Accepted Manuscript

The JAK2 inhibitors CEP-33779 and NVP-BSK805 have high P-gp inhibitory activity and sensitize drug-resistant cancer cells to vincristine
J.I. Hyun Cheon, Kyeong Seok Kim, Mihyun Kim, Hyung Sik Kim, Sungpil Yoon

PII: S0006-291X(17)31309-8
DOI: 10.1016/j.bbrc.2017.06.178
Reference: YBBRC 38084

To appear in: Biochemical and Biophysical Research Communications

Received Date: 23 June 2017
Accepted Date: 28 June 2017

Please cite this article as: J.I.H. Cheon, K.S. Kim, M. Kim, H.S. Kim, S. Yoon, The JAK2 inhibitors CEP-33779 and NVP-BSK805 have high P-gp inhibitory activity and sensitize drug-resistant cancer cells to vincristine, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.06.178.

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The JAK2 inhibitors CEP-33779 and NVP-BSK805 have high P-gp inhibitory activity and sensitize drug-resistant cancer cells to vincristine
JI HYUN CHEON1, KYEONG SEOK KIM1, MIHYUN KIM2, HYUNG SIK KIM1,*, and SUNGPIL YOON1,*

1 School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Republic of Korea.
2 College of Pharmacy, Gachon University, Incheon, 21936, Republic of Korea.
* Correspondence to: Sungpil Yoon, PhD (e-mail: [email protected]) and Hyung Sik Kim, PhD (e-mail: [email protected]), School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 16419, Republic of Korea. Tel: +82 312907789, Fax: +82 312928800
Key Words: JAK2, NVP-BSK805, CEP-33779, Cancer, P-gp, drug-resistance Running title: P-gp inhibition by JAK2 inhibitors in drug-resistant cancer
Notes: Manuscript information: 18 text pages, 4 figures, 4,597 words

 

CEP-33779,CEP; NVP-BSK805, NVP; vincristine, VIC; Verapamil, VER; Dimethylsulfoxide, DMSO; p-glycoprotein, P-gp; Cleaved ploy ADP ribose polymerase, C- PARP; Fluorescence-activated cell sorting, FACS

Abstract

P-glycoprotein (P-gp) is overexpressed in cancer cells in order to pump out chemotherapeutic drugs, and is one of the major mechanisms responsible for multidrug resistance (MDR). It is important to identify P-gp inhibitors with low toxicity to normal cells in order to increase the efficacy of anti-cancer drugs. Previously, a JAK2 inhibitor CEP-33779 demonstrated inhibitory actions against P-gp and an ability to sensitize drug- resistant cancer cells to treatment. In the present study, we tested another JAK2 inhibitor NVP-BSK805 for P-gp inhibitory activity. In molecular docking simulation modeling, NVP-BSK805 showed higher binding affinity docking scores against a P-gp member (ABCB1) than CEP-33779 did.
Furthermore, we found that lower doses of NVP-BSK805 are required to inhibit P-gp in comparison with that of CEP-33779 or verapamil (an established P-gp inhibitor) in KBV20C cells, suggesting that NVP-BSK805 has higher specificity. NVP-BSK805, CEP-33779, and verapamil demonstrated similar abilities to sensitize KBV20C cells to vincristine (VIC) treatment. Our results suggested that the JAK2 inhibitors were able to inhibit P-gp pump- action via a direct binding mechanism, similar to verapamil. However, JAK2 inhibitor- induced sensitization was not observed in VIC-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells.
FACS, western-blot, and annexin V analyses were used to further investigate the mechanism of action of JAK2 inhibitors in VIC-treated KBV20C cells. Both CEP-33779 and NVP- BSK805 induced the sensitization of KBV20C cells to VIC treatment via the same mechanisms; they each caused a reduction in cell viability, increased G2 arrest, and upregulated expression of the DNA damaging protein pH2AX when used as co-treatments with VIC. These findings indicate that inhibition of JAK2 may be a promising target in the treatment of cancers that are resistant to anti-mitotic drugs

