Abstract
Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The pro-apoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models we identified an inhibitor of the transcription factor heterodimer CBFβ/RUNX1. By genetic gain and loss of function experiments we validated that the mode of action depends on RUNX1 and NOXA. Of note, RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a novel way to target a therapy resistant PDAC, an unmet clinical need.
Significance Recent evidence demonstrated the existence of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), which resist all current therapies. The paucity of therapeutic options, including a complete lack of targeted therapies, underscore the urgent and unmet medical need for the identification of targets and novel treatment strategies for PDAC. Our study unravels a function of the transcription factor RUNX1 in apoptosis regulation in PDAC. We show that pharmacological RUNX1 inhibition in PDAC is feasible and leads to NOXA-dependent apoptosis. The development of targeted therapies that influence the transcriptional landscape of PDAC might have great benefits for patients who are resistant to conventional therapies. RUNX1 Inhibition as a new therapeutic intervention offers an attractive strategy for future therapies.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease often diagnosed at an advanced stage. The incidence of PDAC is steadily increasing, and PDAC is predicted to be the second leading cause of cancer-related death by 2030 (1). Evasion of apoptosis is a characteristic of PDAC and is often associated with treatment resistance (2, 3). A dysregulated transcription commonly results in apoptosis resistance (4). Therefore, the identification of novel concepts to reactivate apoptosis by disrupting cancerous transcription programs is a promising approach for the effective elimination of PDAC cells (3, 5).
Comprehensive integrated genome analyses from RNA expression profiles in recent years revealed different subtypes of PDAC with variable biology and therapeutic responsiveness (6–9). NOXA (latin for damage; also known as PMAIP1 - Phorbol-12-myristate-13-acetate-induced protein 1) is part of an identifier gene-set for the quasi-mesenchymal subtype of the disease (8). This subtype overlaps with the described basal-like and squamous subtype of the disease, which is particularly resistant to the currently used chemotherapeutics (7, 9).
NOXA belongs to the BCL-2 homology (BH) BH3-only subgroup of the B-cell lymphoma 2 (BCL2) protein family which is essential for the regulation of cell intrinsic apoptosis (10). BCL2 proteins are divided into sensor, effector and protector proteins (10). The classical anti-apoptotic protector proteins including Myeloid cell leukemia 1 (MCL1) inhibit effector proteins (e.g. Bcl-2-associated X protein) thereby blocking apoptosis. Pro-apoptotic sensor proteins including NOXA, which directly binds to MCL1, neutralize the anti-apoptotic function of the protector proteins (10), leading to the initiation of apoptotic cell death. In PDAC, NOXA is tightly regulated at the transcriptional level, and transcriptional activation of NOXA by HDAC-inhibitors or proteasome inhibitors contribute to induce the cell intrinsic apoptosis pathway (11–13). Furthermore, NOXA is regulated by multiple transcription factors including p53 and is involved in apoptosis under genotoxic stress (14, 15).
Runt-related (RUNX) proteins are master regulators involved in a broad range of biological processes including proliferation, differentiation and apoptosis (16). DNA binding of these transcription factors is mediated by heterodimerization of a core DNA binding factor alpha chain (CBFα), composed of one of the three RUNX family members, RUNX1-3, to the non-DNA binding core binding factor beta (CBFβ). Each of the three RUNX family members play important roles in different stages of tumor development (17). As has been shown in mouse models, knockouts of any of the three RUNX transcription factors exhibit significant developmental defects: RUNX1 plays an important role in hematopoiesis (18), RUNX2 in bone development (19) and RUNX3 in the gastrointestinal tract (20) and in neurogenesis (21). RUNX expression patterns are highly dynamic and depend on the stage of differentiation, development and environmental conditions (22). In addition, RUNX transcription factors are expressed in almost all cancers (23). Besides its implication in leukemogenesis (24), RUNX1 is strongly expressed in a broad spectrum of solid tumors (25) and is associated with poor prognosis in PDAC (26). Depending on the cellular context, RUNX1 can act both oncogenic and tumor suppressive in solid tumors (27). RUNX1 interacts with various co-factors to shape gene expression. RUNX1-dependent activation of target genes is mediated through an interaction with CBP/p300 (28) and the protein arginine methyltransferase 1 (PRMT1) (29). The inhibitory function of RUNX1 is achieved by interaction with co-repressors such as the Sin3A-HDAC corepressor complex (30).
In this study, we aimed at identifying novel strategies affecting the delicate balance of NOXA expression to drive cell death in an aggressive subtype of PDAC. We found that inhibition of RUNX1 led to a global gain of H3K27ac enrichment contributing to the activation of the proximal NOXA promoter region and suggest a strategy to overcome treatment resistance in an aggressive subtype of PDAC with inferior prognosis.
