Heart failure drug proscillaridin A targets MYC overexpressing leukemia through global loss of lysine acetylation
Cardiac glycosides are approved for the treatment of heart failure as Na+/K+ pump inhibitors. Their repurposing in oncology is currently investigated in preclinical and clinical studies. However, the identification of a specific cancer type defined by a molecular signature to design targeted clinical trials with cardiac glycosides remains to be characterized. Here, we demonstrate that cardiac glycoside proscillaridin A specifically targets MYC overexpressing leukemia cells and leukemia stem cells by causing MYC degradation, epigenetic reprogramming and leukemia differentiation through loss of lysine acetylation.
Proscillaridin A anticancer activity was investigated against a panel of human leukemia and solid tumor cell lines with different MYC expression levels, overexpression in vitro systems and leukemia stem cells. RNA-sequencing and differentiation studies were used to characterize transcriptional and phenotypic changes. Drug-induced epigenetic changes were studied by chromatin post-translational modification analysis, expression of chromatin regulators, chromatin immunoprecipitation, and mass-spectrometry.
At a clinically relevant dose, proscillaridin A rapidly altered MYC protein half-life causing MYC degradation and growth inhibition. Transcriptomic profile of leukemic cells after treatment showed a downregulation of genes involved in MYC pathways, cell replication and an upregulation of hematopoietic differentiation genes. Functional studies confirmed cell cycle inhibition and the onset of leukemia differentiation even after drug removal. Proscillaridin A induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27) and 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 acetyltransferases. Importantly, proscillaridin A demonstrated anticancer activity against lymphoid and myeloid stem cell populations characterized by MYC overexpression.
Overall, these results strongly support the repurposing of proscillaridin A in MYC overexpressing leukemia.
KeywordsCardiac glycosides Proscillaridin A MYC Leukemia Lysine acetylation Chromatin remodelling Lysine acetyltransferase Leukemia stem cells
Acute myeloid leukemia
Half-maximal inhibitory concentration
Leukemic stem cells
T-cell acute lymphoblastic leukemia
MYC (c-MYC) transcription factor is a driver of oncogenic programs. It contributes to gene deregulation in cancer by promoting expression of genes involved in cell proliferation . High MYC expression drives tumor initiation, progression, and maintenance and is associated with aggressive cancers and poor prognoses . MYC is a potent driver in leukemia inducing cell proliferation and blocking cell differentiation . Moreover, MYC contributes to long-term self-renewal of leukemic stem cells . Conversely, genetic suppression of MYC in transgenic mouse models induces differentiation and cell growth arrest of leukemic cells [5, 6]. 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 [7, 8, 9]. 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 [10, 11]. 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 . 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 . Pharmacological inhibition of KATs represents 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 .
Recently, we reported that cardiac glycosides, which are Na+/K+ pump inhibitors and approved for heart failure treatment, exhibit significant epigenetic and anticancer effects [15, 16]. Cardiac glycosides, including digitoxin, digoxin, lanatoside C, ouabain and proscillaridin A, triggered reactivation of epigenetically silenced tumor suppressor genes, supporting their repurposing potential . Interestingly, all cardiac glycosides produced synergistic responses when used in combination with the epigenetic drug decitabine (demethylating agent), further supporting their epigenetic activity . Several epidemiological studies argue in favor of repurposing cardiac glycosides in oncology. Indeed, patients treated with cardiac glycosides for heart failure have a lower rate of cancer diagnosis as compared to the general population and upon cancer diagnosis; these patients exhibit generally a less aggressive disease and respond better to therapy . However, the repurposing of cardiac glycosides in oncology is limited by their narrow therapeutic window. Indeed, maximal plasmatic level is around 10 nanomolar, due to cardiac toxicities [17, 18, 19]. 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 [19, 20, 21]. 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 A, which was identified as the most potent cardiac glycoside in our previous screens [15, 16]. Proscillaridin A produced antiproliferative effects with a preferential selectivity towards MYC overexpressing leukemia cells. We demonstrated that proscillaridin A 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 A 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 were used in the study. Leukemia cell lines MOLT-4 and REH were transduced with MYC lentivirus and hTERT/SV40ER-immortalized human primary fibroblasts were transduced with MYC, RASV12 or MYC and RASV12 lentiviruses. MYC lentivirus MYC_pLX307 was a gift from William Hahn & Sefi Rosenbluh (Addgene plasmid # 98363). RASV12 lentivirus was a gift from Dr. Christian Beauséjour laboratory. Cell types and culture conditions are described in Additional file 1 Supplementary Materials and Methods. Proscillaridin A was purchased from Santa Cruz Biotechnologies (CAS number: 466–06-8; purity ≥90%) and cycloheximide was purchased from Acros Organics (CAS: 66–81-9; purity ≥95%). 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. Densitometric analysis was performed using ImageJ software. ANOVA tests were performed with GraphPad Prism software. Antibodies are listed in the Additional file 1 Supplementary 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 6000 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 [22, 23]. 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 results from publicly available dataset (GEO: GSM2037790) were associated with transcriptomic data. GSEA analysis of 8227 AML fractions and the leukemia stem cell (LSC) signatures were 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 . Briefly, 4 biological replicates of untreated and proscillaridin A-treated MOLT-4 cells (5 nM, 48 h) were digested with trypsin. Peptides were analyzed by mass-spectrometry and data were extracted with the MaxQuant software package (version 18.104.22.168) 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) [25, 26]. Peptides were searched with SwissProt human protein database.