1. Introduction

Anti-mitotic drugs, which target different binding sites on tubulin, are widely used for the treatment of different types of cancer[1, 2]. These compounds inhibit mitosis by targeting microtubules and preventing their polymerization and depolymerization [1-4]. Patients often develop resistance to anti-mitotic drugs [5]; therefore, it is important to identify the mechanisms that underlie tumor cell desensitization in order to develop more effective treatments.
P-glycoprotein (P-gp) overexpression is a well-known mechanism for anti-mitotic drug resistance in tumor cells [6]. P-gp is a component of the cell membrane that pumps out anti- mitotic drugs, and its overexpression is one of major mechanisms for multi-drug resistance (MDR) [6, 7]. Although P-gp inhibitors have been developed, the toxicity of these agents to normal cells has resulted in the failure of clinical tests [7-10]. Although the toxicity of first- generation P-gp inhibitors has prevented their use, there have been several attempts to improve their safety for further clinical trials [7, 9, 10]. The purpose of the present study was to identify and investigate the efficacy of novel P-gp inhibitors with low toxicity to drug- resistant cancer cells.
Janus kinase 2 (JAK2) is a protein that contributes to drug-resistant cancer [11, 12], therefore JAK2 inhibitors have been developed to increase the efficacy of anti-cancer drugs. Previously, the JAK2 inhibitor CEP-33779 demonstrated P-gp inhibitory activity and drug-sensitization effects in P-gp overexpressing drug-resistant cancer cells [13]. In this study, we investigated another JAK2 inhibitor NVP-BSK805 for P-gp inhibitory activity. Our results indicated a conserved P-gp inhibitory mechanism of the JAK2 inhibitor that resulted in sensitization of vincristine (VIC)-treated, P-gp overexpressing, drug-resistant KBV20C cancer cells. These results will therefore contribute to the development of JAK2 inhibitor-based therapies for the co-treatment of drug-resistant cancers.

 

2. Materials and methods

2.1. Reagents and cell culture.

Reagents and cell lines [14-16] in this study are provided in the Supporting Information.

2.2. Microscopic observation

The detailed method is described in the Supporting Information.

2.3. Rhodamine uptake tests

The detailed method is described in the Supporting Information.

2.4. Fluorescence-activated cell sorting (FACS) analysis

FACS analysis was performed as previously described [17-20]. The detailed method is described in the Supporting Information.
2.5. Annexin V analysis

The detailed method is described in the Supporting Information.

2.6. Western blot analysis

The detailed method is described in the Supporting Information.

2.7. Cellular viability assay

The detailed method is described in the Supporting Information.

2.8. Statistical analysis

The detailed method is described in the Supporting Information.

2.9. Molecular docking studies

A suitable model of the human homolog of ABCB1 (model_1) was selected and demonstrated that the 3D structure of ABCB1 obtained by simulation were reasonable and appropriate for use in further investigations [21-23]. The detailed method is described in the Supporting Information.

