High-Throughput Drug Screening of Primary Tumor Cells Identifies Therapeutic Strategies for Treating Children with High-Risk Cancer.

For one-third of patients with pediatric cancer enrolled in precision medicine programs, molecular profiling does not result in a therapeutic recommendation. To identify potential strategies for treating these high-risk pediatric patients, we performed in vitro screening of 125 patient-derived samples against a library of 126 anticancer drugs. Tumor cell expansion did not influence drug responses, and 82% of the screens on expanded tumor cells were completed while the patients were still under clinical care. High-throughput drug screening (HTS) confirmed known associations between activating genomic alterations in NTRK, BRAF, and ALK and responses to matching targeted drugs. The in vitro results were further validated in patient-derived xenograft models in vivo and were consistent with clinical responses in treated patients. In addition, effective combinations could be predicted by correlating sensitivity profiles between drugs. Furthermore, molecular integration with HTS identified biomarkers of sensitivity to WEE1 and MEK inhibition. Incorporating HTS into precision medicine programs is a powerful tool to accelerate the improved identification of effective biomarker-driven therapeutic strategies for treating high-risk pediatric cancers. SIGNIFICANCE: Integrating HTS with molecular profiling is a powerful tool for expanding precision medicine to support drug treatment recommendations and broaden the therapeutic options available to high-risk pediatric cancers.

The potential of PARP as a therapeutic target across pediatric solid malignancies.

BACKGROUND: Pediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers. METHODS: Using the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations. RESULTS: Using published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines. CONCLUSION: Collectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies.

MEK inhibition causes BIM stabilization and increased sensitivity to BCL-2 family member inhibitors in RAS-MAPK-mutated neuroblastoma.

Introduction: Mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition in vitro. However, these inhibitors alone do not lead to tumor regression in vivo, indicating the need for combination therapy. Methods and results: Via high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members. In vitro validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway. In vivo combination of trametinib with BCL-2 inhibitors led to tumor inhibition in NRAS-mutant and NF1-deleted xenografts. Conclusion: Together, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients.

Chromosome 11q loss and MYCN amplification demonstrate synthetic lethality with checkpoint kinase 1 inhibition in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

High-Throughput Drug Library Screening in Primary KMT2A-Rearranged Infant ALL Cells Favors the Identification of Drug Candidates That Activate P53 Signaling.

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) represents an aggressive type of childhood leukemia characterized by a poor clinical outcome with a survival chance of <50%. Implementing novel therapeutic approaches for these patients is a slow-paced and costly process. Here, we utilized a drug-repurposing strategy to identify potent drugs that could expeditiously be translated into clinical applications. We performed high-throughput screens of various drug libraries, comprising 4191 different (mostly FDA-approved) compounds in primary KMT2A-rearranged infant ALL patient samples (n = 2). The most effective drugs were then tested on non-leukemic whole bone marrow samples (n = 2) to select drugs with a favorable therapeutic index for bone marrow toxicity. The identified agents frequently belonged to several recurrent drug classes, including BCL-2, histone deacetylase, topoisomerase, microtubule, and MDM2/p53 inhibitors, as well as cardiac glycosides and corticosteroids. The in vitro efficacy of these drug classes was successfully validated in additional primary KMT2A-rearranged infant ALL samples (n = 7) and KMT2A-rearranged ALL cell line models (n = 5). Based on literature studies, most of the identified drugs remarkably appeared to lead to activation of p53 signaling. In line with this notion, subsequent experiments showed that forced expression of wild-type p53 in KMT2A-rearranged ALL cells rapidly led to apoptosis induction. We conclude that KMT2A-rearranged infant ALL cells are vulnerable to p53 activation, and that drug-induced p53 activation may represent an essential condition for successful treatment results. Moreover, the present study provides an attractive collection of approved drugs that are highly effective against KMT2A-rearranged infant ALL cells while showing far less toxicity towards non-leukemic bone marrow, urging further (pre)clinical testing.

Target Actionability Review: a systematic evaluation of replication stress as a therapeutic target for paediatric solid malignancies.

