10	>10
MOLM-14	AML(FLT3-ITD)	2.6 ± 0.032	>10	9.5 ± 0.029
MV4-11	AML(FLT3-ITD)	9.4 ± 0.34	5.8 ± 0.04	1.3 ± 0.047
U937	AML	4.6 ± 0.036	>10	1.2 ± 0.032
CHL	Normal Chinese hamster lung cell	>10	>10	7.4 ± 0.051
CHO	Normal Chinese hamster ovary cell	>10	>10	>10
Patient 1	CML(BCR-ABLwt +)	0.498	0.167	0.099
Patient 2	CML(BCR-ABLwt +)	1.62	1.55	0.968
CD34+ PBMC	Normal cell	4.87	6.38	2.26

^a all GI50s were obtained from triple test (except Patient cell lines and CD34+ PBMC cell lines)

Figure 3.

Characterization of Cellular effect of CHMFL-ABL-039. (a) Anti-colony formation effect of CHMFL-ABL-039 against K562, KU812, MEG-01, and BaF3-BCR-ABL-V299L cells. (b) Signaling pathway examination of CHMFL-ABL-039 in K562, KU812, MEG-01 and BaF3-BCR-ABL-V299L cells. (c) Cell cycle progression examination of CHMFL-ABL-039 in K562, KU812, MEG-01, and BaF3-BCR-ABL-V299L cells. (d) Apoptotic effect of CHMFL-ABL-039 in K562, KU812, MEG-01, and BaF3-BCR-ABL-V299L cells.

In order to further confirm on target effect of the CHMFL-ABL-039, we then examined its effect on the ABL mediated signaling pathways. Not surprisingly, CHMFL-ABL-039 could dose dependently inhibit the ABL Y245 phosphorylation and the subsequent downstream signaling mediators such as pSTAT5 Y694, pERK T202/204 in K562, KU812, MEG-01, and BaF3-BCR-ABL-V299L. (Figure 3b) However, unlike Imatinib, which has been reported to not affect phosphorylation of CRKL, CHMFL-ABL-039 could also dose dependently and potently block phosphorylation of CRKL Y207 site, which might contribute to the better anti-proliferative effect of CHMFL-ABL-039 than Imatinib since CRKL is one of the direct downstream substrates of ABL kinase in the BCR-ABL driven CML cell lines. In addition, at 24 h, CHMFL-ABL-039 could effectively arrest cell cycle at the G0/G1 phase. Furthermore, this drug also induced the late stage apoptosis in these tested cell lines in a dose-dependent manner by examining the cleavage PAPR and Caspase-3 and this was also confirmed in the more sensitive Annexin V flow cytometry analysis. (Figure 3(c, d) and Supplemental Figure 4)

CHMFL-ABL-039 suppressed the tumor progression in the ABL wt/V299L mutant cells mediated xenograft models

We next tested CHMFL-ABL-039 anti-tumor efficacy in vivo. We first evaluated the PK properties of CHMFL-ABL-039 in mice. i.p. administration of drug was chosen for the in vivo efficacy study since it exhibited better drug exposure (AUC_0-t: 6168 hr*ng/ml) than i.v. and p.o. (Supplemental Table 2). In both K562 cell and BaF3-BCR-ABL-V299L mutant cells inoculated mouse xenograft model, CHMFL-ABL-039 did not exhibit any apparent general toxicity and did not affect the mouse weight upon IP administration. (Figure 4a) CHMFL-ABL-039 could dose dependently suppress the tumor progression for both models at the dosage of 25, 50 and 100 mg/kg. (Figure 4b) Interestingly, even 25 mg/kg daily administration of CHMFL-ABL-039 could achieve 77% tumor growth inhibition (TGI) in K562 mediated models and 100 mg/kg dosage even almost completely eliminated the tumor (TGI: about 100%). (Figure 4c and Supplemental Figure 5a) However, with this IP administration, Imatinib only achieved 38% of TGI. In the Imatinib insensitive BaF3-BCR-ABL-V299L mutant cells mediated xenograft model, 25 mg/kg dosage of CHMFL-ABL-039 displayed similar efficacy as 100 mg/kg Imatinib (TGI: 44.6%). One hundred mg/kg dosage could achieve 94.4% TGI which was much better than Imatinib (TGI: 44.0%). In order to confirm the on-target effect of CHMFL-ABL-039 in the tumor tissues, we then isolated the tumor cells from the tumor tissues after sacrificed the animal at the final drug treatment day and examined the ABL mediated signaling pathways. The results showed that all of the pABL, pSTAT5, pCRKL and pERK were inhibited as expected in the dose-dependent manner, and a similar trend was observed as to the in vitro phenotype. (Figure 4d) Immunohistochemistry staining also demonstrated a dose dependent anti-proliferative (Ki67) and apoptosis induction (TUNEL) effect in the tumor tissues. (Supplemental Figure 5b)