3. Results

3.1. NVP-BSK805 had higher binding affinity for ABCB1 than CEP-33779 did in molecular docking simulations
Previously, the JAK2 inhibitor CEP-33779 demonstrated P-gp binding affinity in a molecular docking simulation. In the present study, we tested and compared another JAK2 inhibitor, NVP-BSK805, for P-gp inhibitory activity. In order to determine the molecular intercommunication of the ABCB1 protein with the two compounds (NVP-BSK8052HCL and CEP-33779), the 3D structure of the ABCB1 protein was predicted through homology modeling [13] and deliberated with the Schrodinger Glide program. The docking results for CEP-33779 against the ABCB1 protein indicated a low binding affinity, with a docking score of -7.423 (Fig. 1A), and indicated the formation of a H-bond of length 2.3Å with the side chain of the polar neutral amino acid residue Gln-990 (glutamine). Moreover, the protonated tertiary amine of the piperazine ring showed pi-pi interaction with the hydroxyl group of Tyr310 and Phe-983 (- NH–OH-Tyr310, 5.2Å and 5.3). According to the results of our molecular docking approach, the chemical nature of amino acid residues associated with the CEP-33779 binding site (within a radius of 4A˚) was basic (polar, hydrophobic, positive charged), for example, Val-982, (valine); nucleophilic (polar, hydrophobic) for example, Leu- 339, Leu-65 (Leucine), Ile-306, Ile-340 (isoleucine); aromatic (hydrophobic), that is, Tyr-307, Tyr-953, Tyr-310 (tyrosine), Phe-728, Phe-303, Phe-343, Phe-983, Phe-336, Phe- 978(Phenylalanine); polar neutral amino acid residue for example, Met-69, Met-986, (methionine), Asn-839, Asn-842 (asparagine); Gln-990, Gln-725 (glutamine); hydrophobic aliphatic, that is, Ala-987, (alanine).
Similarly, the docking results for NVP-BSK8052HCL against the ABCB1 receptor protein indicated high binding affinity, with a docking score of -9.104 (Fig. 1B). The docking studies revealed intermolecular hydrogen bonding interactions between the hydrogen molecules of

Tyr-307(-NH–OH-Tyr307, 2.0Å) and the pyrrolidine and piperidine residues of the NVP- BSK8052HCL side-chain. According to the ligand docking pose, the chemical nature of amino acid residues within a radius of 4A˚ from the binding site was aromatic (polar, hydrophobic, and positively charged), that is, Tyr-307, Tyr-953, Tyr-310 (tyrosine), Phe-728, Phe-303, Phe-343, Phe-983, Phe-336, Phe-978(Phenylalanine); basic (polar, hydrophobic, positive charged), for example, Val-982, (valine); hydrophobic aliphatic, that is, Ala-987, (alanine); polar neutral amino acid residue for example, Met-69, Met-986, (methionine), Asn- 839, Asn-842 (asparagine); Gln-990, Gln-725 (glutamine); nucleophilic (polar, hydrophobic) for example, Leu-339, Leu-65 (Leucine), Ile-306, Ile-340 (isoleucine). Thus, NVP-BSK805 showed a high binding affinity due to strong hydrophobic interactions, which may lead to higher stability and increased anti-tumor activity in comparison with those of CEP-33779.
3.2. JAK2 inhibitors, CEP-33779 and NVP-BSK805 have high P-gp inhibiting activity.

Next, we tested the P-gp inhibitory activity of CEP-33779 and NVP-BSK805 in P-gp- overexpressing KBV20C cells [14, 15]. We evaluated whether JAK2 inhibitors increased the inhibition of P-gp substrate efflux. Rhodamine123, a well-known P-gp substrate, was used to measure P-gp inhibition [14-16]. In this experiment, cellular accumulation of green fluorescence was indicative of rhodamine123 intracellular accumulation. As shown in Fig. 1C, CEP-33779 and NVP-BSK805 showed high P-gp inhibitory activity, while VIC, used as a negative control, did not inhibit P-gp. The known P-gp inhibitor verapamil was used as a positive control [14, 15], and upon comparison, we found that a four-fold lower dose of either of JAK2 inhibitors could induce equivalent P-gp inhibitory activity. This suggests that JAK2 inhibitors may bind to P-gp with higher specificity than verapamil does. This may also indicate that drug toxicity can be reduced by the use of JAK2 inhibitors.
3.3. Co-treatment with CEP-33779, NVP-BSK805, or verapamil increased sensitization of drug-resistant KBV20C cancer cells to VIC treatment

