ABSTRACT
Targeting MYC oncogene remains a major therapeutic goal in cancer chemotherapy. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment, causing Na+/K+ pump inhibition, targets efficiently MYC overexpressing cancer cells. At clinically relevant doses, proscillaridin induced rapid downregulation of MYC protein level, and produced growth inhibition preferentially against MYC overexpressing leukemic cell lines including lymphoid and myeloid stem cell populations. Transcriptomic profile of leukemic cells after treatment showed a downregulation of gene sets involved in MYC pathways, cell replication and an upregulation of genes involved in hematopoietic differentiation. Gene expression changes were associated with an epigenetic remodeling of chromatin active marks. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27). In addition, loss of lysine acetylation was observed also in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferase proteins (such as CBP and P300) involved in histone and MYC acetylation. Overall, these results strongly support the repurposing of proscillaridin in MYC overexpressing leukemia and suggest a novel strategy to target MYC by inducing the downregulation of histone acetyltransferases involved in its stability.
INTRODUCTION
MYC (c-MYC) transcription factor is a major driver of oncogenic transcriptional programs. It contributes to gene dysregulation in cancer by promoting expression of genes involved in cell proliferation (1). High MYC expression drives tumor initiation, progression, and maintenance and is associated with aggressive cancers and poor prognoses (2,3). MYC is a potent driver in leukemia inducing cell proliferation and blocking cell differentiation (4). Moreover, MYC contributes to long-term self-renewal of leukemic stem cells (5). Conversely, genetic suppression of MYC in transgenic mouse models induces differentiation and cell growth arrest of leukemic cells (6-8). Therefore, targeting MYC addiction in leukemia is a major therapeutic goal. Since MYC lacks a catalytic site, its direct inhibition has been extremely challenging. Indirect MYC inhibition demonstrated therapeutic efficacy with bromodomain inhibitors (such as JQ1 or THZ1), by blocking MYC transcriptional effects (9-13). Unfortunately, cancer cells, such as leukemia, breast and ovarian cancers, develop resistance to these inhibitors by compensatory mechanisms using other bromodomain containing proteins or kinome reprogramming (14-16). Together, these studies highlight the need to develop new strategies to abrogate MYC addiction in cancer.
MYC stability is regulated by post-translational modifications and MYC acetylation increases its stability (17,18). The deposition of acetyl groups on lysine residues is catalyzed by lysine acetyltransferases (KATs), which acetylate also histone proteins causing chromatin opening and gene activation (19). Lysine acetyltransferase pharmacological inhibition represent an interesting strategy to target indirectly MYC by blocking upstream mechanisms involved in its stability. However, KATs have overlapping targets and commercially available KAT inhibitors require further optimization (20).
While screening more than 1,000 FDA-approved drugs for repurposing in oncology, we reported that cardiac glycosides, which are approved for heart failure treatment, exhibit significant epigenetic and anticancer effects (21,22). Cardiac glycosides, including digitoxin, digoxin, lanatoside, ouabain and proscillaridin, triggered reactivation of epigenetically silenced tumor suppressor genes (22). Moreover, all cardiac glycosides produced synergistic responses when used in combination with the epigenetic drug decitabine (demethylating agent), further supporting their epigenetic activity (21). Several epidemiological studies argue in favor of repurposing cardiac glycosides in oncology. Indeed, several reports showed that patients treated with cardiac glycosides for heart failure have a lower rate of cancer diagnosis as compared to the general population (23). Upon cancer diagnosis, these patients exhibit generally a less aggressive disease and responds better to therapy (23,24).
Repurposing cardiac glycosides in oncology is limited by their narrow therapeutic window, for which maximal plasmatic level is around 10 nanomolar, due to cardiac toxicities (23, 25-27). Several in vitro and in vivo studies tested their anticancer activity at supra-pharmacological doses, which are not reachable in humans; in particular, in rodents who can tolerate high doses of these drugs due to structural differences in Na+/K+ pump as compared to human (26,28,29). Since the repurposing of cardiac glycosides is restricted to the low nanomolar range, we sought to identify cancer types highly sensitive to these drugs. To do so, we screened a panel of human cancer cell lines with proscillaridin, which was identified as the most potent cardiac glycoside in our previous screens (21,22). Proscillaridin produced antiproliferative effects with a preferential selectivity towards MYC overexpressing leukemia cells. We demonstrated that proscillaridin produced a global loss of acetylation in chromatin and MYC itself, producing epigenetic effects and MYC downregulation. These results provide compelling evidence for the repurposing of cardiac glycoside proscillaridin against leukemia driven by MYC oncogenic signature.
