The mTORC1 inhibitor everolimus prevents and treats Eμ-Myc lymphoma by restoring oncogene-induced senescence

MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in EμMyc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of EμMyc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established EμMyc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes

Genomic characterisation of Eμ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene

The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.We acknowledge the following funding agencies: Leukaemia Foundation of Australia, Arrow Bone Marrow Transplant Foundation, National Health and Medical Research Council Australia, Cancer Council Victoria, Victorian Cancer Agency, Australian Cancer Research Foundation, Peter MacCallum Cancer Centre Foundation, National Institutes of Health

Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity

Abstract: It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies

Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas

RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut

Disruption of RNA Polymerase I transcription as a strategy for the treatment of cancer

© 2011 Dr. Megan Julie BywaterElevated rates of ribosomal RNA (rRNA) synthesis have long been considered a common characteristic of cancer cells. However, the questions as to whether cancer cells are dependant on this hyperactivation of RNA Polymerase I (Pol I) transcription, and whether this dependency could be exploited for therapeutic gain have until now remained unanswered. Through the explosion of high content gene expression technology over the past decade it has become apparent that the transcriptional regulator c-MYC has a global regulatory role in the process of ribosome biogenesis, and indeed transcription of the rRNA genes. Consequently we reasoned that a MYC-driven model of malignancy would be the ideal setting to address these outstanding questions concerning the role of ribosome biogenesis in cancer. To this end the studies in this thesis employed the Eμ-Myc transgenic mouse model of lymphoma in which disease spontaneously develops as a consequence of MYC over expression in cells of the B-lymphocyte lineage. B-cells purified from these transgenic mice displayed dysregulated growth and ribosome biogenesis characterised by an increase in the percentage of transcriptionally competent rDNA repeats, and an increased rate of rRNA synthesis. The hyperactivation of Pol I transcription in Eμ-Myc transgenic B-cells was most likely a consequence of the over expression of a cohort of Pol I specific transcription factors, including UBF and RRN3. This aspect of the malignant phenotype was targeted by reducing the expression of UBF or RRN3 by RNA interference in Eμ-Myc lymphoma cell lines generated from spontaneously arising tumours. Knockdown of UBF and RRN3 in Eμ-Myc lymphoma cell lines decreased rRNA synthesis rates and resulted in a selective disadvantage in cell survival. This disadvantage was mediated by the induction of apoptosis. Furthermore, UBF knockdown cells appeared to be under greater selective pressure, possibly due to the reduced percentage of transcriptionally competent rDNA repeats in these cells. As the relatively modest reduction in rRNA synthesis observed in UBF and RRN3 knockdown cells was so rapidly selected against in culture, it was hypothesised that Eμ-Myc lymphoma cell lines are extremely sensitive to perturbations in Pol I transcription. This was confirmed with the use of the selective small molecule inhibitor of Pol I transcription, CX-5461. Sensitivity to CX-5461 was shown to be dependant on the expression of wild type p53, and most likely mediated by the induction of the ribosomal protein-MDM2-p53 ribosome surveillance pathway. Furthermore, the dependence of Eμ-Myc lymphoma cells on high rates of rDNA transcription was exploited therapeutically with CX-5461 showing marked efficacy in the elimination of transplanted tumour cells, resulting in an increased survival time in mice. In summary, this work demonstrates that Eμ-Myc lymphomas require the hyperactivation of Pol I transcription for their maintained malignant phenotype. Consequently inhibition of this process can be targeted therapeutically in the selective elimination of these tumour cells in vitro and in vivo, identifying a novel strategy in the treatment of cancer

Disruption of RNA Polymerase I transcription as a strategy for the treatment of cancer

© 2011 Dr. Megan Julie BywaterElevated rates of ribosomal RNA (rRNA) synthesis have long been considered a common characteristic of cancer cells. However, the questions as to whether cancer cells are dependant on this hyperactivation of RNA Polymerase I (Pol I) transcription, and whether this dependency could be exploited for therapeutic gain have until now remained unanswered. Through the explosion of high content gene expression technology over the past decade it has become apparent that the transcriptional regulator c-MYC has a global regulatory role in the process of ribosome biogenesis, and indeed transcription of the rRNA genes. Consequently we reasoned that a MYC-driven model of malignancy would be the ideal setting to address these outstanding questions concerning the role of ribosome biogenesis in cancer. To this end the studies in this thesis employed the Eμ-Myc transgenic mouse model of lymphoma in which disease spontaneously develops as a consequence of MYC over expression in cells of the B-lymphocyte lineage. B-cells purified from these transgenic mice displayed dysregulated growth and ribosome biogenesis characterised by an increase in the percentage of transcriptionally competent rDNA repeats, and an increased rate of rRNA synthesis. The hyperactivation of Pol I transcription in Eμ-Myc transgenic B-cells was most likely a consequence of the over expression of a cohort of Pol I specific transcription factors, including UBF and RRN3. This aspect of the malignant phenotype was targeted by reducing the expression of UBF or RRN3 by RNA interference in Eμ-Myc lymphoma cell lines generated from spontaneously arising tumours. Knockdown of UBF and RRN3 in Eμ-Myc lymphoma cell lines decreased rRNA synthesis rates and resulted in a selective disadvantage in cell survival. This disadvantage was mediated by the induction of apoptosis. Furthermore, UBF knockdown cells appeared to be under greater selective pressure, possibly due to the reduced percentage of transcriptionally competent rDNA repeats in these cells. As the relatively modest reduction in rRNA synthesis observed in UBF and RRN3 knockdown cells was so rapidly selected against in culture, it was hypothesised that Eμ-Myc lymphoma cell lines are extremely sensitive to perturbations in Pol I transcription. This was confirmed with the use of the selective small molecule inhibitor of Pol I transcription, CX-5461. Sensitivity to CX-5461 was shown to be dependant on the expression of wild type p53, and most likely mediated by the induction of the ribosomal protein-MDM2-p53 ribosome surveillance pathway. Furthermore, the dependence of Eμ-Myc lymphoma cells on high rates of rDNA transcription was exploited therapeutically with CX-5461 showing marked efficacy in the elimination of transplanted tumour cells, resulting in an increased survival time in mice. In summary, this work demonstrates that Eμ-Myc lymphomas require the hyperactivation of Pol I transcription for their maintained malignant phenotype. Consequently inhibition of this process can be targeted therapeutically in the selective elimination of these tumour cells in vitro and in vivo, identifying a novel strategy in the treatment of cancer

MPN: The Molecular Drivers of Disease Initiation, Progression and Transformation and their Effect on Treatment

Myeloproliferative neoplasms (MPNs) constitute a group of disorders identified by an overproduction of cells derived from myeloid lineage. The majority of MPNs have an identifiable driver mutation responsible for cytokine-independent proliferative signalling. The acquisition of coexisting mutations in chromatin modifiers, spliceosome complex components, DNA methylation modifiers, tumour suppressors and transcriptional regulators have been identified as major pathways for disease progression and leukemic transformation. They also confer different sensitivities to therapeutic options. This review will explore the molecular basis of MPN pathogenesis and specifically examine the impact of coexisting mutations on disease biology and therapeutic options