Materials and Methods
Cell culture, cell viability assay
Cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, #41965062) or Roswell Park Memorial Institute (RPMI) 1640 Medium (Thermo Fisher Scientific, #21875091) supplemented with 10% FBS (Thermo Fisher Scientific, #10270106) and 1% Penicillin/Streptomycin (Thermo Fisher Scientific, #15070063). They were passaged up to 14 times in a 1:10 dilution every 3-4 days. Murine PDAC cell lines were generated from KrasG12D driven mouse models as described (13). For all cell lines used, PCR-based mycoplasma tests were performed at regular intervals. Cell viability was measured by MTT-Test (Sigma-Aldrich, #M5655). Detailed information on the procedures cell viability, drug screening and colony formation assay in Supplementary Materials and Methods.
Patient-derived organoids
PDAC biopsies and tissues were received from endoscopy punctures or surgical resection. 3D organoids were collected, propagated, and analyzed in agreement with the declaration of Helsinki. This study was approved by the ethical committee of TUM (Project 207/15). Written informed consent from the patients for research use of tumor material was obtained prior to the use. Detailed information in Supplementary Materials and Methods.
In vivo drug efficacy analysis in mice, IHC
Xenograft assays were performed by EPO (Experimental Pharmacology and Oncology, Berlin-Buch). All animal experiments were approved by the local responsible authorities and performed in accordance with the German Animal Protection law. Detailed information in Supplementary Materials and Methods.
Statistical Analysis, qChIP, ChIPseq, RNAseq, ATAC seq, 4C, qPCR, Western blot, CRISPR-editing
Detailed information on the procedures and data analyses in Supplementary Materials and Methods and in STable 5.
Results
High NOXA mRNA expression is associated with an aggressive PDAC subtype
As a stress response, tumor cells may express pro-apoptotic effectors that can be neutralized by anti-apoptotic counterparts, thus dampening the apoptotic response. Such tumor cells expose apoptotic potential. To investigate, if classical pro-apoptotic BH3-only proteins may contribute to this phenotype, we analyzed the mRNA expression of BH3-only genes (6) and filtered for transcriptome profiles of human PDAC tumors (Fig. 1A). From this data, we extracted indicated classical BH3-only genes, performed a hierarchical clustering based on the mRNA expression of the BH3-only genes and subdivided PDAC patient samples into the squamous subtype and combined the ADEX-, classical- and the immunogenic subtypes as “other” subtypes. We subsequently determined if the mRNAs of the listed classical BH3-only members are significantly enriched in these two groups. Of note, specifically high expression of NOXA was significantly (p < 0.001) associated with the squamous subtype (Fig. 1A and SFig1A), which is in line with the described expression in quasi-mesenchymal (QM) cancers (8). In accordance with this finding, high NOXA expression (>75th percentile) characterized a PDAC patient cohort with inferior survival as compared to patients with low NOXA expression (<25th percentile) in ICGC/Bailey et al. (Fig. 1B and SFig1B) and TCGA datasets (SFig. 1C). We hypothesized that the high expression of NOXA indicates that these tumors harbor an apoptotic potential, and that shifting the balance of apoptosis regulators to a pro-apoptotic state may constitute a therapeutic strategy.
Identification of a synthetic lethal interaction of NOXA and inhibition of RUNX1
By analyzing transcriptome profiles, we selected human PDAC cell lines of the quasi-mesenchymal subtype (8) (SFig 1D) and KrasG12D-driven murine PDAC cell lines that exhibit relatively high basal Noxa mRNA expression (SFig. 1E) for cross-species validation to identify drugs that affect the apoptotic balance. To investigate vulnerabilities specifically created by NOXA expression, we generated human and murine isogenic PDAC cell line models with genetically defined NOXA status (SFig. 2A-D). To prove NOXA deficiency, we performed western blotting of the two human PDAC cell lines PSN1 and MiaPaCa-2. NOXA protein was absent in NOXAko cell lines (Fig. 1C). Importantly, NOXA deficiency did not influence the proliferation of the isogeneic cell lines (Fig. 1D).
To identify vulnerabilities associated with NOXA, we next performed a drug screening with a total of 1842 compounds in NOXA-proficient (parental) and NOXA-deficient (NOXAko) cells and measured viability (Fig. 1E). Drug testing and viability assays were performed with a single concentration of 600 nM, as previously described (13). For identification of effects due of the NOXA status, we used a cut-off of 10% difference in viability (Fig. 1F). Out of the 1842 compounds we identified 50 drugs that showed higher efficiency in parental cell lines compared to NOXAko cell lines. Importantly, within the hits found, we identified topoisomerase and proteasome inhibitors, which is in line with data from previous studies (11–13), underlining the robustness of our screening experiment (STable 1). From our screening hits, we selected for specific targeted molecules (target specificity) and novelty (Fig. 1E) and further validated 12 hits with multi-dose experiments (Fig. 1F). Only one of these twelve compounds analyzed in the validation experiments, AI-10-49, showed no growth inhibition in NOXAko cell lines whereas viability of NOXA-proficient cells was dose-dependently reduced (Fig. 1F). AI-10-49 was originally designed to inhibit the interaction of the oncofusion protein CBFβ-SMMHC with RUNX1 (32). The lead compound of the bivalent AI-10-49 has been shown to inhibit RUNX1/CBFβ (33). Co-immunoprecipitations with either RUNX1 or CBFβ revealed AI-10-49 as RUNX1/CBFβ inhibitor in MiaPaCa-2 cells (SFig. 2E). Additionally, using the SwissTargetPrediction tool (34), CBFβ was a predicted target of this compound (STable 2). In summary, our data suggest that impairment of RUNX1 activity may affect NOXA-dependent execution of cell death in PDAC.