MATERIALS AND METHODS
Cell Culture and Drug Treatments
A panel of 14 human cancer cell lines and hTERT/SV40ER-immortalized human primary fibroblasts transformed with MYC, RASV12 or MYC and RASV12 were used in the study. Cell types and culture conditions are described in Supplementary Materials and Methods. Proscillaridin was purchased from Santa Cruz Biotechnologies. IC50 values were calculated with GraphPad Prism software.
Protein and Histone Extractions
Whole cell proteins were extracted using cold whole-cell lysis buffer (50 mM Tris-Cl pH 7.4, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton, 0.1 mM Na3VO4, and 1 mM PMSF), supplemented with Complete™ Protease Inhibitor Cocktail (Roche). Histones were harvested using acid-extraction method with cold Triton Extraction Buffer (TEB; 0.5% Triton, 2 mM PMSF, 0.02% NaN3, 10 mM sodium butyrate), supplemented with protease inhibitor cocktail. Protein extracts were separated by SDS-PAGE and transferred onto a polyvinyl difluoride membrane. All experiments were performed in triplicate. Antibodies are listed in the Supplemental Materials and Methods section.
RNA Extraction, Sequencing and Analysis
QIAshredder was used to homogenize cell lysates and eliminate debris prior to RNA extraction using RNeasy Mini Kit. Briefly, 10 μg of purified RNA was treated with DNAse and quantified by Agilent RNA 6,000 Nano kit bioanalyser chips. 1 μg of mRNA was used for library preparation with TruSEq Stranded mRNA LT. RNA sequencing was performed using HiSeq 2500. Experiments were performed in triplicate. Reads were aligned to human genome (hg19) using STAR v2.4.2 and differential gene expression analysis between untreated and treated cells was done using DESeq2 v1.10.1 (30,31). For bioinformatics analyses, data were processed using gene set enrichment analyses (GSEA, broadinstitute.org/gsea), metascape (metascape.org) and gene mania (genemania.org). MOLT-4 cells H3K27ac ChIP-seq data from publicly available dataset (GEO: GSM2037790) were used in associated with transcriptomic data. GSEA analysis of 8227 AML fractions and the LSC signatures was performed using the control sample data from GSE55814. GEO2R was used to generate a ranked list of LSC-related genes (6 LSC CD34+CD38- samples vs 12 non-LSC CD34- samples) used in GSEA analysis.
Acetylation Analysis by Immunoprecipitation and Mass Spectrometry
Whole cell protein extracts were incubated overnight with 5 μg/ml of MYC antibody (Abcam, AB32072). After immunoprecipitation and transfer, proteins were probed with lysine pan-acetyl antibody (1:2500 Cell Signaling 9681). For acetylome analysis by mass spectrometry, samples were prepared as previously described (32). Briefly, 4 biological replicates of untreated and proscillaridin-treated MOLT-4 cells (5 nM, 48h) were digested with trypsin. Peptides were analyzed by mass-spectrometry and data were extracted with the MaxQuant software package (version 1.5.5.1) and subsequently analyzed using an in-house computational pipeline for statistical analysis of relative quantification with fixed and/or mixed effect models, implemented in the MSstats Bioconductor package (version 3.3.10) (33,34). Peptides were searched with SwissProt human protein database.
