Abstract
Purpose
FLT3 mutations occurred in approximately one third of patients with acute myeloid leukemia (AML). FLT3-ITD mutations caused the constitutive activation of the RAS/MAPK signaling pathway. Ribosomal S6 Kinases (RSKs) were serine/threonine kinases that function downstream of the Ras/Raf/MEK/ERK signaling pathway. However, roles and mechanisms of RSKs inhibitor LJH-685, and combinational effects of LJH-685 and FLT3 inhibitor FF-10101 on AML cells were till unclear.
Methods
Cell viability assay, CFSE assay, RT-qPCR, Colony formation assay, PI stain, Annexin-V/7-AAD double stain, Western blot, and Xenogeneic transplantation methods were used to used to investigate roles and mechanisms of LJH-685 in the leukemogenesis of AML.
Results
LJH-685 inhibited the proliferation and clone formation of AML cells, caused cell cycle arrest and induced the apoptosis of AML cells via inhibiting the RSK-YB-1 signaling pathway. MV4-11 and MOLM-13 cells carrying FLT3-ITD mutations were more sensitive to LJH-685 than that of other AML cell lines. Further studies suggested that LJH-685 combined with Daunorubicin or FF- 10101 synergistically inhibited the cell viability, promoted the apoptosis and caused cycle arrest of AML cells carrying FLT3-ITD mutations. Moreover, in vivo experiments also indicated that LJH-685 combined with FF-10101 or Daunorubicin prolonged the survival time of NSG mice and reduced the leukemogenesis of AML.
Conclusion
Thus, these observations demonstrated combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 showed synergism anti-leukemia effects in AML cell lines with FLT3-ITD mutations via inhibiting MAPK-RSKs-YB-1 pathway and provided new targets for therapeutic intervention especially for AML with FLT3-ITD mutations and Daunorubicin-resistant AML.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13402-022-00703-7.
Keywords: AML, RSKs, FLT3-ITD, LJH-685, FF-10101, Proliferation
Introduction
Acute myeloid leukemia (AML) is a malignant disorder of haemopoietic stem cells characterized by clonally expansion of abnormally differentiated blasts of myeloid lineage [1]. AML is the most common acute leukemia in adults, posing a serious threat to human health. Although the standard induction chemotherapy and hematopoietic stem cell transplantation (HSCT) have greatly improved the survival of AML patients, the curative effect is not satisfactory [2]. In younger patients, complete remission (CR) rates of ≥80% may be reached, with 5-year overall survival (OS) ~40%, while only around 10% of patients over 60 years old survive [3–5]. In addition, the pretreatment of HSCT and the adjuvant effect of chemotherapy make AML survivors often in chronic health conditions, resulting in a decline in the quality of life [6]. Therefore, it is essential to screen novel therapeutic targets and drugs for the treatment of AML to prolong the survival time and improve the patients’ quality of life.
In order to screen key genes for the occurrence of AML, our team selected AML primary cells for RNA-Seq, which indicated that ribosomal S6 Kinases (RSKs) was overexpressed and hyperactivation in AML primary cells. Recently study also suggested that RSKs overexpression predicted poor prognosis [7], suggesting that RSKs may be a potential target for AML treatment, but the mechanism of RSKs in the leukemiagenesis of AML is still unclear.
RSKs are a family of Serine/Threonine protein kinases involved in the regulation of biological behaviors such as transcription, translation, cell cycle regulation, and cell survival [7–9]. Previous studies showed that the Ras/Raf/MEK /ERK pathway constitutively activated in more than 50% of AML and acute lymphocytic leukemia (ALL), which indicated the poor prognosis and faster resistance to chemotherapy [10–12]. RSKs were the most downstream and critical effectors of the MAPK pathway which might play an important role in AML pathogenesis and progression [7]. RSK1/2 was the predominant isoforms expressed in AML, the high expression and phosphorylation levels of RSK1/2 were associated with poor survival [7, 13]. Downregulation of RSKs expression reduced the pathology of tumors, indicating that RSKs was a potential therapeutic target for tumor therapy.
The internal tandem duplication mutation in FLT3 (FLT3-ITD) is the most frequent mutation in AML found in 25-30% of cases and associated with a poor prognosis [14, 15]. FLT3-ITD leads to constitutive activation of the Ras/Raf/MEK/ERK pathway [12, 15]. As a downstream regulator of this pathway, RSKs played vital roles in the pathogenesis and myeloid lineage determination of FLT3/ITD- induced hematopoietic transformation [16]. RSK1 downregulated the pro-apoptotic Bcl-2 family members Bad and BIM to promote proliferation and prevented the apoptosis of AML cells [17]. Besides, FLT3-ITD activated RSK1 to enhance proliferation and survival of AML cells by activating mTORC1 and eIF4 [18]. These observations suggested that combined inhibition of FLT3 and RSKs may be a viable therapeutic strategy to cure AML patients with FLT3-ITD. However, roles and mechanisms of combinational effects of RSK inhibitors and FLT3 inhibitors on the leukemiagenesis of AML have not been reported.
In this study, LJH-685 [19], the RSK inhibitor with smaller IC50, and FLT3 inhibitor FF-10101 were selected to investigate roles and mechanisms of their combinational effects on the biological behavior of AML cells. Our results indicated that LJH-685 inhibited the proliferation and clone formation of AML cells, caused cell cycle arrest and induced the apoptosis of AML cells via RSK-YB-1 signaling pathway. MV4-11 and MOLM-13 cells carrying FLT3-ITD mutations were more sensitive to LJH-685 than that of other AML cell lines. Thus, MV4-11 and MOLM-13 cells were used to explore roles and mechanisms of combinational effects of LJH-685 and FF-10101 on the leukemiagenesis of AML with FLT3-ITD mutations. LJH-685 combined with FF-10101 or Daunorubicin synergistically inhibited the proliferation of MV4-11 and MOLM-13 cells, promoted the apoptosis and caused cycle arrest of AML cells. In Vivo experiments also suggested that LJH-685 combined with FF-10101 or Daunorubicin prolonged the survival time of NSG mice and inhibited the leukemogenesis of AML. Thus, these observations demonstrated combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 showed synergism anti-leukemia effects in FLT3-ITD AML cell lines via inhibiting MAPK-RSKs-YB-1 pathway and provided new targets for therapeutic intervention especially for AML with FLT3-ITD mutations and Daunorubicin-resistant AML.