Induction of NOXA by CBFβ/RUNX1 inhibition
To investigate whether the AI-10-49-induced drop in viability is mediated by induction of the apoptotic process, we performed fluorometric analysis of Annexin V/PI stained PDAC cells. Indeed, the reduced viability in parental cell lines upon AI-10-49 treatment was clearly associated with a significant induction of cell death in parental cells, whereas only marginal apoptosis induction was observed in NOXAko cells (Fig. 1G).
We next investigated whether an altered expression of other BCL2 family members such as MCL1, BCL2, BCLxL, BIM, BID, BAK or BAX mediates AI-10-49-induced apoptosis. We detected PARP cleavage but did not detect differential expression of these BCL2 family proteins 6 hours and 24 hours after AI-10-49 treatment (Fig. 1H, SFig. 3A, B), a marked induction of NOXA protein (Fig. 1I) and NOXA mRNA expression (Fig. 1I) was observed upon treatment with AI-10-49. In HCT116 cells harboring wild-type p53, a DNA damage stimulus induced both RUNX1 and p53 and activated p53 target genes, including NOXA (35). To test if p53 is involved in AI-10-49 induced NOXA expression we treated murine cell lines harboring either wiltype, mutant or deleted p53- (SFig. 3C) with AI-10-49 and the topoisomerase II inhibitor etoposide (SFig. 3D). While Noxa induction was highest in wildtype p53 cells upon etoposide treatment, we observed a rather uniform induction of Noxa in each of these cell lines after AI-10-49 treatment (SFig. 3D). Since p53 is mutated in the human PDAC cell lines PSN1 and MiaPaCa-2 we also analyzed the expression of the p53 family member p63, but did not detect significant regulation (SFig. 3E). Both p63 and mutant p53 showed no difference in expression at early time points after treatment with AI-10-49, but tended to show decreased expression after 48 h and 72 h, respectively (SFig. 3B,E). Our data show that both p53mut and RUNX1 are not induced upon AI-10-49 treatment (SFig. 3B, E). Contrary, we rather observed a decreased expression of RUNX1 after AI-10-49 treatment in MiaPaCa-2 and PSN1 cells (SFig. 3E).
Next, to substantiate caspase-induced apoptosis, we examined AI-10-49 together with the pan-caspase inhibitor zVAD-FMK by Annexin V/PI FACS. Here, we observed a significant rescue in apoptosis induction (Annexin V+/PI− fraction) (SFigure 3F). Both fractions, i.e. Annexin V+/PI− and Annexin V+/PI+, remained unaffected in NOXAko cells, indicating the relevance of NOXA in cell death (SFigure 3F).
To further investigate the role of NOXA in AI-10-49 induced cell death, we generated MiaPaCa-2 cells stably expressing the CRISPR activator (CRISPRa) dCas9-MS2-p65-HSF1 (MpH), and an established NOXA sgRNA to endogenously overexpress NOXA (SFig. 2E, SFig. 3G). NOXA-CRISPRa cells phenocopied AI-10-49 treated cells in clonogenic assays and, more importantly, NOXA-CRISPRa cell growth was drastically inhibited when treated with AI-10-49 (SFig. 3H, I).
Together, our data show that inhibition of RUNX1 by AI-10-49 induces NOXA mRNA and protein expression and thereby drives NOXA-dependent apoptosis.
RUNX1 is upregulated in pancreatic cancer and suppresses NOXA expression
Understanding the mode of action of drugs is critical for the implementation of patient stratification strategies. To address this question, we next investigated how AI-10-49 induces NOXA expression. Since AI-10-49 inhibits the interaction between CBFβ and a DNA binding α subunit encoded by RUNX proteins (16), we tested whether loss of RUNX expression affects NOXA expression in knockouts of all three RUNX genes RUNX1, RUNX2 and RUNX3 in MiaPaCa-2 cells (SFig 2F). Remarkably, we observed an induction of NOXA mRNA solely in RUNX1 knockout cells (Fig. 2A), arguing for a RUNX1-specific repression of the NOXA gene. We validated this effect by siRNA-mediated RUNX1 depletion in Panc1, AsPC1 and MiaPaCa-2 cells (SFig. 4A). In addition to the induction of NOXA mRNA, we also observed a significant NOXA induction in RUNX1ko cells at the protein level (Fig. 2B). To analyze whether RUNX1ko cells have a growth disadvantage, we compared colony formation of RUNX1ko cells to the parental cell line. Here, we observed significantly reduced formation of colonies (Fig. 2C), which phenocopied the effects observed in AI-10-49 treated cells (SFig. 3H, I). In addition, RUNX1ko cells showed an increased cell death rate at basal levels, as demonstrated by Annexin V flow cytometry (Fig 2D). To demonstrate the specificity of AI-10-49 in this context, we applied AI-10-49 in two RUNX1ko clones as well as in RUNX1 siRNA-treated MiaPaCa-2 cells. Although we did not observe a consistent effect in the RUNX1 knockout clones, which is probably related to the difficult cultivation of these cells, the use of siRNA, on the other hand, showed reproducible effects equivalent to a doubling of the GI50 value, confirming the dependence on RUNX1 (SFig. 4B). These genetic experiments identify RUNX1 as negative regulator of NOXA gene expression.
