GANT61 surmounts drug resistance of ADR by upregulating lysosome activities and reducing BCL2 expression in HL-60/ADR cells

Abstract

Background

Drug resistance remains a significant obstacle to Acute myeloid leukemia (AML) successful treatment, often leading to therapeutic failure. Our previous studies demonstrated that Glioma-associated oncogene-1 (GLI1) reduces chemotherapy sensitivity and promotes cell proliferation in AML cells. GANT61, an inhibitor of GLI1, emerges as a promising candidate in AML treatment. This study aims to explore the effects of the combination of GANT61 and Adriamycin (ADR) on AML cells resistance and elucidate the mechanisms through which GANT61 may potentiate the sensitivity of AML cells to ADR.

Methods

AML cell lines and AML primary cells were studied to evaluate effects and mechanisms of GANT61. Flow cytometry assays were used to verify apoptosis. Cell Counting Kit-8 (CCK-8) and EDU⁺ staining were used to observe changes in cell viability and the cytotoxic effect to different drugs. The transcriptomic profiles of HL-60/ADR cells with or without GANT61 treatment were compared via RNA-Seq analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and Gene Set Enrichment Analysis (GSEA) were performed for differentially expressed genes (DEGs) to reveal the underlying mechanisms of GANT61 in AML cells. GLI1, BCL2, Bax protein and mRNA expression levels were assessed by Western blot and Real-time polymerase chain reaction (RT-PCR).

Results

Our studies found that the combination of GANT61 and ADR synergistically inhibits proliferation while enhancing apoptosis in HL-60/ADR cells, and does not significantly exacerbate myelosuppression. Mechanistically, GSEA revealed enrichment of the gene set associated with the KEGG term “Apoptosis” and “Lysosome” in GANT61 treated cells. Meanwhile, “Apoptosis” was identified as the third most relevant pathway enriched by lysosomal DEGs, and BCL2 expression showed a negative correlation with these lysosomal DEGs in AML patients. RT-PCR and Western blot analysis disclosed that GANT61 significantly restrained BCL2 expression in AML cells. Lastly, we proved that venetoclax, a BCL2 inhibitor, co-treatment with GANT61 improved ADR sensitivity in HL-60/ADR cells.

Conclusions

GANT61 effectively reversed ADR resistance in HL-60/ADR cells by upregulating lysosome activities and downgrading BCL2 expression, providing a new treatment strategy with acceptable toxicity for AML-resistant patients.

Background

Acute myeloid leukemia (AML) is a life-threatening and hardly curable hematologic malignancy. Recent technological and supportive therapy advances, in conjunction with the approval of novel molecularly targeted therapeutics, have substantially improved AML outcomes [1]. However, the prognosis for refractory/relapsed AML remained poor, with a 5-year survival rate of less than 30% [2]. Drug resistance is a key factor contributing to mortality in AML patients, accounting for over 50% of deaths arising from the ability of drug-resistant cells to reinitiate the disease, ultimately leading to patient fatality [3, 4]. Hence, there is an urgent medical need to elucidate the possible molecular mechanism underlying drug resistance and apply innovative therapeutic agents, improving the outcome of AML patients.

Glioma-associated oncogene-1 (GLI1) is a transcription factor that serves as a terminal effector in the Hedgehog (Hh) signaling pathway and is instrumental in tumorigenesis [5]. Our previous studies demonstrated elevated expression of GLI1 in refractory/relapse AML patients. And GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells [6]. Therefore, we hypothesize that targeting GLI1 might offer a promising strategy for overcoming drug resistance in AML.

GANT61 has been the first small molecule reported to inhibit GLI1 activity. It suppresses the reporter signal induced by exogenously upregulated GLI1 and is therefore anticipated to be effective against cancers where GLI1 is over-expressed [7, 8]. Multiple studies have shown that GANT61 attenuates chemotherapeutic resistance and has a synergistic anti-tumor effect in treating various tumors via multiple mechanisms including inhibiting p21 mediated cell cycle progression, promoting cell apoptosis, repressing DNA damage repair and so on [9]. For instance, GANT61 re-sensitized of 5-fluorouracil (5-FU) resistant BRAF^V600E colorectal cancer to treatment by diminishing Nijmegen breakage syndrome-1 (NBS1) expression and increasing DNA damage/apoptosis [10]. GANT61 enhanced the sensitivity of temozolomide (TMZ) in glioma cells by increasing DNA damage effect, decreasing MGMT expression and the Notch1 pathway [11]. Additionally, a study displayed that GANT61 treatment sensitized Human-derived gastric cancer organoids (huTGOs) to epirubicin, oxaliplatin, and 5-fluorouracil through downgrading programmed cell death ligand 1 (PD-L1) expression [8, 12]. Besides, GANT61 also appears to be a promising therapeutic strategy for hematologic malignancies including myelodysplastic syndromes, Multiple Myeloma and AML [9, 13, 14]. GANT61 dramatically restrained the GLI1 protein expression level in drug-resistant Myeloma Cells, and GANT61 combined with Valproic Acid synergistically inhibited cell proliferation in Multiple Myeloma Cells [15, 16]. In AML cells, GANT61 suppressed cell proliferation and colony formation, caused apoptosis, and enhanced the antitumor effects of Ara-c and rapamycin [6, 17, 18]. However, there have been few studies reporting the impact of GANT61 on addressing AML resistance.