Materials and Methods
Cell cultures and reagents
The human AML cell lines KG-1, THP-1, MV4-11, NB4, HEL, MOLM13 and HL-60 were preserved in our laboratory. HL-60 was originated from the acute promyelocytic leukemia patient with normal Karyotype and cultured in IMDM medium (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Other cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS. KG-1 cells were originated from acute myelogenous leukemia patient with FGFR1OP2 -FGFR1 fusion gene. HEL cell was obtained form erythroleukemia patient with normal Karyotype. MV4-11 cells were originated from Biphenotypic B myelomonocytic leukemia patient with MLL-AF4 fusion gene and FLT3-ITD mutation. MOLM13 were originated from acute myeloid leukaemia patient with MLL-AF9 fusion gene and FLT3-ITD mutation. THP-1 cells were originated from acute monocytic leukemia patient with MLL-AF9 fusion gene. The MV4-11 and MOLM-13 cell lines with FLT3-ITD mutations were proper to selected as the tool to investigate roles and mechanisms of combinational effects of RSK inhibitors and FLT3-ITD inhibitors on the AML cells with FLT3-ITD mutations. All cultures were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The RSK inhibitor LJH-685 was purchased from Selleck Chemicals (Houston, USA). The FLT3 inhibitor FF-10101 and Daunorubicin were purchased from MCE (New Jersey, USA).
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from AML cells using Trizol reagent (Invitrogen). The cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara, Kyoto, Japan). Quantitative analysis of RSK mRNA expression was evaluated by qPCR using SYBR® Green One-Step qRT-PCR kit (Roche) and β-actin was used as an endogenous control.
Cell viability assay
The cell counting kit-8 (CCK-8) assay was performed to determine cell viability. AML cells were seeded into 96-well plates (2 × 104 cells/well, in triplicate), and 10 μL of CCK-8 was added to each well after the indicated time of drug exposure. After incubating for 3 h, the absorbance was measured at a wavelength of 450 nm.
Determination of LJH-685 inhibition of the proliferation of AML cells by CFSE
The MV4-11 and MOLM-13 cells were harvested and washed with PBS once. After that, the cells were resuspended with PBS. CFSE was added to the system at a final concentration of 5 μmol/L. The liquid was mixed gently and incubated for 10 min at 37 °C. After removal of the liquid, 5 times the volume of pre-cooled FBS was added, centrifuged, washed twice with PBS, and the cell density was adjusted to 105/mL. Then cells were treated with indicated concentrations of LJH-685. After treatment for 1, 2, and 3 days, cells were collected and washed twice with PBS. The AML cells were assessed by flow cytometry using the Alexa Fluor 488. The analysis was performed on the FACScan cytometer (BD) using CellQuest software.
Analysis of the cell cycle and apoptosis by flow cytometry (FCM)
AML cells were seeded into 12-well culture plates at a density of 2×105 cells/well in 1.5 mL medium and treated with indicated concentrations of LJH-685 for 48 h. The AML cells were fixed overnight with 75% ethanol, then removed the ethanol and incubated in the PI buffer (BD) for 30 min to detect the cell cycle. AML cell apoptosis was detected using the Annexin-V-APC and 7-AAD kit (BD). The analysis was performed on the FACScan cytometer (BD) using CellQuest software.
Western Blot
AML cells were seeded into 6-well culture plates at a density of 1.5×106 cells/well in 2 mL medium and treated with indicated concentrations of LJH-685 for indicated time. Then the inhibitor induced changes of MAPK signal pathway and cell-cycle related proteins were detected. The antibodies used were as followings: P-p90RSK, RSK1/2/3, CDK4, Cyclin E, Cyclin B, CDK1, p27 and c-Myc (Cell Signaling Technology), RSK3 (Santa Cruz), P-YB1, YB-1, p-STAT3, STAT3 and GAPDH (Bioworld), p-MEK1/2, MEK1/2, p-ERK1/2, ERK1/2 (Proteintech).
Colony formation assay
AML cells were seeded into 12-well culture plates at a density of 300 cells/well in 1 mL methylcellulose semi-solid medium (Stem cell technologies) and treated with indicated concentrations of LJH-685 for 14 d. Then the colonies were observed using Giemsa staining and pictured at 100 magnifications under microscope (Olympus, Tokyo, Japan).
Xenogeneic transplantation in NSG mice
Luciferase-GFP expressing leukemia cell line MV4-11 were injected intravenously (i.v.) into sublethally irradiated (220 cGy) 6-8 week old NSG mice. MV4-11-GFP-Luc was constructed by stable expression of PLKO.1-GFP-Luc plasmid into the MV4-11 cells via lentivirus mediated transduction. To evaluate engraftment by luciferase-expressing leukemic cell line, mice were injected with 150 mg/kg D-luciferin (Promega, WI, USA) intraperitoneally and bioluminescence was examined using IVIS Spectrum in vivo imaging system (Perkin-Elmer, MA, USA). For survival experiments, engraftment was confirmed on day 21 following transplantation when human CD45.+ cells in the blood reached 1% to 5%. Engrafted mice were subsequently randomized into groups to receive control, LJH-685, FF-10101 or combination of LJH-685 and FF-10101 groups (eight mice each group), and bioluminescence was reassessed at the indicated time. Mice were bled weekly to assess leukemia burden. Some mice were killed at day 14 or 36 to assess leukemia burden in marrow and spleens.
Statistical analysis
The results are expressed as the mean ± SD of three independent experiments. The data were analyzed by SPSS version 25.0 or GraphPad Prism version 7.03, and Student’s t-test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups. All tests were performed as two-sided, and P < 0.05 was considered statistically significant.
