NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

Somatostatin analogues have been examined as a treatment for somatostatin receptor overexpressing tumors for years; specifically, octreotate (TATE) and octreotide (TOC). Several versions of these analogues coupled to beta or gamma nuclides are currently used as imaging agents, as treatments with peptide receptor radionuclide therapy (PRRT) for patients with neuroendocrine tumors or are being explored in preclinical and clinical settings. Our study describes the use of 212 Pb-DOTAMTATE, the octreotate analogue, in combination with 212 Pb, the parent of an alpha emitter. Preclinical studies demonstrated tumor targeting of 212 Pb-DOTAMTATE of >20% ID/g up to 24 hours post drug injection. The addition of kidney protection agents, including L-lysine and L-arginine decreases drug accumulation in the kidneys and the addition of ascorbic acid to the chelation mixture reduces oxidation of the drug product. 212 Pb-DOTAMTATE displays a favorable toxicity profile with single-dose injections of 20 mCi showing 100% survival and with nontoxic cumulative doses up to 45 mCi, when fractionated into three smaller doses of 15 mCi. In an initial efficacy study, a single 10 mCi injection of 212 Pb-DOTAMTATE extended the mean survival 2.4-fold. Efficacy was enhanced by giving three treatment cycles of 212 Pb-DOTAMTATE and reducing the time between injections to two weeks. Efficacy was optimized further by the addition of a chemo-sensitizing agent, 5-fluorouracil, given in combination with three cycles of 10 mCi 212 Pb-DOTAMTATE. These conditions led to 79% of the animals being tumor free at the end of the 31-week study suggesting that 212 Pb-DOTAMTATE alone or in combination with a chemotherapeutic may have positive clinical implications.

DNA double-strand breaks (DSBs) are generated by exogenous DNA damage stimuli (e.g., ionizing radiation) and endogenous metabolic intermediates, such as collapsed replication forks. Upon DSB generation, NBS1 together with MRE11 and RAD50 form the MRN complex (26). MRE11, which contains two DNA binding domains, mediates the MRN complex binding to the exposed DNA ends. The adjacent MRNassociated DNA ends could be further tethered together through the coiled-coil domain (zinc hook) of RAD50 (19,43). The chromatin loading of MRN stimulates the ATM kinase that subsequently phosphorylates the downstream effectors, such as p53 and CHK2, for cell cycle regulation, apoptosis, and repair initiation (9,27,40). In addition to transducing the repair signal, the NBS1/MRN complex participates in modulating DNA damage response (DDR) pathways by promoting error-free homology-directed repair (HDR) while repressing nonhomologous end joining (NHEJ) to minimize the generation of errant, therefore potentially "dangerous," DNA joints in S-and G 2 /M-phase cells (47).Mutations in any of the MRN complex proteins increase genomic instability and cause human genomic instability disorders. NBS1 hypomorphism causes the autosomal recessive disorder Nijmegen breakage syndrome (NBS; OMIM 251260).NBS patients show multisystemic defects, among which immunodeficiency and a predisposition to lymphoid malignancies originating from B-or T-cell lineages are the major hallmarks (11,22). NBS patients are prone to opportunistic infections due to agammaglobulinemia, primarily IgA and IgG. Other immune defects include a reduced total number of CD3 ϩ and CD4 ϩ but not CD8 ϩ single-positive (SP) T cells (11,22,30). The MRN complex is therefore proposed to play an important role in the sensing (as a DDR component) and repairing (as a component of NHEJ) of V(D)J-generated DSBs and thereby to have its regulatory function in lymphoid development (2, 13). However, NBS patient cells carrying hypomorphic mutations of the NBS1 gene fail to demonstrate a direct role of NBS1 in V(D)J recombination (15,20,48).During lymphocyte development, G 1 -phase-specific endonucleases RAG1/2 generate the DSBs at recombination signal sequences (RSS) flanking functional V, D, and J gene segments, which are in turn joined mainly by the NHEJ machinery (13, 28). Recent studies using intrachromosomal recombination substrates in the MRN hypomorphic mouse B cells or a small interfering RNA (siRNA) knockdown of MRN in cell lines demonstrate that the MRN complex stabilizes the DSB ends generated by RAG1/2 to promote canonical NHEJ (C-NHEJ; LigIV-Xrcc4 dependent) and also alternative NHEJ (A-NHEJ; LigIV-Xrcc4 independent) if C-NHEJ is not available (8,10,12,17,32,34,44,46). NBS1 consists of multiple domains and interacts with several key DDR molecules at both the early or late stages of the DDR cascade (49). While hypomorphic mutation supports cell and mouse survival, the complete loss of any of the MRN components causes embryonic lethality, suggesting a vital role of Nbs1

Relapse of chronic lymphocytic leukaemia and non-Hodgkin's lymphoma after standard of care treatment is common and new therapies are needed. The targeted alpha therapy with 212 Pb-NNV003 presented in this study combines cytotoxic α-particles from 212 Pb, with the anti-CD37 antibody NNV003, targeting B-cell malignancies. The goal of this study was to explore 212 Pb-NNV003 for treatment of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin's lymphoma in preclinical mouse models.An anti-proliferative effect of 212 Pb-NNV003 was observed in both chronic lymphocytic leukaemia (MEC-2) and Burkitt's lymphoma (Daudi) cells in vitro. In biodistribution experiments, accumulation of 212 Pb-NNV003 was 23%ID/g and 16%ID/g in Daudi and MEC-2 tumours 24 h post injection. In two intravenous animal models 90% of the mice treated with a single injection of 212 Pb-NNV003 were alive 28 weeks post cell injection. Median survival times of control groups were 5-9 weeks. There was no significant difference between different specific activities of 212 Pb-NNV003 with regards to therapeutic effect or toxicity. For therapeutically effective activities, a transient haematological toxicity was observed. This study shows that 212 Pb-NNV003 is effective and safe in preclinical models of CD37 positive chronic lymphocytic leukaemia and non-Hodgkin's lymphoma, warranting future clinical testing.

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