Research ArticleExperimental Studies

The CGRP Receptor Antagonist MK0974 Induces EVI1high AML Cell Apoptosis by Disrupting ERK Signaling

AKIRA SUEKANE, TOMONAGA ICHIKAWA, YUSUKE SAITO, SHINGO NAKAHATA and KAZUHIRO MORISHITA
Anticancer Research October 2022, 42 (10) 4743-4752; DOI: https://doi.org/10.21873/anticanres.15979

Abstract

Background/Aim: Acute myeloid leukemia (AML) with high expression of the oncogenic transcription factor ecotropic viral integration site-1 (EVI1) (EVI1high AML) is refractory, and there is an urgent need to develop treatment for EVI1high AML. We previously showed that calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein 1 (RAMP1) is highly expressed in EVI1high AML and participates in calcitonin gene-related peptide (CGRP)-induced stress hematopoiesis. This study examined whether MK0974 (a CGRP antagonist) acts as a therapeutic agent in CRLR/RAMP1high AML cell lines. Materials and Methods: An in vitro experimental system was used to determine the effect of MK0974 on EVI1high AML cell lines. The expression of CRLR and RAMP1-3 in EVI1high and EVI1low AML lines was evaluated by reverse-transcription polymerase chain reaction (RT–PCR). Next, MK0974 was added to the AML cell lines, and cell proliferation, cell cycle and apoptosis assays were carried out using flow cytometry (FCM). Proteins were evaluated using western blot analysis. We also generated AML cell lines with CRLR knockdown and evaluated whether the effect of MK0974 was reduced. Results: Apoptosis was induced by adding MK0974 to the EVI1high AML cell line. In the EVI1high AML cell line, the addition of MK0974 attenuated the phosphorylation of ERK and p38. These effects were also attenuated by CRLR knockdown. Conclusion: MK0974, a CGRP receptor antagonist, inhibits the CRLR/RAMP1 complex and induces apoptosis, making it a potential therapeutic agent for CRLR/RAMP1high AML.

Key Words:

The transcription factor ecotropic viral integration site-1 (EVI1) is highly expressed in approximately 10% of patients with acute myeloid leukemia (AML). Since most AML patients with high EVI1 expression (EVI1high AML) present with refractory AML, which has a poor prognosis, the development of a novel treatment strategy is desired (1-6). Although EVI1 plays an important role in maintaining hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) (7-16), the exact molecular role of EVI1 in stem cells has not been elucidated (17-19). Our recent studies examining EVI1 regulatory genes and a group of surface antigens that are highly expressed in EVI1high AML identified G protein-coupled receptor 56 (GPR56), CD52, integrin α6 and angiopoietin-1, which are important for the maintenance of HSCs and LSCs (20-24).

As a member of one of these gene clusters, we found that calcitonin receptor-like receptor (CRLR) is highly expressed in EVI1high AML (24). CRLR works together with receptor activity modifying protein 1 (RAMP1) as a receptor for calcitonin gene-related peptide (CGRP) or with RAMP2 or RAMP3 as a receptor for adrenomedullin (ADM) (25). Since we found that RAMP1 was specifically expressed in EVI1high AML cell lines (26), CGRP was considered a specific ligand for CRLR in EVI1high AML. CGRP, a neuropeptide secreted under stress conditions, such as mechanical stimulation, infection, ischemia, and pain, binds transient receptor potential vanilloid receptor-1 (TRPV1), which is distributed in unmyelinated nerve fibers (group C nerve fibers) (27-29). Moreover, we found that CGRP is secreted into the bone marrow and is important for the maintenance of hematopoiesis during transient exposure to proliferative stress in RAMP1-deficient mice (25).

In addition to EVI1high AML, high CRLR expression has been found in many other types of AML, and high CRLR expression in AML has been reported to be associated with resistance to anticancer drugs (30). In this study, we found high expression of CRLR and RAMP1 in EVI1high AML cells and investigated the effects of inhibition of the CGRP/CRLR+RAMP1 signaling pathway on AML cells in vitro and in vivo using CRLR knockdown and a CRLR antagonist (MK0974). MK0974 inhibited AML cell proliferation via apoptosis in the absence of CGRP stimulation, suggesting that CRLR antagonists have potential as therapeutic agents for CRLR/RAMP1high AML.