In this study, we sought to explore the impact of GANT61 on ADR resistance and elucidate the hidden mechanism of GANT61 in HL-60/ADR cells.

Materials and methods

Drugs

GNAT61 was purchased from APExBIO (USA, A1615). ADR (China, HY-15142), venetoclax (China, HY-15531), idarubicin (China, HY-17381 A) and cytarabine (China, HY-13605) were purchased from MedChemExpress. liposomal Mitoxantrone (China, H20220001) was purchased from Cspc Pharmaceutical Group Limited.

Cell lines and culture conditions

The HL-60/ADR cell line was obtained from the Cell Resource Center (Xiangya Medical College, Central South University, Hunan, China). The wild type HL-60 (HL-60/WT) and THP-1 cell lines were purchased from Procell Life Science & Technology Company (Wuhan, China). They were cultured in RPMI-1640 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (Biological Industries Inc., CA, USA) and a 1% antibiotic solution of penicillin and streptomycin (Corning Inc., NY, USA) in a humidified atmosphere containing 5% CO2 at 37 °C.

Cytotoxicity assay

Cell viability was determined by a Cell Counting Kit-8 (CCK-8, Dojin Laboratories, Kumamoto, Japan) after treatment to evaluate the cell response to different drugs. AML cell lines (HL-60 and HL-60/ADR) or primary cells, including hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from de novo AML patients, were seeded in 96-well culture plates at a density of 5 × 10⁴ cells/ml and incubated with GANT61, ADR, and Venetoclax for 24–48 H. At designated time points, 10 µl CCK-8 solution was added to each well and incubated for 3 H at 37℃. A spectrophotometer (Bio Tek Instruments, US) was used to measure the absorbance at the wavelength of 450 nm. Combination index (CI) using CompuSyn software was calculated. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively.

5-Ethynyl-2’-deoxyuridine (EDU)+ staining

DNA synthesis was measured using an EDU-based cell proliferation kit (Cell-Light EDU Apollo567 In Vitro Kit, Ribobio, Guangzhou, China), following the manufacturer’s instructions. All steps were performed at room temperature.

Apoptosis assessment

A total of 1 × 10⁶ cells were treated with GANT61, ADR, with or without Venetoclax and harvested after 24–48 H incubation, followed by 2 times washes with cold PBS. Subsequently, the cells were resuspended in 100ul binding buffer, stained with Annexin-V and 7-AAD at a dilution of 1:20 and 1:10 (Becton Dickinson, CA, USA), incubated in the dark on ice for 15 min, and subjected to flow cytometry and fluorescence microscope analysis (Becton Dickinson, CA, USA).

Western blot analysis

Equal amounts of protein were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with PBS-T containing 5% BSA (Bio Sharp Sigma A-4612) for 1 H at room temperature, followed by overnight incubation with primary antibodies at 4℃. After incubation with secondary antibodies, the protein bands were visualized using a Chei DocTMMP system (Bio-Rad). The antibody dilutions used were as follows: GLI1 1:1000 (Cell Signaling Technology, 3538, USA), BCL2 1:1000 (Wanleibio, WL01556, Shenyang, China), Bax 1:1000 (Wanleibio, WL01637, Shenyang, China), PUMA 1:1000 (ABclonal, A3753, Wuhan, China), TP53 1:1000 (Wanleibio, WL01919, Shenyang, China), P21 1:1000 (Wanleibio, WL0326, Shenyang, China), and GAPDH 1:5,000 (Proteintech, SA00001-2, Wuhan, China).

