Both NF-?B and Stat3 are abnormally activated in leukemic blasts and are implicated in drug-resistance and poor prognosis, suggesting they could be potential targets for therapy. We found that inactivation of both NF-?B and Stat3 signaling pathways synergistically represses self-renewal and drug-resistance in leukemia stem cells (LSCs), suggesting a compensatory role for these two pathways in the pathogenesis of leukemia. Stat3? and Stat3? are two major splicing isoforms. Active Stat3? promotes tumor growth by regulating target gene expression (functions as a transcription factor) and controlling mitochondrial production of ATP and ROS (functions as a regulator of the electron transport chain), while Stat3? lacks a transactivation domain and functions as a dominant-negative to Stat3?. All currently used inhibitors of Stat3 only repress its transcriptional activity without taking consideration of its mitochondrial activity, which might explain why these inhibitors failed to repress leukemia in patients. It was reported that induction of the switch from Stat3? to Stat3? provides a better tumor repressive effect than inhibition of both isoforms. We found that we can induce such a switch by inhibiting the serine/threonine-protein kinases receptor-interacting protein kinase 1 (Rip1) and Rip3. Rip3 and NF-?B are parallel downstream signaling pathways of Rip1, mediating cytokine-induced kinase- dependent and -independent activities of Rip1. We found that a moderate level of activation of Rip1-Rip3 kinase signaling exists in acute myeloid leukemia (AML) cells with MLL1-rearrangement (MLL-r) or NPM1 mutation (NPM1c+). Rip1-Rip3 signaling plays distinct roles in normal hematopoietic stem/progenitor cells?HSPCs?and AML cells. In HSPCs, Rip1-Rip3 signaling mediates TNF? and IL1?-induced necroptosis, while in AML cells, the moderate activation of such signaling is required for maintaining the levels of Stat3? by inhibition of calpain (CAPN), a family of proteolytic enzymes. CAPN reduces Stat3? and enhances Stat3? by specifically cleaving Stat3? protein and also SFRS5, a splice regulator for alternative splicing for Stat3?. Inhibition of Rip1-Rip3 kinase signaling results in depletion of Stat3? and an increase of Stat3?. Our study suggested that, as with co-inhibition of Stat3 and NF-?B, co-inhibition of Rip1-Rip3 signaling and NF-?B also compromises self-renewal of LSCs and sensitizes AML to standard chemotherapy. We want to test our novel combination treatment regimen in primary human AML cells using xenograft models. We also intend to elucidate the molecular mechanisms by which Stat3 and NF-?B regulate self-renewal and drug-resistance in LSCs as well as the molecular mechanism by which Rip3 signaling regulates CAPN-dependent Stat3 isoform switch. The expected results of this study will allow us to determine whether combinations of currently known inhibitors of Rip1/Rip3 and NF-kB signaling could improve treatment for MLL-r and NPM1c+ AML when combined with standard chemotherapy. The mechanistic studies will provide detailed information allowing us to more effectively target the Rip3-CAPN-Stat3 pathway to treat AML.

Public Health Relevance

Inflammatory cytokine-stimulated Rip1-Rip3 kinase pathway induces necrosis in normal tissue cells but promotes the growth and drug-resistance in leukemia cells. Co-inhibition of the Rip1-Rip3 pathway and NF-?B, a well-known survival factor for leukemia stem cells, sensitizes leukemic cells to standard chemotherapy and at the same time provides protection to normal tissue cells. We want to evaluate such novel combination treatments in human primary leukemic cells using xenograft animal models.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA223194-03
Application #
9922242
Study Section
Tumor Cell Biology Study Section (TCB)
Program Officer
O'Hayre, Morgan
Project Start
2018-05-01
Project End
2023-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Loyola University Chicago
Department
Pathology
Type
Schools of Medicine
DUNS #
791277940
City
Maywood
State
IL
Country
United States
Zip Code
60153