To further investigate the relevance of RUNX1 in PDAC, we analyzed RUNX1 expression in human and murine datasets. We found that RUNX1 indeed is transcriptionally upregulated in pancreatic cancer in all three datasets investigated (Fig. 2E, SFig4C). In addition, RUNX1 was expressed in pancreatic cancer, whereas it was not detected in normal pancreas on protein level (Fig. 2F). In premalignant KrasG12D driven murine pancreatic epithelial cells Runx1 mRNA (Fig. 2G) and RUNX1 target gene signatures (Fig. 2H) were significantly enriched compared to normal pancreatic epithelial cells, displaying specificity for RUNX1 expression in non-stromal tumor-initiating cells. During PDAC progression RUNX1 expression is maintained. Suspecting a RUNX1-mediated repression of the NOXA gene, we re-analyzed the ICGC/Bailey et al. (6) and Collisson et al. (8) transcriptome datasets and indeed observed a negative correlation trend of RUNX1 and NOXA expression in the squamous/quasi-mesenchymal PDAC subtype (SFig. 4D). To investigate whether RUNX1 expression was the main contributing factor to NOXA expression, we performed a shRNA-mediated knockdown of NOXA in RUNX1ko cells (SFig. 4E). Congruent with our initial findings, the basal apoptosis rates were restored (SFig. 4F).
Taken together, these data argue for a RUNX1-mediated repression of NOXA expression.
RUNX1 inhibition induces NOXA through amplification of active chromatin marks
To understand the immediate effect of AI-10-49 treatment, we performed RNAseq analyses of MiaPaCa2 cells 6 h upon treatment in comparison to vehicle treated cells. Apart from NOXA, which was upregulated in treated cells, less than 300 genes, were found to be differentially expressed, suggesting a high specificity of AI-10-49 (Fig. 3A). Gene set enrichment analyses revealed a significant apoptosis signature and a negative enrichment score for RUNX1 targets (Fig. 3B, STable 3) upon AI-10-49 treatment. To analyze the impact of RUNX1 inhibition on chromatin accessibility we performed assays for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). We observed reduced chromatin accessibility after AI-10-49 treatment, compared to the vehicle control 6 h after AI-10-49 treatment (Fig. 3C). In addition, a comparison of parental MiaPaCa-2 cells with isogenic RUNX1 deficient cells showed a an increased accessibility of the chromatin.
To investigate the association of RUNX1 inhibition with regulation of chromatin dynamics, we performed H3K27ac ChIPseq experiments to detect transcriptionally active chromatin. A cross-coverage and fingerprint plot showed adequate signal strength in enriched regions (SFig. 5A, B). Globally, H3K27ac signal was increased in both replicates (Fig. 3D, SFig. 5C), arguing for a neutralization of the repressor activity of RUNX1.
