Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

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

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS

CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity

Homeostatic trafficking of lymphocytes through extralymphoid tissues has been recently observed and a potential role in immune surveillance and the establishment of peripheral tolerance is considered. However, the mechanisms regulating lymphocyte recirculation through peripheral tissues under non-inflammatory conditions are not well understood. Here, we demonstrate that the chemokine receptor CCR7 controls not only lymphocyte trafficking to and within secondary lymphoid organs, but also homeostatic recirculation of T and B lymphocytes through non-lymphoid tissues. Lack of CCR7 results in a massive accumulation of lymphocytes in epithelial tissues. In particular, the gastrointestinal mucosal tissue of CCR7(-/-) mice is highly permissive for the formation of lymphoid aggregates, which develop into ectopic follicular structures with major topological characteristics of lymph nodes. Flow cytometry analysis of CD4(+) T cells derived from ectopic follicles revealed that CD44(hi)CD62L(lo) effector memory T cells predominate in the gastric lymphoid aggregates. In aged mice, lack of CCR7 induced age-dependent histomorphological changes in the stomach with profound cystic hyperplasia and an increased rate of mucosal proliferation resembling Menetrier's disease. Thus, CCR7 regulates the cellular organization of visceral tissue by governing life-long recirculation of naive and memory lymphocytes under homeostatic conditions

A Novel NAMPT Inhibitor Based Antibody Drug Conjugate Payload Class for Cancer Therapy

Inhibition of intracellular nicotinamide phosphoribosyltransferase NAMPT represents a new mode of action for cancer targeting antibody drug conjugates ADCs with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure activity relationship driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a LYPD3 , HER2 c erbB2 , or B7H3 CD276 and tested on antigen positive and negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi ADCs demonstrated potent in vivo antitumor efficacy in target antigen expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate to our knowledge for the first time the generation of NAMPTi payload metabolites from the NAMPTi ADCs in vitro and in vivo. In conclusion, NAMPTi ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancer

Treating Cancer by Spindle Assembly Checkpoint Abrogation Discovery of Two Clinical Candidates, BAY 1161909 and BAY 1217389, Targeting MPS1 Kinase

Inhibition of monopolar spindle 1 MPS1 kinase represents a novel approach to cancer treatment instead of arresting the cell cycle in tumor cells, cells are driven into mitosis irrespective of DNA damage and unattached misattached chromosomes, resulting in aneuploidy and cell death. Starting points for our optimization efforts with the goal to identify MPS1 inhibitors were two HTS hits from the distinct chemical series triazolopyridines and imidazopyrazines . The major initial issue of the triazolopyridine series was the moderate potency of the HTS hits. The imidazopyrazine series displayed more than 10 fold higher potencies; however, in the early project phase, this series suffered from poor metabolic stability. Here, we outline the evolution of the two hit series to clinical candidates BAY 1161909 and BAY 1217389 and reveal how both clinical candidates bind to the ATP site of MPS1 kinase, while addressing different pockets utilizing different binding interactions, along with their synthesis and preclinical characterization in selected in vivo efficacy model