Figure 4.

CHMFL-ABL-039’s antitumor efficacy in K562 and BaF3-BCR-ABL-V299L cells inoculated xenograft mouse model. Female nu/nu mice bearing established K562/BaF3-BCR-ABL-V299L tumor xenografts were treated with CHMFL-ABL-039 at 25, 50, and 100 mg/kg/d dosage, 100 mg/kg/d Imatinib, or vehicle. Daily Intraperitoneal injection (IP) administration was initiated when tumors had reached a size of 200−400 mm³. Data, mean ± SEM, (a) body weight, and (b) tumor size measurements. Initial body weight and tumor size were set as 100%. (c) Comparison of the final tumor weight in each group. Numbers in columns indicate the mean tumor weight in each group. *p < 0.05. (d) Signaling pathway examination of tumor cells isolated from drug-treated tumors tissues on the final day.

Discussion

With the successful application of the target therapies in the clinic as well as the development of the high throughput gene sequencing technology, more and more primary and secondary gain-of-function mutants or the drug-resistant mutants were identified from the patients, though the frequency of some of the mutants may be rare. In order to overcome these rare but oncogenic and lethal mutants, more and more chemical diverse pharmacophores would be highly desired to fulfill the anti-tumor armory. In addition, in the precision medicine era, the highly selective drugs especially the mutants selective drugs which bears different mutants sensitive spectrum would be highly valuable since these agents would not only provide the potential precise treatment for the proper patients but also furnish the research tool to precisely study the mutant associated targets structural biology and pathology, etc.. ABL-V299L mutants have been found in the Imatinib, Dasatinib and Bosutinib treated patients and have exhibited both the primary and secondary resistance to those drugs⁸. Although the frequency of the ABL-V299L mutant was considered rare, the median survival rate of it was poor and its mutation related biology and pathology was still poorly understood yet.

Comparing to the current BCR-ABL inhibitors used in the clinic, CHMFL-ABL-039 exhibited better selectivity among the other protein kinases. It only potently inhibit BCR-ABL and PDGFRα/β and completely abolished the activity of structurally similar KIT kinase, which are the targets for Imatinib, Sunitinib, Regorafenib, and Nilotinib. In addition, among different ABL mutants, CHMFL-ABL-039 was only highly potent to ABL wt and V299L, which made it a valuable research tool to dissect the V299L mutant associated biology and pathology. In this aspect, it would be better than the mutants unselective Nilotinib, though Nilotinib was highly effective for the V299L mutant bearing patients.¹⁹ In summary, CHMFL-ABL-039 was a highly potent and mutants selective BCR-ABL wt/V299L mutant kinase inhibitor, which may reserve as a good pharmacological tool to study V299L associated tumorigenesis.

Funding Statement

This work was supported by the National Natural Science Foundation of China [81471773]; the National Natural Science Foundation of China [81473088]; the National Natural Science Foundation of China [2140207]; the Natural Science Foundation of Anhui province [1508085MB23]; the Natural Science Foundation of Anhui province [1608085QH180]; the Natural Science Foundation of Anhui province [1708085MH208].

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.