Quantitative RT-PCR

The HL-60/ADR cells were collected. Total RNA was isolated from 1 × 10⁶ cells using Trizol reagent (TaKaRa, Kusatsu, Japan). Total cDNA was prepared using a two-step reverse transcription kit (TaKaRa, Japan). Primers for real-time PCR were obtained from Integrated DNA Technologies. Primers were (GLI1/forward) 5’-AACGCTATACAGATCCTAGCTCG-3’; (GLI1/reverse) 5’-GTGCCGTTT GGT CACATGG-3’; (BCL2/forward) 5’-GTCTTCGCTGCGGAGATCAT-3’; (BCL2/ reverse) 5’-CATTCCGATAT ACGCTGGGAC-3’; (Bax/forward) 5’-CCCGAGAGGT CTTTTTCCGAG-3’; (Bax/reverse) 5’-CCAGCCCATGATGGTTCTGAT-3’; (PUMA /forward) 5’-GCCAGATTTGTGAGACAAGAGG-3’; (PUMA/reverse) 5’-CAGGC ACCTAATTGGGCTC-3’; (TP53/forward) 5’-AACTGCGGGACGAGACAGA-3’; (TP53/reverse) 5’-AGCTTCAAGAGCGACAAGTTTT-3’; (P21/forward) 5’-TGTC CGTCAGAACCCAT GC-3’; (P21/reverse) 5’-AAAGTCGAAGTTCCATCGCTC-3’; (GAPDH/forward) 5’-CTTTGTCAAGCTCATTTCCTGG-3’; (GAPDH/reverse) 5’-T CTTCCTCTTGTGCTCTTGC-3’. Reactions were on an ABI StepOnePlus machine (Applied Biosystems, USA). The transcript levels were normalized to GAPDH.

Lentiviral infection of cell lines

Mock infection and GLI1 overexpression (GLI1/OE) lentiviruses were purchased from the company GeneChem (Shanghai, china). HL-60 and THP-1 cells were resuspended in enhanced infection solution. A total of 5 × 10⁴ cells/ml were seeded in 96-well plates (three sub-wells for each cell line). The lentiviruses at a titer of 1 × 10⁸ TU/ml were added to the corresponding wells (MOI: 20–50), and polybrene was added at 1:1000. Stable cells were selected with puromycin at a concentration of 2 µg/ml⁶.

Drug sensitivity analysis

The Cancer Therapeutics Response Portal (CTRP) databases were utilized to analyze the correlation between GLI1 expressions and drug sensitivity. FDR < 0.05 was defined as significant.

RNA-Seq analysis

Approximately 1 × 10⁷ HL-60/ADR cells (including control and 24 H GANT61 treatment groups) were collected for three replicate, and suspended in Trizol (TaKaRa, Kusatsu, Japan). Then, the cells were sent to Heyuan Biotechnology (Shanghai, China) for RNA isolation and sequencing.

Gene expression profiles

The gene expression profile dataset GSE107465 (24 AML complete remission (CR).

and 6 AML relapsed and refractory (RR) patient samples), GSE111678 (260 AML patient samples) used in this study was downloaded from the Xiantao web (https://www.xiantaozi.com/). The Cancer Genome Atlas (TCGA)-LAML gene expression profiling (150 AML patients samples) was downloaded from (https://portal.gdc.cancer.gov).

Functional enrichment and pathway enrichment analysis

The Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed for DEGs based on the Xiantao web. p < 0.05 was deemed significant.

Protein-protein interaction network analysis

STRING ((http://string-db.org/) database was used to construct the protein-protein interaction (PPI) network, displaying the Nodes with interaction confidence above 0.4. Then, the PPI network was constructed by Cytoscape software (version 3.7.2).

Statistical analysis

Data are represented as the mean ± standard deviation of three replicate experiments. Differences between groups were analyzed using Student’s t-test or one-way analysis of variance, as appropriate. Statistical analyses were performed using the GraphPad Prism software (version 9.0) and. A p-value of < 0.05 was considered statistically significant.

Results

The combination of GANT61 and ADR synergistically inhibits proliferation, enhances apoptosis in HL-60/ADR cells