BACKGROUND: Owing to the high numbers of paediatric cancer-related deaths, advances in therapeutic options for childhood cancer is a heavily studied field, especially over the past decade. Classical chemotherapy offers some therapeutic benefit but has proven long-term complications in survivors, and there is an urgent need to identify novel target-driven therapies. Replication stress is a major cause of genomic instability in cancer, triggering the stalling of the replication fork. Failure of molecular response by DNA damage checkpoints, DNA repair mechanisms and restarting the replication forks can exacerbate replication stress and initiate cell death pathways, thus presenting as a novel therapeutic target. To bridge the gap between preclinical evidence and clinical utility thereof, we apply the literature-driven systematic target actionability review methodology to published proof-of-concept (PoC) data related to the process of replication stress. METHODS: A meticulous PubMed literature search was performed to gather replication stress-related articles (published between 2014 and 2021) across 16 different paediatric solid tumour types. Articles that fulfilled inclusion criteria were uploaded into the R2 informatics platform [r2.amc.nl] and assessed by critical appraisal. Key evidence based on nine pre-established PoC modules was summarised, and scores based on the quality and outcome of each study were assigned by two separate reviewers. Articles with discordant modules/scores were re-scored by a third independent reviewer, and a final consensus score was agreed upon by adjudication between all three reviewers. To visualise the final scores, an interactive heatmap summarising the evidence and scores associated with each PoC module across all, including paediatric tumour types, were generated. RESULTS AND CONCLUSIONS: 145 publications related to targeting replication stress in paediatric tumours were systematically reviewed with an emphasis on DNA repair pathways and cell cycle checkpoint control. Although various targets in these pathways have been studied in these diseases to different extents, the results of this extensive literature search show that ATR, CHK1, PARP or WEE1 are the most promising targets using either single agents or in combination with chemotherapy or radiotherapy in neuroblastoma, osteosarcoma, high-grade glioma or medulloblastoma. Targeting these pathways in other paediatric malignancies may work as well, but here, the evidence was more limited. The evidence for other targets (such as ATM and DNA-PK) was also limited but showed promising results in some malignancies and requires more studies in other tumour types. Overall, we have created an extensive overview of targeting replication stress across 16 paediatric tumour types, which can be explored using the interactive heatmap on the R2 target actionability review platform [https://hgserver1.amc.nl/cgi-bin/r2/main.cgi?option=imi2_targetmap_v1].

Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma.

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.

Organoid-based drug screening reveals neddylation as therapeutic target for malignant rhabdoid tumors.

Malignant rhabdoid tumors (MRTs) represent one of the most aggressive childhood malignancies. No effective treatment options are available, and prognosis is, therefore, dismal. Previous studies have demonstrated that tumor organoids capture the heterogeneity of patient tumors and can be used to predict patient response to therapy. Here, we perform drug screening on patient-derived normal and tumor organoids to identify MRT-specific therapeutic vulnerabilities. We identify neddylation inhibitor MLN4924 as a potential therapeutic agent. Mechanistically, we find increased neddylation in MRT organoids and tissues and show that MLN4924 induces a cytotoxic response via upregulation of the unfolded protein response. Lastly, we demonstrate in vivo efficacy in an MRT PDX mouse model, in which single-agent MLN4924 treatment significantly extends survival. Our study demonstrates that organoids can be used to find drugs selectively targeting tumor cells while leaving healthy cells unharmed and proposes neddylation inhibition as a therapeutic strategy in MRT.

The important role of routine cytopathology in pediatric precision oncology.

BACKGROUND: The development of high-throughput drug screening (HTS) using primary cultures provides a promising, clinically translatable approach to tailoring treatment strategies for patients with cancer. However, this has been challenging for solid tumors because of often limited amounts of tissue available. In most cases, in vitro expansion is required before HTS, which may lead to overgrowth and contamination by non-neoplastic cells. METHODS: In this study, hematoxylin and eosin staining and immunohistochemical staining were performed on 129 cytopathology cases from 95 patients. These cytopathology cases comprised cell block preparations derived from primary tumor specimens or patient-derived xenografts as part of a pediatric precision oncology trial. Cytopathology cases were compared with the morphology and immunohistochemical staining profile of the original tumor. Cases were reported as tumor cells present, equivocal, or tumor cells absent. The HTS results from cytopathologically validated cultures were incorporated into a multidisciplinary tumor board report issued to the treating clinician to guide clinical decision making. RESULTS: On cytopathologic examination, tumor cells were present in 77 of 129 cases (60%) and were absent in 38 of 129 cases (29%), whereas 14 of 129 cases (11%) were equivocal. Cultures that contained tumor cells resembled the tumors from which they were derived. CONCLUSIONS: Cytopathologic examination of tumor cell block preparations is feasible and provides detailed morphologic characterization. Cytopathologic examination is essential for ensuring that samples submitted for HTS contain representative tumor cells and that in vitro drug sensitivity data are clinically translatable.

High-Throughput Screening Identifies Idasanutlin as a Resensitizing Drug for Venetoclax-Resistant Neuroblastoma Cells.

Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.

High-throughput drug screening reveals Pyrvinium pamoate as effective candidate against pediatric MLL-rearranged acute myeloid leukemia.