We also performed RUNX1 ChIPseq to assess the impact of RUNX1 inhibition on RUNX1 binding. This analysis showed a peak downstream of the NOXA gene, which we hypothesized to be an enhancer region and coined it downstream binding site 1 (dBS1). To substantiate our findings, we analyzed RUNX1 ChIPseq from K562 and MCF10A cells. Indeed, we found an overlap of our identified peak in both ChIPseq datasets and were thus able to validate the peak identified in MiaPaCa-2 cells (SFig. 6A). In both, ChIPseq and qChIP experiments, we observed a drop in RUNX1 binding at downstream binding site 1 (dBS1) and an increased acetylation of H3K27 at the NOXA promoter upon AI-10-49 treatment (Fig. 3E, F), indicating an activation of the gene by acetylation of the proximal promoter region of NOXA, subsequently leading to increased gene expression. To analyze the spatial organization of the NOXA region, we performed chromosome conformation capture assays (4C) to capture interactions between the NOXA locus (view point) and all other genomic loci. Here, we found a hitherto unknown interaction with a downstream region of the NOXA gene, which is abrogated upon AI-10-49 treatment and in RUNX1ko cells (Fig. 3E, arrow/dBS1, SFig. 6A). Binding of the nuclear protein CCCTC-binding factor (CTCF), which marks insulator regions to prevent crosstalk between active and inactive chromatin, was unaffected (Fig. 3E). Additionally, RUNX1 peaks in the vehicle control (indicated by an arrow in Fig. 3E) and at the NOXA gene, arguing for a spatial interaction, which is mediated by RUNX1 (Fig. 3E, G). The dBS1 region was the only region within the CTCF boundaries where both RUNX1 binding and DNA-DNA interaction had disappeared after AI-10-49 (Fig. 3G, SFig. 6A). This spatial interaction could also be found in public Hi-C data of Panc1, K562 cells, and to a lower extend in the epithelial cell line MCF10A (Fig. 3F, SFig. 6B) (36). Taken together, these data suggest that RUNX1 binding to the dBS1 region actively represses the NOXA gene. To identify which histone deacetylases are responsible for this effect, we used class I HDAC inhibitors. Here, in particular, inhibition of HDAC1/2 by Merck60 showed a significant induction of NOXA mRNA expression (SFig. 6C) as well as an induction of the H3K27ac mark at the NOXA promoter (SFig. 6D). Additionally, murine PDAC cells harboring a dual recombinase system and a 4-hydroxy-tamoxifen inducible Cre to knockout alleles for either Hdac1 (PPT-F3641) or Hdac2 (PPT-F1648) (SFig. 6E), displayed a Noxa induction only in Hdac2 deleted cells (SFig. 6F), which is in line with a previous study showing that HDAC2 is responsible for the repression of NOXA in PDAC (11). Therefore, a HDAC2-RUNX1/CBFβ axis might be responsible for NOXA repression. This requires further validation.
To prove that the dBS1 region is causative for the repression of NOXA, we first screened this region for evolutionary conserved RUNX1 binding motifs, and indeed identified conserved RUNX1 consensus sequences (SFig.6G). We next performed a CRISPR/Cas9-mediated knockout of the dBS1 region (SFig. 6H) to genetically demonstarte its involvement in NOXA repression. Indeed, loss of the dBS1 region led to increased NOXA mRNA (Fig. 3H) and protein levels (Fig. 3I). This demonstrates the repressive function of this RUNX1 binding site. In contrast to parental cells, AI-10-49 treatment did not further affected NOXA expression (Fig. 3H, I). Furthermore, in the dBS1Δ/wt clone NOXA expression still was induced upon AI-10-49 treatment, albeit to a lesser extent (Fig. 3H, I).
In summary, we describe a previously unknown mechanism of a RUNX1-mediated repression of the NOXA gene in PDAC. Through enrichment of an active chromatin mark at the NOXA gene itself, its expression is significantly increased (Fig. 3J), thereby inducing apoptosis. This mechanism could be crucial for therapeutic interventions that depend on a NOXA-induced cell death program.
RUNX1 inhibition by AI-10-49 is effective in vivo and in patient derived organoids
To validate whether RUNX1 inhibition could be effective in vivo, we first examined the efficacy of AI-10-49 in mice carrying MiaPaCa-2 PDAC xenografts (Fig. 4A). AI-10-49 treatment resulted in a significant decrease in tumor volume (Fig. 4B) and proliferative capacity (Ki67, Fig. 4C). Importantly, AI-10-49 treatment induced apoptosis measured by cleaved caspase 3 (CC3) positivity in the tumor compared to the vehicle control in parental cells (Fig. 4B, C). Whereas parental cells did not change in tumor volume after AI-10-49 treatment, NOXAko cells still grew upon treatment, supporting NOXA as an essential contributor of AI-10-49 efficacy (Fig. 4B). Additionally, no significant difference between control and treatment was observed in either K67 or CC3 stainings in NOXAko cells (Fig. 4D). Taken together, these data indicate that apoptosis induction by AI-10-49 treatment is also dependent on NOXA in vivo.
We next isolated seven human patient-derived organoids (PDOs) from PDAC patients to investigate RUNX1 inhibition by AI-10-49. First, we performed transcriptome profiling (STable 4), and sorted PDOs for high and low NOXA mRNA expression (Fig. 4E). Gene set enrichment analysis revealed a significant (q<0.001) accumulation of an apoptosis signature in the PDOs with a high NOXA expression (Fig. 4F). PDOs with a high NOXA expression showed the strongest growth inhibition towards AI-10-49 treatment, which further supports our previous findings (Fig. 4G).
Overall, these findings show that RUNX1 inhibition might be a novel therapeutic option to treat PDAC.