ADR-resistant (HL-60/ADR) cells exhibited reduced susceptibility to Adriamycin (ADR) compared to wild type HL-60 (HL-60/WT) cells. The IC₅₀ of ADR in HL-60/ADR cells was significantly higher than that in HL-60/WT cells at both 24 (HL-60/ADR, IC₅₀ = 102.1µM; HL-60/WT, IC₅₀ = 0.74µM) and 48 (HL-60/ADR, IC₅₀ = 5.82µM; HL-60/WT, IC₅₀ = 0.16µM) hours (Figure S1A and B). Besides, HL-60/WT and HL-60/ADR cells were incubated with clinically used chemotherapeutic drugs, including idarubicin, liposomal mitoxantrone, and cytarabine (Arc-C), for 24 h. And the IC₅₀ were calculated after CCK-8 assay. The IC₅₀ of all tested chemotherapeutic drugs on HL-60/ADR cells were remarkably higher compared to those on HL-60/WT cells (Figure S1C-E). These results suggest the presence of cross-resistance among ADR, idarubicin, liposomal mitoxantrone, and Arc-C in HL-60/ADR cells. Moreover, our findings revealed that the cell growth rate and EDU⁺ labeling rate were significantly higher in HL-60/ADR compared with HL-60/WT cells, indicating rapid proliferation of HL-60/ADR cells (Figure S1F-H).

Prior researches validated that GLI1 overexpression (GLI1/OE) promotes cell proliferation and decreases ADR sensitivity in THP-1 and U937 cell lines [6]. After treatment with ADR for 24 to 48 h, the relative cell viability and IC₅₀ values of HL-60/OE cells were higher than those of the MOCK cells (Fig. 1A). Additionally, the CTRP databases was employed to analyze GLI1 sensitivity to drug therapy, and the results revealed a negative correlation between ADR drug sensitivity and GLI1 expression (Fig. 1B). These results imply that GLI1 overexpression may also diminish ADR sensitivity in HL-60 cells, and targeting GLI1 could achieve anti-tumor effects through drug synergistic therapy. Therefore, we investigated the effects of GANT61, a GLI1 inhibitor, in HL-60/ADR cells. HL-60/ADR cells were treated with GANT61 for 24 and 48 h, resulting in IC₅₀ values of 35.39µM and 24.15µM, respectively (Fig. 1C). Moreover, our results suggested that GANT61 inhibited cell proliferation in a dose-dependent manner in HL-60/ADR cells (Fig. 2D). Further, HL-60/ADR cells were treated with ADR alone or in combination with GANT61 for 24 h to explore whether GANT61 synergizes with ADR. Our results uncovered that the combination of ADR (8µM) and GANT61 (16µM) significantly suppressed cell viability, with a combination index (CI) value of 0.639 (Fig. 1E). Consistent with the cell viability, the combination of ADR and GANT61 also significantly decreased EDU⁺ labeling of HL-60/ADR cells (Fig. 1F-G). A markedly elevated apoptosis was observed in HL-60/ADR cells following co-treatment with GANT61 and ADR for 24 and 48 h (Fig. 1H-I and Figure S2A). These data collectively prove that GANT61 and ADR synergistically promoted apoptosis and reduced cell proliferation in HL-60/ADR cells. Namely, GANT61 could effectively reverse ADR resistance in HL-60/ADR cells. Moreover, we found that the combination of GANT61 with idarubicin, liposomal mitoxantrone, or Arc-C also remarkably inhibited the viability of HL-60/ADR cells compared to the single-agent treatments (Figure S2B-D).

Fig. 1

GANT61 enhanced drug sensitivity of ADR in HL-60/ADR. (A) Relative cell viability and the IC₅₀ values in HL-60/OE and HL-60/MOCK cells treated with ADR for 24 to 48 H. (B) Analysis of the correlation between drug sensitivity and GLI1 expression using CTRP database. (C) Cell viability IC₅₀ curve after gradient concentrations of GANT61 (0–64µM) treatment for 24 to 48 H in HL-60/ADR cells. (D) The cell growth rate of HL-60/ADR cells treated with different concentration of GANT61 (0, 8, 16, and 32µM) for 24 to 72 H. (E) The cell viability of HL-60/ADR cells treated with varying concentrations of ADR or GANT61, either alone or in combination, for 24 h was analyzed using the CCK-8 assay. Combination Index (CI) values were calculated using CompuSyn software. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic effects, respectively. (F-G) The percentage of EDU⁺ after ADR (8µM) and GANT61 (16µM) treatment for 24 H in HL-60/ADR cells. (H-I) The percentage of apoptotic cells after ADR (8µM) and GANT61 (16µM) treatment for 24 (H) and 48 H (I) in HL-60/ADR cells. The left panel shows the flow cytometry scatter plot. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant

Co-treatment of GANT61 and ADR were effective in primary AML patient cells

In order to assess the effectiveness and cytotoxicity of GANT61, we assessed the toxicity profiles of GANT61 and ADR in healthy donor cells, observing a dose-dependent increase in cytotoxicity for both compounds (Figure S3A-B). Based on our findings, we subsequently selected a less cytotoxic dose of the drugs for the combination experiments in primary AML cells and healthy donor cells. Importantly, the addition of GANT61 did not result in an obvious decrease in the cell viability of healthy donor cells when compared to monotherapy with either GANT61 or ADR alone (Fig. 2A and Figure S3C). In contrast, GANT61 combined with ADR evidently reduced cell viability in primary AML cells (Fig. 2B). These findings suggested that GANT61 could enhance the chemosensitivity of ADR in primary AML patient cells without further increasing cytotoxicity in healthy donor cells.