Cardiac glycoside Proscillaridin A targets MYC-driven leukemic cells
To evaluate MYC contribution to proscillaridin A therapeutic efficacy in cancer cells, 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. After transduction, 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 (Additional file 2: Figure S1C). High levels of MYC and RAS protein were detected after transductions when compared to non-transduced cells (Fig. 1b). Using a wide range of proscillaridin A concentrations (from 0.01 nM to 100 μM) for 48 h, we measured cell viability and calculated IC50 values (Fig. 1c). Untransformed fibroblasts were fully resistant to proscillaridin A. Likewise, RASV12-transformed cells were mildly affected by the treatment where proscillaridin A at high doses failed to impact cell viability by more than 50%. Conversely, MYC transformed fibroblasts were highly sensitive to proscillaridin A 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, we demonstrated that MYC overexpression is causing proscillaridin A sensitivity in transformed fibroblasts. In comparison, after 48 h treatment, MOLT-4 and NALM-6 cells (MYC overexpressing leukemic cells) showed IC50 values of 2.3 nM and 3 nM, respectively (Fig. 1d). Interestingly, both after 24 h (Fig. 1a) and 48 h (Fig. 1d) treatment, proscillaridin A produced IC50 values below 10 nM, which correspond to clinically achievable concentrations measured in the plasma of heart failure patients. To further confirm the role of MYC protein levels to proscillaridin A sensitivity, we transduced high MYC expressing (MOTL-4) and low MYC expressing (REH) leukemia cells, with MYC oncogene. After transduction and selection steps, MOLT-4 cells had similar MYC protein levels than WT cells, most likely due to an increased cell death upon additional MYC expression (Additional file 2: Figure S1D). In these cells, proscillaridin A sensitivity was unchanged (Additional file 2: Figure S1D). However, MYC-transduced REH cells expressed higher MYC levels, which increased proscillaridin A sensitivity (Additional file 2: Figure S1E). Altogether, these results support proscillaridin A repurposing against MYC overexpressing leukemia.
To explore the mechanism by which MYC overexpression in cancer cells correlates with proscillaridin A 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 A (5 nM; 48 h) significantly reduced MYC protein level by more than 50% in MOLT-4 and NALM-6 cells but not in SW48 and A549 cells (Fig. 1e). Time-course experiments with both leukemic cell lines (8 h to 96 h) showed that proscillaridin A induced a significant (up to 80%) and rapid MYC downregulation (Fig. 1f; Additional file 2: Figure S1F). These results demonstrate that low dose proscillaridin A inhibits efficiently leukemia growth causing rapid MYC downregulation. Lastly, we performed a cycloheximide-chase assay in MOLT-4 cells treated with proscillaridin A at low dose (5 nM; 16 h). We observed that drug treatment produced almost a 50% reduction in MYC protein half-life (MYC t1/2: 41 min in untreated cells vs 23 min in proscillaridin A treated cells; Fig. 1g and h). Therefore, proscillaridin A targets MYC overexpressing leukemia cells by reducing MYC protein half-life, causing its rapid degradation.
