Acute induction of oncogenic Ras provokes cellular senescence involving the retinoblastoma (Rb) pathway, but the tumour suppressive potential of senescence in vivo remains elusive. Recently, Rb-mediated silencing of growth-promoting genes by heterochromatin formation associated with methylation of histone H3 lysine 9 (H3K9me) was identified as a critical feature of cellular senescence, which may depend on the histone methyltransferase Suv39h1. Here we show that Emicro-N-Ras transgenic mice harbouring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T-cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wild-type ('control') animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient, lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras.

p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16(INK4a). Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone-respond poorly to cyclophosphamide therapy in vivo. Moreover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16(INK4a), and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics. Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs. This type of metabolic Achilles' heel will allow us and others to more effectively "starve" the CSC population.

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