Fig. 2

Effects of GANT61 and ADR on the cell viability of primary cells. (A-B) The relative cell viability after treatment of ADR (1.6µM), GANT61 (20µM), or both for 24 H in primary cells from healthy donors (A) or de novo patients with AML (B). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant

GANT61 regulated the expression of apoptosis-related genes in HL-60/ADR

We next sought to explore the molecular mechanism underlying how GANT61 affects ADR sensitivity in HL-60/ADR cells. Based on our RNA-seq data of HL-60/ADR cells with or without GANT61 treatment, we identified 715 DEGs (with a P-value < 0.05 and an absolute log2 fold-change ≥ 0.585), namely 605 up-regulated and 110 down-regulated genes in HL-60/ADR cells following treatment with GANT61 (Fig. 3A). KEGG and GSEA enrichment analyses of the DEGs from RNA-seq data and the GSE107465 dataset were performed using the Xiantao web tool to identify the potential biological functions of GANT61 affecting AML cell resistance. The results indicated that the DEGs were significantly enriched in “Apoptosis”, and the apoptosis-related genes were mostly up-regulated in GANT61 treated cells and down-regulated in AML relapsed and refractory (AML-RR) patients compared with their respective control groups (Fig. 3B-E and Figure S4A-B). Subsequently, the mRNA expression levels of key apoptotic regulators in the RNA-Seq data were assessed. It was observed that GANT61 restricted the mRNA expression of the anti-apoptotic molecule BCL2 (Fig. 3F). In the GSE107465 dataset, the mRNA expression levels of BCL2, GLI1 and BAD were elevated in RR compared to those in complete remission (CR) patients (Fig. 3G). These results revealed that GANT61 may conquer ADR resistance in HL-60/ADR cells through regulating the expression of BCL2.

Fig. 3

KEGG and GSEA results of RNA-seq data and the GSE107465 dataset. (A) Volcano plot of differential genes from RNA-seq of DMSO and GANT61 treatment HL-60/ADR cells. Red are up-regulated, Blue are down-regulated, and gray are no difference. (B) The DEGs were enriched to the top 10 enriched by KEGG from analysis of RNA-seq. (C) A GSEA plot showing enrichment of the gene set associated with the KEGG term “Apoptotic Cleavage of Cellular Proteins” in RNA-seq data. (D) The DEGs were enriched to the top 10 enriched by KEGG in from the GSE107465 dataset. (E) A GSEA plot showing enrichment of the gene set associated with the KEGG term “Apoptosis” in GSE107465 dataset. (F) The mRNA expression levels of apoptosis-related genes in HL-60/ADR cells treated with and without GANT61 were compared through an analysis of RNA-seq data. (G) A comparative analysis of apoptosis-related gene expression profiles between CR and RR patients in the GSE107465 dataset. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant

Then, the STRING online tool was utilized to construct protein-protein interaction (PPI) network of GLI1 and apoptosis-related genes (Fig. 4A). Employing the Xiantao online tool, we generated gene co-expression heatmaps and scatter plots to investigate the expression correlation between GLI1 and crucial apoptosis-regulating genes (such as BCL2, MCL1, BIM, Bax, BAD, BID, CASP3/7/8/9/10, and TP53) within the GSE107465, GSE111678, and TCGA datasets (Fig. 4B-D and Figure S4C-D). Our findings displayed that GLI1 was positively correlated with BCL2 in AML patients. Meanwhile, we evidenced that GLI1 overexpression significantly elevated BCL2 expression and decreased Bax expression in AML cell lines (Fig. 4E). Conversely, GANT61 suppressed BCL2 expression and enhanced Bax protein expression in GLI1/OE cells and HL-60/ADR cells, without notably affecting the levels of PUMA, TP53, and P21 (Fig. 4F and Figure S5A-B). Likewise, our results showed that GANT61 restrained BCL2 protein expression in primary AML cells (Fig. 4G). These above data illustrated that GANT61 re-sensitized HL-60/ADR cells to ADR maybe by modulating the expression of BCL2 and Bax.

