Pancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to... Show morePancreatic ductal adenocarcinoma (PDAC) is a highly therapy-resistant tumor entity of unmet needs. Over the last decades, radiotherapy has been considered as an additional treatment modality to surgery and chemotherapy. Owing to radiosensitive abdominal organs, high-precision proton beam radiotherapy has been regarded as superior to photon radiotherapy. To further elucidate the potential of combination therapies, we employed a more physiological 3D, matrix-based cell culture model to assess tumoroid formation capacity after photon and proton irradiation. Additionally, we investigated proton- and photon-irradiation-induced phosphoproteomic changes for identifying clinically exploitable targets. Here, we show that proton irradiation elicits a higher efficacy to reduce 3D PDAC tumoroid formation and a greater extent of phosphoproteome alterations compared with photon irradiation. The targeting of proteins identified in the phosphoproteome that were uniquely altered by protons or photons failed to cause radiation-type-specific radiosensitization. Targeting DNA repair proteins associated with non-homologous endjoining, however, revealed a strong radiosensitizing potential independent of the radiation type. In conclusion, our findings suggest proton irradiation to be potentially more effective in PDAC than photons without additional efficacy when combined with DNA repair inhibitors. Show less
Warmerdam, D.O.; Alonso-de Vega, I.; Wiegant, W.W.; Broek, B. van den; Rother, M.B.; Wolthuis, R.M.F.; ... ; Smits, V.A.J. 2020
The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following... Show moreThe cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Borjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint. Show less
Warmerdam, D.O.; Alonso-de Vega, I.; Wiegant, W.W.; Broek, B. van den; Rother, M.B.; Wolthuis, R.M.F.; ... ; Smits, V.A.J. 2019
The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following... Show moreThe cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Borjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint. Show less
Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as... Show more Adenoviral vectors (AdVs) constitute powerful gene delivery vehicles. However, so far, their potential for genome editing has not been extensively investigated. By tailoring AdVs as carriers of designer nucleases and donor DNA sequences, the research presented in this thesis expands the utility of the AdV platform to genome editing. In particular, in the first part of this thesis, AdVs are exploited for tackling two of the major bottlenecks of genome editing: (i) developing improved methods for delivering the sizable gene-editing tools, such as RNA-guided nuclease complexes, into target cells, and (ii) increasing the specificity and fidelity of the gene-editing procedures. In the second part of the thesis, the insights derived from these studies are further exploited for testing AdVs encoding nucleases as repairing agents of defective DMD alleles in muscle cell populations derived from patients with Duchenne muscular dystrophy (DMD). Finally, the application of AdVs as gene editing tools for repairing endogenous DMD alleles is discussed in the context of other viral vector-based DMD editing strategies. Taken together, the findings reported in this work are expected to aid in the designing and testing of new therapeutic interventions for tackling DMD and are anticipated to be applicable to other genetic disorders. Show less
The genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a... Show moreThe genetic code of life is stored in DNA molecules that consist of two parallel strands of coupled nucleotides that form a DNA double helix. One of the most deleterious forms of DNA damage is a DNA double-strand break (DSB) in which both strands of the helix are broken. When not repaired adequately DSBs can lead to extensive loss of genetic information and/or genomic rearrangements, ultimately fueling genome instability, cellular dysfunction and malignant transformation. This thesis describes several studies conducted to examine how living organisms preserve their genetic material and how different DNA repair pathways influence genome stability. To study these questions the nematode C. elegans was used as a model organism, as it allows efficient genetic manipulation as well as in-depth genetic analysis of mutagenic processes. We exploited these unique attributes to i) convert these animals into in vivo sensors of DNA damage ii) identify factors not implicated in genome stability before, iii) unveil mechanisms that dictate DNA repair pathway choice, and iv) determine the biological consequences of endogenous barriers that impede DNA replication. Show less