RESULTS
Cardiac Glycoside Proscillaridin Targets MYC-Driven Leukemic Cells
To identify cancer types with high sensitivity to proscillaridin in order to obtain concentrations within their therapeutic window, we screened a panel of 14 human cancer cell lines and measured cancer cell proliferation after a 24h treatment. Upon calculating the half-maximal inhibitory concentration (IC50), we noticed a 2,800-fold difference in IC50, with leukemic cells being more sensitive to proscillaridin (Supplementary Figure 1a). To explore the cause of this striking difference, we hypothesized that the oncogenic context might influence drug efficacy. By comparing protein expression of MYC oncogene (in untreated cancer cells) with proscillaridin IC50 values, we found that there was a significant inverse correlation (p = 0.0172; Supplementary Figure 1b). Proscillaridin produced a more potent growth inhibition in cells expressing high levels of MYC protein, such as acute lymphoblastic T-cell (MOLT-4) and B-cell (NALM-6) leukemia while being less effective in colorectal (SW48) and lung (A549) cancer cells expressing low levels of MYC (Figure 1a).
To evaluate MYC contribution in cancer cell sensitivity to proscillaridin, we investigated drug response using an isogenic cell system consisting of hTERT/SV40ER-immortalized human primary fibroblasts, transformed with different oncogenes including MYC, RASV12 or the combination of both oncogenes. This system allowed exploring the effect of oncogenic transformation within the same genetic background. We choose this approach as opposed to MYC overexpression or knockout genetic manipulations in leukemic cell lines since MYC overexpression was shown to amplify more robustly already MYC-dependent genes, and MYC knockout in leukemia leads to proliferation arrest, apoptosis and/or senescence (6,35,36). After transfection, MYC-transformed fibroblasts had a small and round phenotype whereas RASV12-transformed cells displayed increased vacuole formation and large cytoplasm. MYC and RASV12-transformed cells exhibited a round phenotype with vacuole formation in the cytoplasm (Supplementary Figure 1c). High levels of MYC and RAS protein levels were detected after transfections when compared to non-transfected cells (Figure 1b). Using a wide range of proscillaridin concentrations (from 0.01 nM to 100 μM) for 48h, we measured cell viability and calculated IC50 values (Figure 1c). Untransformed fibroblasts were fully resistant to proscillaridin. Likewise, RASV12 transformed cells were mildly affected by the treatment where proscillaridin at high doses failed to impact cell viability by more than 50%. Conversely, MYC transformed fibroblasts were highly sensitive to proscillaridin with an IC50 value of 70 nM. Moreover, MYC and RASV12 transformed fibroblasts (referred as to RASV12+MYC) had a low IC50 value (132 nM) despite the presence of RASV12. Consequently, MYC overexpression was driving proscillaridin sensitivity in transformed fibroblasts. In comparison, after 48h treatment, MOLT-4 and NALM-6 cells (MYC overexpressing leukemic cells) showed IC50 values of 2.3 nM and 3 nM, respectively, which correspond to clinically achievable concentrations (Supplementary Figure 1d).
To explore the mechanism by which MYC overexpression in cancer cells correlates with proscillaridin sensitivity, we compared its effects between MYC driven leukemic cells (MOLT-4 and NALM-6) and low expressing MYC cancers driven by KRAS mutations (SW48 colon and A549 lung cancer cells). We found that proscillaridin (5 nM; 48h) significantly reduced MYC protein level by more than 50% in MOLT-4 and NALM-6 cells but not in SW48 and A549 cells (Figure 1d). Time-course experiments with both leukemic cell lines (8h to 96h) showed that proscillaridin induced a significant (up to 80%) and rapid MYC downregulation (Figure 1d; Supplementary Figure 1e). These results demonstrate that low dose proscillaridin inhibits efficiently leukemia growth causing rapid MYC downregulation.
Proscillaridin Efficiently Targets MYC-Driven Leukemic Stem Cell Populations
We sought to determine whether proscillaridin could target leukemic stem cells (LSCs) (37-39). To explore this possibility, we used two LSC models, a mouse model of T-ALL and a LSC model of human acute myeloid leukemia (AML) (37,40,41). First, pre-LSCs T-ALL cells were isolated from a transgenic mouse model that closely reproduces human T-ALL (42). We previously showed that these pre-LSCs are driven by the SCL/TAL1 and LMO1 oncogenes, which depend on NOTCH1-MYC pathways, and are resistant to chemotherapeutic drugs used against leukemia (doxorubicin, camptothecin and dexamethasone) (5,37). Low concentrations (3-10 nM) of proscillaridin significantly decreased pre-LSC T-ALL viability by 70% after 4 days of treatment (Figure 2a). Despite being resistant to chemotherapeutic drugs, these pre-LSCs (T-ALL) were sensitive to proscillaridin at clinically relevant doses (37).