In the next phase of our investigation, we tested whether co-treatment with JAK2 inhibitors increased sensitization of KBV20C cells to VIC-induced mitotic-arrest. We compared the effects of each of our agents with the well-known P-gp inhibitor, verapamil [14, 15]. As seen in Fig. 2A-B, 10 M of CEP-33779, NVP-BSK805, or verapamil was similarly efficacious when applied with VIC in decreasing KBV20C cell viability. This suggests that each agent functions as a P-gp inhibitor. We also observed that 10 M of CEP-33779 or NVP-BSK805 was more efficacious than a 5 µM dose (Fig. 2A-B), suggesting a dose-dependent relationship between JAK2 inhibitors and cancer-cell sensitization to anti-mitotic drug treatment.
Neither KB nor KBV20C cells responded to individual treatment with CEP-33779 or NVP- BSK805 alone, while VIC sensitized only parent KB cells (Fig. 2C). We observed a slight increase in sensitization to VIC upon co-treatment with JAK2 inhibitors in sensitive parent KB cells (Fig. 2D). Those results for KB cells indicate that JAK2 inhibitors, CEP-33779 and NVP-BSK805 have only P-gp inhibiting functions in KB or KBV20C cells.
JAK2 inhibitors are known to reduce STAT3 activation [24, 25]; therefore, we tested whether co-treatment with CEP-33779 or NVP-BSK805 can reduce STAT3 activation in VIC-treated KBV20C cells. As seen in Fig. 3E and 3F, STAT3 activation significantly decreased after treatment with either of the JAK2 inhibitors alone. However, co-treatment with VIC did not further reduce STAT3 activation. The STAT3 and JAK2 total protein levels did not decrease in cells after co-treatments (Fig. 3E and 3F). This suggested that co-treatment of VIC-treated KBV20C cells with CEP-33779 or NVP-BSK805 increased sensitization to mitotic-arrest, independent to the activation of the JAK/STAT signaling pathways.
Altogether, we found that co-treatment with JAK2 inhibitors sensitized KBV20C cells to VIC treatment, with similar efficacy as that of verapamil. STAT3 inhibition, however, did not appear to be responsible for this mechanism. This suggests that P-gp inhibition by JAK2

inhibitors is a more important factor for the sensitization of KBV20C cells to co-treated anti- mitotic drug.
3.4. Co-treatment of KBV20C cells with NVP-BSK805 and VIC induces G2-arrest and increases DNA damage.
In order to confirm the efficacy of JAK2 inhibitors as modulators of cell-cycle arrest in VIC- treated KBV20C cells, we performed microscopic observation in cells co-treated with NVP- BSK805 and VIC. As seen in Fig. 3A, we confirmed that co-treatment with 5 µM or 10 M NVP-BSK805 resulted in decreased VIC-treated KBV20C cell numbers, compared with that observed for singular treatment with each of the inhibitors. We also confirmed microscopic observation in another JAK2 inhibitor CEP-33779 (data not shown).
In order to further clarify the mechanism of action of NVP-BSK805, we performed fluorescence-activated cell-sorting (FACS) analyses. As shown in Fig. 3B and Supplementary Fig. 1A, co-treatment with NVP-BSK805 and VIC increased the number of cells in G2 arrest compared with that observed for singular treatments with either agent. A positive relationship was found between the dose of NVP-BSK805 and the proportion of VIC-treated cells in G2 arrest.
In order to further investigate the expression of proteins involved in G2 arrest [15, 20], we performed western-blot analysis. As seen in Fig. 3C, there were no significant differences in cyclin protein expression; however, we found that the protein marker of DNA damage pH2AX was largely increased following co-treatments, suggesting that DNA damage may increase G2 arrest in NVP-BSK805-VIC co-treated KBV20C cells.
3.5. Co-treatment of KBV20C cells with NVP-BSK805 and VIC increases apoptosis in a dose-dependent manner.
As seen in Fig. 3B, co-treatment of cells with VIC and NVP-BSK805 increased pre-G1 region in a dose-dependent manner, suggesting that the combinatory use of these agents

