The DNA helicase recql4 is required for normal osteoblast expansion and osteosarcoma formation.

RECQL4 mutations are associated with Rothmund Thomson Syndrome (RTS), RAPADILINO Syndrome and Baller-Gerold Syndrome. These patients display a range of benign skeletal abnormalities such as low bone mass. In addition, RTS patients have a highly increased incidence of osteosarcoma (OS). The role of RECQL4 in normal adult bone development and homeostasis is largely uncharacterized and how mutation of RECQL4 contributes to OS susceptibility is not known. We hypothesised that Recql4 was required for normal skeletal development and both benign and malignant osteoblast function, which we have tested in the mouse. Recql4 deletion in vivo at the osteoblastic progenitor stage of differentiation resulted in mice with shorter bones and reduced bone volume, assessed at 9 weeks of age. This was associated with an osteoblast intrinsic decrease in mineral apposition rate and bone formation rate in the Recql4-deficient cohorts. Deletion of Recql4 in mature osteoblasts/osteocytes in vivo, however, did not cause a detectable phenotype. Acute deletion of Recql4 in primary osteoblasts or shRNA knockdown in an osteoblastic cell line caused failed proliferation, accompanied by cell cycle arrest, induction of apoptosis and impaired differentiation. When cohorts of animals were aged long term, the loss of Recql4 alone was not sufficient to initiate OS. We then crossed the Recql4fl/fl allele to a fully penetrant OS model (Osx-Cre p53fl/fl). Unexpectedly, the Osx-Cre p53fl/flRecql4fl/fl (dKO) animals had a significantly increased OS-free survival compared to Osx-Cre p53fl/fl or Osx-Cre p53fl/flRecql4fl/+ (het) animals. The extended survival was explained when the Recql4 status in the tumors that arose was assessed, and in no case was there complete deletion of Recql4 in the dKO OS. These data provide a mechanism for the benign skeletal phenotypes of RECQL4 mutation syndromes. We propose that tumor suppression and osteosarcoma susceptibility are most likely a function of mutant, not null, alleles of RECQL4.

Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA.

Osteosarcoma is the most common primary cancer of bone and one that predominantly affects children and adolescents. Osteoblastic osteosarcoma represents the major subtype of this tumor, with approximately equal representation of fibroblastic and chondroblastic subtypes. We and others have previously described murine models of osteosarcoma based on osteoblast-restricted Cre:lox deletion of Trp53 (p53) and Rb1 (Rb), resulting in a phenotype most similar to fibroblastic osteosarcoma in humans. We now report a model of the most prevalent form of human osteosarcoma, the osteoblastic subtype. In contrast to other osteosarcoma models that have used Cre:lox mediated gene deletion, this model was generated through shRNA-based knockdown of p53. As is the case with the human disease the shRNA tumors most frequently present in the long bones and preferentially disseminate to the lungs; feature less consistently modeled using Cre:lox approaches. Our approach allowed direct comparison of the in vivo consequences of targeting the same genetic drivers using two different technologies, Cre:lox and shRNA. This demonstrated that the effects of Cre:lox and shRNA mediated knock-down are qualitatively different, at least in the context of osteosarcoma, and yielded distinct subtypes of osteosarcoma. Through the use of complementary genetic modification strategies we have established a model of the most common clinical subtype of osteosarcoma that was not previously represented and more fully recapitulated the clinical spectrum of this cancer.