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

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]

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

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. As novel and improved immunotherapies may fill this need, we dissected the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 25 tumors (10 pre- and 15 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas were infiltrated by NK, T and B cells, and immunosuppressive myeloid populations. NK cells showed reduced cytotoxicity and T cells had a dysfunctional profile. Interaction analysis revealed a vast immunoregulatory network and identified NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduced neuroblastoma growth, with complete responses in vivo. Moreover, addition of TIGIT blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model significantly improved survival. Concluding, our integrative analysis of neuroblastoma’s vast immunoregulatory network provides novel targets and a rationale for immunotherapeutic combination strategies

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential

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

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

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

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]