Materials and Methods

Cell lines. Detailed information regarding the 15 human AML cell lines used for detecting the mRNA expression of EVI1 and CRLR has been reported previously (24). Four AML cell lines (UCSD/AML1, HNT34, Kasumi-3 and MOLM1) have chromosome 3 abnormalities with high EVI1 expression, and 11 AML cell lines do not have chromosome 3 abnormalities. Two AML cell lines (UCSD/AML1 and HNT34) are factor-dependent; these cell lines were cultured in RPMI 164 medium supplemented with 10% fetal calf serum (FBS) and 1 ng/ml human granulocyte-macrophage (GM) colony-stimulating factor. Other factor-independent AML cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS, and the human embryonic kidney cell line 293GP was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS.

RT–PCR. The expression levels of CRLR, RAMP1-3 and β-actin in AML cell lines were assessed by semiquantitative RT–PCR. Total RNA was extracted from leukemia cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and 1 μg of total RNA was reverse-transcribed to obtain first-strand cDNA using an RNA-PCR kit (Takara Bio Inc., Kusatsu, Japan). The cDNA fragments were amplified by PCR using the primers listed in Table I.

View this table:
Table I.

Primer list for RT–PCR.

Quantitative real-time RT–PCR. The levels of CRLR expression in AML cell lines was assessed by quantitative real-time RT–PCR using cDNA. Quantitative real-time RT–PCR was performed using GeneAce SYBR qPCR Mix (Nippon Gene, Tokyo, Japan) on a StepOne Real-time PCR System (Applied Biosystems, Waltham, MA, USA). The amplification data were analysed using StepOne software (Applied Biosystems). PCR was performed in triplicate, and the expression levels were normalized to those of β-actin. The primer sequences used are listed in Table II.

View this table:
Table II.

Primer list for quantitative real-time RT–PCR.

Establishment of AML cells with stable CRLR knockdown. Small hairpin RNA (shRNA) with two different oligonucleotide DNA sequences against CRLR was cloned into the BamHI–EcoRI site of the RNAi-Ready-pSIREN-RetroQ-ZnGreen vector (Takara Bio USA, Mountain View, CA, USA) (shCRLR1,2) (Table III). The control shRNA vector against luciferase was purchased from Takara Bio USA. shRNA vectors were co-transfected into 293GP cells along with the envelope plasmid pVSV-G using HilyMax reagent (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Six hours post-transfection, the medium was changed, and the cells were incubated for 48 h in DMEM with 10% FBS and 10 μM forskolin (Sigma–Aldrich, St. Louis, MO, USA). The supernatant containing the retrovirus was collected using polyethylene glycol (PEG; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) purification. Two days after retroviral infection of the AML cells, positive cells were sorted using a JSAN cell sorter (Bay Bioscience, Kobe, Japan), and knockdown of gene expression was confirmed by quantitative real-time RT–PCR.

View this table:
Table III.

The sequences used to construct expression vectors with shRNA sequences specific for calcitonin receptor-like receptor (CRLR).

Western blotting. Western blotting was performed as described previously (26). Briefly, protein extraction from leukemia cells was performed using NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, and 1% NP-40) supplemented with a proteinase inhibitor cocktail (Sigma–Aldrich) and phosphatase inhibitor tablets (Roche, Penzberg, Germany). The protein concentrations of the supernatants collected from centrifuged lysates were measured using a bovine serum albumin (BSA) standard. Equal amounts of protein were separated by electrophoresis using SDS-polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membrane blocking was performed using PBS–Tween (0.1%) (PBST) with 1% BSA or 5% non-fat dried milk. All primary antibodies listed in Table IV were used at a dilution of 1:1,000 in PBST-BSA, 5% non-fat dried milk, or Can Get Signal Buffer (TOYOBO, Osaka, Japan). Band detection was performed using a Lumi-Light Plus kit (Roche) and LAS-3000 imager (Fujifilm, Tokyo, Japan).

View this table:
Table IV.

The list of the antibodies used in this study.

Cell cycle and apoptosis analysis. For cell cycle and apoptosis analysis, UCSD/AML1 and MOLM1 cells were incubated for 48 h with MK0974. The treated cells were collected and analyzed for apoptosis and the cell cycle using FCM. After propidium iodide staining in vitro, BrdU incorporation was measured by FCM using an APC BrdU Flow Kit (BD Biosciences Pharmingen, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. For apoptosis analysis, cells were stained using an Annexin V Apoptosis Detection Kit I (BD Biosciences Pharmingen) following the manufacturer’s instructions. FCM and cell sorting were performed using a JSAN cell sorter.

Statistics. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA). The unpaired 2-tailed Student’s t-test was used when two groups were compared. One-way or two-way ANOVA, Dunnett’s multiple comparisons test, and Tukey’s multiple comparisons test were used for multiple comparisons.