Results
The RSK was high expression in AML cells
RT-qPCR and western blotting were used to detect the expression of RSKs in AML cell lines at the mRNA and protein levels, respectively. RSKs was expressed in all AML cell lines, and the expression of RSKs was higher in the AML cell lines than that of normal human peripheral blood mononuclear cells (PBMC) (Fig. 1a, b). Similarity, the patients with high expression of RSKs displayed the low overall survival (OS) and event free survival (EFS) (Fig. 1c–e), these results indicated that the high expression of RSKs might play key roles in the leukemogenesis of AML. There were many AML well-known prognostic mutations and gene alterations which could indicate the poor prognosis and faster resistance to chemotherapy. The hyperactivation of Ras/Raf/MEK/MAPK/ERK signal pathway caused by N-RAS or K-RAS mutations were important for the pathology of many type of cancers [20], thus, the association between RSKs high expression and the N-RAS or K-RAS mutations were evaluated. Our results indicated that either N-RAS or K-RAS mutations combined with the RSKs high expression predicted the low OS and EFS (sFig. 1a, b). Besides, FLT3-ITD can also activate the Ras/Raf/MEK/MAPK/ERK signal pathway [12, 15], thus, FLT3-ITD combined with the RSKs high expression on the OS and EFS of AML patients were also evaluated. Our results suggested that FLT3-ITD combined with the RSKs high expression foretold the low OS and EFS (sFig. 1c).
Fig. 1.
The RSKs were high expressed in AML cell lines and AML patients which indicating the low OS and EFS. (a, b) The expression of RSKs was detected by real-time quantitative PCR and western blotting in AML cell lines in which the normal human PBMC was used as control. (c-e) The high expression of RSK1, RSK2 and RSK3 predicted the low OS and PFS
LJH-685 inhibit the proliferation of AML cells
Effects of LJH-685 on the viability of AML cells were determined by CCK-8 assay. The activity of AML cells decreased with the increasing concentrations of LJH-685, compared with the control group. The IC50 values of LJH-685 were 13.42 ± 2.67 μM, 18.72 ± 2.77 μM, 6.99 ± 0.91 μM for KG-1, THP-1 and MV4-11, respectively (Fig. 2a). MV4-11 was more sensitive to LJH-685 compared with KG-1 and THP-1 at the same concentration. Moreover, the viability of AML cells decreased with LJH-685 treatment time prolong, when the LJH-685 concentration was 20 μM (Fig. 2b). Thus, LJH-685 decreased the viability of AML cells in the dose-dependent and time-dependent manners. In order to investigate effects of LJH685 on the proliferation of AML cell lines, the CSFE assays were done. Our results indicated that LJH685 effectively inhibited the proliferation of MV4-11 and MOLM-13 cell lines at 24 h and 48 h, while it did not show inhibition effects on the proliferation of MV4-11 and MOLM-13 cells at 72 h, when the LJH-685 concentration was 5 μM (Fig. 2c). These observations indicated that LJH685 could effectively inhibit the proliferation of AML cells.
Fig. 2.
LJH-685 inhibited the proliferation and the colony formation of AML cell lines. (a) KG-1, THP-1 and MV4-11 were incubated with the indicated concentrations of LJH-685 for 72 h, and then CCK-8 assay was performed to assess cell viability. (b) KG-1, THP-1 and MV4-11 were incubated with a concentration of 20 μM of LJH-685 for 0, 24, 48, 72 and 96 h, then CCK-8 assay was performed to assess cell viability. (c) LJH-685 inhibited the proliferation of MV4-11 and MOLM-13 as detected by the CFSE assay. (d) Images of colonies stained by Giemsa. (e) Images of colonies formed by KG-1, THP-1 and MV4-11 under the microscope (100 ×) after 14 d drug exposure. (f) Statistical analysis of the number of colonies formed by the AML cells (Data from Fig. 2d). Error bars, mean ± SD. Compare with the control group, ns, not statistically significant, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001
LJH-685 suppress the colony formation of AML cells
The colony formation assay was determined to evaluate effects of LJH-685 on colony formation of AML cell lines. After treatment with LJH-685 at the indicated concentration for 14 d, colonies were observed and pictured under the microscope. As shown in the Fig. 2d and e, LJH-685 reduced the number and volume of colonies in a dose-dependent manner, compared with the control group. After the 10, 20, and 40 μM concentrations LJH-685 treatment, the relative numbers of colonies formed by KG-1 cells was 77.91% ± 8.14%, 61.69% ± 10.23%, 51.90% ± 12.70%, respectively, compared with the control group (Fig. 2f). Similarity, the relative numbers of colonies formed by THP-1 cells was 74.71% ± 12.55%, 64.70% ± 28.29%, 41.21% ± 14.51%, respectively, compared with the control group. And that of MV4-11 was 87.94% ± 3.96%, 73.04% ± 6.69%, 68.24% ± 14.48%, respectively, compared with the control group (Fig. 2f). This result indicated that LJH-685 inhibited the colony formation of AML cells.
LJH-685 promoted the apoptosis of some AML cell lines
Effect of LJH-685 on the apoptosis of AML cells was determined by means of Annexin V-APC/ 7-AAD double staining method. The LJH-685 significantly induced the apoptosis of THP-1 and MV4-11, but had no effect on the apoptosis of KG-1 cells (sFig. 2a). The cell apoptosis rate of THP-1 and MV4-11 was increased with the elevated concentrations of LJH-685. And the apoptosis of THP-1 and MV4-11 were 14.26% ± 4.62%, 25.05% ± 3.40%, respectively, when treated with 40 μM LJH-685 (sFig. 2b). Consistent with CCK-8 assay, MV4-11 was also more sensitive to LJH-685 than that of KG-1 and THP-1 cells. Thus, this result suggested that LJH-685 induced the apoptosis of some AML cell lines, especially AML cells carrying FLT3-ITD mutations.
LJH-685 led to G1 phase arrest and regulated the expression of cell cycle related proteins
In order to investigate the mechanism of LJH-685 inhibiting the proliferation of AML cells, flow cytometry was performed to analyze effects of LJH-685 on the cell cycle distribution of AML cells. The cell population in G0/G1 phase increased from 49.25% to 63.53% for KG-1, 46.39% to 59.89% for THP-1 and 69.87% to 96.16% for MV4-11, accompanied with the population in S phase decreased (Fig. 3a, b). Effects of LJH-685 on the expression of cell cycle related proteins were detected by western blot to elucidate the mechanism of LJH-685 caused G1 phase arrest. As shown in the Fig. 3c, the expression of c-Myc, CDK4 and Cyclin E were decreased, and p27 was increased in AML cells, suggesting that LJH-685 affected G1-S phase transformation. At the same time, the expression of Cyclin B, CDK1 were decreased, indicating that LJH-685 may inhibit the cell cycle process via affecting the G2/M checkpoint. Thus, LJH-685 caused G1 phase arrest by regulating the expression of cell cycle related protein and then inhibited proliferation of AML cells.
