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INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases.Significance:The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes.See related commentary by Eggermont et al., p. 2677.This article is highlighted in the In This Issue feature, p. 2659

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Supplementary Table from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

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Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option. Mol Cancer Ther; 10(10); 1846–56. ©2011 AACR.

Supplementary Figure from The Pediatric Precision Oncology INFORM Registry: Clinical Outcome and Benefit for Patients with Very High-Evidence Targets

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Flow cytometry is an important diagnostic tool in childhood acute lymphoblastic leukemias (ALL), flow cytometry data analysis is limited by multiple sources of bias and variation. We present a unified machine learning framework for automated analysis of a standardized diagnostic pediatric leukemia staining that can overcome these challenges. We applied our framework in a large cohort of ALL flow cytometry samples and demonstrated how it can robustly extract the frequencies of cell lineage populations with minimal expert intervention. This work provides a proof of concept that our method meets the needs of an automated analysis tool for diagnostic flow cytometry data.

Children with other extramedullary relapse of acute lymphoblastic leukemia are currently poorly characterized. We aim to assess the prevalence and the clinical, therapeutic and prognostic features of extramedullary localizations other than central nervous system or testis in children with relapse of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) treated on a relapsed ALL protocol. Patients and Methods: Patients with relapse of ALL and LBL, treated according to the multicentric ALL-REZ BFM trials between 1983 and 2015, were analyzed for other extramedullary relapse (OEMR) of the disease regarding clinical features, treatment and outcome. Local treatment/irradiation has been recommended on an individual basis and performed only in a minority of patients. Results: A total of 132 out of 2323 (5.6%) patients with ALL relapse presented with an OEMR (combined bone marrow relapse n = 78, isolated extramedullary relapse n = 54). Compared to the non-OEMR group, patients with OEMR had a higher rate of T-immunophenotype (p &lt, 0.001), a higher rate of LBL (p &lt, 0.001) and a significantly different distribution of time to relapse, i.e., more very early and late relapses compared to the non-OEMR group (p = 0.01). Ten-year probabilities of event-free survival (pEFS) and overall survival (pOS) in non-OEMR vs. OEMR were 0.38 ± 0.01 and 0.32 ± 0.04 (p = 0.0204) vs. 0.45 ± 0.01 and 0.37 ± 0.04 (p = 0.0112), respectively. OEMRs have been classified into five subgroups according to the main affected compartment: lymphatic organs (n = 32, 10y-pEFS 0.50 ± 0.09), mediastinum (n = 35, 10y-pEFS 0.11 ± 0.05), bone (n = 12, 0.17 ± 0.11), skin and glands (n = 21, 0.32 ± 0.11) and other localizations (n = 32, 0.41 ± 0.09). Patients with OEMR and T-lineage ALL/LBL showed a significantly worse 10y-pEFS (0.15 ± 0.04) than those with B-Precursor-ALL (0.49 ± 0.06, p &lt, 0.001). Stratified into standard risk (SR) and high risk (HR) groups, pEFS and pOS of OEMR subgroups were in the expected range whereas the mediastinal subgroup had a significantly worse outcome. Subsequent relapses involved more frequently the bone marrow (58.4%) than isolated extramedullary compartments (41.7%). In multivariate Cox regression, OEMR confers an independent prognostic factor for inferior pEFS and pOS. Conclusion: OEMR is adversely related to prognosis. However, the established risk classification can be applied for all subgroups except mediastinal relapses requiring treatment intensification. Generally, isolated OEMR of T-cell-origin needs an intensified treatment including allogeneic stem cell transplantation (HSCT) as a curative approach independent from time to relapse. Local therapy such as surgery and irradiation may be of benefit in selected cases. The indication needs to be clarified in further investigations.

PURPOSE Off-label use of vemurafenib (VMF) to treat BRAFV600E mutation–positive, refractory, childhood Langerhans cell histiocytosis (LCH) was evaluated. PATIENTS AND METHODS Fifty-four patients from 12 countries took VMF 20 mg/kg/d. They were classified according to risk organ involvement: liver, spleen, and/or blood cytopenia. The main evaluation criteria were adverse events (Common Terminology Criteria for Adverse Events [version 4.3]) and therapeutic responses according to Disease Activity Score. RESULTS LCH extent was distributed as follows: 44 with positive and 10 with negative risk organ involvement. Median age at diagnosis was 0.9 years (range, 0.1 to 6.5 years). Median age at VMF initiation was 1.8 years (range, 0.18 to 14 years), with a median follow-up of 22 months (range, 4.3 to 57 months), whereas median treatment duration was 13.9 months (for 855 patient-months). At 8 weeks, 38 complete responses and 16 partial responses had been achieved, with the median Disease Activity Score decreasing from 7 at diagnosis to 0 ( P < .001). Skin rash, the most frequent adverse event, affected 74% of patients. No secondary skin cancer was observed. Therapeutic plasma VMF concentrations (range, 10 to 20 mg/L) seemed to be safe and effective. VMF discontinuation for 30 patients led to 24 LCH reactivations. The blood BRAFV600E allele load, assessed as circulating cell-free DNA, decreased after starting VMF but remained positive (median, 3.6% at diagnosis, and 1.6% during VMF treatment; P < .001) and was associated with a higher risk of reactivation at VMF discontinuation. None of the various empirical therapies (hematopoietic stem-cell transplantation, cladribine and cytarabine, anti-MEK agent, vinblastine, etc) used for maintenance could eradicate the BRAFV600E clone. CONCLUSION VMF seemed safe and effective in children with refractory BRAFV600E-positive LCH. Additional studies are needed to find effective maintenance therapy approaches.

INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. Significance: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes. See related commentary by Eggermont et al., p. 2677 . This article is highlighted in the In This Issue feature, p. 2659

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell- based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibitionin vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenograftsin vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identifiedFOSgene family members as inducers of resistance against ATR inhibitors. Mechanistically,FOSgene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell-based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

BACKGROUND TRK fusions are oncogenic drivers of a variety of tumors, many of which can involve the central nervous system (CNS). Larotrectinib is a selective TRK inhibitor FDA-approved for the treatment of TRK fusion cancers (Drilon et al., NEJM 2018). Here we report on the clinical activity of larotrectinib in an expanded set of TRK fusion-positive primary CNS tumors. METHODS Patients with primary CNS tumors harboring a TRK fusion treated with larotrectinib on two clinical trials (NCT02637687 and NCT02576431) were identified by local molecular testing. Larotrectinib was administered until disease progression, withdrawal, or unacceptable toxicity. Disease status was investigator assessed (RANO). Data cutoff: February 19, 2019. RESULTS 18 patients with various histological types of glial tumors (11 high-grade, 4 low-grade, 3 unknown) were identified. The patients had gene fusions involving NTRK2 (n=13), NTRK1 (n=2) and NTRK3 (n=2); one was not determined. Median age was 10 years (range 1–79); 14 patients were pediatric (< 18). In 14 evaluable patients, the objective response rate was 36% (2 CR, 3 PR), with responses seen in high- and low-grade disease and across histologies. Nine patients had SD. The 24-week disease control rate was 71%. The duration of treatment ranged from 0.03+ to 16.6+ months. One patient (3.7 years old) with glioblastoma progressed after 5.5 months on larotrectinib. Sequencing revealed a solvent front mutation and the patient was subsequently enrolled in compassionate use protocol for BAY2731954 (formerly known as LOXO-195). CONCLUSION Larotrectinib is active in patients with TRK fusion cancer with intracranial disease. Confirmed responses and durable disease control were seen in primary CNS tumors of various grades and histologies. These results further support expanded testing for NTRK gene fusions in patients with primary CNS tumors.

