Leukemia-Associated HSC Vascular Niche Is Negatively Regulated By PERK of Unfolded Protein Response (UPR)

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from early T cell progenitors. Diffuse infiltration of the bone marrow by T-ALL is associated with worse prognosis. We previously reported that actively proliferating leukemia cells inhibit normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. We found that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. However, the underlying molecular mechanism that leads to the suppression of hematopoiesis and decreased HSPC in the vascular niche is unclear. It has been demonstrated that rapid cellular proliferation associated with oncogenic activity such as MYC in T-ALL leads to a global increase in protein synthesis and an increase in misfolded/unfolded polypeptides in the endoplasmic reticulum (ER), referred to as unfolded protein response (UPR) or ER stress. Elevated ER stress leads to activation of at least three types of ER stress transducers through the release of inhibitory binding by glucose-regulated chaperone protein (GRP78/BIP): the protein kinase RNA-like ER kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription factor 6 (ATF6). Activation of PERK phosphorylates eIF2 to repress global translation with the exception of a small number of proteins including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis and genes in apoptosis. Here, we studied the role of UPR in the regulation of HSC niche function in the setting of T-ALL progression.

Using in vitro assays in which T-ALL leukemia cells driven by activated Notch1 (ICN1) were co-cultured with endothelial cells (MILE SVEN 1, MS1), and in vivo ICN1-driven T-ALL model, we found that PERK-eIF2a-ATF4 pathway was activated in both MS1 cells and BM endothelial cells isolated from T-ALL mice, while IRE1 and ATF6 pathways were only mildly altered. The activation of PERK was accompanied with the increased expression of Jagged1 and suppressed expression of CXCL12 in both cultured endothelial cells and bone marrow endothelial cells from leukemia mice. PERK inhibitor (GSK2606414) treatment of co-cultured cells largely restored CXCL12 expression, which was also negatively regulated by Jagged1, and accelerated the leukemia cell apoptosis as indicated by the enhanced annexin staining. These findings suggest that PERK is the upstream regulator of Jagged1 and CXCL12 in the endothelial cells; however, the function of cell-autonomous PERK on leukemia cell survival needs to be further clarified. To understand the role of PERK in bone marrow endothelium during leukemia development in vivo, we examined T-ALL leukemia progression and its effect on vascular niche function in VE-Cadherin^ERT2/PERK^F/F mice in which Perk was specifically deleted in endothelial cells. Consistent with in vitro findings, T-ALL development induced endothelial PERK-eIF2a-ATF4 activation, while up-regulated Jagged1 and down-regulated CXCL12 were also identified in isolated BM endothelial cells. Compared to the wild type mice, VE-Cadherin^ERT2/PERK^F/F mice showed attenuated leukemia progression, increased HSPC (Lin^-Sca-1⁺c-kit⁺) frequency, and improved survival.

Taken together, our findings suggest that PERK activation in BM endothelial cells is a key regulator of the leukemia vascular niche to promote leukemia progression and to suppress normal hematopoiesis. Therefore, targeting PERK may offer an effective strategy in restoring normal HSPC homeostasis and limiting leukemia progression.