The combination of venetoclax and quercetin exerts a cytotoxic effect on acute myeloid leukemia

doi:10.21203/rs.3.rs-4807182/v1

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The combination of venetoclax and quercetin exerts a cytotoxic effect on acute myeloid leukemia

https://doi.org/10.21203/rs.3.rs-4807182/v1

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Venetoclax is a BH3 mimetic and approved recently for Acute mteloid leukemia (AML) treatment. However, venetoclax’s effect on AML is still restrictive and a novel strategy is required. We demonstrate here for the first time that by a combination with a kind of naturally occurring flavonoid quercetin, the cytotoxic effect of venetoclax drastically increased. Combined treatment with venetoclax and quercetin changed most of AML cell KG-1 to a condensed morphology. Cell cycle analysis revealed that the combination strongly induced cell death. Caspase inhibitor blocked the cell death and the combination induced PARP cleavage, indicating that apoptosis was mainly induced. These effects were also observed in another AML cell Kasumi-1 but not in chronic myeloid leukemia (CML) cell K562. Public data analysis demonstrated that Bcl-2 expression is increased in AML cells compared to other malignant tumors and the survival and the growth of AML cell line depends on Bcl-2. Bcl-2/Bax ratio is a determining factor in whether or not to induce apoptosis. We found that quercetin increased Bax expression in KG-1. Our study provides a novel function of quercetin and a promising strategy for AML treatment using venetoclax

Health sciences/Diseases/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia

Health sciences/Oncology/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia

Health sciences/Medical research/Drug development

Acute myeloid leukemia (AML) is the most common type of adult leukemia among hematological malignancies [1-3]. AML has genetic diversity with various gene mutations and is a heterogeneous group of diseases [4, 5]. The 5-year overall survival rate of AML is still less than 50% and AML remains high mortality rate [6, 7]. Moreover, most of AML patients frequently become relapse and chemotherapy resistance and these lead to a poor prognosis [8-10]. Thus, new strategies of AML treatment are expected to be developed.

Venetoclax (ABT-199) is a mimetic of the BH3 domain and selectively inhibits Bcl-2 [11, 12], without a thrombocytopenic side effect which is caused other BH3 mimetic, navitoclax (ABT-263). Venetoclax was first tested in phase III clinical trials for CLL in combination with rituximab and the progression-free period increased to 57.3% in the combination of venetoclax and rituximab compared with 4.6% in rituximab treatment only and the overall survival also increased to 85.3% in the combination of venetoclax and rituximab compared with 66.8% in rituximab treatment only, after 4 years [13]. Thus, venetoclax has been approved for with relapsed or refractory CLL by The U. S. Food and Drug administration (FDA) in 2016. In the case of clinical study for AML patients, phase III trials have been conducted in combination of venetoclax with azacitidine and the median overall survival has been prolonged (14.7 months in azacytidine+venetoclax group against 9.6 months in azacytidine alone group) [14]. In addition, the combined treatment of venetoclax and low-dose cytarabine (LDAC) in phase III trials demonstrated that the median overall survival has been prolonged (7.2 months in venetoclax+LDAC group against 4.1 months in LDAC alone group)[15]. Venetoclax has been also approved for patients age 75 and older with newly diagnosed AML or who have complications that made intensive induction chemotherapy ineligible by the FDA in 2018. The effects of venetoclax treatment with azacitidine or cytarabine in AML are still restrictive comparted with CLL. New combination treatments with venotoclax have been attempted for AML treatment [16, 17]. We also recently reported that sodium butyrate enhanced venetoclax efficacy on AML cells [18]. Therefore, new strategies to strengthen the venetoclax’s efficacy are urgently required.