Fig. 3.
LJH-685 induced cell cycle arrest and regulated the expression of cell cycle proteins. (a) After KG-1, THP-1, and MV4-11 being incubated with the indicated concentrations of LJH-685 for 48 h, the cells were harvested, and fixed overnight with 75% ethanol, then removed the ethanol, stained with the PI buffer, and analyzed the cell cycle by flow cytometry. (b) Percentages of subpopulation of AML cells at different cell cycle phases. (c) KG-1, THP-1, and MV4-11 were incubated with the indicated concentrations of LJH-685 for 12 h, and then assessed the expression of p27 and c-Myc by western blot; cells were incubated for 48 h, and then assessed the expression of CDK4, Cyclin E, Cyclin B, and CDK1 by western blotting. All data obtained from at least three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001
LJH-685 inhibited the expression and activity of MAPK pathway and STAT3
Effects of LJH-685 on the activity of MAPK pathway and STAT3 was determined to investigate the mechanism of LJH-685 affected the biological behaviors of AML cells. LJH-685 reduced the expression of p-p90RSK, RSK3, RSK1/2/3 and p-YB1 in a dose-dependent manner (Fig. 4a). Besides, LJH-685 also reduced the expression of p-STAT3 and STAT3 (Fig. 4b), suggesting that LJH-685 might inhibit the activity of STAT3 via down-regulating the expression of RSK, and was important for the leukemogenesis of AML.
Fig. 4.
LJH-685 inhibited the expression and activity of MAPK pathway and STAT3 in AML cells. After KG-1, THP-1, and MV4-11 being incubated with the indicated concentrations of LJH-685 for 72 h, the cells were harvested, and (a) the protein level changes of P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 and (b) the protein level changes of P-STAT3, STAT3 were determined by Western blotting
FF-10101 combined with LJH-685 synergistically inhibited the cell viability of MV4-11 and MOLM-13 cell lines
The foregoing results showed that the MV4-11 (carrying FLT3-ITD mutations) was more sensitive to RSKs inhibitor than other AML type cells. CCK-8 assay was determined to explore whether the combination of LJH-685 and FF-10101 had synergistic effects on the MV4-11 cells. Our results showed that the cell viability of the combined drug group was lower than that of the single drug treatment group, and combination index (CI) values obtained by CompuSyn software was less than 1 at all concentrations examined (Fig. 5a), suggesting that the combination of LJH-685 and FF-10101 synergistically inhibited the cell viability of MV4-11.At the same time, the effect of combination of LJH-685 and FF-10101 on the cell proliferation of MOLM-13 was determined by the cell count. Our results indicated that the combination of LJH-685 and FF-10101 synergistically inhibited the proliferation of MOLM-13 (sFig. 3).
Fig. 5.
FF-10101 combined with LJH-685 synergistically inhibited the viability and induced the apoptosis of MV4-11 and MOLM-13. (a) MV4-11 were incubated with the indicated concentrations of LJH-685 and FF-10101 for 72 h, then CCK-8 assay was performed to assess cell viability, and CompuSyn software was used to analyze the combination index (CI). (b) After MV4-11 being incubated with the indicated combination of 5 μM LJH-685 and 0.5 nM FF-10101 for 48 h, the cells were harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (c) Percentages of MV4-11 apoptosis were determined from at least three independent experiments. (d) After MOLM-13 cells being incubated with the indicated combination of 10 μM LJH-685 and 1 nM FF-10101 for 48 h, the cells was harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (e) Percentages of MOLM-13 apoptosis were determined from at least three independent experiments. Error bars, mean ± SD. ***P < 0.005, ****P < 0.001
FF-10101 combined with LJH-685 synergistically promoted the apoptosis and caused G1 phase arrest of MV4-11 and MOLM-13 cells
The APC-Annexin V/7-AAD double stained was used to detect effects of combination of 5 μM LJH-685 and 0.5 nM FF-10101 on the apoptosis of MV4-11 by flow cytometry. The apoptosis of LJH-685, FF-10101, and the combined group were 6.72%±0.20%, 22.12%±0.19% and 47.47±1.29% (Fig. 5b), respectively. Similarity, effects of combination of 10 μM LJH-685 and 1 nM FF-10101 on the apoptosis of MOLM-13 were also determined by flow cytometry. As shown in Fig. 5, The apoptosis of LJH-685, FF-10101, and the combined group were 7.38%±1.52%, 2.45%±0.51% and 11.88±1.48% (Fig. 5d, e), respectively. These observations indicated that FF-10101 combined with LJH-685 was more effectively in inducing the apoptosis of MV4-11 and MOLM-13 than that of single drug treatment group. Moreover, the cell populations in G0/G1 phase increased and the populations in S phase decreased in the combined group both in the MV4-11 and MOLM-13 cells, compared with the control group and single drug treatment groups (Figs. 6a, b, 4a, b). These results suggested that FF-10101 combined with the LJH-685 synergistically promoted the G1 phase arrest of MV4-11 and MOLM-13 cells.
Fig. 6.