Proscillaridin A downregulates cell proliferation programs and induces T-cell differentiation
Gene ontology analysis also revealed that upregulated genes were enriched for hematopoietic and lymphoid organ development, suggesting the onset of leukemia differentiation (Fig. 2f and Additional file 3: Figure S2E). 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 48 h treatment and remained expressed for 2 days after drug removal (Fig. 2g) . 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 (Fig. 2h and i) . Upregulation of these differentiation markers were in the same range than the levels measured after TPA treatment, a well-known inducer of leukemia differentiation (Fig. 2h and i) . Altogether, proscillaridin A treatment produced a transcriptomic shift from a proliferative program to the induction of T-cell leukemia differentiation.
Proscillaridin A induces global loss of histone H3 acetylation
We next asked if there was a correlation between loss of H3 acetylation and gene expression changes after proscillaridin A treatment. To address this question, we combined our RNA-Seq data pre- and post-treatment with H3K27ac ChIP-seq data of untreated MOLT-4 since this mark is associated with transcribed regions and is globally lost after treatment . Among 7097 genes marked at their promoters with H3K27ac (− 500 to + 500 bp) in untreated MOLT-4 cells, 2169 genes were differentially expressed after proscillaridin A treatment. Seventy-four percent of those (1608 genes), marked by H3K27ac in untreated cells, were significantly downregulated after treatment (Fig. 3b), which is consistent with the loss of this active epigenetic mark (Fig. 3c). Gene ontology analysis of these 1608 downregulated genes showed a significant relationship with metabolism and proliferation processes (Fig. 3d; Additional file 6: Figure S5A). Among these genes, all MYC PATHWAY genes (n = 30) previously described (Fig. 2c) were marked by H3K27ac in untreated MOLT-4 cells and were all downregulated by treatment (Fig. 3c 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 A on MYC pathway (Additional file 7: Figure S6A and S6B). By contrast, upregulated genes marked by H3K27ac in untreated cells were associated with apoptosis, negative regulation of proliferation and cell differentiation (Additional file 6: Figure S5B) corroborating our functional analyses. Collectively, these results demonstrate that proscillaridin A produces global loss of H3 acetylation, which was associated with silencing of genes involved in proliferation and MYC pathway.
Proscillaridin A induces loss of lysine acetylation in MYC target genes and chromatin regulators
To further characterize the extent of acetylation loss, we conducted an acetylome study by mass spectrometry on untreated and proscillaridin A-treated (5 nM; 48 h) MOLT-4 cells (Fig. 4b). Two distinct MYC peptides showed a significant reduction in lysine acetylation after treatment, which confirmed our immunoprecipitation results (Additional file 8: Figure S7A). Mass spectrometry analysis showed that 28 peptides (including MYC) had a significant loss of lysine acetylation after treatment, associated with chromatin organization (Fig. 4c). Among them, 8 are known MYC target proteins and 6 are involved in chromatin organization (Fig. 4d and e). Networks analysis showed that these 28 proteins are generally co-expressed, suggesting a connection between their acetylation and expression levels (Additional file 8: Figure S7B). Interestingly, 8 out of 28 proteins were MYC target proteins, including MYC itself and 6 out of 28 are involved in histone acetylation regulation (Fig.4d and e). Altogether, proscillaridin A reduces lysine acetylation of MYC, its protein partners and several histone acetylation regulators.
Proscillaridin A efficiently downregulates histone acetyltransferases involved in MYC acetylation
Since KATs have overlapping enzymatic activities, we asked if the extent KAT protein downregulation was associated with proscillaridin A sensitivity. We compared KAT protein levels before and after proscillaridin A 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; Fig. 5d; Additional file 10: Figure S9A). Cancer cell lines were treated at 5 nM for 48 h, which was clinically relevant and close the IC50 values of leukemic cells. Transformed fibroblasts were treated at 70 nM for 48 h, which was the IC50 value of MYC transduced fibroblasts as described in Fig. 1c. 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 A 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 (Fig. 5d). 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 [33, 39, 40, 41]. In stark contrast, proscillaridin A failed to downregulate more than one KATs in low MYC expressing cancer cells (SW48, A549 and RASV12 transformed fibroblasts, Fig. 5d). Thus, proscillaridin A-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 (Additional file 10: Figure S9B and S9C). Proscillaridin A 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 A-resistant and low expressing MYC cancer cells, histone acetylation levels were unchanged in SW48 cells after treatment, which correlated with our previous report (Additional file 9: Figure S8B and S8C) . 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) (Additional file 10: Figure S9B and S9C). These data suggest that proscillaridin A sensitivity is not entirely dependent on histone acetylation loss, suggesting the importance of non-histone acetylation. In summary, proscillaridin A antiproliferative effect was associated with its ability to downregulate simultaneously several KATs resulting in loss of acetylation on histone and non-histone proteins (Fig. 5e).