Venetoclax specifically targets Bcl-2 which is an anti-apoptotic protein localized on mitochondria with little affects for other Bcl-2 family proteins [11, 12]. Apoptotic signals stimulate mitochondria and cause mitochondrial outer membrane permeability (MOMP) through pro-apoptotic factors such as Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak) [19, 20]. MOMP triggers cytochrome C release from mitochondria leading to cleavage and activation of caspases, resulting in execution of apoptosis accompanied with degradation of various substrates by caspases. Bcl-2 interacts with BH3 domain of Bax or Bak and inhibits apoptotic induction. Not only Bcl-2 but also inhibitor of apoptosis (IAP) family blocks apoptosis induction through the binding and inactivation of caspases [21]. To facilitate the venetoclax effects, regulations of apoptosis-related factors are considered to be effective.

Quercetin is a plant flavonoid containing in fruits and vegetables such as onion, apple, red grape and broccoli [22]. Intake of quercetin is considered to be safe since several human intervention studies have been reported that no severe adverse events were detected [23-25]. Quercetin possesses many biological functions such as anti-inflammatory, anti-oxidant and anti-malignant tumor effects [26, 27]. The-anti-malignant tumor effects of quercetin have been reported by many studies and depend on cell cycle arrest and apoptosis induction [28-30]. The cell growth inhibitions by quercetin are regulated through cellular signal transduction such as phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway [31, 32], Wnt/b-catenin pathway [33], Janus kinase (JAK)/signal transducer and transcription activator (STAT) pathway [34, 35], p53 pathway [36, 37] and microRNAs (miRNAs) regulation [38]. To increase the absorption and the anti- malignant tumor activities of quercetin, quercetin encapsuled in nanoparticles [39, 40] and chemical changed isoquercetin are developed [41, 42]. Thus, quercetin is a promising agent for anti-malignant tumor treatments. Quercetin has already been thoroughly investigated in many studies, especially for cancers, however, it has not been examined whether quercetin affects venetoclax efficacy. Therefore, we analyzed the effect of quercetin on venetoclax.

The present study here demonstrates that the combination of venetoclax and quercetin strongly induces apoptosis in AML cells.

Cell culture

KG-1 acute myeloid leukemia (AML) cell line was obtained from Riken BioResource Center (Ibaraki, Japan) and maintained in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% Fetal bovine serum (FBS) (Sigma-Aldrich; Merck KGaA, Burlington, MA). Kasumi-1 AML cell line was obtained from the JCRB cell bank (Osaka, Japan) and maintained in RPMI-1640 supplemented with 10% FBS. K562 chronic myeloid leukemia (CML) cell line was obtained from Riken BioResource Center and maintained in RPMI-1640 supplemented with 10% FBS. These cells were cultured at 37℃ in humidified air with 5% CO₂.

Reagents

Venetoclax (also called ABT-199) was purchased from Selleckchem (Houston、TX) and dissolved in Dimethyl sulfoxide (DMSO). Pan-caspase inhibitor Q-VD-OPh was purchased from MedChemExpress (Monmouth Junction, NJ), dissolved in DMSO and added to cells at 20 mM as a final concentration. Quercetin was obtained from Wako (FUJIFILM Wako Pure Chemical), dissolved in DMSO and used at described concentrations.

Western blotting

KG-1 or Kasumi-1 cells were lysed in RIPA buffer which was contained aprotinin. The cells suspended in RIPA buffer was centrifuged and the supernatant was collected as cell lysate. The cell lysate was dissolved in 7.5 % sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and proteins were transferred to a nitrocellulose filter. The filter was reacted to poly (ADP-ribose) polymerase (PARP) antibody (#9542) (Cell signaling technology, Danvers, MA) or b-actin antibody (PM053) (Medical and Biological Laboratories, Tokyo, Japan). The signal was detected by ECL Western blotting detection reagents and image quant LAS500 (GE healthcare, Chicago, IL). Band intensities of cleaved PARP and actin were quantified using Image J software (https://imagej.net/ij/).

