The Interferon Consensus Sequence Binding Protein (Icsbp) is an interferon regulatory transcription factor that functions as a tumor-suppressor (also referred to as Irf8). During this Merit Review project, we used high throughput screening approaches to identify Icsbp-target-genes that mediate tumor-suppressor activity. We identified an Icsbp-target-gene set that is enriched for genes which control Fas-induced apoptosis and/or bcatenin activity. This is of interest, because decreased Icsbp-expression, Fas-resistance, and increased bcatenin activity are associated with poor prognosis in chronic myeloid leukemia (CML). Insensitivity of cancer/leukemia stem cells (CSC/LSC) to Fas-induced apoptosis is associated with development of drug resistance in CML, but does not correlate with decreased Fas/FasL. Increased catenin activity in CML- CSC/LSC precedes progression to blast crisis (BC), but does not correlate with Wnt expression or CTNNB1 transcription. Tyrosine kinase inhibitors (TKI) that target Bcr-abl (the CML oncogene) induce remission in the majority of patients. However, the CSC/LSC population expands during remission, preventing cure with TKIs. CSC/LSC persistence is hypothesized to be due to dysregulation of Fas and/or bcatenin. During the previous funding period, we identified Icsbp-target-genes that explain Fas-resistance and increased bcatenin activity in CML. We found that Icsbp represses PTPN13; the gene encoding Fap1. Fap1 interacts with and inhibits Fas, and we found Icsbp/Fap1-dependent Fas-resistance in cells expressing Bcr-abl. Fap1 also interacts with Apc, and we found inhibition of Gsk3b and stabilization of bcatenin in these cells. We identified GAS2 as another relevant Icsbp-target-gene. Gas2 inhibits calpain; a serine protease with substrates that include bcate-nin, Stat3, Stat5 and Xiap. We found a Gas2-dependent decrease in calpain activity and increase in bcatenin in Bcr-abl+ or Icsbp-/- murine bone marrow cells. Stat3, Stat5 and Xiap proteins are also increased in Icsbp-/- cells in a Gas2/calpain-dependent manner. Interestingly, we found that Stat5 represses the IRF8 promoter in a Gas2/calpain-dependent manner. Xiap inhibits caspase 3, contributing to Fas-resistance. We identified RASSF5, the gene encoding Nore1, as another Icsbp-target-gene. Nore1 activates Mst1; a kinase that facilitates caspase cleavage and therefore Fas-induced apoptosis. The hypothesis of these studies is that decreased expression of the tumor-suppressor Icsbp results in Fas-resistance and increased bcatenin activity. We also hypothesize that Icsbp-target-genes or cognate pathways would be rational therapeutic targets to prevent overt drug resistance and progression to BC in CML. This hypotheses will be pursued through three Aims;
AIM 1 : Define the role of Icsbp-dependent expression of Nore1 and Xiap in CML-LSC expansion. The contribution of Nore1a or Xiap to Fas-resistance and cooperation with Fap1 will be studied in CML-LSC. The impact of targeting these pathways on LSC expansion in an in vivo CML murine model will be determined.
AIM 2 : Determine if Icsbp contributes to disease progression by inhibiting Gsk3 or increasing Stat3 in CML-LSC. Contribution of Gsk3 and Stat3 to bcatenin activity will be studied in vitro and in vivo, as above. The effect of targeting these pathways on disease progression will be studied in an in vivo CML murine model.
AIM 3 : Identify the roles of Stat5 and Aml1 in IRF8 transcription and leukemogenesis. IRF8 transcriptional regulation by Bcr-abl-dependent Stat5 activation will be studied in studies with myeloid leukemia cell lines and with primary murine bone marrow cells. The role of targeting calpain to decrease Stat5 protein stability, and thereby increase Icsbp expression, will be investigated in murine models in vitro and in vivo. The goal of these studies is to identify molecular mechanisms for Icsbp tumor suppressor activity. Targeting these mechanisms may lead to cure in CML by abolishing the CSC/LSC, and suggest therapeutic approaches to other forms of cancer with dysregulation of Fas and bcatenin.

Public Health Relevance

Icsbp (Interferon Consensus Sequence Binding Protein) is a tumor suppressor that acts through mostly undefined molecular mechanisms. We used chromatin immuno-precipitation based high throughput screening to identify Icsbp-target-genes involved in this tumor suppressor effect. We defined an Icsbp-target-gene-set that is enriched for genes that control the activities of Fas and bcatenin. Previous studies determined that decreased Icsbp-expression contributes to the pathogenesis of chronic and acute myeloid leukemia. The goal of these studies is to use this information to identify therapeutic targets for the leukemia stem cell.

National Institute of Health (NIH)
Veterans Affairs (VA)
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Oncology A (ONCA)
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Jesse Brown VA Medical Center
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Hu, Liping; Huang, Weiqi; Bei, Ling et al. (2018) TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice. J Immunol 200:2129-2139
Shah, Chirag A; Broglie, Larisa; Hu, Liping et al. (2018) Stat3 and CCAAT enhancer-binding protein ? (C/ebp?) activate Fanconi C gene transcription during emergency granulopoiesis. J Biol Chem 293:3937-3948
Huang, Weiqi; Bei, Ling; Hjort, Elizabeth E et al. (2017) Decreased calpain activity in chronic myeloid leukemia impairs apoptosis by increasing survivin in myeloid progenitors and xiap1 in differentiating granulocytes. Oncotarget 8:50629-50641
Huang, Weiqi; Bei, Ling; Hjort, Elizabeth E et al. (2017) Decreased Calpain activity in chronic myeloid leukemia impairs apoptosis by increasing Survivin in myeloid progenitors and xiap1 in differentiating granulocytes. Oncotarget :
Hu, Liping; Huang, Weiqi; Hjort, Elizabeth E et al. (2016) The Interferon Consensus Sequence Binding Protein (Icsbp/Irf8) Is Required for Termination of Emergency Granulopoiesis. J Biol Chem 291:4107-20
Hjort, Elizabeth E; Huang, Weiqi; Hu, Liping et al. (2016) Bcr-abl regulates Stat5 through Shp2, the interferon consensus sequence binding protein (Icsbp/Irf8), growth arrest specific 2 (Gas2) and calpain. Oncotarget 7:77635-77650
Shah, Chirag A; Bei, Ling; Wang, Hao et al. (2016) Cooperation between AlphavBeta3 integrin and the fibroblast growth factor receptor enhances proliferation of Hox-overexpressing acute myeloid leukemia cells. Oncotarget 7:54782-54794
Huang, W; Luan, C-H; Hjort, E E et al. (2016) The role of Fas-associated phosphatase 1 in leukemia stem cell persistence during tyrosine kinase inhibitor treatment of chronic myeloid leukemia. Leukemia 30:1502-9
Wang, Hao; Bei, Ling; Shah, Chirag A et al. (2015) HoxA10 Terminates Emergency Granulopoiesis by Increasing Expression of Triad1. J Immunol 194:5375-87
Huang, Weiqi; Bei, Ling; Eklund, Elizabeth A (2013) Fas-associated phosphatase 1 mediates Fas resistance in myeloid progenitor cells expressing the Bcr-abl oncogene. Leuk Lymphoma 54:619-30

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