Then, we used primary human AML 8227 cells, which contain functional LSCs within the CD34+ sub-population, and non-LSC cells characterized by CD34- with or without CD15+ expression (Figure 2b) (40,41). Gene set enrichment analysis from transcriptomic data published by Lechman et al. revealed that the LSCs-enriched fraction (CD34+/CD38-) in AML 8227 are enriched for MYC target genes expression as compared to non-LSCs (CD34-) (Figure 2c) (40). After 6 days of proscillaridin treatment, bulk AML 8227 cells had an IC50 of 29 nM (Figure 2d). Likewise, CD34- with or without CD15+ non-LSC cells had IC50 of 38 nM and 29 nM, respectively. In contrast, all CD34+ AML cells (CD34+, CD34+/CD38+ and CD34+/CD38-) were more sensitive to proscillaridin with IC50 values of 15 nM. Altogether, proscillaridin efficiently targets LSC-enriched populations, in both T-ALL and AML models marked by high MYC expression, further supporting its repurposing against MYC-dependent leukemia.
Proscillaridin Downregulates Cell Proliferation Programs and Induces T-Cell Differentiation
To gain insight into proscillaridin effects against MYC-driven leukemic cells, we investigated drug-induced gene expression changes in T-ALL cells (MOLT-4). By quantitative RT-PCR (qPCR), we found that proscillaridin significantly downregulated MYC mRNA after 16h treatment and up to 90% after 48h (Figure 3a). Then, we used RNA-sequencing to explore transcriptomic effects of proscillaridin (5 nM; 48h) in MOLT-4 cells. After drug treatment, transcriptome analysis showed a downregulation of 2,759 genes (log2FC < 0.5; P-value adjusted < 0.05) and concomitant upregulation of 3,271 genes (log2FC > 1; P-value adjusted < 0.05; Supplementary Figures 2a and b). Using Metascape, gene ontology analysis revealed that downregulated genes were involved in DNA replication, biosynthesis and metabolic processes (Figure 3b; Supplementary Figure 2c). Consistent with qPCR results, MYC transcript was significantly downregulated in our RNA-sequencing data set (Figure 3c). Gene Set Enrichment Analysis showed that MYC PATHWAY (which includes 30 MYC target genes) was significantly downregulated (Figure 3c). Notably, these transcriptomic effects correlated with a 25% decrease of S-phase cells as measured by BrdU staining (Figure 3d; Supplemental Figure 2d). Proscillaridin also significantly downregulated 11 T-cell leukemia master transcription factors (Figure 3e) (43-45). These data support that proscillaridin efficiently inhibits proliferation programs in MYC-driven leukemia.
Gene ontology analysis also revealed that upregulated genes were enriched for hematopoietic or lymphoid organ development, suggesting the onset of leukemia differentiation (Figure 3f and Supplemental Figure 2e). To probe the functional significance of this change, we measured T-cell differentiation markers in MOLT-4 cells before and after treatment. By qPCR, mRNA levels of T-cell differentiation markers NOTCH3 and its target HES1 were upregulated after 48h treatment and remained expressed for 2 days after drug removal (Figure 3g) (46,47). By flow cytometry, we measured a significant increase in TCR and CD3 expression, which lasted up to 4 days after drug removal, suggesting the onset of normal T-cell activation. Upregulation of these differentiation markers were in the same range than the levels measured after TPA treatment, a well-known inducer of leukemia differentiation (Figures 3h and i) (48,49). Altogether, proscillaridin treatment produced a transcriptomic shift from a proliferative program to the induction of T-cell leukemia differentiation.
