Kristina Aaltonen

High-risk neuroblastoma is an aggressive pediatric malignancy characterized by a high proliferative capacity, intrinsic replication stress, and poor clinical outcomes. Although mitotic inhibitors show preclinical efficacy, their clinical use is limited by dose-dependent toxicity. In this study, we performed high-throughput combinatorial drug screening using MYCN-amplified (MNA) human neuroblastoma tumoroids to identify drugs with orthogonal mechanisms that synergize with low-dose mitotic inhibition. Promising drug combinations were prioritized using a multifactorial approach including dose–response matrices across multiple neuroblastoma tumoroids and CD34+ cord blood cells, synergy area scores, and machine learning–based response predictions. Several therapeutic classes were selected, including kinase inhibitors, differentiation agents, and hormone therapies. Notably, combined ataxia telangiectasia and Rad 3–related (ATR) inhibition by elimusertib and mitotic kinesin spindle protein (KSP) inhibition showed additive to mild synergy and induced neuroblastoma cell death. This combination reduced tumor growth and prolonged survival in neuroblastoma MNA, chemoresistant patient-derived xenografts. Furthermore, alternating dosing schedules were well tolerated and maintained stable disease control in vivo. Collectively, these findings establish combined ATR and low-dose mitotic inhibition as a promising therapeutic strategy for MNA neuroblastoma.

Background: Neuroblastoma (NB) is a childhood cancer with a high relapse rate despite intensive treatment. TRPA1 is a pain‐sensing ion channel with downstream impacts on proliferative and pro‐apoptotic pathways. Here, we evaluated TRPA1 expression in NB and performed pharmacological inhibition in preclinical models to assess its potential as a therapeutic target in NB.Methods: TRPA1 protein levels were assessed in NB patient tumors on tissue microarrays. Bulk and single‐cell gene expression data were retrieved from publicly available databases. The effects of three TRPA1 inhibitors (AP‐18, A967079, and Bay 390) on NB cell viability and cell death were evaluated using NB patient‐derived xenograft (PDX)‐derived organoids. In vivo testing was performed in a MYCN‐amplified NB PDX model. Drug combination testing was performed using combination or sequential treatments and evaluated using drug synergy scores.Results: TRPA1 is widely expressed in NB patient tumors and preclinical patient‐derived NB models. Pharmacological TRPA1 inhibition decreased NB cell viability and increased cell death. In vivo TRPA1 inhibition alone did not significantly affect NB tumor growth. Pretreatment with TRPA1 inhibition prior to chemotherapy resulted in synergistic effects in vitro.Conclusions: TRPA1 is expressed in NB tumors, and pharmacological TRPA1 inhibition can be effective in vitro and synergistic when used as pretreatment to chemotherapy. However, the tested inhibitors did not show in vivo efficacy, at least as monotherapy.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Background Metastatic breast cancer (MBC) is the main cause of breast cancer-related death. The outcome of MBC varies, and there is a lack of biomarkers to aid in prognostication. The primary aim of this study was to evaluate the prognostic value of gene expression (GEX) signatures in the primary tumor (PT) and distant metastasis (DM) for progression-free survival (PFS) and overall survival (OS). The secondary aim was to describe GEX changes through MBC evolution and to identify MBC subtypes. Methods RNA was extracted from the PT, lymph node metastases (LNM), and DM from MBC patients in a prospective observational study (n = 142; CTC-MBC NCT01322893) and was subjected to GEX analysis retrospectively using the NanoString Breast Cancer 360™ panel. 31 continuous GEX variables in DMs and PTs were analyzed for PFS and OS by Cox regression analysis and Kaplan-Meier estimates. Multivariable Cox regressions were adjusted for number of DM sites and CTCs, visceral metastasis, ECOG status, age at MBC diagnosis and, in additional analyses, PAM50 subtype. Differential GEX analyses and Euclidean distances were used to describe subgroup differences and visualize within-patient heterogeneity. Results Compared to DM GEX, GEX of the PT was at least equally useful for prognosticating MBC outcome. The strongest marker for a favorable PFS, both when expressed in the PT and the DM was AR, even after adjustment for prognostic markers including PAM50. GEX signatures related to hormone responsiveness, including ESR1, FOXA1, PGR, and AR were favorable prognostic markers, and the p53 signature was unfavorable for PFS when expressed in PT or DM. The previously published PAM50MET signature was prognostic for both PFS and OS. We established five distinct DM GEX profiles where two associated with liver and bone metastases, respectively. Finally, we identified four DM GEX profiles able to identify MBCs with poor OS in this cohort. Conclusion GEX of both DM and PT are useful in MBC prognostication. GEX of AR adds prognostic information for MBC. Our descriptive analyses illuminate the biological differences between MBCs in relation to outcome and metastatic site.