induced apoptosis. Furthermore, annexinV staining was analyzed as a marker of apoptosis [15, 16]. As shown in Fig 4A and Supplementary Fig. 1B, co-treatment with NVP-BSK805 and VIC increased the number of cells in early phases of apoptosis compared with that reported for treatment with NVP-BSK805 alone. However, we detected no increase cells in a late stage of apoptosis in co-treated cells, suggesting that early apoptosis mainly plays a role increased sensitization in NVP-BSK805-VIC co-treatments. In order to confirm increased apoptosis in co-treated cells by the presence of molecular markers, we measured C-PARP production [15, 16]. As seen in Fig. 4B, C-PARP expression increased in NVP-BSK805-VIC co-treated cells. We also determined an increase in pRB levels in co-treated cells [26], indicating that an increase in cell cycle arrest stimulated early apoptosis. Altogether, co- treatment of NVP-BSK805 with VIC increased apoptosis of drug-resistant KBV20C cells via cell cyclic arrest.
3.6. NVP-BSK805 and CEP-33779 sensitize VIC-treated KBV20C cells via similar mechanisms of action.
Comparisons were made between the effects of CEP-33779 and NVP-NSK805 on VIC- treated KBV20C cells in order to determine their mechanism of action. As seen in Fig. 4C, we found that co-treatment of cells with CEP-33779-VIC increased C-PARP, pRB, and pH2AX expression, suggesting that CEP-33779 induces DNA damage, G2 arrest, and apoptosis. Equivalent effects were also induced by NVP-BSK805-VIC co-treatment.
Altogether, we found that JAK2 inhibitors have actions that are independent of the JAK/STAT signaling pathway. Therefore, co-treatment of cells with JAK2 inhibitors strongly prevented P-gp-mediated efflux of VIC and thereby may increase the efficacy of anti-mitotic drugs.
4. Discussion

The selective JAK2 inhibitor CEP-33779 has recently demonstrated P-gp inhibitory activity via direct binding [13]. By molecular docking, we identified another JAK2 inhibitor NVP- BSK805 with a higher predicted affinity for P-gp than CEP-33779. We subsequently proceeded to investigate the mechanism of action of JAK2 inhibitors on P-gp function in an in vitro model of drug resistance. Our results indicated that both JAK2 inhibitors directly bound to P-gp in drug-resistant KBV20C cancer cells, preventing P-gp-mediated drug efflux. Our measurements additionally indicated that CEP-33779 and NVP-BSK805 showed equal efficacy to the established P-gp inhibitor verapamil at comparably lower doses, implying that the two novel agents have a higher binding affinity. We therefore postulate that drug toxicity can be reduced via the use of JAK2 inhibitors against drug-resistant cancers. Comparison among verapamil, CEP-33779, and NVP-BSK805 showed that all three exhibited similar drug-sensitization effects with the same dose in VIC-treated KBV20C cells. Considering that verapamil is a representative and well-known P-gp inhibitor [14, 15], both JAK2 inhibitors are regarding as members of P-gp inhibitors.
The molecular mechanism of cancer cell suppression induced by JAK2 inhibitors in combination with VIC is unresolved, and may be different between CEP-33779 and NVP- BSK805. However, based on our analyses we found that both JAK2 inhibitors have similar mechanisms for the sensitization of KBV20C cells to VIC treatment. It seems to be useful that both JAK2 inhibitors have similar sensitization-mechanisms, when combined with VIC. Since different structure of JAK2 inhibitors can be used for clinics as alternative backup inhibitors for resistant cancer cells. According to our results, the marker of DNA-damage pH2AX, cell cycle protein pRb, and pro-apoptotic protein C-PARP were increased by both CEP-33779 and NVP-BSK805 co-treatment with VIC. This is indicative of a mechanism involving G2 arrest via DNA damage and an increase of cells in the early phase of apoptosis. This suggests that the JAK2 inhibitors only prevented P-gp function in our model. Further

