Target actionability review to evaluate CDK4/6 as a therapeutic target in paediatric solid and brain tumours

BACKGROUND: Childhood cancer is still a leading cause of death around the world. To improve outcomes, there is an urgent need for tailored treatment. The systematic evaluation of existing preclinical data can provide an overview of what is known and identify gaps in the current knowledge. Here, we applied the target actionability review (TAR) methodology to assess the strength and weaknesses of available scientific literature on CDK4/6 as a therapeutic target in paediatric solid and brain tumours by structured critical appraisal. METHODS: Using relevant search terms in PubMed, a list of original publications investigating CDK4/6 in paediatric solid tumour types was identified based on relevancy criteria. Each publication was annotated for the tumour type and categorised into separate proof-of-concept (PoC) data modules. Based on rubrics, quality and experimental outcomes were scored independently by two reviewers. A third reviewer evaluated and adjudicated score discrepancies. Scores for each PoC module were averaged for each tumour type and visualised in a heatmap matrix in the publicly available R2 data portal. RESULTS AND CONCLUSIONS: This CDK4/6 TAR, generated by analysis of 151 data entries from 71 publications, showed frequent genomic aberrations of CDK4/6 in rhabdomyosarcoma, osteosarcoma, high-grade glioma, medulloblastoma, and neuroblastoma. However, a clear correlation between CDK4/6 aberrations and compound efficacy is not coming forth from the literature. Our analysis indicates that several paediatric indications would need (further) preclinical evaluation to allow for better recommendations, especially regarding the dependence of tumours on CDK4/6, predictive biomarkers, resistance mechanisms, and combination strategies. Nevertheless, our TAR heatmap provides support for the relevance of CDK4/6 inhibition in Ewing sarcoma, medulloblastoma, malignant peripheral nerve sheath tumour and to a lesser extent neuroblastoma, rhabdomyosarcoma, rhabdoid tumour and high-grade glioma. The interactive heatmap is accessible through R2 [r2platform.com/TAR/CDK4_6]

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]

Systematic target actionability reviews of preclinical proof-of-concept papers to match targeted drugs to paediatric cancers

BACKGROUND: Children with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. METHODS: A sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for paediatric tumour type and categorised into nine separate PoC data modules. Each experimental finding was scored for experimental outcome and quality independently by two reviewers; discrepancies were assessed by a third reviewer and resolved by adjudication. Scores corresponding to one PoC module were merged for each tumour type and visualised in a heat map matrix in the publicly available R2 data portal [r2.amc.nl]. RESULTS AND CONCLUSIONS: To test our TAR methodology, we conducted a pilot study on MDM2 and TP53. The heat map generated from analysis of 161 publications provides a rationale to support drug development in specific paediatric solid and brain tumour types. Furthermore, our review highlights tumour types where preclinical data are incomplete or lacking and for which additional preclinical testing is advisable

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

Systematic target actionability reviews of preclinical proof-of-concept papers to match targeted drugs to paediatric cancers

Background: Children with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. Methods: A sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for paediatric tumour type and categorised into nine separate PoC data modules. Each experimental finding was scored for experimental outcome and quality independently by two reviewers; discrepancies were assessed by a third reviewer and resolved by adjudication. Scores corresponding to one PoC module were merged for each tumour type and visualised in a heat map matrix in the publicly available R2 data portal [r2.amc.nl]. Results and conclusions: To test our TAR methodology, we conducted a pilot study on MDM2 and TP53. The heat map generated from analysis of 161 publications provides a rationale to support drug development in specific paediatric solid and brain tumour types. Furthermore, our review highlights tumour types where preclinical data are incomplete or lacking and for which additional preclinical testing is advisable

Target actionability review to evaluate CDK4/6 as a therapeutic target in paediatric solid and brain tumours

Background: Childhood cancer is still a leading cause of death around the world. To improve outcomes, there is an urgent need for tailored treatment. The systematic evaluation of existing preclinical data can provide an overview of what is known and identify gaps in the current knowledge. Here, we applied the target actionability review (TAR) methodology to assess the strength and weaknesses of available scientific literature on CDK4/6 as a therapeutic target in paediatric solid and brain tumours by structured critical appraisal. Methods: Using relevant search terms in PubMed, a list of original publications investigating CDK4/6 in paediatric solid tumour types was identified based on relevancy criteria. Each publication was annotated for the tumour type and categorised into separate proof-of-concept (PoC) data modules. Based on rubrics, quality and experimental outcomes were scored independently by two reviewers. A third reviewer evaluated and adjudicated score discrepancies. Scores for each PoC module were averaged for each tumour type and visualised in a heatmap matrix in the publicly available R2 data portal. Results and conclusions: This CDK4/6 TAR, generated by analysis of 151 data entries from 71 publications, showed frequent genomic aberrations of CDK4/6 in rhabdomyosarcoma, osteosarcoma, high-grade glioma, medulloblastoma, and neuroblastoma. However, a clear correlation between CDK4/6 aberrations and compound efficacy is not coming forth from the literature. Our analysis indicates that several paediatric indications would need (further) preclinical evaluation to allow for better recommendations, especially regarding the dependence of tumours on CDK4/6, predictive biomarkers, resistance mechanisms, and combination strategies. Nevertheless, our TAR heatmap provides support for the relevance of CDK4/6 inhibition in Ewing sarcoma, medulloblastoma, malignant peripheral nerve sheath tumour and to a lesser extent neuroblastoma, rhabdomyosarcoma, rhabdoid tumour and high-grade glioma. The interactive heatmap is accessible through R2 [r2platform.com/TAR/CDK4_6]

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. Nonresistant 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 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. Nonresistant 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