Pediatric MLL-rearranged acute myeloid leukemia (AML) has a generally unfavorable outcome, primarily due to relapse and drug resistance. To overcome these difficulties, new therapeutic agents are urgently needed. Yet, implementing novel drugs for clinical use is a time-consuming, laborious, costly and high-risk process. Therefore, we applied a drug-repositioning strategy by screening drug libraries, comprised of >4000 compounds that are mostly FDA-approved, in a high-throughput format on primary MLL-rearranged AML cells. Here we identified pyrvinium pamoate (pyrvinium) as a novel candidate drug effective against MLL-rearranged AML, eliminating all cell viability at <1000 nM. Additional screening of identified drug hits on non-leukemic bone marrow samples, resulted in a decrease in cell viability of ∼50% at 1000 nM pyrvinium, suggesting a therapeutic window for targeting leukemic cells specifically. Validation of pyrvinium on an extensive panel of AML cell lines and primary AML samples showed comparable viabilities as the drug screen data, with pyrvinium achieving IC50 values of <80 nM in these samples. Remarkably, pyrvinium also induced cell toxicity in primary MLL-AF10+ AML cells, an MLL-rearrangement associated with a poor outcome. While pyrvinium is able to inhibit the Wnt pathway in other diseases, this unlikely explains the efficacy we observed as ß-catenin was not expressed in the AML cells tested. Rather, we show that pyrvinium co-localized with the mitochondrial stain in cells, and hence may act by inhibiting mitochondrial respiration. Overall, this study shows that pyrvinium is highly effective against MLL-rearranged AML in vitro, and therefore represents a novel potential candidate for further studies in MLL-rearranged AML.

Combined targeting of the p53 and pRb pathway in neuroblastoma does not lead to synergistic responses.

BACKGROUND: Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutation but rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. METHODS AND RESULTS: We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. In vivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. CONCLUSION: We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas.

Whole genome, transcriptome and methylome profiling enhances actionable target discovery in high-risk pediatric cancer.

The Zero Childhood Cancer Program is a precision medicine program to benefit children with poor-outcome, rare, relapsed or refractory cancer. Using tumor and germline whole genome sequencing (WGS) and RNA sequencing (RNAseq) across 252 tumors from high-risk pediatric patients with cancer, we identified 968 reportable molecular aberrations (39.9% in WGS and RNAseq, 35.1% in WGS only and 25.0% in RNAseq only). Of these patients, 93.7% had at least one germline or somatic aberration, 71.4% had therapeutic targets and 5.2% had a change in diagnosis. WGS identified pathogenic cancer-predisposing variants in 16.2% of patients. In 76 central nervous system tumors, methylome analysis confirmed diagnosis in 71.1% of patients and contributed to a change of diagnosis in two patients (2.6%). To date, 43 patients have received a recommended therapy, 38 of whom could be evaluated, with 31% showing objective evidence of clinical benefit. Comprehensive molecular profiling resolved the molecular basis of virtually all high-risk cancers, leading to clinical benefit in some patients.

Therapeutic vulnerabilities in the DNA damage response for the treatment of ATRX mutant neuroblastoma.

BACKGROUND: In neuroblastoma, genetic alterations in ATRX, define a distinct poor outcome patient subgroup. Despite the need for new therapies, there is a lack of available models and a dearth of pre-clinical research. METHODS: To evaluate the impact of ATRX loss of function (LoF) in neuroblastoma, we utilized CRISPR-Cas9 gene editing to generate neuroblastoma cell lines isogenic for ATRX. We used these and other models to identify therapeutically exploitable synthetic lethal vulnerabilities associated with ATRX LoF. FINDINGS: In isogenic cell lines, we found that ATRX inactivation results in increased DNA damage, homologous recombination repair (HRR) defects and impaired replication fork processivity. In keeping with this, high-throughput compound screening showed selective sensitivity in ATRX mutant cells to multiple PARP inhibitors and the ATM inhibitor KU60019. ATRX mutant cells also showed selective sensitivity to the DNA damaging agents, sapacitabine and irinotecan. HRR deficiency was also seen in the ATRX deleted CHLA-90 cell line, and significant sensitivity demonstrated to olaparib/irinotecan combination therapy in all ATRX LoF models. In-vivo sensitivity to olaparib/irinotecan was seen in ATRX mutant but not wild-type xenografts. Finally, sustained responses to olaparib/irinotecan therapy were seen in an ATRX deleted neuroblastoma patient derived xenograft. INTERPRETATION: ATRX LoF results in specific DNA damage repair defects that can be therapeutically exploited. In ATRX LoF models, preclinical sensitivity is demonstrated to olaparib and irinotecan, a combination that can be rapidly translated into the clinic. FUNDING: This work was supported by Christopher's Smile, Neuroblastoma UK, Cancer Research UK, and the Royal Marsden Hospital NIHR BRC.

Decitabine mildly attenuates MLL-rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo-sensitizer.

MLL-rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL-rearranged ALL cells in vitro. Here, we assessed the in vivo anti-leukemic potential of low-dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL-rearranged ALL. Furthermore, we explored whether prolonged exposure to low-dose decitabine could chemo-sensitize MLL-rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic-based and anti-neoplastic compounds. Our data reveal that decitabine prolonged survival in xenograft mice of MLL-rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out-growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low-dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL-rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic-based or anticancer drugs using high-throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line-dependent manner.

Enhancer of zeste homologue 2 plays an important role in neuroblastoma cell survival independent of its histone methyltransferase activity.

Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein.