The endoplasmic reticulum (ER) is an intracellular organelle for protein folding, lipid synthesis and Ca²⁺ storage. It is also responsible for the transportation for most of the secretory and transmembrane proteins. When the protein load exceeds the ER folding capacity, the ER undergoes stress and activates a set of signaling cascades that is termed the unfolded protein response (UPR). The multifunctional GRP78 is the major ER molecular chaperone with protein folding abilities and the master regulator of the UPR, and recently has been shown that a subfraction of it is localized on the cell surface acting as a co-receptor for various signaling pathway activation. ❧ Traditionally GRP78 is regarded as protective against hypoxia and nutrient starvation prevalent in the microenvironment of solid tumors, thus, its role in the development of hematologic malignancies remains to be determined. In this thesis, elevated GRP78 expression was detected in leukemic blasts of adult patients, leukemia cell lines and inversely correlates with time to relapse in childhood acute lymphocytic leukemia. To directly elucidate the requirement of GRP78 in leukemogenesis, we created a biallelic conditional knockout mouse model of GRP78 and PTEN in the hematopoietic system. Strikingly, heterozygous knockdown of GRP78 in PTEN null mice is sufficient to restore the hematopoietic stem cell (HSC) population back to the normal percentage and suppress leukemic blast cell expansion. AKT/mTOR activation in PTEN null bone marrow cells is potently inhibited by Grp78 heterozygosity, corresponding with suppression of the PI3K/AKT pathway by GRP78 knockdown in leukemia cell lines. This is the first demonstration that GRP78 is a critical effector of leukemia progression, at least in part through control of oncogenic AKT signaling. Furthermore, overexpression of GRP78 renders human leukemic cells more resistant to AraC-induced apoptosis whereas knockdown of GRP78 sensitizes them, suggesting GRP78 is a novel potent therapeutic target for leukemia. ❧ Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression and interactions with extrinsic factors in the microenvironment. GRP78 has been shown to be critical for the maintenance of cellular homeostasis and prevention of apoptosis. Homozygous knockout mice of GRP78 are embryonic lethal at E3.5, indicating GRP78 is essential for embryonic cell growth and pluripotent cell survival. However, this has not been investigated in adult hematopoietic stem cells. Here we generated a conditional knockout mouse model that acutely deletes GRP78 in the hematopoietic system. GRP78 deficiency results in a significant reduction of HSCs, progenitor and lymphoid cell populations yet an increase in myeloid lineage granulocytes and monocytes in cKO mice. The GRP78-null induced reduction of the HSC pool can be attributed to enhanced apoptosis. In agreement, GRP78 deficient BM cells exhibited activated UPR signaling in all three branches and induced expression of pro-apoptotic CHOP and caspase activation. Transplanting Grp78 KO hematopoietic cells into WT microenvironment also yielded a decrease in HSCs, suggesting that GRP78 in hematopoietic cells is required for HSC survival and homeostasis. In addition, a multiplex cytokine assay reveals alteration in several cytokine serum levels in cKO mice, suggesting GRP78 plays a pleiotropic role and is essential in regulating hematopoietic system homeostasis and maintenance.
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.