Proscillaridin Induces Global Loss of Histone H3 Acetylation
Since proscillaridin induced gene expression and phenotypic changes, we hypothesized that it triggers epigenetic effects in high MYC-driven leukemia. We analyzed histone H3 and H4 post-translational modifications by western blotting after 16h to 96h of proscillaridin treatment (5 nM). We found that proscillaridin produced a significant time-dependent reduction (by 75%) of lysine acetylation at H3K9, H3K14, H3K18, H3K27 residues and global loss of H3 acetylation in MOLT-4 cells (Figure 4a; Supplemental Figure 3a). The dramatic reduction in H3K27ac level was confirmed by chromatin immunoprecipitation where H3K27ac antibody pulled-down similar levels of DNA than IgG after treatment (Supplemental Figure 3b). Similar results were obtained in NALM-6 cells after proscillaridin treatment (Supplemental Figure 3c). No change was detected on H4 acetylation or H3 methylation marks (Supplemental Figures 4a and b; and data not shown). Interestingly, loss of H3 acetylation induced global chromatin reorganization in MOLT-4 cells after treatment, as shown by DAPI staining (Supplemental Figure 4c).
We next asked if there was a correlation between loss of H3 acetylation and gene expression changes after proscillaridin treatment. To address this question, we combined our RNA-Seq data pre- and post-treatment with H3K27ac ChIP-seq data of untreated MOLT-4 (50) since this mark is associated with transcribed regions and is lost globally after treatment (Figure 4a) (23). Among 7,097 genes marked at their promoters with H3K27ac (−500 to +500 bp) in untreated MOLT-4 cells, 2,169 genes were differentially expressed after proscillaridin treatment. Seventy-four percent of those (1,608 genes), marked by H3K27ac in untreated cells, were significantly downregulated after treatment (Figure 4b), which is consistent with the loss of this active epigenetic mark (Figure 4c). Gene ontology analysis of these 1,608 downregulated genes showed a significant relationship with metabolism and proliferation processes (Figure 4d; Supplemental Figure 5a). Among these genes, all MYC PATHWAY genes (n=30) previously described (Figure 3c) were marked by H3K27ac in untreated MOLT-4 cells and were all downregulated by treatment (Figures 4 c and e). Network analysis showed that these MYC target genes are co-expressed simultaneously, and are known to exhibit protein-protein interactions with MYC, confirming the global effect of proscillaridin on MYC pathway (Supplemental Figures 6a and b). By contrast, upregulated genes marked by H3K27ac in untreated cells were associated with apoptosis, negative regulation of proliferation and cell differentiation (Supplemental Figure 5b), corroborating our transcriptomic and functional analyses. Collectively, these results demonstrate that proscillaridin produces global loss of H3 acetylation, which was associated with silencing of genes involved in proliferation and MYC pathway.
Proscillaridin Induces Loss of Lysine Acetylation in MYC Target Genes and Chromatin Regulators
We then asked whether depletion of lysine acetylation was extended to non-histone proteins after treatment. First, we measured MYC acetylation levels after 8, 16 and 24h of proscillaridin treatment (5 nM) in MOLT-4 cells, since this posttranslational modification plays a role in its stability (17,51). After MYC immunoprecipitation and probing with a pan-acetyl antibody, we measured a time dependent decrease (up to 75%) of MYC total acetylation (Figure 5a).
To further characterize the extent of acetylation loss, we conducted an acetylome study by mass spectrometry on untreated and proscillaridin-treated (5 nM; 48h) MOLT-4 cells (Figure 5b). Two distinct MYC peptides showed a significant reduction in lysine acetylation after treatment, which confirmed our immunoprecipitation results (Supplemental Figure 7a). Mass spectrometry analysis showed that 28 peptides (including MYC) had a significant loss of lysine acetylation after treatment, associated with chromatin organization (Figure 5c). Among them, 8 are known MYC target proteins and 6 are involved in chromatin organization (Figures 5d and e). Networks analysis showed that these 28 proteins are generally co-expressed, suggesting a connection between their acetylation and expression levels (Supplemental Figure 7b). Interestingly, 8 out of 28 proteins were MYC target proteins, including MYC itself and 6 out of 28 are involved in histone acetylation regulation (Figures 5d and e) (52). Altogether, proscillaridin reduces lysine acetylation of MYC, its protein partners and several histone acetylation regulators.