Fig. 4

GANT61 regulated the expression of BCL2 and Bax. (A) Protein–protein interaction (PPI) network analysis to predict proteins interacted between GLI1 and apoptosis-related genes based on STRING database. (B-C) Heatmap of co-expression genes between GLI1 and apoptosis-related genes in AML patients from the GES107465 (B) and GSE111678 (C) datasets. The high expression represented red, and blue represented the low expression. (D) Scatter plot shows the expression correlation between GLI1 and BCL2 in AML patients from the GES107465 and GSE111678 datasets. (E) Western blot for BCL2 and Bax protein levels in MOCK and GLI1/OE HL-60 and THP-1 cell lines. (F) HL-60/OE, THP-1/OE and HL-60/ADR cells were treated with GANT61 (16µM) for 24 H. Cell lysates were analyzed by Western blotting for the BCL2 and Bax protein levels. (G) The protein levels of BCL2 after GANT61 (20µM) treatment for 24 H in AML primary cells

GANT61 upregulates lysosome activities in HL-60/ADR

Notably, the KEGG enrichment analysis of these DEGs in RNA-seq data and GSE107465 dataset showed a remarkable enrichment not only in “Apoptosis” but also in “Lysosome” (Fig. 3B and D). Utilizing GSEA, we observed that the lysosome activities was up-regulated in GANT61-treated cells and down-regulated in AML-RR patients compared with their respective control groups (Fig. 5A-B). A total of 26 overlapping lysosomal DEGs between the RNA-seq data and the GSE107465 dataset were identified (Fig. 5C). And KEGG analysis indicated the third top pathophysiologically relevant, significantly enriched pathway by these intersecting lysosomal genes was “Apoptosis” (Fig. 5D). It has been shown that the down-regulation of lysosomal protein expression hinders lysosome-mediated apoptosis and the release of lysosomal cathepsins facilitates cell death by promoting BCL2 protein degradation [19, 20]. So, we exploited STRING online tool to map the protein-protein interactions between BCL2 and the aforementioned overlapping lysosomal genes (Fig. 5E), and employed gene expression profiles from the GSE107465, GSE111678, and TCGA datasets to create a co-expression heatmap of both in AML patients. The correlation heatmap disclosed that BCL2 was significantly negatively correlated with lysosomal genes such as ASAH1, GNS, NPC2, and SLC17A5 (Fig. 5F-G and Figure S5C). Taken together, we speculated that GANT61 downgrade BCL2 expression by raising lysosomal activities in AML cells.

Fig. 5

The expression correlation analysis of BCL2 and lysosome related genes in AML cells. (A-B) GSEA enrichment of “KEGG-Lysosome” in RNA-seq data (A) and GSE107465 dataset (B). (C) The intersection of lysosome related genes from RNA-seq data and GSE107465 dataset. (D) The 26 DEGs of lysosomal were enriched by KEGG. (E) Protein–protein interaction (PPI) network analysis to predict proteins interacted between BCL2 and lysosome related genes based on STRING database. (F-G) Heatmap of co-expression genes between BCL2 and lysosome function-related genes in AML patients from the GES107465 (F) and GSE111678 (G) datasets

GANT61 and BCL2 inhibitor collaboratively reinforced the drug sensitivity in HL-60/ADR cells

Deeply, HL-60/ADR cells were exposed to venetoclax, a BCL2 inhibitor, for 24 and 48 h to assess its effects. The IC₅₀ values of venetoclax in HL-60/ADR cells were determined to be 9.47µM and 6.11µM, respectively (Fig. 6A). A significant inhibition of HL-60/ADR cell viability by venetoclax (Fig. 6B). Next, we evaluated the effects of combined treatment of GANT61 and venetoclax. HL-60/ADR cells were treated with GANT61 (16µM) w/o venetoclax (4µM) for 24 h. ADR was further added to the combination to test whether its addition could result in a more significant inhibitory effect on cell apoptosis and viability. Elevation was observed in the apoptosis of HL-60/ADR cells when GANT61 was co-administered with venetoclax. The most significant promotion level was achieved when ADR was co-administered with GANT61 and venetoclax for 24 h (Fig. 6C-D). Consistent with the apoptotic cells, the percentage of cell viability in the three-drug combinations treatment group decreased significantly compared with the other treatment groups for HL-60/ADR cells (Fig. 6E). At last, we assessed the effects of combined treatment of venetoclax and ADR. HL-60/ADR cells were treated with venetoclax w/o ADR at various concentrations for 24 h. All CI values (CI < 1) demonstrated that venetoclax and ADR synergistically inhibited cell viability in HL-60/ADR cells (Fig. 6F). These above results suggested that the combination of GANT61 and BCL2 inhibitor strengthened the anti-proliferation and pro-apoptotic effects of ADR in HL-60/ADR cells.