Discussion
Molecular tumor profiling and functional studies have led to the identification and validation of genes and signaling pathways that are dysregulated or mutated in PDAC (7, 37–39). Based on comprehensive molecular characterization of PDACs (6–9), it might thus be possible to define personalized treatment strategies (38). An imbalance of signaling pathways, such as cell death-associated pathways, promote tumor maintenance and treatment resistance in PDAC (3). In this study, we analyzed publicly available transcriptome profiles of PDAC patients and found that increased NOXA mRNA expression defines an aggressive squamous/quasi-mesenchymal subtype. In contrast to NOXA, which is tightly regulated at the transcriptional level in PDAC (11), one of its anti-apoptotic counterparts, MCL1 (10), is mostly regulated at the protein level (40). Since NOXA mRNA and protein expression do not correlate strongly, as has been shown in mantle cell lymphoma (41) and in PDAC (11), it is important to identify substances that can induce apoptosis by taking advantage of a NOXA-associated vulnerability. We therefore performed drug-screening experiments in isogenic cell models with NOXA-deficient and NOXA-proficient counterparts to search for compounds that can exploit this vulnerability. We unexpectedly found a substance that inhibits the core binding alpha units RUNX1, RUNX2 or RUNX3 with CBFβ.
The functions of RUNX1 are highly specific depending on the tissue and cell type. Deletion of Runx1 in a mouse model of T-cell acute lymphoblastic leukemia (T-ALL)(42), silencing of RUNX1 in human T-ALL cells (42) and silencing of RUNX1 in SW480 human colon cancer cells (43) all triggered apoptosis. In contrast, in Kasumi-1 t(8;21) leukemia cells RUNX1 overexpression induced apoptosis by eliciting expression of the cyclin-dependent kinase inhibitor p57Kip2 (44). In line with the biological effects observed in T-ALL (42) and in colon cancer cells (43), we observed an induction of apoptosis both through the pharmacological CBFβ/RUNX1 inhibition by the compound AI-10-49 and in CRISPR/Cas9 mediated knockouts of RUNX1. We observed a significant induction of NOXA mRNA expression, which was exclusive to RUNX1 knockout cells and could not be observed in RUNX2 or RUNX3 knockout cells, arguing for a non-redundant function for RUNX1.
In a chemical high-throughput screen, which was performed to identify compounds that disrupt the interaction between RUNX1 and the CBFβ-MYH11/SMMHC fusion protein first a lead molecule was identified, which exhibited low selectivity for the fusion protein (33). Therefore, a bivalent derivate of this compound was generated. AI-10-49 inhibits CBFβ-MYH11/SMMHC with an increased selectivity and restores the formation of wild-type CBFβ-RUNX1 (33). In cells, lacking the CBFβ-MYH11/SMMHC fusion protein, AI-10-49 acts like the monomeric lead molecule and inhibits wild-type CBFβ/RUNX1. In fact, both AI-10-49 treatment as a putative pharmacological CBFβ/RUNX1 inhibitor, as well as a genetic knockout of RUNX1 unexpectedly showed an induction of NOXA, similarities in transcriptional regulation at a genome-wide scale and an associated apoptosis induction in PDAC cells, arguing for RUNX1 as a repressor of NOXA gene expression. We could show that the pharmacological CBFβ/RUNX1 inhibition leads to a global enrichment of H3K27ac, a marker for active chromatin, including the NOXA gene. We observed an unexpected punctual interaction of a NOXA downstream RUNX1 binding site and the NOXA promoter. How exactly RUNX1 exercises its repressive function in PDAC has to be addressed in detail in further studies. An analysis of different data sets showed a high expression of RUNX1 in pancreatic cancer compared to normal pancreas. Of note, knockdown of RUNX1 in PDAC cells was shown to suppress the invasive/aggressive phenotype via regulation of miR-93 (45). In particular, high RUNX1 expression and RUNX1 target gene signatures were observed in premalignant KrasG12D driven murine pancreatic epithelial cells, which together indicate a largely unexplored and possibly unexploited relevance of RUNX1 in PDAC.
Since the apoptosis machinery in PDAC cells retains its functionality (3), the strategy of directly inhibiting pro-survival BCL2 proteins such as the NOXA antagonist MCL1 appears extremely attractive (46). One of the first selective MCL1 inhibitors with in vivo activity, S63845, showed massive apoptosis induction in multiple myeloma and acute myeloid leukemia, but many solid tumors were resistant to S63845 monotherapy (47). Combination therapies such as S63845 with the SRC kinase inhibitor Dasatinib reduced cell viability in PDAC models and even lead to a reduction in metastasis formation (48). This data shows that the development of new therapeutic strategies, especially with regard to apoptosis evasion for PDAC, are extremely promising in improving the current clinical regimens. Whether compounds like S63845 synergistically combine with AI-10-49 is currently under investigation.
Unraveling the transcriptional regulation of apoptosis-associated genes and the interplay of transcription factors, such as RUNX1, is important to understand the tumor biology of PDAC. The development and improvement of compounds that can inhibit transcription factors, such as the CBFβ/RUNX1 inhibitor used here, perhaps in combination with proteolysis-targeting chimeras (PROTAC) technology (49), could provide new approaches for PDAC treatment. Therefore, our mechanistic work, demonstrating a control of NOXA by a repressive facet of RUNX1 opens a novel research direction into potent RUNX1 inhibitors and a novel way to target this deadly disease.