FF-10101 combined with LJH-685 induced cell cycle arrest and inhibited the MAPK pathway of MV4-11. (a) FF-10101 combined with LJH-685 caused cell cycle arrest, after MV4-11 being incubated with the indicated combination of 5 μM LJH-685 and 0.5 nM FF-10101 for 48 h, the cells were harvested, and fixed overnight with 75% ethanol, then removed the ethanol, stained with the PI buffer, and analyzed the cell cycle by flow cytometry. (b) Percentages of subpopulation of MV4-11 at different cell cycle phases. (c) FF-10101 combined with LJH-685 inhibited the MAPK pathway. After MV4-11 being incubated with the indicated combination of 5 μM LJH-685 and 0.5 nM FF-10101 for 72 h, the cells were harvested and protein level changes of P-MEK, MEK, P-ERK, ERK, P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 were determined by Western blotting. All data obtained from at least three independent experiments. (d, e) The expressions of proteins were quantified by the Image J software. Error bars, mean ± SD. *P < 0.05, **P < 0.01
FF-10101 combined with LJH-685 inhibited the activity of MAPK pathway in the MV4-11 and MOLM-13 cells
FLT3-ITD and TKD mutations caused the constitutive activation of FLT3 and its downstream signaling pathways, including PI3K/AKT/mTOR, Ras/MAPK, and STAT5 [15, 20]. RSKs were a downstream effector of MAPK pathway. Given this, the expression of RSKs and the MAPK pathway related proteins were detected to clarify whether the combined application of LJH-685 and FF-10101 had a synergistic inhibitory effect on the MAPK pathway in the MV4-11 and MOLM-13 cells. Our results showed that the expression of P-p90RSK, RSK1/2/3, P-YB1, and YB-1 in the MV4-11 and MOLM-13 cells were down-regulated in the combined treatment group, compared with the control group and the single drug treatment groups (Figs. 6c, 4c).
Effects of Daunorubicin combined with LJH-685 on biological behavior of AML cells
Daunorubicin (DNR) or Idarubicin (IDA) combined with cytarabine is still the first-line treatment for AML, but the complete CR of this treatment was still not satisfactory [21]. Previous studies indicated that drug resistance was an important factor of treatment failure and one of the key reasons for the short survival of AML [22]. Effects of the combination of Daunorubicin and LJH-685 on the biological behavior of AML cells were investigated to clarify whether RSKs inhibitor combined with Daunorubicin can overcome Daunorubicin resistance. Our results showed that the cell viability of KG-1, THP-1 and MV4-11 cells in the Daunorubicin and LJH-685 combination treatment groups were significantly lower than that of the single drug treatment groups, and the CI values were all less than 1 (sTable 1), suggesting that the combined application of LJH-685 and Daunorubicin can synergistically inhibit the cell viability of AML cells. Similarity, the percentage of apoptosis and the cell population in G2/M phase were increased in the combination group (sFigs. 5, 7a, b). Moreover, the expression of Cyclin B and CDK1 were decreased (Fig. 7c). Finally, effects of the combination of Daunorubicin and LJH-685 on MAPK pathway were also determined, no significantly different was observed between the combined group and the single drug treatment groups (sFig. 6), and the mechanism of the combined action remains to be studied.
Fig. 7.
DNR combined with LJH-685 induce cell cycle arrest of AML cells. (a) After KG-1 and THP-1 being incubated with the indicated combination of 10 μM LJH-685 and 10 nM DNR for 48 h, the cells were harvested, and fixed overnight with 75% ethanol, then removed the ethanol, stained with the PI buffer, and analyzed the cell cycle by flow cytometry. (b) Percentages of subpopulation of AML cells at different cell cycle phases based on three independent experiments. Error bars, mean ± SD. *P < 0.05, ****P < 0.001. (c) KG-1, THP-1, and MV4-11 were incubated with the indicated concentrations of LJH-685 for 48 h, and then assessed the expression of Cyclin B, CDK1 by western blotting
LJH-685 combined with FF-10101 or DNR enhanced the survival rate of Xenogeneic transplantation mice
Luciferase-GFP expressing leukemia cell line MV4-11 were injected intravenously (i.v.) into sublethally irradiated (220 cGy) 6-8 week old NSG mice to obtain the AML Xenogeneic transplantation mice model. The combination of FF-10101 and LJH-685 treatment significantly prolonged the survival time of the mice, compared with the mice treated with single inhibitors or control (Fig. 8a, b). Untreated humanized mice succumbed to AML between 46 and 66 days after engraftment (Fig. 8b). Both the single inhibitor treatment groups and the combination of FF-10101 and LJH-685 treatment group showed longer survival times than that of control group. The proportion of human CD45+ cells were also detected to evaluate the progression of AML, both single inhibitors decreased the percentage of CD45+ cells in the AML mice, the combined treatment groups significantly reduced the percentage of CD45 + cells (Fig. 8c). At the same time, the contain of L-GMP cells were also determined to evaluate effects of inhibitor on the LSC, our result indicated that both single inhibitor treatment groups and the combination treatment group showed lower L-GMP cells than that of control group, especially the combination treatment group (Fig. 8d). Similarity, the combination of DNR and LJH-685 also significantly prolonged the survival time of the mice than that of mice treated with single inhibitors or control, at the same time, both the single inhibitor treatment groups and the combination treatment group showed lower L-GMP cells than that of control group (sFig. 7). This result might suggest that the survival benefit seen from the low AML cells and the clearance of LSC.
Fig. 8.
LJH-685 combined with FF-10101 enhanced survival rate of Xenogeneic transplantation mice. (a) Luciferase-GFP expressing leukemia cell line MV4-11 were injected intravenously (i.v.) into sublethally irradiated (220 cGy) 6-8 week old NSG mice. The mice were treated by the control, LJH-685, FF-10101 or combination of LJH-685 and FF-10101 for 14 days, each group had eight mice and the picture was captured by IVIS. The words before and after means that pictures captured before drug treatment and pictures captured after drug treatment. (b) Single inhibitor LJH-685, FF-10101 treatment or combination of LJH-685 and FF-10101 treatment enhanced the survival rate of Xenogeneic transplantation mice. (c) Single inhibitor LJH-685, FF-10101 treatment or combination of LJH-685 and FF-10101 treatment inhibited contain of human CD45+ AML cells. (d) Single inhibitor LJH-685, FF-10101 treatment or combination of LJH-685 and FF-10101 treatment inhibited percentage of L-GMP cells. Error bars, mean ± SD. **P < 0.01, ***P < 0.005
Discussion
RSKs participate in the regulation of cell behaviors by phosphorylation and activation of a variety of substrates [13, 23, 24]. The hyperactivation of RSKs have a closely relationship with many types of malignant tumors [13]. Katayama et al. found that RSKs are overexpressed and hyperactivated in AML, and that high levels confer an adverse prognosis [25], suggesting that RSKs may be a potential target for AML treatment. FLT3-ITD led to constitutive activation of the Ras/Raf/MEK/ERK pathway [12, 15]. As a downstream regulator of this pathway, RSKs played vital roles in the pathogenesis of AML carrying FLT3-ITD mutations [12, 16]. Although previous studies suggested that combined inhibition of FLT3 and RSKs may be a viable therapeutic strategy to cure AML patients with FLT3-ITD mutations. However, roles and mechanisms of combinational effects of RSKs inhibitors and FLT3 inhibitors on the leukemiagenesis of AML carrying FLT3-ITD mutations have not been reported. In this study, RSKs inhibitor LJH-685 was used to investigate roles and mechanisms of RSKs inhibitor or the combination of RSKs inhibitor LJH-685 and FLT3 inhibitor FF-10101 on the biological behavior of AML cells. Our results suggested that LJH-685 treatment inhibited clone formation ability, induced cell apoptosis, and caused AML cells in the G0/G1 phase arrest. Combination of FLT3 and RSKs inhibitors in AML cell lines with FLT3-ITD mutations showed synergism in their anti-leukemia effects.