Proscillaridin A efficiently targets MYC-driven leukemic stem cell populations
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 (Fig. 6b) [44, 45]. Gene set enrichment analysis of data from Lechman et al., revealed that the LSC-enriched fraction (CD34+/CD38−) in AML 8227 is enriched for MYC target genes expression as compared to non-LSCs (CD34−) (Fig. 6c) . After 6 days of proscillaridin A treatment, bulk AML 8227 cells had an IC50 of 29 nM (Fig. 6d). 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 A with IC50 values of 15 nM. Altogether, proscillaridin A 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.
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 cancer, 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) . However, none of these clinical studies are designed to use cardiac glycosides against cancer types driven by specific oncogene, such as MYC-driven cancers; although the effect of cardiac glycosides on MYC inhibition has been previously reported [48, 49]. Here, we demonstrate that the anticancer activity of proscillaridin A correlates positively with MYC oncogenic expression in leukemia, suggesting its repurposing potential against cancer types defined by a specific molecular signature. Deregulated MYC is found in more than half of hematological malignancies, including T-cell and B-cell neoplasms, lymphomas and myeloid leukemia. Chromosomal translocation, gene amplification, and gene activation contribute to MYC overexpression, which often correlates with a poor prognosis [3, 50]. Interestingly, proscillaridin A exhibited a strong anticancer activity on both lymphoid and myeloid leukemic stem cell populations overexpressing MYC, indicating the potential of controlling leukemia self-renewal capacity.
Proscillaridin A induced a rapid loss of MYC protein expression in MYC-driven leukemia cells through the downregulation of series of KATs (KAT2A, KAT3A, KAT3B, KAT5 and KAT6A) known to be involved in MYC protein stability (by lysine acetylation) and activation of MYC transcriptional programs [33, 39, 40, 41, 51]. Interestingly, KAT downregulation induced by proscillaridin A was associated with a significant reduction of MYC protein half-life. Since KATs have overlapping activity and targets, it may be relevant to target these enzymes simultaneously to ensure an efficient reduction in lysine acetylation of MYC and its partners [12, 39, 41, 51]. This may represent an alternative strategy as opposed to pharmacological inhibition of a specific KAT to overcome the redundant activity of KATs .
Proscillaridin A treatment also resulted in a significant loss of H3 acetylation levels in MYC overexpressing cells. This effect was associated with the loss of epigenetic active marks (including H3K27ac) in promoters of MYC-dependent genes associated with cell proliferation programs. Interestingly, MYC-driven leukemic cells started to differentiate during treatment and after drug removal, suggesting the onset of a stable epigenetic reprogramming. The contribution of global histone acetylation loss to the anticancer effects of proscillaridin A remains to be better understood since loss of histone acetylation was observed in some proscillaridin A-resistant cells, suggesting that loss of H3 acetylation may not be sufficient to modulate cell viability in these low MYC expressing cells.
Here, we demonstrated that proscillaridin A treatment in MYC overexpressing leukemia cells, led to the downregulation of several KATs, induced significant acetylation loss, produced MYC degradation, and induced persistent epigenetic effects and 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, to overcome the redundant activity of KATs. The mechanism implicated in proscillaridin A-induced KATs downregulation is under investigation. Overall, we conclude that proscillaridin A has a promising potential to be repurposed as an epigenetic drug in personalized oncology, particularly in MYC overexpressing leukemia.
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.
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, G M-B, 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. All authors read and approved the final manuscript.
We acknowledge the Cole Foundation for the Transition Award (to N. J-M. R) and a PhD fellowship (E.M D.C.), the Charles-Bruneau immuno-hemato-oncology Unit, the Cancer Research Society, the Canadian Foundation for Innovation for funding, and the Fond de Recherche du Québec en Santé.
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The authors declare that they have no competing interests.
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