RT-qPCR

Total RNA was extracted from cells using Isogen II reagent (Nippongene, Tokyo, Japan). Equal amount of total RNA was reverse-transcribed to cDNA by superscript IV reverse transcriptase (Thermo Fisher scientific, Waltham, MA). Real-time PCR was performed with the cDNA, Thunderbird SYBR qPCR mix (Toyobo Life Science, Osaka, Japan), and specific primers by StepOne plus real-time PCR system (Thermo Fisher scientific). The thermocycling condition was 95^oC for 10 sec, followed by 40 cycles of 95^oC for 15 sec and 60^oC for 1 min, then 95^oC for 15 sec, 60^oC for 1 min and 95^oC for 15 sec. Sequences of specific primers are following. Human BAX (Forward; 5’-TCAGGATGCGTCCACCAAGAAG, reverse; 5’-TGTGTCCACGGCGGCAATCATC), human BAK1 (Forward; 5’-ATGGTCACCTTACCTCTGCAA, reverse; 5’-TCATAGCGTCGGTTGATGTCG), human Survivin (Forward; 5’-ATGGGTGCCCCGACGTTG, reverse; 5’- AGAGGCCTCAATCCATGG), human XIAP (Forward; 5’-GCAAGAGCTGGATTTTATGCTTTAGG, reverse; 5’-CAGATATTTGCACCCTGGATACCAT), human cIAP2 (Forward; 5’-TTCCGGGTACAGAAAACAGTGGA, reverse; 5’-TGGTAGGAACTTCTCATCAAGGCAGA), human cIAP1 (Forward; 5’-TCAGCTAGTCTGGGATCCACCTC, reverse; 5’-AGGGTTTGGAGAAAGGCTGGAGT), human Bcl-2 (Forward; 5’-TCGCCCTGTGGATGACTGAG , reverse; 5’-CAGAGTCTTCAGAGACAGCCAGGA). human Actin (Forward; 5’-GCTGTGCTACGTCGCCCTG , reverse; 5’-GGAGGAGCTGGAAGCAGCC).

Giemsa staining

Cells were attached to glass slide by cytospin 3 (Shandon; Thermo Fisher Scientific). Then May-Gruenwald-Giemsa staining was done and cell morphological changes were observed under an Eclipse E800 optical microscope (Nikon, Tokyo, Japan). The magnification was x400.

Cell counts

Cultured-cell solution was mixed with the same amount of 0.5% trypan blue/PBS solution and the mixture was applied to Burker-Turk counting chamber (Erma, Saitama, Japan). Cells stained with blue or not were observed under an Olympus CK40 inverted light microscope (Olympus, Tokyo, Japan). We regarded cells stained blue by trypan blue as dead cells since dead cells could not emit trypan blue dye.

Flow cytometry analysis

Cells were collected by centrifugation and suspended in propidium iodide (PI) solution, containing PBS, 0.1% triton-x100 and 10 mg/ml of propidium iodide. We analyzed cells stained with PI for DNA contents using BD FACS Canto II and BD FACS diva (BD Biosciences, Franklin Lakes, NJ).

Bioinformatics analysis

Bcl-2 expression in bone marrow samples from leukemia patients was analyzed by surveying Leukemia MILE study data set (GSE13159) [43] of the BloodSpot (https://www.fobinf.com) [44]. Bcl-2 expression in various types of malignant tumors were analyzed by cBioPortal for cancer genomics (https://www.cbioportal.org) using data of The Cancer Genome Atlas (TCGA) database (https://www.cancer.gov/ccg/research/genome-sequencing/tcga). Characteristics of the patients, age, sex and stage of the disease of TCGA data are summarized in [45]. Expression of Bcl-2 and cell dependency on Bcl-2 in leukemia cell lines were analyzed by depmap (https://depmap.org/portal/).

Statistical analysis

For comparisons among multiple groups, one-way ANOVA followed by Tukey’s post hoc test with GraphPad Prism 10 (Dotmatics, Boston, MA). A two-tailed, unpaired Student’s t-test was used in order to analyze the data obtained from Bloodspot analysis.

Combination of venetoclax and quercetin changed cell morphology of KG-1 cells. We investigated the effect of venetoclax, which is used for AML treatment in clinic, on AML cell line KG-1 cells. Cell morphology was not change by treatment of venetoclax alone (Figure 1A). Quercetin alone treatment also did not mostly affect KG-1cells. On the other hand, the combination of venetoclax and quercetin increase condensed morphology in a quercetin dose-dependent manner. May-Gruenwald-Giemsa staining clearly showed cell and nuclei condensation when venetoclax simultaneously treated with quercetin (Figure 1B). These results indicated quercetin affected venetoclax efficacy on KG-1 AML cells.

The combination of venetoclax and quercetin induced cell death.

We examined the reason why KG-1 cells became cell and nuclei condensation by the combined treatment with venetoclax and quercetin. We hypothesized that KG-1 cells died and cell cycle analysis was performed by flow cytometry. As shown in Figure 2 A and B, only quercetin treatment slightly increased G1 phase at 2 and 5 mM and then increased sub-G1 phase which indicates dead cells containing low DNA contents by DNA fragmentation, at 10 mM. On the other hand, venetoclax treatment alone weakly increased sub-G1 and decreased G1 phase. Combination of venetoclax and quercetin drastically increased sub-G1 phase and decreased other phases in a quercetin dose-dependent manner. Venetoclax (200 nM) plus quercetin (10 mM) induced sub-G1 phase in more than 70% of cells, which was equivalent to the condensed cell number observed in Figure 1. These results indicated that when quercetin was added, venetoclax efficiently killed KG-1 AML cells.

The combination of venetoclax and quercetin induced caspase-dependent apoptosis.

Since cells were condensed by the treatment with venetoclax and quercetin, the cell death type was considered to be apoptosis but not necrosis. Thus, we used Q-VD-OPh which is a potent pan-caspase inhibitor [46]. First, we performed a trypan blue dye exclusion assay to investigate cell growth and cell death (Figure 3A). In this assay, live cells can exclude the dye out of cells and looks white without staining in blue. On the other hand, dead cells cannot exclude the dye and are stained in blue. After treatment of venetoclax in KG-1 cells, we counted the number of white or blue cells. When KG-1 cells were incubated for 48 hours without venetoclax nor quercetin, KG-1 cells markedly proliferated compared to cell number of day 0. Only venetoclax or quercetin treatment weakly blocked the proliferation and increased dead cells. The combination of venetoclax and quercetin remained only 20 % live cells and most of cells died. Q-VD-OPh almost completely blocked the cell death and recovered live cells to the level of only quercetin treatment. Cell cycle analysis also showed that sub-G1 population increased by the combination of venetoclax and quercetin was completely blocked by Q-VD OPh (Figure 3B and C). In the observation under a microscope, QVD was able to return the condensed cell image to the control state (Figure 3D). These results indicated that quercetin functioned to enhance the apoptosis-inducing property of venetoclax.

The combination of venetoclax and quercetin induced apoptosis in Kasumi-1 cells.

We employed another AML cell line Kasumi-1. Quercetin alone partly increased condensed cells and the combination of venetoclax and quercetin facilitated the effect (Figure 4A). Q-VD-OPh could block the condensed morphology of cells. The combination of venetoclax and quercetin induced sub-G1 in about 50 % of cells and Q-VD-OPh completely inhibited the increase of sub-G1 phase (Figure 4B and C). To confirm apoptosis induction, we examined cleavage of PARP, which is a caspase substrate and cleaved by activated caspases when apoptosis is induced. Only venetoclax or quercetin showed weak band of the cleaved PARP in both KG-1 and Kasumi-1 cells. The cleaved band was strengthened by the combination of venetoclax and quercetin. These results indicated that quercetin enhances venetoclax effect on apoptosis induction in Kasumi-1 as well as KG-1 cells.

