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Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2 antibody dinutuximab beta has become a standard treatment for neuroblastoma. The combinatorial therapy of haplo SCT and dinutuximab may potentiate the efficacy of the immunotherapy. To gain further understanding of the synergistic effects, functional immunomonitoring was assessed during the clinical trial CH14.18 1021 Antibody and IL2 After haplo SCT in Children with Relapsed Neuroblastoma (NCT02258815). Rapid immune reconstitution of the lymphoid compartment was confirmed, with clinically relevant dinutuximab serum levels found in all patients over the course of treatment. Only one patient developed human anti-chimeric antibodies (HACAs). In-patient monitoring revealed highly functional NK cell posttransplant capable of antibody-dependent cellular cytotoxicity (ADCC). Degranulation of NK cell subsets revealed a significant response increased by dinutuximab. This was irrespective of the KIR receptor–ligand constellation within the NK subsets, defined by the major KIR receptors CD158a, CD158b, and CD158e. Moreover, complement-dependent cytotoxicity (CDC) was shown to be an extremely potent effector-cell independent mechanism of tumor cell lysis, with a clear positive correlation to GD2 expression on the cancer cells as well as to the dinutuximab concentrations. The ex vivo testing of patient-derived effector cells and the sera collected during dinutuximab therapy demonstrated both high functionality of the newly established lymphoid immune compartment and provided confidence that the antibody dosing regimen was sufficient over the duration of the dinutuximab therapy (up to nine cycles in a 9-month period). During the course of the dinutuximab therapy, proinflammatory cytokines and markers (sIL2R, TNFa, IL6, and C reactive protein) were significantly elevated indicating a strong anti-GD2 immune response. No impact of FcGR polymorphism on event-free and overall survival was found. Collectively, this study has shown that in-patient functional immunomonitoring is feasible and valuable in contributing to the understanding of anti-cancer combinatorial treatments such as haplo SCT and antibody immunotherapy.

The ends of linear eukaryotic chromosomes resemble double strand breaks, susceptible to the vigilant cellular DNA damage response machinery. Telomeres are specialised nucleoprotein structures found at the ends of human chromosomes that function as a protective cap safeguarding the integrity of the genome. In normal human somatic cells, telomeres shorten with each replicative cell division. Eventually, shortened telomeres trigger cellular senescence and apoptosis. Cancer cells overcome this proliferative barrier by acquiring a telomere maintenance mechanism. Alternative Lengthening of Telomeres (ALT) is a homologous recombination (HR) mediated telomere maintenance mechanism utilised by approximately 10-15% of all cancers. ALT status in certain cancer subtypes, such as osteosarcomas and astrocytomas, often predicts aggressive nature and poor prognosis. Therefore, the ALT pathway presents an attractive avenue for developing novel cancer therapeutics. To date, ALT specific therapeutic targets are lacking. Our lab has previously identified the zinc finger protein ZNF827 as a promising molecular target in the ALT pathway. ZNF827 recruits the nucleosome remodelling and histone deacetylase (NuRD) complex to ALT telomeres, and collaboratively facilitates multifaceted functions to promote HR-mediated telomere synthesis. However, the precise molecular mechanisms underlying the important roles of ZNF827 at ALT telomeres have not been fully elucidated. Prior to the study by our lab, ZNF827 was a protein of unknown function. This thesis has focused on the functional characterisation of ZNF827, with an ultimate goal of validating it as a therapeutic target in cancers utilising the ALT pathway. We have characterised ZNF827 from several perspectives. First, we have explored the biological functions of ZNF827 as a transcription factor and discovered novel roles of ZNF827 in cellular processes including embryonic development and immune response. Second, we have gained new insights into the recruitment of ZNF827 to ALT telomeres, characterised structurally and functionally the interaction interface between ZNF827 and the NuRD complex, and implicated ZNF827 SUMOylation in promoting ALT activity. We have also discovered that ZNF827 is novel ssDNA binding protein implicated in HR-directed repair of replication-associated DNA damage at telomeres as well as genome-wide, and that its role as a DNA damage response and repair protein is likely to be mediated through the ATR-mediated DNA signalling pathway. Finally, we have provided preliminary evidence that supports synthetic lethality between ZNF827 inhibition and the topoisomerase I inhibitor topotecan, implicating ZNF827 as a potential molecular target for ATR inhibition. Taken together, our findings have substantially expanded our knowledge on the zinc finger protein ZNF827 and provided further support for its therapeutic potential in the development of novel cancer therapies.

The telomerase protein hTERT has a noncanonical role in promoting telomere protection by heat shock protein 70., Telomerase is a ribonucleoprotein complex that catalyzes addition of telomeric DNA repeats to maintain telomeres in replicating cells. Here, we demonstrate that the telomerase protein hTERT performs an additional role at telomeres that is independent of telomerase catalytic activity yet essential for telomere integrity and cell proliferation. Short-term depletion of endogenous hTERT reduced the levels of heat shock protein 70 (Hsp70-1) and the telomere protective protein Apollo at telomeres, and induced telomere deprotection and cell cycle arrest, in the absence of telomere shortening. Short-term expression of hTERT promoted colocalization of Hsp70-1 with telomeres and Apollo and reduced numbers of deprotected telomeres, in a manner independent of telomerase catalytic activity. These data reveal a previously unidentified noncanonical function of hTERT that promotes formation of a telomere protective complex containing Hsp70-1 and Apollo and is essential for sustained proliferation of telomerase-positive cancer cells, likely contributing to the known cancer-promoting effects of both hTERT and Hsp70-1.