Chemotherapy resistance and relapses are common in high-risk neuroblastoma (NB). Here, we developed a clinically relevant in vivo treatment protocol mimicking the first-line five-chemotherapy treatment regimen of high-risk NB and applied this protocol to mice with MYCN -amplified NB patient-derived xenografts (PDXs). Genomic and transcriptomic analyses were used to reveal NB chemoresistance mechanisms. Intrinsic resistance was associated with high genetic diversity and an embryonic phenotype. Relapsed NB with acquired resistance showed a decreased adrenergic phenotype and an enhanced immature mesenchymal–like phenotype, resembling multipotent Schwann cell precursors. NBs with a favorable treatment response presented a lineage-committed adrenergic phenotype similar to normal neuroblasts. Novel integrated phenotypic gene signatures reflected treatment response and patient prognosis. NB organoids established from relapsed PDX tumors retained drug resistance, tumorigenicity, and transcriptional cell states. This work sheds light on the mechanisms of NB chemotherapy response and emphasizes the importance of transcriptional cell states in chemoresistance.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

Neuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system that accounts for 15% of childhood cancer deaths. High-risk (HR) NB tumors are treated with an aggressive multimodal therapy, including the COJEC induction protocol which is a combination of five different chemotherapeutic agents. However, around 15% of primary tumors are refractory from the start and over 60% present treatment resistant relapses. In this project we aim to decipher the mechanisms involved in treatment resistance in HR-NB. Using various NB patient-derived xenograft (PDX) models we designed and tested a clinically-relevant protocol that mimics COJEC treatment and surgical resection. RNA-sequencing, SNP analysis, and histology, were used to explore the molecular mechanisms involved in treatment response. With our COJEC-like protocol we were able to replicate the original patient response in our PDX models. NB PDX1 presented upfront progression and a lack of morphological differentiation. At a molecular level it was characterized by genome duplication and high number of chromosomal aberrations, as well as a high expression of early development/mesenchymal and cell cycle genes. In contrast, NB PDX3 presented an overall good response, leading to a 22% relapse rate after surgery. Relapsed tumors displayed high expression of early development/mesenchymal genes, similar to those seen in refractory PDX1. Meanwhile, the PDX3 tumors collected during surgery that did not relapse (cured) showed low MYCN expression, downregulation of cell cycle and DNA repair genes, along with a strong upregulation of nervous system/nor-adrenergic genes. These genetic signatures derived from the cured PDX3 samples also correlated with good prognosis in large cohorts of HR-NB patients. Despite observing a convergent/parallel evolution of specific genetic aberrations across our PDXs, their presence or absence does not predict prognosis. Instead, the high RNA expression levels of those specific genes correlates with treatment response both in our PDX models and in patient cohorts. Finally, we established 3D tumor organoids derived from treated PDX tumors and confirmed they maintain the molecular characteristics of the parental tumor as well as the acquired COJEC resistance and the tumorigenic capacity, making them an excellent tool to test novel therapeutics against HR-NB. In conclusion, we designed a clinically-relevant COJEC-like protocol that can mimic patient response in PDX models. Early developmental gene signatures appear to correlate with treatment resistance in HR-NB, while the expression of nor-adrenergic signatures after chemotherapy are associated with good prognosis, independently of the accumulation of copy number aberrations. Based on our results, we propose that a combination of DNA and RNA analysis at the surgical resection time point after COJEC could be of clinical interest to evaluate COJEC treatment response and predict future relapses. Citation Format: Adriana Mañas, Kristina Aaltonen, Natalie Andersson, Karin Hansson, Alexandra Seger, Hiroaki Yasui, Katarzyna Radke, Javanshir Esfandyari, David Lindgren, David Gisselsson, Daniel Bexell. Modeling and deciphering COJEC resistance in high risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3901.

KSP inhibition results in cell death through mitotic arrest and causes regression of neuroblastoma PDX tumors in mice.