The combination of venetoclax and quercetin effects on another type of leukemia cells. Next, we investigated the effect of the combination of venetoclax and quercetin in another type of leukemia cells. We used chronic myeloid leukemia (CML) cell line K562. In K562 cells, quercetin alone increased sub-G1 at about 20 % (Figure 5A and B). When venetoclax was combined with quercetin, sub-G1 population was not almost changed, less than 30%. Trypan blue dye exclusion assay showed that cell proliferation was weakly blocked and sub-G1 was increased very little by quercetin treatment, however addition of venetoclax did not alter the quercetin’s effect. Moreover, cell morphologies were almost not changed by treatment of venetoclax with or without quercetin. These results indicated that the combined effect was minor in CML cell line K562 compared with AML cell lines KG-1 and Kasumi-1.

Quercetin up-regulated Bax expression.

To analyze the mechanism of the combined effect, we examined expressions in apoptosis related genes. First, we searched and re-analyzed TCGA data. Bcl-2 expression was increased in leukemia patient samples compared to other types of cancer (Figure 6A). Another analysis of TCGA data showed AML more highly expressed Bcl-2 than another malignant tumor samples (Supplementary Figure 1). Bloodspots analysis of Microarray Innovations in LEukemia (MILE) study with 2096 blood or bone marrow samples of acute and chronic leukemia patients demonstrated that all types of AML possessed higher Bcl-2 expression compared to pre-AML syndrome, myelodysplastic syndrome (MDS) with significance (Supplementary Figure 2). Moreover, Bcl-2 expression was increased in AML cell line KG-1 and Kasumi-1 among total malignant tumors (Supplementary Figure 3A). On the other hand, CML cell line K562 had the lowest expression of Bcl-2 among 62 leukemia cell lines. Depmap analyzed dependency of cell survival and growth on Bcl-2 by CRISPR knockdown. Kasumi-1 depended on Bcl-2 but K562 did not depend on Bcl-2 (Supplementary Figure 3B). These results indicated AML cells had increased Bcl-2 expression and Bcl-2 contributed to those survival and growth. Apoptosis induction is decided by Bax/Bcl-2 ratio. We hypothesized that quercetin increased Bax expression and sensitized to venetoclax in AML cells. We performed RT-qPCR of Bax and showed increase of Bax by quercetin treatment (Figure 6B). On the hand, another pro-apoptotic member of Bcl-2 family, Bak was not affected by quercetin treatment. Bcl-2 was slightly decreased by quercetin treatment. Apoptotic induction is also determined by IAP family which directly bind to caspases and inhibit their function. Quercetin treatment was not changed survivin. Another members IAP-1, IAP-2 and XIAP did not decrease, but rather increased (Figure 6C).

In the present study, we showed that the combination of venetoclax and quercetin exerted strong cytotoxic effect against AML cells compared with venetoclax alone. Venetoclax was recently approved for AML treatment but the effect was partial. We demonstrated here a novel strategy to overcome the problem of venetoclax treatment.

Re-analysis of TGCA database demonstrated that leukemia (especially AML) possesses high level expression of Bcl-2 compared to other cancer types. Theas results indicates that the Bcl-2 expression level is the mark of venetoclax sensitivity. In addition, the re-analysis data suggests that the promising other targets for venetoclax are ocular melanoma, renal carcinoma, thymic epithelial tumor, thyroid cancer, breast invasive carcinoma, synovial sarcoma, thymoma and uveal melanoma, since these tumors have high Bcl-2 expression as the same of that of AML. Moreover, re-analysis of MILE study also demonstrated that all type AMLs more highly express Bcl-2 than pre-leukemic MDS but the Bcl-2 expression level is lower than that of CLL. CLL was first approved using venetoclax treatment by FDA. In clinical trial, venetoclax showed a higher response rate against CLL than AML. In order for venetoclax to be sufficiently effective in AML treatment, a strategy such as combining it with quercetin, as we have reported in the present study, will be necessary. In AML, some cells have been reported to be resistance to venetoclax [47, 48]. Mutation of p53 is one of the mechanisms of venetoclax resistance. Bax is a target gene of p53 and an executer of p53-induced apoptosis. KG-1 cells possess a 5-base insertion between codons 224 and 225 of the p53 coding sequence [49]. It has been reported that no constitutive expression of p53 was detected and irradiation did not induce p53 in KG-1 cells [50]. Thus, the combination of venetoclax and quercetin is efficient for AML cells acquiring venetoclax resistance caused by p53 mutation.