Fig. 6

BCL2 inhibitor Venetoclax enhanced the pro-apoptosis effect of GANT61 in HL-60/ADR cells. (A) Relative cell viability IC₅₀ after gradient concentrations of Venetoclax (0–16µM) treatment for 24 to 48 H in HL-60/ADR cells. (B) The Relative cell viability after gradient concentrations of Venetoclax (0–16µM) treatment for 24 to 48 H in HL-60/ADR cells. (C-D) Representative flow cytometry scatter plots showing the apoptotic rates after treatment with GANT61 (16µM), Venetoclax (4µM), GANT61 + Venetoclax (4µM), GANT61 + ADR (8µM), or combined treatment with GANT61, ADR and Venetoclax for 24 H in HL-60/ADR cells (C). Statistical analysis of apoptotic cells in (D). (E) The relative cell viability of HL-60/ADR cells in response to different combinations of drugs. ADR, GANT61, and Venetoclax were added at concentrations of 8, 16, and 4µM for 24 H. (F)The cell viability of HL-60/ADR cells treated with varying concentrations of ADR or Venetoclax, either alone or in combination, for 24 h was analyzed using the CCK-8 assay. Combination Index (CI) values were calculated using CompuSyn software. Vene, Venetoclax. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant

Discussion

Chemoresistance, either innate or acquired, has become the main obstacle in AML treatment. In this project, we found that HL-60/ADR cells were resistant to ADR, which is an anthracycline chemotherapy drug used as the first-line treatment for newly diagnosed AML patents. GNAT61, a GLI1 inhibitor, enhanced the inhibitory cell viability and pro-apoptotic effects of ADR in HL-60/ADR cells. We further demonstrated that HL-60/ADR cells were also resistant to idarubicin, liposomal mitoxantrone and Ara-C, and GANT61 improved the inhibitory effects of these drugs on the viability of HL-60/ADR cells (Figure S1C-E and S2B-D). Additionally, Lin et al. previously reported that HL-60/ADR cells were resistant to Ara-C, ADR, vincristine, daunorubicin, mitoxantrone, pirarubicin, homoharringtonine and etoposide [21]. Thus, we concluded that cross-resistance exists in HL-60/ADR cells, and that the resistance mechanisms of different chemotherapeutic agents exhibit some similarities. GANT61 co-administration with chemotherapeutic agents (ADR, idarubicin, liposomal mitoxantrone, or Ara-C) may be a plausible strategy to resensitize the anti-tumor potency of these drugs in AML cells. However, further validation of the effectiveness and clinical application of GANT61 in treating cell line derived xenograft (CDX) and patient derived xenograft (PDX) mouse models and is needed in future studies.

GANT61 has been reported to exhibit anticancer activity against various malignancies, including AML [6], multiple myeloma [9], breast cancer [22], colorectal cancer [23], oral squamous cell carcinoma [24] and pancreatic cancer [25]. Hu et al. previously reported that GANT-61 inhibited pancreatic cancer stem cell tumor growth which was associated with suppression of GLI1, GLI2, BCL2, CCND2 and Zeb1 expression in tumor tissues [25]. Results from Zhang et al. indicated GANT61 was found to promote apoptosis marker Caspase 3, Bax expression and decrease BCL2 expression in both time- and dose-dependent manners in multiple myeloma [9]. Oladapo et al. reported that GANT61 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in breast cancer cells via regulating cell cycle (cyclin D and E) and BCL2 expression [26]. In medulloblastoma cells, GANT61 was found to reduced mRNA levels of the oncogene BCL2 [27]. In addition, the dysregulation of the balance between anti-apoptotic and pro-apoptotic factors is intricately linked to drug resistance in cancer. Chang et al. suggested that ADR resistance has been attributed to the expression of P-glycoprotein (P-gp) efflux pumps, the upregulation of BCL2, and the downregulation of Bax [28]. When the therapeutic siRNA silences the BCL2 gene, it increases the curative effect of ADR and reduces the side effect of ADR by tumor-targeted delivery [29]. Here, we confirmed that GANT61 prevented cell proliferation and induced apoptosis in HL-60/ADR cells by downregulating BCL2 expression. BCL2 inhibitor and ADR synergistically reduced the viability of HL-60/ADR cells.