Results

MK0974 alone suppressed the proliferation of CRLR-expressing AML cell lines. First, the expression of CRLR and the heterodimeric RAMP protein group (1 to 3) was analyzed using semiquantitative RT–PCR in various AML cell lines: 11 cell lines with low EVI1 expression and four cell lines with high EVI1 expression. All four EVI1high AML cell lines (UCSD/AML1, HNT-34, Kasumi-3, and MOLM-1) expressed high levels of CRLR and RAMP1 but low levels of RAMP2 or RAMP3; among the 11 EVI1low AML cell lines, CRLR and members of the RAMP family were expressed at low levels in most of the cell lines; however, CRLR was highly expressed in the KG-1-cell line, and RAMP1 was highly expressed in the NH cell line (Figure 1A).

Figure 1.
Figure 1.

Expression of calcitonin receptor-like receptor (CRLR) and RAMP family members and the effects of CGRP and/or CRLR inhibitors on AML cell lines. (A) The expression levels of CRLR, RAMP1, RAMP2, and RAMP3 in 15 AML cell lines, including ten EVI1low and five EVI1high AML cell lines, were determined using semiquantitative RT–PCR. The expression of β-actin is shown at the bottom as a control. (B) The growth of two EVI1high AML cell lines (UCSD/AML1 and MOLM1) was measured at the indicated time points after the addition of CGRP (100 nM), CGRP+MK0974 (100 nM), MK0974 (100 nM) or DMSO (control). (C) The growth of the EVI1low AML cell line (U937) was measured at the indicated time points after the addition of CGRP (100 nM), CGRP+MK0974 (100 nM), MK0974 (100 nM) or DMSO (control). Viable cells were counted using a light microscope after trypan blue staining. Data are shown as the mean±SD of triplicate reactions. ****p<0.0001 compared with the DMSO-treated control (two-way ANOVA and Tukey’s multiple comparisons test).

To examine whether CGRP stimulates the growth of CRLR/RAMP1high AML cells, we initially assessed the effect of CGRP treatment on CRLR/RAMP1high AML cell growth. Two CRLR/RAMP1high AML cell lines (UCSD/AML1 and MOLM1) and one CRLR/RAMP1low AML cell line (U937) were treated with CGRP. CGRP treatment significantly enhanced the growth of both CRLR/RAMP1high AML cell lines. Additionally, MK0974 (telcagepant), which was developed for migraine treatment, is a CGRP receptor antagonist that potently blocks human α-CGRP-stimulated cAMP responses in human CGRP receptor-expressing HEK293 cells (31). Therefore, we assessed the effect of MK0974 on the growth of two CRLR/RAMP1high AML cell lines (UCSD/AML1 and MOLM1) and one CRLR/RAMP1low AML cell line (U937). The proliferation of the two CRLR/RAMP1high AML cell lines was enhanced by CGRP administration but suppressed by the simultaneous addition of MK0974. In the absence of CGRP, MK0974 alone inhibited proliferation to the same degree as that observed in CRLR/RAMP1high AML cells (Figure 1B). In contrast, U937 cells (CRLR/RAMP1low AML cells) showed no change in proliferation when treated with CGRP, MK0974, or both CGRP and MK0974 (Figure 1C). Thus, the proliferation of CRLR/RAMP1high AML cells was inhibited by MK0974, a CGRP receptor antagonist, even in the absence of CGRP.

MK0974 induced apoptosis of CRLR/RAMP1high AML cell lines. To examine the mechanism of the inhibitory effect of MK0974 on the growth of CRLR/RAMP1high AML cell lines, the cell cycle distribution of CRLR/RAMP1high AML cell lines was examined on the second day after MK0974 administration. MK0974 treatment significantly increased the sub-G1-phase cell population and decreased the S-and G2/M-phase cell populations compared to those in untreated CRLR/RAMP1high AML cells (UCSD/AML1 and MOLM1) (Figure 2A). Furthermore, to confirm that the change in the sub-G1-phase cell population was due to apoptosis, cells were stained with Annexin V, and the apoptotic fraction was examined by FCM. Before MK0974 treatment, only a few percent of USCD/AML1 cells were stained with Annexin; however, after treatment, over 20% of the cells were positive for Annexin staining (Figure 2B). This finding indicates that MK0974 treatment induced apoptosis.

Figure 2.
Figure 2.

