Abstract 1264: Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence

The INK4b-ARF-INK4a locus on human chromosome 9p21 is a hot spot genomic region that undergoes frequent inactivation or deletion in a wide spectrum of human cancers. The locus encodes three cell cycle inhibitory proteins: p15INK4b encoded by CDKN2b, p16INK4a encoded by CDKN2a and p14ARF (p19Arf in mice) encoded by an alternative reading frame (ARF) of CDKN2a. We have recently identified a homologous INK4 genomic locus in zebrafish that is surprisingly devoid of ARF sequences, and encodes a single zebrafish ink4ab gene that functions to activate stress-induced senescence. Therefore, evolution of the mammalian INK4 locus to include multiple tumor suppressors sharing regulatory features could attribute to stronger mechanisms of tumor suppression in these longer-lived vertebrates. Oxidative and/or oncogenic stress provokes cellular senescence involving the retinoblastoma (Rb) and ARF/p53 pathways, leading to silencing of growth-promoting genes by methylation of histone H3 lysine 9 (H3K9me) with the histone methyltransferase Suv39h1. We found that ink4ab deficient zebrafish had deregulated Rb signaling and senescence responses, and reduced suv39h1 expression levels that led to poor overall survival. Ink4ab deficient zebrafish had increased lymphocyte proliferation, splenomegaly, and developed multiple spontaneous tumors including metastatic melanoma, osteosarcoma, hepatocellular adenoma, leukemias and myelodysplastic disorders. Moreover, zebrafish heterozygous for both p53 and ink4ab mutations displayed significantly higher tumor incidence compared to p53 heterozygotes with wild-type ink4ab, indicating that ink4ab haploinsufficiency promotes tumorigenesis. To this end, the combined deficiency of ink4ab and p53 accelerates tumor latency, likely by reversing cellular senescence responses. To further examine ARF-independent senescence regulatory roles of INK4 products in a mammalian model, we have generated conditional mice deficient for all three INK4 open reading frames in the adult hematopoietic system. Within seven months, homozygous Ink4a/Ink4b/ARF-/- mice exhibited marked splenomegaly and developed spontaneous tumors, including leukemias and soft tissue sarcomas. Peripheral blood, splenic and bone marrow analyses of homozygous mutant mice showed deregulated lymphocyte development, uncontrolled proliferation, apoptosis and Suv39h1-dependent senescence when compared to heterozygous and wild type mice. These results identify ARF-independent and Suv39h1-dependent senescence regulatory and tumor suppressor mechanisms whose inactivation permits tumor formation in response to oncogenic and/or oxidative stress. Our models offer the opportunity to reveal novel paradigms for promoting cellular senescence as an alternative strategy for cancer prevention and therapy. Citation Format: Kathleen Flaherty, Daniel Jones, Shamila Yussuf, Stephani Davis, Eric Huselid, Wei Wang, Monica Bartucci, Hatem E. Sabaawy. Conditional mouse and zebrafish models of INK4-mediated tumor suppression reveal ARF-independent regulation of cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2015-1264