Previous studies indicated that many types of malignant tumors’ patients with the high expression of RSKs displayed the low OS and EFS [13, 26]. Our study also indicated that RSKs were high expressed in the AML cell lines in protein levels, but the relative expression mRNA levels of RSKs were not consistent with the expression of its protein (Fig. 1a, b). Analyzing the reason, this difference might be caused by the different expression of TRAF6 and Ltn1 which medicated the ubiquitin-proteasomal degradation of RSK1/RSK2 [27, 28]. Consistent with previous results, our further study also suggested that the relative mRNA expression levels of TRAF6 and Ltn1 was higher in the control (PBMC), THP-1 and HEL than that of MV4-11, KG-1 and HL-60 (sFig. 8). Thus, the difference expression of TRAF6 and Ltn1 which medicated the ubiquitin-proteasomal degradation of RSK1/RSK2 was responsible for the difference between mRNA and protein of RSKs in the AML cell lines. Previous studies indicated that the RSK1/RSK2 were the predominate isoforms in AML cell lines [7], combined with our results, the post-translational regulation might be more possible responsible for the difference of gene expression than that of expression of different isoforms.
The hyperactivation of Ras/Raf/MEK /MAPK/ERK signal pathway caused by the N-RAS or K-RAS mutations played vital roles in the pathology of many type of cancers [10, 20], thus the association between RSKs high expression and the N-RAS or K-RAS mutations were also evaluated. Our results indicated that either N-RAS or K-RAS mutations combined with the RSKs high expression predicted the low OS and EFS which was consistent with the newly published article [29], while was opposite with some previous studies which reported that both N-RAS and K-RAS mutations had no effects on the OS and EFS of AML patients [10, 30]. Besides, effects of FLT3-ITD combined with the RSKs high expression on the OS and EFS of AML patients were also evaluated, our results suggested that FLT3-ITD combined with the RSKs high expression foretold the low OS and EFS. This result was also consistent with previous studies [12, 16]. These observations might provide stratification for patients which both had RSK high expression and FLT3-ITD mutations.
The inhibition of cell proliferation can be caused by disrupting cell cycle progression or promoting the apoptosis of cells. Our results indicated that LJH-685 inhibited the proliferation of AML cells by causing the G1 phase arrest and altering the expression of cell cycle related genes (Figs. 2, 3). RSK1/2 regulated the G phase process by inhibiting the CDK2 inhibitor p27kip1 or promoted the activation of CDK1 through direct phosphorylation and activation of Cdc25C [31, 32]. Our results also demonstrated that LJH-685 inhibited the expression of c-Myc, CDK4 and Cyclin E, while promoted the expression of p27 (Fig. 3c). These results suggested that LJH-685 might affect G1-S phase transformation and arrest cell cycle in G0/G1 phase. At the same time, LJH-685 decreased the expression of Cyclin B and CDK1 which implied that it might affect the G2/M checkpoint and thus affect cell cycle progression (Fig. 3). Consistent with previous studies [31, 32], our study revealed that LJH-685 might cause G1 phase arrest by decreased the expression of c-Myc and inhibited the conversion of p27 from Cyclin E-CDK2 to Cyclin D-CDK4/6 complex
YB-1 is a downstream effector of RSKs, the high expression of YB-1 has a closely relationship the progression of many types of cancers [33, 34]. Our study showed that LJH-685 decreased the expression of p-YB1, which inhibited the activation and nuclear transfer of YB-1 by reducing the expression of RSKs, and ultimately decreased the proliferation of AML cells (Fig. 4). Pervious study also showed that RSKs mediated the transfer of YB-1 to the nucleus and promoted the progression of T-ALL [34]. Lee et al. indicated that knockdown the expression of MEK1/2 and RSK2 inhibited the transcriptional activity of STAT3 in melanoma cells, and thus decreased the expression of Cyclin D1, leading to cell cycle arrest [35]. In this study, we found that LJH-685 inhibited the activity of STAT3 in AML cells by inhibiting the expression of RSKs (Fig. 4b). However, whether LJH-685 impact the expression of Cyclins in AML cells by inhibiting the activity of STAT3 remains to be studied.
AML patients with FLT3-ITD mutations usually respond poorly to conventional therapies, and always caused resistant to FLT3 tyrosine kinase inhibitors (TKIs) [36]. Ras-MAPK was always activated by the FLT3-ITD and TKD mutations caused constitutive activation of FLT3 [37, 38]. In this study, our results suggested that FF-10101 combined with LJH-685 could more effectively inhibit the proliferation, promote the apoptosis and cause G0/G1 phase arrest than that of single drug treatment groups (Figs. 5, 6, 4). Compared with control group and single drug treatment groups, the expression of P-p90rsk, RSK3, RSK1/2/3 and the downstream P-YB1, YB1 were significantly decreased in the combined drugs treatment group (Figs. 6c, 4c). These observations suggested that FF-10101 enhanced the inhibition of LJH-685 on RSKs and YB-1 by inhibiting MAPK pathway in AML cells carrying FLT3-ITD mutations. Consistent with previous studies, our results also suggested that LJH-685 could inhibit the proliferation of FLT3-ITD AML by both inhibited the cell cycle progression and promoted the cell apoptosis through regulating the expression of p53 and Bax [37, 38].
