Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biology

Abstract

Leukemia Stem Cells (LSCs) from Acute Myeloid Leukemia (AML) require the activity of the transcription factor NF-kB to maintain stemness and drive tumor formation. Blocking NF-kB can preferentially eliminate LSCs in vitro with minimal effects on healthy Hematopoietic Stem and Progenitor Cells (HSPCs), making NF-kB a compelling target for anti-leukemia therapies. However, blocking NF-kB in vivo can only extend survival for a short period of time before transplanted mice succumb to the disease. I propose this is due to components of the in vivo niche supporting LSC survival and compensating for the inhibition of NF-kB.

I observed patients with partially differentiated blast-like AML (subtypes M3, M4, and M5) have significantly elevated levels of pro-inflammatory cytokines TNFa and IL-1a/b; (TNF and IL1) circulating in their peripheral blood. Further study revealed these cytokines are primarily produced by LSCs because such cells express several times more of TNF and IL1 than their healthy tissue counterparts. I found that TNF and IL1 stimulate the growth and expansion of LSCs while inducing cell death in HSPCs. Also, LSC-conditioned media alone is sufficient to drive apoptosis/necroptosis in HSPCs that can be prevented by blocking TNF and IL1, suggesting a mechanism for hematopoietic repression commonly observed in AML cases.

TNF and IL1 drive their pro-inflammatory effects on target cells through activation of cellular signaling networks. Both TNF and IL1 are potent activators of NF-kB in almost all cells studied, connecting the ability of these cytokines to drive LSC growth with the need of NF-kB for survival. In addition, TNF and IL1 also stimulate activation of JNK signaling which operates in parallel to NF-kB in LSCs and HSPCs. I found that JNK stimulation results in cell death in HSPCs by subsequent inactivation of pro-survival Bcl-2 family proteins by phosphorylation. However, LSCs convert JNK-mediated cell death signals into proliferation/survival signals by both limiting the signal duration to <60 min through dephosphorylation by Map Kinase Phosphatase 5, and also by upregulating JNK's nuclear target c-Jun. Such short duration of JNK activation correlates to activation of JNK's nuclear targets without activating the death signal.

I determined that concurrent inhibition of NF-kB and JNK has two major effects: (1) combined inhibition specifically targets LSCs in vitro and in vivo, and (2) the toxicity in healthy HSPCs due to loss of NF-kB signaling is mediated by JNK, making the combined treatment protective. I can substantially increase survival in AML-transplanted mice if they are treated with combination of NF-kB and JNK inhibition in vivo. I can further extend survival in leukemic mice when I treat with blockers to upstream pro-inflammatory cytokines: anti-TNF, anti-IL1, and NF-kB inhibitor. These data provide a strong rationale to treat AML patients by combined inhibition of both TNF/IL1- JNK and NF-kB signaling.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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