Conflict of Interest
The authors declare no competing interests.
Availability of Data and Materials
RNAseq (cell lines), ChIPseq, ATACseq and 4C accession No.: PRJEB39828. RNAseq (organoids) gene expression matrix is shown in the STable 4.
Supplements
SFigure 1 Classical BH3 only mRNA expression and survival analysis in PDAC
A) mRNA expression of BH3-only differentially expressed in PDAC subtypes classified in squamous and other. Fisher’s exact test, * p < 0.01, ** p < 0.001, *** p < 0.0001. B) Simple Cox Regression models were constructed using the expression of all “classical BH3 family members”. Genes with p-values < 0.1 were used to construct a multiple Cox regression model. Out of all members, only NOXA significantly correlated with inferior survival (HR = 1.9, p < 0.001), while increasing expression of BBC3/PUMA was associated with higher overall survival ((HR = 0.73, p = 0.095). C) Survival of PDAC patients with a low (lower quartile) and a high (upper quartile) NOXA mRNA expression derived from curated TCGA datasets of PDAC patients as described (13). Log rank test as indicated. D) Human PDAC cell line subtypes according to Collisson et al. (8). QM: quasi-mesenchymal.
SFigure 2 CRISPR/Cas9 knockout and CRISPRa strategies and co-immunopreciptitation of RUNX1 and CBFb in AI-10-49 treated MiaPaCa-2 cells
A) Scheme of the human NOXA gene (parental and knockout) with indicated primer binding sites. B) Genotyping PCR of indicated human PDAC cells to screen for a NOXA knockout. C) Scheme of the murine NOXA gene (parental and knockout) with indicated primer binding sites. D) Genotyping PCR of indicated murine PDAC cells to screen for a NOXA knockout. E) Co-immunoprecipitation of RUNX1 and CBFβ in MiaPaCa-2 cells. Cells were treated for 6h with 3μM of AI-10-49 or DMSO as vehicle control. Either a RUNX1 or a CBFβ antibody was used for the Co-IP as indicated. An IgG pulldown was used as negative control. F) Schematic representation of dCas9-VP64-MS2-HSF1 driven NOXA overexpression. One sgRNA drives dCas9 towards the NOXA promoter region to induce its expression. G) Schematic representation of RUNX gene family knockout. For RUNX1 knockout, 2 sgRNAs were designed to excise the gene region from exon 3 to exon 4. For RUNX2 knockout, 2 sgRNAs were designed to target excision of beginning of exon 8 until stop codon. For RUNX3 knockout, 2 sgRNAs target upstream exon 1 and end of exon 2.
SFigure 3 RUNX1 inhibition by AI-10-49 induces NOXA associated apoptosis
A) Western blot analysis of indicated BCL2 family members and cleaved PARP upon 6h and 24h treatment with 3μM of AI-10-49 in MiaPaCa-2 and PSN1 cells. Actin served as loading control. B) Protein expression analysis of MCL1, p53, BCL-xL, BIM and BAX upon 24h and 72h treatment with 3μM of AI-10-49 and the pan-caspase inhibitor zVAD-FMK in parental and NOXAko MiaPaCa-2 cells. GAPDH served as loading control. C) p53 Western Blot of indicated murine PDAC cell lines to determine basal p53 expression of n=3 biological replicates. Actin served as loading control. D) Relative Noxa mRNA expression in murine PDAC cells harboring wild type p53 (PPT-5123), mutant p53 (PPT-5436) and deleted p53 (PPT-6554 and PPT-W22). Cells were treated for 6h with 12.5μg/ml Etoposide, 3μM AI-10-49 and DMSO as vehicle control. Actin served as housekeeping gene. E) Western blot of RUNX1 and p63α upon 6, 24 and 48h treatment with 3μM of AI-10-49 in parental and NOXAko MiaPaCa-2 cells and parental PSN1 cells. GAPDH served as loading control. F) AnnexinV/PI FACS of parental and NOXAko MiaPaCa-2 cells upon 24h treatment with 3μM AI-10-49 and 50μM zVAD-FMK. Upper panel: AnnexinV+/PI− fraction. Lower panel: Annexin V+/PI+ fraction. G) Western blot of NOXA protein in MiaPaCa-2 cells. Vinculin served as loading control. H) Representative image of clonogenic assay in MiaPaCa-2 parental and NOXA-CRISPR-dCas9-VP64-MS2-HSF1-mediated activation (NOXA-CRISPRa). Cells were treated for 3 weeks with vehicle (DMSO) or 400 nM AI-10-49. n=4; all biological replicates were performed as technical triplicates. I) Quantification of clonogenic assay in parental and NOXA-CRISPRa. Each treatment was quantified and normalized against its DMSO control. Depicted is the number of colonies in % per treatment compared to vehicle. P value of ANOVA test ***p<0.001.