There are several doubts in our study. Firstly, we discovered that GANT61 increased the protein expression level of Bax without significantly affecting its mRNA expression level (Fig. 4F and S5A). As we know, the molecular protein level is governed by the dynamic equilibrium between protein synthesis and degradation. Protein synthesis is not only regulated at the transcriptional level but also critically dependent on post-transcriptional modifications [30]. Additionally, the anti-apoptotic protein BCL2 prevents apoptosis by inhibiting or inactivating apoptotic proteins (BAK, Bax, and BOK) [31]. Consequently, we formulated a speculation that GANT61 may diminish the inhibitory effect of BCL2 on Bax through downregulation of BCL2 expression or regulate Bax protein levels via non-transcriptional mechanisms. Secondly, we found that GANT61 did not alter the expression levels of the pro-apoptotic proteins BAD and BIM (Figure S5A). So far, the BCL2 family has expanded to include over 25 members. Based on the composition of these four motifs (BH1, BH2, BH3, and BH4), the BCL2 family are be divided into three categories: the anti-apoptotic proteins (BCL2, MCL-1, BCL2L1, BCL2L2, BCL2A1, etc.), the pro-apoptotic proteins (Bax, BAK1, BOK, etc.), and the pro-apoptotic BH3 only proteins (BAD, BMF, BID, BIM, BIK, etc.).Each subclass exhibited unique protein sources and occupies distinct locations within cellular pathways, thereby contributing distinctively to the regulation of various apoptosis mechanisms [32]. Such as, anti-apoptotic BCL2 family proteins exert their anti-apoptotic effects by binding to BH3 domain [33]. Pro-apoptotic proteins mediate the permeabilization of the outer mitochondrial membrane, leading to the release of cytochrome c and ultimately triggering apoptosis [34]. BH3-only pro-apoptotic proteins are to promote apoptosis via the inhibition of anti-apoptotic factors [35]. A new study proposed that a region of BID is involved in the ability of the protein to induce MOM polarization (MOMP) independently from BID activation of BAK1 and Bax [36]. Therefore, the differential effects of GANT61 on the expression of various BCL2 family molecules may be attributed to the differences in their molecular structures and complex molecular mechanisms of action. Lastly, we noted that an increase in both the anti-apoptotic protein BCL2 and pro-apoptotic protein BAD in AML-RR patients (Fig. 3G). BCL2 was at a high level in Multi-drug resistance cancer cells and highly express the BCL2 gene to resist the apoptosis [29]. BAD is a BH3-only pro-apoptotic proteins, participating in cellular apoptosis, invasion and chemosensitivity of cancers. Numerous studies have consistently demonstrated that the expression levels of BAD are decreased in various cancers, including hepatocellular carcinoma, small cell lung carcinoma and breast cancer. Furthermore, the level of BAD expression closely correlates with overall survival, prognosis, and disease staging in these patients [37,38,39]. Interestingly, Zhu et al. previously reported that the expression levels of both BCL2 and BAD were elevated in clinical cases of salivary gland adenoid cystic carcinoma, and a significant association was observed between the expression level of BAD and distant metastasis [40]. Unfortunately, they did not elucidate the specific underlying mechanism that led to the upregulation of BAD expression. Hayakawa et al. revealed that cisplatin treatment induces BAD phosphorylation (p-BAD) in breast cancer cells, and that elevated levels of p-BAD are involved in the evolution of cisplatin resistance [41]. Liu et al. reported that inhibition of protein kinase AMP-activated catalytic subunit α1 (AMPK) upregulates the expression level of BAD in AMPK-driven hematologic cancer [42]. Hence, we hypothesize that in AML-RR patients, the administration of chemotherapeutic agents may ultimately result in the upregulation of BAD expression. This effect may occur through the promotion of BAD phosphorylation feedback mechanisms or alterations in the activities of intracellular signaling pathways. Overall, more realistic and in-depth experiments are needed to validate the aforementioned hypothesis in the future.

Conclusions

In conclusion, the present study demonstrated that HL-60/ADR cells were resistant not only to ADR but also to idarubicin, liposomal mitoxantrone, and Ara-C. GANT61 restores ADR chemosensitivity in HL-60/ADR cells without significantly exacerbating myelosuppression. This effect is achieved by downregulating the expression of BCL2 and upregulating lysosomal activities. Additionally, GANT61 synergized with the BCL2 inhibitor venetoclax to resensitize the HL-60/ADR cells to ADR. This study facilitates the prospective translation of GANT61 to relapsed/refractory AML patients.