We also investigated another gene ablation in AML in the present study. AML cells belonging to the French–American–British (FAB) classification M2-subtype such as Kasumi-1 carry RUNX1-ETO fusion gene because of t(8;21) chromosomal translocation [51, 52]. RUNX1-ETO protein abuses normal hematopoietic function by wild type RUNX1 which is a transcription factor with an ability of specific DNA-binding. Moreover, Kasumi-1 has a p53 mutation Arg248Gln [53]. We showed that venetoclax combined with quercetin effectively killed Kasumi-1 cells even carrying RUNX1-ETO expression and p53 mutation. On the hand, CML cell line K562 was little affected the combined treatment. Our analysis with public data revealed that Bcl-2 expression was low in K562 cells and the survival and the growth of K562 did not depend on Bcl-2 gene. These Bcl-2 status are indicated to be a determining factor for sensitivity against the combined treatment of venetoclax and quercetin. Venetoclax was also conducted for phase II clinical trials in B-cell lymphoma and breast cancer [54] and phase III clinical trial in multiple myeloma [55]. The combination of venetoclax and quercetin may be also useful for treatments of these malignant tumors.

We showed here that quercetin increased Bax expression. Apoptosis induction requires apoptotic stimuli as well as Bcl-2 inhibition. Bax up-regulation may easily stimulates mitochondria because of Bcl-2 inhibition by venetoclax, resulting in strong apoptosis induction. We suggested a mechanism that quercetin strengthen the venetoclax’s efficacy by Bax induction. Recent study has reported that quercetin directly causes uncoupling of mitochondria and induces mitochondrial Ca²⁺ overload [56]. These effects may also contribute to facilitate the efficacy of venetoclax. Taken together, quercetin enhanced venetoclax’s cytotoxic effect against AML. These results provide a novel function of quercetin and a new strategy for AML treatment using venetoclax.

Acknowledgments

This study was partly supported by the Salt Science Research Foundation for 2022.

Author Contributions

R.K., K.T., K.Y. and T.Y. performed experiments and analyzed data. R.K., K.T., K.Y., Y.K. and T.Y. wrote and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Data availability statement

For original data, please contact [email protected].

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No competing interests reported.

SupFig1.tif
Supplementary Figure 1. Bcl-2 expression of TCGA data. Bcl-2 expression was analyzed by cBioPortal using the TCGA Pan Cancer Atlas Studies 10071 patient samples. Bcl-2 expression in AML was shown in a red box.
SupFig2.tif
Supplementary Figure 2. Bcl-2 expression of MILE study data. Bcl-2 expression was analyzed by BloodSpot using Leukemia MILE study data set ( href="http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13159" target="_blank">GSE13159). Student’s t-test was used for statical analysis compared to MDS.
SupFig3.tif
Supplementary Figure 3. Bcl-2 expression and dependency of cell lines. (A) Bcl-2 expression in leukemia cell lines were analyzed by depmap. Positions of KG-1, Kasumi-1 and K562 cells were shown. (B) Depmap analysis was used to demonstrate the dependence of Bcl-2 on the survival and the growth of leukemia cells in which the Bcl-2 gene was knocked out using CRISPR. 0 means independent. The smaller the value, the greater the degree of dependence. -1 corresponds to an essential gene.
SupFig4.tif
Supplementary Figure 4. Full-length blots of Figure 3E and 4D.

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The combination of venetoclax and quercetin exerts a cytotoxic effect on acute myeloid leukemia

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