SFigure 4 RUNX1 is upregulated in pancreatic cancer and suppresses apoptosis via NOXA repression
A) Relative RUNX1 and NOXA expression in MiaPaCa-2, Panc-1 and AsPC1 cells upon treatment with a pool of specific siRNAs targeting RUNX1. Cells were treated for 72h with either scrambled siRNAs (Ctrl) or RUNX1 specific siRNAs. Actin was used as housekeeping gene (ΔΔCt). B) GI50 values generated in viability assays using CellTiterGlo in parental, RUNX1ko and cells, determined 72h after AI-10-49 treatment. In addition cells were treated 48h with siRNA (Ctrl and RUNX1) and treated for additional 72 h with AI-10-49. C) RUNX1 mRNA expression of patient samples from normal pancreas and pancreatic cancer GEO accession no. GSE15471, GSE16515 D) Pearson correlation (incl. linear regression with 95% confidence bands) of PDAC patient samples from the squamous subtype (6) and from microdissected PDAC patient samples (8). E) Western blot of RUNX1 and NOXA in parental and RUNX1ko MiaPaCa-2 cells. RUNX1ko cells were stably transduced with either a control shRNA or a NOXA specific shRNA. Actin served as loading control. F) Annexin V FACS analysis of cells as indicated in E).
SFigure 5 Quality assessment of H3K27ac ChIP-seq.
A) Fingerprint plot of all replicates including the input controls. Samples with uniform distribution across the genome (e.g. input controls) should plot across the diagonal line, while samples with enrichment across small genomic regions show a steep rise towards the end of the plot. B) Correlation plot calculated using the Spearman coefficient quantified by the horizontal bar at the bottom of the plot. C) Average signal profile across peaks calculated with macs2 callpeak. The profile was generated with ChIPQC.
SFigure 6 RUNX1 suppresses NOXA via the dBS1 downstream enhancer in PDAC cells
A) Chromosome conformation capture (4C, spatial chromatin organisation) and ChIPseq analysis for RUNX1 in indicated cell lines. MiaPaCa-2 cells were treated with vehicle (DMSO)- or 3μM AI-10-49 for 6h. RUNX1 ChIPseq of MCF10A breast epithelial cells (GSE121370) and K562 CML cells (GSE96253) are displayed. The NOXA downstream binding site 1 (dBS1) is depicted. B) Heatmap of Hi-C data from K562 and MCF10A cells accessed via http://3dgenome.fsm.northwestern.edu/ displaying spatial chromatin organization. C) Relative NOXA mRNA expression in MiaPaCa-2 and PSN1 cells. Cells were treated with 4μM Entinostat, 10μM Merck60 and 10μM RGFP966 for 24h. Expression was determined by ΔΔCt method. Actin served as housekeeping control. D) ChIP-qPCR analysis at the NOXA promoter for H3K27ac and IgG in DMSO- or Merck60 treated MiaPaCa-2 cells. E) Western Blot of HDAC1, HDAC2 and HDAC3 in murine PDAC cells harboring a dual recombination system. 600 nM 4-Hydroxytamoxinfen (4-OHT) treatment induced CreERT2 shuttling and excision of either Hdac1 (PPT-F3641) or Hdac2 (PPT-F1648). Actin served as loading control. F) Relative Noxa mRNA expression in murine PPT-F3641 and PPT-F1648 cells. Cells were treated with 600nM 4-OHT for 48h. Actin served as housekeeping control. G) Sequence analysis using ConTra v3 to identify conserved RUNX1 consensus sequences (as indicated) in the dBS1 region. H) Upper Panel: Scheme of the human dBS1 region (parental and knockout) with indicated primer binding sites. Lower Panel: Genotyping PCR of MiaPaCa-2 cells to screen for a dBS1 knockout. wt: wildtype; ko: knockout.
STable 1 Mean fold change of drug hits from drug screening experiment
STable 2 SwissTargetPrediction of AI-10-49
STable 3 Enriched gene sets of AI-10-49 versus vehicle control treated MiaPaCa-2 cells
STable 4 PDO Gene Expression Matrix
STable 5 Primer sequences, sgRNAs, Antibodies, murine cell lines
Acknowledgements
We thank Martin Schlensog and Yakup Yasar for performing the IHC staining. We thank Dieter Saur for providing murine PDAC cell lines and thank Ernesto Acevedo for providing the p53(1C12) antibody. Funding: Else-Kröner-Fresenius Stiftung (2016_A43 to M.W.); Walter Schulz Stiftung to M.W.; Deutsche Forschungsgemeinschaft (DFG) [SFB1321 (Project-ID 329628492) C13 to G.S. and SCHN 959/6-1 and SCHN 959/3-2 to G.S.] and SFB 1335 project P3 to U.K.; Wilhelm-Sander-Stiftung (2017.048.2 to G.S. and U.K.); Deutsche Krebshilfe project 70114425 to U.K.; Stiftung Charité to U.K..