Despite progress with immunotherapy and targeted agents, treatment of refractory disease remains challenging, in particular for patients with T-ALL. Identification of genomic lesions defining actionable targets had limited impact on patient care so far. The complexity of biological systems highlights the need to develop complementary functional approaches. We and others have established platforms with a library of 120 drugs based on current treatment and (pre-)clinical development, to detect ex-vivo drug response phenotypes on leukemia samples at single cell resolution by high content image analysis. We demonstrated that drug response profiling (DRP) identifies dependencies not predicted by genetic alterations adding a functional information layer for clinicians. Here we report first correlations with clinical outcome using DRP in a non-interventional setting. From 2016 to 2019 we performed DRP in the framework of European ALL first- and second- line protocols upon request by treating centers. Here we analyze retrospectively treatment decisions and outcome for 23 T- and 50 BCP-ALL patients. To evaluate drug responses, we compared dose response curves of individual patients to data recorded for all patients. Sensitivity and resistance were defined based on the IC50 outlier analysis using cut-offs depending on distribution (normal gaussian vs. skewed). From 73 patients tested, clinical outcome data has been available for 36 BCP- and 15 T-ALL patients. NGS data provided by the INFORM registry has been available in 8 BCP- and 2 T-ALL patients. In first line BCP-ALL patients, ex-vivo Dexamethasone response predicted clinical response to prephase prednisone (d8) and minimal residual disease (MRD) reduction measured by flow cytometry at d15 of first line AIEOP BFM 2009 induction (Fig. 1). For refractory and relapsed ALL we observed an association of DRP and response to targeted agents in 14 out of 16 patients (87.5 %; Table 1). Data for the r/r BCP-ALL cohort is limited because most patients underwent CD19 and / or CD22-directed immunotherapy. Sensitivity and resistance to Calicheamicin correlated with clinical response to Inotuzumab, suggesting functional testing to be evaluated in future studies. In contrast, lack of correlation of ex-vivo sensitivity to MEK-inhibitors with presence of RAS-pathway alterations caution the exclusive use of molecular information to predict response to these agents. Most therapeutic decisions based on DRP information were made for patients with r/r T-ALL. Bortezomib ex-vivo sensitivity correlated with clinical responses in 5 T-ALL patients (Fig. 2). Both patients predicted to respond to Bortezomib and treated on Bortezomib + Venetoclax experienced good MRD response providing a bridge to stem cell transplantation (SCT). However, these patients relapsed after SCT emphasizing the need for additional consolidative therapeutic elements for heavily pretreated patients. In line with previous reports, we confirmed a T-ALL with high sensitivity to Dasatinib (IC50 1.9 nM; Fig. 3). Dasatinib monotherapy induced a molecular remission. A 2nd T-ALL showing an ex- vivo Dasatinib IC50 at 80 nM was refractory to treatment with Dasatinib + Daunoxome-FLAG. A 3rd ABL1-fusion positive T-ALL, ex-vivo resistant to Imatinib and Dasatinib, had only short- term response to Imatinib + chemotherapy. Finally, treating a T-ALL patient based on high sensitivity to the XPO1 inhibitor Selinexor as 4th line monotherapy led to significant decrease of PB blasts from d1 25 G/L to 0.7 G/L at d13 of treatment (Fig. 4). The patient experienced improved quality of life, minimizing need of hospitalization with stable disease for 3 months on maintenance with Selinexor. Given the promising preclinical data with this class of agents and current lack of established biomarkers, we propose that DRP should be evaluated for this class of agents. In conclusion, we established first associations between DRP and clinical response for various agents providing a rationale for the evaluation of DRP in prospective clinical trials. Integration of molecular and functional information may improve the selection of more specific treatment options for patients with resistant disease. The international BFM Study Group and ITCC Consortium are planning an international multiarm early clinical trial for treatment of r/r ALL patients that will include DRP for evaluation in order to improve selection of targeted therapy. Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Hrusak:Amgen: Other: MRD investigations funded by Amgen, Research Funding. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. von Stackelberg:Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau. Jacoby:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lonza: Membership on an entity's Board of Directors or advisory committees. Bourquin:Servier: Other: Travel Support.

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models.

Langerhans cell histiocytosis (LCH) is a diagnostic and therapeutic challenge. We report on a rare case of its primary oral manifestation that was treated successfully with the BRAF-specific agent, vemurafenib, after insufficient standard LCH treatment. This case underlines the importance of proper diagnosis and the evaluation of targeted therapy as a valuable tool in LCH treatment. Furthermore, the close collaboration of surgeons, oncologists, and dentists is mandatory to ensure adequate treatment, restore the stomatognathic system in debilitating post-treatment situations, improve quality of life, and ensure effective disease control in infants and young patients.

Background TRK fusions are oncogenic drivers of a variety of tumors, many of which can involve the central nervous system (CNS). Larotrectinib is an FDA-approved selective TRK inhibitor for the treatment of TRK fusion cancer (Drilon et al., NEJM 2018). Here we report on the clinical activity of larotrectinib in an expanded set of TRK fusion-positive primary CNS tumors. Methods Patients with primary CNS tumors harboring an NTRK gene fusion detected by local molecular testing who were treated with larotrectinib in two clinical trials (NCT02637687 and NCT02576431) were identified. Larotrectinib was administered until disease progression, withdrawal, or unacceptable toxicity. Disease status was investigator assessed (RANO). Data cutoff: February 19, 2019. Results 18 patients with various histological types of glial tumors (11 high grade, 4 low grade, 3 unknown) were identified. The patients had gene fusions involving NTRK2 (n = 13), NTRK1 (n = 3) and NTRK3 (n = 2). Median age was 10.5 years (range 1.3–79.0); 14 patients were pediatric ( Conclusions Larotrectinib is active in patients with TRK fusion cancer with intracranial disease. Objective responses and durable disease control were seen in primary CNS tumors of various grades and histologies. These results further support expanded testing for NTRK gene fusions in patients with primary CNS tumors. Clinical trial identification NCT02637687 and NCT02576431. Legal entity responsible for the study Bayer. Funding Bayer. Disclosure All authors have declared no conflicts of interest.

Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option. Mol Cancer Ther; 10(10); 1846–56. ©2011 AACR.