Besides FLT3 inhibitor, Daunorubicin combined with LJH-685 also synergistically inhibited the activity of AML cells, induced apoptosis, caused cell cycle arrest, and enhanced the sensitivity of AML cells to Daunorubicin (sFigs. 5, 7). However, these results were not completely consistent with previous study which suggested that the combined of RSKs inhibitor BI-D1870 with Daunorubicin had an additive effect on the survival rate of HL-60 cells [25]. Analyzing the reason, the traditional drugs combined with RSKs inhibitors might have different effects on different AML subtypes. But whether the combination of traditional drugs with RSKs inhibitors had synergistic effects, and which AML subtypes would be more effective, still need to be further explored.
In conclusion, this study suggested that LJH-685 inhibited the proliferation and clone formation of AML cells, caused cell cycle arrest and induced apoptosis of AML cell lines through inhibiting RSK-YB-1 pathway, especially the AML cell lines with FLT3-ITD mutations. When LJH-685 combined with FLT3 inhibitor FF-10101 or Daunorubicin, they synergistically inhibited the proliferation of AML cells, promoted the apoptosis and cycle arrest of AML cell lines with FLT3-ITD mutations. Thus, this study provided new clues that combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 showed synergism anti-leukemia effects in AML cell lines with FLT3-ITD mutations via inhibiting MAPK-RSKs-YB-1 pathway and provided new targets for therapeutic intervention especially for AML with FLT3-ITD mutations and Daunorubicin-resistant AML.
Supplementary information
(RAR 357717 kb)
AML patients had high expression of RSKs with K-RAS or N-RAS or FLT-ITD mutations indicating the low OS and EFS. (a) RSKs high expression with the N-RAS mutations displayed low OS and EFS. (b) RSKs high expression with the K-RAS mutations displayed low OS and EFS. (c) RSKs high expression with the FLT3-ITD mutations displayed low OS and EFS (PNG 139 kb)
LJH-685 induced the apoptosis of some AML cells. (a) After KG-1, THP-1, and MV4-11 being incubated with the indicated concentrations of LJH-685 for 48 h, the cells were harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (b) Percentages of apoptosis of THP-1 and MV4-11 were determined from three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 (PNG 311 kb)
FF-10101 combined with LJH-685 inhibited the proliferation of MOLM-13 detected by the cell count. (a) MOLM-13 were incubated with 5μ M LJH-685 and 0.5 nM FF-10101 single drug treatment or the combination drugs treatment for 24 h, 48 h, 72h and 96 h, then cell numbers was obtained by cell counter. (b) MOLM-13 were incubated with 10 μM LJH-685 and 1 nM FF-10101 single drug treatment or the combination drugs treatment for 24 h, 48 h, 72h and 96 h, then cell numbers was obtained by cell counter (PNG 204 kb)
FF-10101 combined with LJH-685 induced cell cycle arrest and inhibited the MAPK pathway of MOLM-13. (a) FF-10101 combined with LJH-685 caused cell cycle arrest, after MOLM-13 being incubated with the indicated combination of 10 μM LJH-685 and 1 nM FF-10101 for 48 h, the cells were harvested, and fixed overnight with 75% ethanol, then removed the ethanol, stained with the PI buffer, and analyzed the cell cycle by flow cytometry. (b) Percentages of subpopulation of MOLM-13 at different cell cycle phases. (c) FF-10101 combined with LJH-685 inhibited the MAPK pathway. After MOLM-13 being incubated with the indicated combination of 10 μM LJH-685 and 1 nM FF-10101 for 72 h, the cells were harvested and protein level changes of P-MEK, MEK, P-ERK, ERK, P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 were determined by Western blotting. All data obtained from at least three independent experiments. (d, e) The expressions of proteins were quantified by the Image J software. Error bars, mean ± SD. *P < 0.05, **P < 0.01 (PNG 609 kb)
DNR combined with LJH-685 induced the apoptosis of AML cells. (A) After KG-1, THP-1 and MV4-11 being incubated with the indicated combination of 10 μM LJH-685 and 5 nM DNR for 48 h, the cells were harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (B) Percentages of apoptosis of AML cells were determined from three independent experiments. Error bars, mean ± SD. * P < 0.05, ****P < 0.001 (PNG 281 kb)
DNR combined with LJH-685 inhibited the MAPK pathway. After KG-1, THP-1 and MV4-11 being incubated with the indicated combination of 10 μM LJH-685 and 10 nM DNR for 72 h, the cells were harvested and protein level changes of P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 were determined by Western blotting (PNG 283 kb)
LJH-685 combined with DNR enhanced survival rate of Xenogeneic transplantation mice. (a) Single inhibitor LJH-685, FF-10101 treatment or combination of LJH-685 and DNR treatment enhanced the survival rate of Xenogeneic transplantation mice. (b) Single inhibitor LJH-685, DNR treatment or combination of LJH-685 and DNR treatment inhibited percentage of L-GMP cells. Error bars, mean ± SD. **P < 0.01, ***P < 0.005 (PNG 248 kb)
The relative mRNA expression levels of TRAF6 and Lnt1 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. (a) The relative mRNA expression level of TRAF6 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. (b) The relative mRNA expression levels of Lnt1 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. Compared with PBMC group, *P < 0.05 (PNG 176 kb)
(DOC 35 kb)
Abbreviations
- AML
Acute myeloid leukemia
- CR
Complete remission
- OS
Overall survival
- RSK
90-kDa ribosomal S6 kinase
- FMK
Fluoromethylketone
- FLT3
FMS-like tyrosine kinase 3
- ITD
internal tandem repeats
- CCK-8
Cell counting kit-8
- EFS
Event free survival
- TKIs
tyrosine kinase inhibitors
- DMSO
Dimethyl sulfoxide
- 7-AAD
7-Aminoactinomycin D
- PI
Propidium Iodide
- BCA
Bicinchoninic acid
- STAT3
Signal transducers and activators of transcription 3
- HSCT
hematopoietic stem cell transplantation
- HRP
Horse radish peroxidase
- ECL
Enhanced chemiluminescence
- LSC
Leukemia stem cell
Authors' contributions
Q.-Y.W. and S.Z. wrote the main manuscript text and Z.-Y. L., S.Z., and J. L. prepared figures. All authors reviewed the manuscript Conceptualization, Q.-Y.W., K.-L.X., F. L.; data curation, S. Z., J.L., Z.-Y. L.; formal analysis, J. L., Y.-T. X., X.-R. M.; methodology, S. Z., J.L., Z.-Y. L.; supervision, Q.-Y.W., K.-L.X., F. L.; writing— original draft, Q.-Y.W., K.-L.X., F. L.; writing—review and editing, X.-R. M., M.-M.M., Z.-Y. L.; J. C., All authors have read and agreed to the published version of the manuscript.