Proscillaridin Efficiently Downregulates Histone Acetyltransferases Involved in MYC Acetylation
We next investigated whether acetylation loss was due to a dysregulation of histone acetyltransferases (KATs). We measured, by western blotting, KAT levels before and after proscillaridin treatment (5 nM; 8h-96h) in MOLT-4 cells. Proscillaridin produced a time-dependent reduction (up to 80%) of several KATs including KAT3A (CBP), KAT3B (P300), KAT5 (TIP60), KAT2A (GCN5) and KAT6A (MOZ) (Figure 6a, Supplemental Figure 8a). Expression of KAT2B (PCAF) and KAT7 (HBO1) were not altered by the treatment (Supplemental Figures 8a and b). No significant changes were observed in class I HDACs expression, suggesting that acetylation loss mainly involved KATs downregulation (data not shown). Interestingly, KAT downregulation was observed only at the protein level, since their mRNA levels were not altered after treatment (data not shown). Significant reduction in KAT protein expression including KAT2A, KAT3A, KAT3B and KAT6A, which target histone H3, occurred 8h prior to significant H3 acetylation loss. Despite KAT5 decrease, a KAT known to acetylate H2A, H3 and H4, no changes in H4 acetylation (total or on specific lysines) were measured after treatment (Supplemental Figure 4a) (53-57). This result can be explained by the fact that KAT7 (HBO1) expression, which is also involved in H4 acetylation, was not affected by the treatment (58,59). To confirm the effects of KAT downregulation in MOLT-4 cells, we used KAT3A/B pharmacological inhibitor C646. Similar to proscillaridin treatment, C646 (10 μM; 48h) significantly reduced lysine acetylation (H3K14, H3K18, H3K27, and total H3-acetylation), depleted KATs (KAT3A, and KAT3B) and MYC protein levels (Figures 6b and c; Supplemental Figures 8c and d).
Since KATs have overlapping enzymatic activities, we asked if the extent KAT protein downregulation was associated with proscillaridin sensitivity. We compared KAT protein levels before and after proscillaridin treatment in MYC overexpressing cancer cells (MOLT-4, NALM-6, MYC and RASV12+MYC transformed fibroblasts) versus low MYC expressing cancer cells (SW48, A549, and RASV12 transformed fibroblasts; Figure 6d; Supplemental Figure 9a). Cancer cell lines were treated at 5 nM for 48h, which was clinically relevant and close the IC50 values of leukemic cells. Transformed fibroblasts were treated at 70 nM for 48h, which was the IC50 value of MYC transfected fibroblasts as described in Figure 1b. After treatment, we observed that KAT protein downregulation was more pronounced in drug-sensitive cells with high MYC expression as compared to drug-resistant cells with low MYC expression. Indeed, proscillaridin induced a significant downregulation of 4/7 KATs in MOLT-4 cells, 3/7 KATs in NALM-6 cells, 3/7 KATs in RASV12+MYC transformed fibroblasts, and 7/7 KATs in MYC transformed fibroblasts. Interestingly, downregulated KATs (KAT2A/GCN5, KAT3A/ CBP, KAT3B/P300, KAT5/TIP60 and KAT6A/MOZ) in MYC overexpressing cells, were shown to acetylate MYC and increase its stability (17,51,60-64). In stark contrast, proscillaridin failed to downregulate more than one KATs in low MYC expressing cancer cells (SW48, A549 and RASV12 transformed fibroblasts). Thus, proscillaridin-induced KAT proteins downregulation was more important in high MYC expressing cells, which correlated with IC50 values within its therapeutic range.
Similar analysis was performed on histone H3 acetylation between high MYC expressing cancer cells versus low MYC expressing cancer cells (Supplemental Figures 9b and c). Proscillaridin induced a significant loss of H3 acetylation in drug-sensitive and MYC overexpressing cells (MOLT-4, NALM-6, MYC and RASV12+MYC transformed fibroblasts). In proscillaridin-resistant and low expressing MYC cancer cells, histone acetylation levels were unchanged in SW48 cells after treatment, which correlated with our previous report (Supplemental Figures 9b and c) (22). By contrast, A549 cells lost significantly H3 acetylation after treatment while RASV12 transformed fibroblasts lost acetylation on some sites (K9, K27 and pan-acetyl) and other sites were not affected (K14 and K18) (Supplemental Figures 9b-c). These data suggest that proscillaridin sensitivity is not entirely dependent on histone acetylation loss, suggesting the importance of non-histone acetylation. In summary, proscillaridin antiproliferative effect was associated with its ability to downregulate several simultaneously KATs resulting loss of acetylation in histone and non-histone proteins (Figure 6e).