studies with other JAK2 inhibitors are required to confirm our model, and may further elucidate the link between JAK2 inhibition and P-gp function in drug-resistant cancer cells. As these agents together have yet to be compared in vivo, mouse studies are required before investigating the use of JAK2 inhibitors in clinical trials.
Although we detected increased STAT3 activation in JAK2 inhibitor-treated KBV20C cells, it is possible that other unknown functions for being cancer can be related in STAT3 activation in resistant cancer cells. Further analysis of the impact of JAK2 inhibitors on the long-term survival and malignancy of cancer cells is required to establish their potential use in anti- cancer therapy.
JAK2 inhibitors are promising drugs for the treatment of triple negative breast cancer (TNBC) and leukemia [27, 28]. JAK2 inhibitors have been considered for the treatment of chemotherapeutic-resistant TNBC [27, 28], due to the fact that JAK2 genes may become upregulated in these cancers in response to various treatments. In a clinical setting, we hypothesize that both CEP-33779 and NVP-BSK805 may be applied to increase drug- sensitivity of both JAK2 and P-gp overexpressing cancer cells in heterogeneous tumor cell populations.
Altogether, our results show that drug-resistant KBV20C cells that overexpress P-gp can be sensitized to anti-mitotic treatment with the JAK2 inhibitors CEP-33779 and NVP-BSK805. Because these agents are already used in clinical trial, the urgent need for pharmacological treatments of antimitotic drug-resistant cancers can be efficiently addressed and these drugs may be used to treat P-gp overexpressing resistant patients at a relatively faster pace.
Conflict of Interests

The authors declare no conflict of interests.

Acknowledgements

We thank Yujin Park for help in technical supports and preparation of the manuscript. This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A2B2011071).
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Figures’ legends

Figure 1. NVP-BSK805, a JAK2 inhibitor, has improved P-gp inhibitory activity compared to CEP-33779 and verapamil (A-B) Molecular docking studies were performed between JAK2 inhibitors and a P-gp member (ABCB1) using the Glide module as described in Materials and methods. The top scoring pose-ABCB1 complex structures were then used for graphical analysis. (C) KBV20C cells were grown on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 20 M verapamil, 5 M CEP-33779, 5 M NVP-BSK805, or 0.1% DMSO (Con). After 24 h, all cells were stained with rhodamine123 and examined by using FACS analysis, as described in Materials and Methods.

Figure 2. JAK2 inhibitors sensitize drug-resistant KBV20C cancer cells to vincristine treatment, and have similar efficacy to verapamil
(A-B) KBV20C cells were plated on 96-well plates and grown to 30%-40% confluence. The cells were then stimulated for 72 h with 5 nM vincristine (VIC), 10 M verapmail (VER), 5
M CEP-33779 (CEP-5), 10 M CEP-33779 (CEP-10), 5 M NVP-BSK805 (NVP-5), 10

M NVP-BSK805 (NVP-10), 5 nM vincrstine with 5 M CEP-33779 (VIC+CEP-5), 5 nM

VIC with 10 M CEP-33779 (VICCEP-10), 5 nM vincrstine with 5 M NVP-BSK805 (VIC+NVP-5), 5 nM VIC with 10 M NVP-BSK805 (VIC+NVP-10), or DMSO (Con). A
cell viability assay was performed as described in “Materials and methods”. The data are represented by the mean ± S.D. of at least two experiments repeated in triplicate experiments. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by multiple-comparison test; * P <0.05 compared to the corresponding control. (C) KB and KBV20C cells were grown on 6-well plates and treated with 5 nM vincristine (VIC), 5 M CEP-33779 (CEP-5), 5 M NVP-BSK805 (NVP-5), or 0.1% DMSO (Con). After 2 days, all
cells were observed using an inverted microscope with a 100 magnification (scale bar=100

m). (D) KB cells were plated on 96-well plates. The cells were then stimulated for 24 h with 5 nM vincristine (VIC), 5 M CEP-33779 (CEP-5), 5 M NVP-BSK805 (NVP-5), 5 nM
vincrstine with 5 M CEP-33779 (VIC+CEP), 5 nM vincrstine with 5 M NVP-BSK805 (VIC+NVP), or DMSO (Con). A cell viability assay was performed as described in “Materials and methods”. (E-F) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 5 M CEP-33779 (CEP), 5 M NVP-BSK805 (NVP), 10 M NVP-BSK805 (NVP-10), 5 nM vincrstine with 5 M CEP-33779 (VIC+CEP), 5 nM
vincrstine with 5 M NVP-BSK805 (VIC+NVP), or DMSO (Con). After 24h, Western blot analysis was performed using antibodies against pSTAT3, STAT3, JAK2, and -actin.