Funding
The present investigation was supported by grants from the National Natural Science Foundation of China (82070156, 81570136), “Outstanding Youth” Talents Foundation of Jiangsu Province (BK20160054), Jiangsu Provincial Medical Youth Talent (QNRC2016777), Xuzhou key research and development project (KC21201) and Jiangsu Provincial research and development program (BE2020639).
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Ethical approval and consent to participate
All subjects filled a questionnaire including their informed consent. The study was carried out according to the Helsinki Declaration, and the samples were processed under approval of the written consent statement by the Ethical Committee of the University Hospital of Marche (Ospedali Riuniti di Ancona), Italy, Protocol Number 211226, 23/06/2011. The cell lines used in the study do not require ethic approval
Consent for publication
All authors have agreed to publish this manuscript.
Competing interests
The authors do not have any actual or potential conflicts of interest with other people or organizations.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Sen Zhang, Jun Liu and Zi-Yi Lu contributed equally to this work.
Contributor Information
Feng Li, Email: lifeng1982315@163.com.
Kai-Lin Xu, Email: lihmd@163.com.
Qing-Yun Wu, Email: qywu82@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
(RAR 357717 kb)
AML patients had high expression of RSKs with K-RAS or N-RAS or FLT-ITD mutations indicating the low OS and EFS. (a) RSKs high expression with the N-RAS mutations displayed low OS and EFS. (b) RSKs high expression with the K-RAS mutations displayed low OS and EFS. (c) RSKs high expression with the FLT3-ITD mutations displayed low OS and EFS (PNG 139 kb)
LJH-685 induced the apoptosis of some AML cells. (a) After KG-1, THP-1, and MV4-11 being incubated with the indicated concentrations of LJH-685 for 48 h, the cells were harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (b) Percentages of apoptosis of THP-1 and MV4-11 were determined from three independent experiments. Error bars, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 (PNG 311 kb)
FF-10101 combined with LJH-685 inhibited the proliferation of MOLM-13 detected by the cell count. (a) MOLM-13 were incubated with 5μ M LJH-685 and 0.5 nM FF-10101 single drug treatment or the combination drugs treatment for 24 h, 48 h, 72h and 96 h, then cell numbers was obtained by cell counter. (b) MOLM-13 were incubated with 10 μM LJH-685 and 1 nM FF-10101 single drug treatment or the combination drugs treatment for 24 h, 48 h, 72h and 96 h, then cell numbers was obtained by cell counter (PNG 204 kb)
FF-10101 combined with LJH-685 induced cell cycle arrest and inhibited the MAPK pathway of MOLM-13. (a) FF-10101 combined with LJH-685 caused cell cycle arrest, after MOLM-13 being incubated with the indicated combination of 10 μM LJH-685 and 1 nM FF-10101 for 48 h, the cells were harvested, and fixed overnight with 75% ethanol, then removed the ethanol, stained with the PI buffer, and analyzed the cell cycle by flow cytometry. (b) Percentages of subpopulation of MOLM-13 at different cell cycle phases. (c) FF-10101 combined with LJH-685 inhibited the MAPK pathway. After MOLM-13 being incubated with the indicated combination of 10 μM LJH-685 and 1 nM FF-10101 for 72 h, the cells were harvested and protein level changes of P-MEK, MEK, P-ERK, ERK, P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 were determined by Western blotting. All data obtained from at least three independent experiments. (d, e) The expressions of proteins were quantified by the Image J software. Error bars, mean ± SD. *P < 0.05, **P < 0.01 (PNG 609 kb)
DNR combined with LJH-685 induced the apoptosis of AML cells. (A) After KG-1, THP-1 and MV4-11 being incubated with the indicated combination of 10 μM LJH-685 and 5 nM DNR for 48 h, the cells were harvested, stained with the APC-Annexin V/7-AAD, and analyzed the apoptosis by flow cytometry. (B) Percentages of apoptosis of AML cells were determined from three independent experiments. Error bars, mean ± SD. * P < 0.05, ****P < 0.001 (PNG 281 kb)
DNR combined with LJH-685 inhibited the MAPK pathway. After KG-1, THP-1 and MV4-11 being incubated with the indicated combination of 10 μM LJH-685 and 10 nM DNR for 72 h, the cells were harvested and protein level changes of P-p90rsk, RSK3, RSK1/2/3, P-YB1, YB-1 were determined by Western blotting (PNG 283 kb)
LJH-685 combined with DNR enhanced survival rate of Xenogeneic transplantation mice. (a) Single inhibitor LJH-685, FF-10101 treatment or combination of LJH-685 and DNR treatment enhanced the survival rate of Xenogeneic transplantation mice. (b) Single inhibitor LJH-685, DNR treatment or combination of LJH-685 and DNR treatment inhibited percentage of L-GMP cells. Error bars, mean ± SD. **P < 0.01, ***P < 0.005 (PNG 248 kb)
The relative mRNA expression levels of TRAF6 and Lnt1 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. (a) The relative mRNA expression level of TRAF6 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. (b) The relative mRNA expression levels of Lnt1 in the PBMC, THP-1 and HEL was higher than that of MV4-11, KG-1, HL-60 and MOLM-13. Compared with PBMC group, *P < 0.05 (PNG 176 kb)
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Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.