Effects of calcitonin receptor-like receptor (CRLR) inhibitors on the cell cycle and signaling pathways in AML cell lines. (A) Two days after MK0974 (1 nM) treatment, the cell cycle distribution of the UCSD/AML1 and MOLM1 cell lines was determined using FCM after propidium iodide staining. Data are shown as the mean±SD of triplicate reactions. (B) Two days after MK0974 (1 nM) treatment, apoptotic UCSD/AML1 cells were determined by FCM after Annexin V staining. Data are shown as the mean±SD of triplicate reactions. ****p<0.0001, **p<0.01 compared to parental control cells (Student’s t-test). (C) After MK0974 (1 nM) treatment, the phosphorylation of p38 and ERK in the UCSD/AML1 and MOLM1 cell lines was determined using western blotting.

Since MK0974 treatment induced CRLR/RAMP1high AML cell death even without CGRP stimulation, we investigated its effects on intracellular signaling pathways. CGRP stimulation is known to maintain cardiomyocyte survival through p38/MAPK and ERK activation downstream of CRLR/RAMP1 (32). Therefore, we examined the effects of MK0974 treatment on the phosphorylation of p38/MAPK and ERK. Since MK-0974 is a potent antagonist of the human (Ki=0.77 nM) CGRP receptors in vitro (31), subsequent experiments were conducted using 1 nM of MK 0974. After the addition of MK0974 (1 nM) to two CRLR/RAMP1high AML cell lines (UCSD/AML1 and MOLM1), the cells were collected at set time points, and the amounts of total and phosphorylated ERK and p38/MAPK proteins were determined using western blot analysis. Both ERK and p38/MAK were highly phosphorylated in the CRLR/RAMP1high AML cells before MK0974 treatment, and upon MK0974 treatment, the levels of phosphorylated p38 gradually decreased and disappeared after 6 h. Phosphorylated ERK was almost eliminated 5 min after treatment (Figure 2C). Thus, MK0974 treatment inhibited ERK and p38/MAPK signaling in AML cells, leading to cell death.

CRLR expression is not essential for constitutive activation of the CRLR/RAMP1 signaling pathway in CRLR/RAMP1high AML cells. In CRLR/RAMP1high UCSD/AML1 cells, the CRLR/RAMP1 signaling pathway is constantly activated without CGRP stimulation, and this signaling pathway is inhibited by the CRLR antagonist MK0974. Therefore, we examined whether CRLR molecules are essential for enhanced activation of the CRLR/RAMP1 signaling pathway by suppressing CRLR expression in UCSD/AML1 cells. CRLR expression was suppressed by more than 50% in two shCRLR-transfected cell lines (UCSD/AML1/shCRLR1 and shCRLR2) (Figure 3A). When the growth curves of the four cell lines (parental, control shLuc, shCRLR1, and shCRLR2) were compared, little difference in cell proliferation was observed (Figure 3B). In addition, the activation of various signaling pathways in the four UCSD/AML1 cell lines was examined. Active phosphorylated ERK and p38/MAPK were observed in all cell lines, with no difference in their activation status (Figure 3C). The activation of other signaling pathways, such as JAK/STAT, PI3K/AKT, and NF-Embedded ImageB, was weak and not affected by the suppression of CRLR expression. Therefore, treatment with MK0974 inhibited the ERK and p38 signaling pathways in these cell lines.

Figure 3.
Figure 3.

Effects of suppressed calcitonin receptor-like receptor (CRLR) expression on cell proliferation and signaling pathways in UCSD/AML1 cells. (A) The expression levels of CRLR were determined in parental, shLuc-transfected, and shCRLR-transfected UCSD/AML1 cells using quantitative real-time RT–PCR. Data are shown as the mean±SD of triplicate reactions. *p<0.05, compared with the parental control cells (one-way ANOVA and Dunnett’s multiple comparisons test). (B) The growth of shCRLR- and shLuc-transfected UCSD/AML1 cells was measured at the indicated time points. (C) After CRLR knockdown, the phosphorylation of p38, STAT3, STAT5, AKT, IEmbedded ImageBα and ERK in the UCSD/AML1 cell line was determined using western blotting.

Suppression of CRLR expression weakens the inhibitory effect of MK0974. In the next experiment, CRLR-suppressed CRLR/RAMP1high AML cell lines were treated with 1 nM of MK0974, and its inhibitory activity was assessed. In parental and shLuc-transfected control UCSD/AML1 cells, 1 nM of MK0974 suppressed their proliferation at, whereas in shCRLR cells its effect was attenuated (Figure 4A). Comparing the effect of MK0974 on the phosphorylation levels of ERK and p38/MAPK, in parental and shLuc-AML1 cells, the levels of phosphorylated ERK and p38/MAPK were almost eliminated by MK0974 treatment. In the shCRLR cell line, however, the levels of phosphorylated ERK and p38/MAPK did not change significantly by the MK0974 treatment (Figure 4B). Therefore, the expression of CRLR is necessary to maintain the effect of MK0974, and MK0974 is thought to affect the CRLR/RAMP1 complex.