 

Figure 3. NVP-BSK805 sensitizes drug-resistant KBV20C cancer cells to vincristine- induced DNA damage and G2 arrest. (A) KBV20C cells were grown on 6-well plates and treated with 5 nM vincristine (VIC), 2.5 M NVP-BSK805 (NVP-2.5), 5 M NVP-BSK805 (NVP-5), 5 nM vincrstine with 2.5 M NVP-BSK805 (VIC+NVP-2.5), 5 nM vincrstine with 5M NVP-BSK805 (VIC+NVP-5), or 0.1% DMSO (Con). After 2 days, all cells were observed using an inverted microscope with a 50 magnification (scale bar=100 m). (B) KBV20C cells were grown on 6-well plates and treated with 5 nM vincristine (VIC), 2.5 M NVP-BSK805 (NVP-2.5), 5 M NVP-BSK805 (NVP-5), 5 nM vincrstine with 2.5 M NVP- BSK805 (VIC+NVP-2.5), 5 nM vincrstine with 5M NVP-BSK805 (VIC+NVP-5), or 0.1%
DMSO (Con). After 24 h, FACS analyses were performed as described in Materials and Methods. (C) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 5 M NVP-BSK805 (NVP), 5 nM vincrstine with 5M NVP-BSK805 (VIC+NVP), or 0.1% DMSO (Con). After 24h, Western blot analysis was performed using antibodies against CyclinB1, CDK2, CyclinE1, p21, pH2AX, CDK4, and -actin.

Figure 4. CEP-33779 and NVP-BSK805 act via similar mechanisms to sensitize drug- resistant KBV20C cancer cells to vincristine.
(A) KBV20C cells were grown on 6-well plates and treated with 5 nM vincristine (VIC), 2.5

M NVP-BSK805 (NVP-2.5), 5 M NVP-BSK805 (NVP-5), 5 nM vincrstine with 2.5 M NVP-BSK805 (VIC+NVP-2.5), 5 nM vincrstine with 5M NVP-BSK805 (VIC+NVP-5), or
0.1% DMSO (Con). After 24 h, Annexin V analyses were performed as described in Materials and Methods. (B) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 5 M NVP-BSK805 (NVP), 5 nM vincrstine with 5M NVP-

BSK805 (VIC+NVP), or 0.1% DMSO (Con). After 24h, Western blot analysis was performed using antibodies against C-PARP, pRB, and -actin. (C) KBV20C cells were plated on 60 mm-diameter dishes and treated with 5 nM vincristine (VIC), 5 M CEP-33779 (CEP), 5 nM vincrstine with 5M CEP-33779 (VIC+CEP), or 0.1% DMSO (Con). After 24h, Western blot analysis was performed using antibodies against C-PARP, pRB, pH2AX, CDK4, and -actin.

- Drug-resistant KBV20C cells that overexpress P-gp can be sensitized to anti- mitotic treatment with the JAK2 inhibitors CEP-33779 and NVP-BSK805.

- JAK2 inhibitors directly bound to P-gp in drug-resistant KBV20C cancer cells, preventing P-gp-mediated drug efflux.
- JAK2 inhibitors showed equal efficacy to the established P-gp inhibitor verapamil at comparably lower doses.
- Both CEP-33779 and NVP-BSK805 are regarding NVP-BSK805 as members of P-gp inhibitors.
- Because these JAK2 inhibitors are already used in clinical trial, they may be applied to treat P-gp overexpressing resistant patients at a relatively faster pace.