Abstract 971: Pro-apoptotic Bid preserves hematopoietic stem cell function through restraint of necrosis: implications for myelodysplastic syndrome
Authors: Zinkel Sandra S. 
Published: August 2015

Abstract
Multicellular organisms remove damaged or superfluous cells through a highly regulated cellular process known as programmed cell death. There are two main forms of programmed cell death, apoptosis and necrosis. In apoptotic cell death, caspases digest the cell. In necroptotic cell death, increased Rip kinase signaling effects plasma membrane rupture, and activation of an inflammatory response.
Death receptors, such as the TNF receptor, can activate either apoptotic or necroptotic death. The upstream activators and transducers including Caspase-8, Rip1, and Fadd, are common to both forms of cell death. Interestingly, Caspase-8 and c-FlipL, were recently shown to inhibit the necrotic pathway through the formation of a catalytically active complex. The BH3-only Bcl-2 family member, Bid is a substrate of Caspase-8, and is thus in position to mediate cell death fate.
We developed a mouse model of unrestrained bone marrow necroptosis in which apoptosis is prevented by the deletion of the pro-apoptotic effectors Bax and Bak. We further deleted the upstream activator Bid (VavBaxBakBid TKO mice). These mice die of bone marrow failure with myeloid dysplasia between 3 and 12 months, and some develop leukemia, closely resembling MDS. TKO bone marrow displays necroptotic cells by electron microscopy, and markedly increased Rip1 expression by immunofluorescence. TKO SLAM-HSCs display increased BrdU incorporation, consistent with compensatory HSC proliferation in response to bone marrow cell death. TKO bone marrow outcompetes WT bone marrow in competitive reconstitution experiments, but the mice succumb to bone marrow failure at 5 months. Thus, increased necroptosis results in a cell autonomous stem cell defect. In addition, necroptotic bone marrow kills normal HSCs by feed-forward TNF signaling.
Myeloid progenitor cell lines (MPCs) derived from the bone marrow of BaxBakBid TKO mice, display increased activation (phosphorylation) and increased levels of Rip1 in the pronecrotic complex (Complex II) following LPS treatment. This association is abrogated by reintroduction of Bid by retrovirus into TKO MPCs, demonstrating that Bid inhibits Rip1 association with complex II, to determine cell death fate.
Finally, we found increased RIP1 expression, but not activated caspase 3 in bone marrow samples from patients with the RCMD, RAEB-1, and RAEB-2 subtypes of MDS. We thus demonstrate that increased necroptosis signaling can result in bone marrow failure with dysplasia, and that necroptotic cell death signaling is increased in bone marrow from MDS patients, highlighting the potential importance of this targetable signaling pathway in bone marrow failure disorders such as MDS.
Citation Format: Sandra S. Zinkel. Pro-apoptotic Bid preserves hematopoietic stem cell function through restraint of necrosis: implications for myelodysplastic syndrome. [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 971. doi:10.1158/1538-7445.AM2015-971