Concomitant inhibition of multiple therapeutic targets is an established strategy to improve the durability of clinical responses to targeted therapies. Rhabdomyosarcoma is the most common childhood soft tissue solid tumor and cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and few molecular therapies have been introduced clinically. High-risk rhabdomyosarcoma is driven by expression of the oncogenic fusion protein PAX3-FOXO1, which acts as a transcription factor. Recently, many efforts have been made to identify pharmacological means of inhibiting PAX3-FOXO1, given its central role in rhabdomyosarcoma cell line survival. The BET protein BRD4 has been shown to be required for transcription of PAX3-FOXO1 and its target genes and pharmacological inhibition via BET protein inhibitors exhibits anti-rhabdomyosarcoma effects. Phosphorylation of PAX3-FOXO1 by the polo like kinase 1 (PLK1) increases the stability of the oncoprotein. Based on these previous observations, we hypothesized that simultaneous inhibition of BRD4 and PLK1 could synergistically target PAX3-FOXO1 stability and activity, leading to increased anti-rhabdomyosarcoma effects. Indeed, combination treatment with PLK1 and BRD4 inhibitors exhibited synergistic effects specifically against PAX3-FOXO1-driven rhabdomyosarcoma cell lines. Dual PLK1-BRD4 inhibition suppressed proliferation and induced apoptosis in rhabdomyosarcoma cell lines at low nanomolar concentrations. Our results demonstrate a new strategy towards achieving dual synergistic targeting of BRD4 and PLK1 with a single agent which may lead to clinical translation for therapy of refractory rhabdomyosarcoma. Citation Format: Natalie Timme, Shuai Liu, Hailemichael Yosief, Heathcliff Dorado García, Ian MacArthur, Annabell Szymansky, Constantin Dohna, Georg Seifert, Patrick Hundsdörfer, Andrej Lissat, Angelika Eggert, Johannes Schulte, Wei Zhang, Anton Henssen. Dual PLK1 and BRD4 inhibition has synergistic therapeutic effects against high-risk rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2624.

Synthetic lethal dependencies have recently emerged as tumor-specific vulnerabilities which provide broad therapeutic windows and have successfully been used for rational therapeutic target discovery. Despite intense international efforts over many decades, cure rates for patients with high-risk rhabdomyosarcoma remain dismal. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined. Telomere maintenance is a hallmark of cancer and requires either activation of telomerase (TERT) or alternative lengthening of telomeres (ALT). ALT is active in a large subset of rhabdomyosarcomas and not active in healthy tissues suggesting it is a unique, tumor-specific process. ALT has recently been shown to depend on DNA damage response activation through the ATR kinase, and studies in osteosarcoma have revealed that ALT renders cancer cells hypersensitive to ATR inhibition. This defines ALT as a tumor-specific synthetic lethal determinant for susceptibility to ATR inhibition. Consistent with previous reports, we observed that ALT-dependent rhabdomyosarcoma cells are hypersensitive to ATR inhibition via AZD6738, a small molecule ATR inhibitor currently being tested in clinical trials. Intriguingly, we found that treatment with ATR inhibitors not only induced DNA damage but also resulted in aneuploidy and subsequent apoptosis in ALT-dependent rhabdomyosarcoma cells, suggesting loss of telomere protection and inadequate chromosomal segregation occur during mitosis upon ATR inhibition. To test this, we measured unrepaired DNA double-strand break formation using TUNEL. ALT-dependent cells, but not TERT-dependent cells, showed increased TUNEL labeling, consistent with an increase in double-stranded DNA breaks and unprotected DNA ends. Consistent with reduced telomere maintenance, ATR inhibition significantly reduced the number of C-circles in rhabdomyosarcoma cells relying on ALT. Furthermore, AZD6738 treatment reduced phosphorylation of known ATR targets, and differentially phosphorylated peptides were enriched for proteins involved in alternative lengthening of telomeres as measured using SILAC labeling and LC-MS/MS phospho-proteomic analysis. Finally, treatment with AZD6738 reduced tumor burden in patient-derived primary rhabdomyosarcoma xenografts, which was potentiated by combined treatment with cisplatin. Our findings delineate a therapeutically actionable DNA repair dependency induced at least in part by ALT and may lead directly to clinical translation of ATR inhibitors for therapy of refractory rhabdomyosarcoma. Citation Format: Heathcliff Dorado García, Jennifer von Stebut, Ian MacArthur, Koshi Imami, Natalie Timme, Kerstin Schoenbeck, Annabell Szymansky, Georg Seifert, Patrick Hundsdoerfer, Andrej Lissat, Matthias Selbach, Angelika Eggert, Johannes Schulte, Anton Henssen. Synthetic lethal targeting of ATR in alternative lengthening of telomeres-dependent rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2628.