Figure 4.
Figure 4.

Suppression of calcitonin receptor-like receptor (CRLR) expression and the effect of CRLR inhibitors on UCSD/AML1 cells. (A) The growth of parental, shLuc-transfected and shCRLR-transfected UCSD/AML1 cells was measured at the indicated time points after the addition of 1 nM MK0974. Viable cells were counted using light microscopy after trypan blue staining. Data are shown as the mean±SD of triplicate reactions. ****p<0.0001 compared with the DMSO-treated control (two-way ANOVA and Tukey’s multiple comparisons test). (B) After MK0974 (1 nM) treatment, the phosphorylation of p38 and ERK in shLuc-transfected and shCRLR-transfected UCSD/AML1 cell lines was determined using western blotting.

Discussion

In this study, we found that CRLR and RAMP1 are highly expressed in many EVI1-high AMLs; high CRLR/RAMP1 expression enhances and activates the downstream ERK and p38 signaling pathways without stimulation with its ligand, CGRP. As a result, activation of the ERK and p38 signaling pathways resulted in the maintenance of the growth of CRLR/RAMP1high AML cells. Moreover, MK0974, a CRLR antagonist, inhibits the activity of the ERK and p38 signaling pathways downstream of the CRLR/RAMP1 complex, resulting in the induction of apoptosis. Thus, these results suggest that CRLR antagonists have therapeutic potential against CRLR/RAMP1high AML.

The over-expression of receptor tyrosine kinases (RTKs; EGFR, HER2/Erb2, MET, etc.) by genome amplification has been reported in various cancers. Receptor over-expression leads to increased local concentrations of the receptor, resulting in enhanced RTK signaling and the induction of cell proliferation (33). The over-expression of CRLR/RAMP1 in AML cells is suggested to utilize a similar mechanism; however, the mechanism by which MK0974 inhibits the CRLR/RAMP1 signaling pathway is unknown. Experiments on amino acid substitutions in RAMP1 and CRLR suggest that the receptor-binding site of MK0974 is related to direct ligand interaction at sites containing Trp-74 in RAMP1 and Met-42 in CRLR (34). Trp-74 of RAMP1 and Met-42 of CRLR may be located near the RAMP1/CRLR interface, which may correspond to the CGRP-binding site. That the suppression of CRLR expression interfered with the function of MK0974 in this study clearly indicates that the binding site for that region is involved in inactivation of the receptor, even in the absence of CGRP.

In this study, we show that CGRP receptor antagonists are a potential treatment for treatment-resistant AML, particularly CRLR/RAMP1high AML. Four clinical trials with MK0974 were conducted, and a meta-analysis showed that MK0974 was more effective than placebo in the treatment of pain caused by acute migraine (35). However, the clinical development of MK0974 has been halted owing to concerns about hepatotoxicity, and the development of other CGRP receptor antagonists or CGRP antibodies is underway. Anti-CGRP antibodies are ineffective against AML because they neutralize CGRP itself, but anti-CRLR antibodies can inhibit the CRLR/RAMP1 complex and thus have potential as therapeutic agents for AML.

Acknowledgements

The Authors thank all the members of the Division of Tumor and Cellular Biochemistry and the HTLV-1/ATL Research Facility, University of Miyazaki, for helpful discussions and comments. The Authors are also grateful to all the researchers who kindly provided us with important cell lines and materials. This work was supported by a Grant-in-Aid for Early Career Scientists (20K17855).

Footnotes

  • Authors’ Contributions

    AS performed the experiments, analyzed the data in Figures 1-4, and wrote the manuscript. YS and TI performed all experiments. YS, SN, and TI analyzed the data, engaged in useful discussions, and edited the manuscript. KM designed the experiments, analyzed the data, wrote the manuscript, conceived, and supervised the project.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received July 28, 2022.
  • Revision received August 17, 2022.
  • Accepted August 22, 2022.

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The CGRP Receptor Antagonist MK0974 Induces EVI1high AML Cell Apoptosis by Disrupting ERK Signaling
AKIRA SUEKANE, TOMONAGA ICHIKAWA, YUSUKE SAITO, SHINGO NAKAHATA, KAZUHIRO MORISHITA
Anticancer Research Oct 2022, 42 (10) 4743-4752; DOI: 10.21873/anticanres.15979
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