Discussion
The repurposing potential of cardiac glycosides in oncology has been suggested several decades ago and is currently under intense clinical investigation either alone (in prostate cancers, NCT01162135; breast cancer, NCT01763931; and sarcoma, NCT00017446) or in combination with chemotherapy (digoxin with cisplatin in head and neck cancers, NCT02906800; or with epigenetic drug decitabine, NCT03113071) (65,66). Here, our data provide a strong rationale to repurpose proscillaridin specifically against leukemia with MYC oncogenic dependency.
Proscillaridin induced a rapid loss of MYC protein expression in MYC-driven leukemia cells. Importantly, proscillaridin efficiently targeted MYC-dependent leukemic stem cells, indicating the potential of controlling leukemia self-renewal capacity. We demonstrated that proscillaridin targeted MYC overexpressing leukemic cells by downregulating KATs involved specifically in MYC acetylation and transcriptional program (KAT2A, KAT3A, KAT3B, KAT5 and KAT6A) (17,51,60-64). These KATs are positive regulators of MYC stability and MYC pathway. Importantly, KATs have overlapping activity and targets, suggesting the relevance of targeting these enzymes simultaneously to efficiently reduce acetylation of MYC and its partners (17,61,62,64). Indeed, proscillaridin-induced downregulation of several KATs was observed in proscillaridin-sensitive and MYC overexpressing leukemic cells whereas this effect was sporadic in resistant and low MYC expressing cells. Proscillaridin treatment induced a significant loss of H3 acetylation levels in MYC overexpressing cells whereas loss of histone acetylation was not observed in SW48 cells but was significantly reduced in A549 and in two lysine residues in RASv12-transfected fibroblasts, suggesting that loss of H3 acetylation is not sufficient to modulate cell viability in these low MYC expressing cells. These data highlight the importance of acetylation levels in non-histone proteins as a potential therapeutic target in MYC overexpressing leukemia. Experiments are ongoing to address this specific question.
Lysine acetylation is a dynamic process that can be modulated within minutes and it is maintained on histone and non-histone proteins by the redundant activity of KATs. Therefore, cancer cells may rapidly recover from incomplete pharmacological inhibition or from the specific inhibition of a particular KAT (53). Here, we showed that proscillaridin treatment in MYC overexpressing leukemia cells, led to the downregulation of several KATs, produced MYC inhibition, and induced persistent leukemia cell differentiation, which was maintained for several days after drug removal. Therefore, this study supports a strategy of simultaneously targeting several KATs to reduce efficiently acetylation in histone and in non-histone proteins, which overcome the redundant activity of KATs. The mechanism implicated in proscillaridin-induced KATs downregulation is under investigation. Overall, we conclude that proscillaridin downregulates MYC protein levels and MYC oncogenic pathway in leukemia through the downregulation of several KATs.
Funding
We acknowledge the Cole Foundation for the Transition Award (to N, J-M R) and a PhD fellowship (E,D.C.), the Charles-Bruneau immuno-hemato-oncology Unit, the Canadian Foundation for Innovation for funding, and the Fond de Recherche du Québec en Santé.
Author contributions
E.M.D.C. and N.J.M.R. designed the study and wrote the manuscript. E.M.D.C., G.A., G. McI., A.B., V. B-L., C.R., M. R., M. B., K. E., EE. F-D., A. H., T. H., C. B., and S. McG. performed the experiments. C.R., M.C., P.S.O and D.S performed RNA sequencing experiments and bioinformatics analyses. J.R. J., N. K., Y. S., M. D. performed mass spectrometry experiments and acetylome studies. The authors declare no conflict of interest.
Acknowledgements
We thank Catherine Legros for the graphical help, and Dr André Tremblay, Dr Audrey Claing, Dr Elie Haddad and Dr Carolina Alfieri for providing cell lines and materials.