The Role of Hedgehog,p53 and Yap1 in chronic myelogenous leukemia and Friend vrus erythroleukemia stem cell self renewal
Open Access
- Author:
- Hegde, Shailaja N
- Graduate Program:
- Pathobiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 01, 2013
- Committee Members:
- Robert Paulson, Dissertation Advisor/Co-Advisor
Robert Paulson, Committee Chair/Co-Chair
Pamela Hankey Giblin, Committee Member
Anthony Paul Schmitt, Committee Member
Wendy Hanna Rose, Committee Member - Keywords:
- CML
Hedgehog
p53
Yap1
BCR-ABL - Abstract:
- Leukemia can be viewed as a newly formed, abnormal hematopoietic tissue initiated by a few leukemic stem cells (LSCs) that undergo a poorly regulated differentiation process compared to that of normal hematopoietic stem cells. A key property of LSCs, is the capacity for unlimited self-renewal. Leukemia cells are made up a heterogeneous mixture of cells, with varying potential to proliferate. At the apex of this developmental hierarchy are leukemia stem cells. These cells have ability to self-renew and can generate “bulk” leukemia cells that have lost their stem cell properties and have limited proliferative potential. Leukemia stem cells cause significant problems for conventional chemotherapy in that they are quiescent cells that can evade cytotoxic chemotherapy regimens. They express P-Glycoprotein a multi drug resistance protein, which actively transports drug out of the cell. For example, chronic myelogenous leukemia (CML), is caused by a 9:22 translocation that generates the BCR-ABL fusion gene encoding a constitutive active tyrosine kinase. The routine care for CML relies on tyrosine kinase inhibitors (TKIs) like either imatinib or nilotinib that inhibit BCR-ABL kinase activity. CML stem cells are resistant to killing by these drugs and following treatment, surviving LSC’s lead to disease relapse. Inducing differentiation of LSC’s is a potential means of treatment, but finding a potent differentiation inducer for each leukemia is difficult. Therefore in order to find new therapies that specifically target LSCs and cause no side effects it is important to understand the mechanism of LSC self renewal. The focus of this proposal to determine the gene regulatory network that mediates self renewal in CML LSCs. The significance of this proposal is that further characterization of these self regulatory network in LSCs will identify specific targets which will aid in the development of a therapy that eradicate LSCs. Towards this approach, in the second chapter we included initial studies I have done concerning the isolation and characterization of LSCs in Friend virus erythroluekemia model. Friend virus induced eryhthroleukemia is a robust model for multistage leukemogenesis and abnormal erythropoiesis. During viral pathogenesis early erythriod progenitors are infected and rapidly expanded, a process which is dependent upon interaction between the viral envelope glycoprotein gp55 and the cellular receptors, Sf-STK and Erythropoietin receptor. Previous work from our laboratory demonstrated that Friend virus activates bone morphogenic protein (BMP4) dependent stress erythropoiesis pathway. This leads to rapid expansion of stress erythriod progenitors, which are the targets for Friend virus in the spleen. In this chapter we show that two stages of Friend virus induced disease is caused by infection of distinct stress erythriod progenitors in the spleen. In the initial stage Friend virus target cells will form Epo-independent BFU-Es and in the later stage target cells will develop as LSCs. These later target cells possess intrinsic self renewal ability which is exploited by LSCs. Although mutation of p53 is thought to be an important step in Friend erythro-leukemia progression, we showed that mutation of p53 is not required for disease progression but proviral insertion into Spi1/Pu.1 is necessary. In addition Hedgehog signaling plays very important role. We were able to show that two signals are required for the self-renewal for the self-renewal of Friend virus LSCs, proviral insertional activation of Spi1/PU.1 and Hedgehog-dependent signaling. These data in chapter 2 establish a new model for Friend virus induced erythroluekemia and demonstrate the utility of Friend virus as a model system to study LSC Self-renewal. In the third chapter I extended my study to investigate LSCs in CML. For the first part of this study I established a murine model of BCR-ABL induced leukemia in our lab. We isolated CML LSCs from the spleen by sorting them with cell surface markers. We showed that CML LSC’s were Lin-, Kit+, Sca1+ and CD133+. CML LSC’s were further characterized by cell division status using PKH26 a membrane dye. We were able to isolate LSC population as Lin-Kit+Sca+CD34+CD133+PKH26high cells that are relatively quiescent. Transplantation of these cells caused disease in irradiated recipients, whereas Lin-Kit+Sca+CD34+CD133-PKH26low cells are progenitors which did not cause disease when transplanted. In the second part of this chapter I have focused on investigating the mechanism of the leukemia stem cell self-renewal. Here we show that Yap1, Yes associated protein 1 a human ortholog of the Drosophila Yorkie, plays an important role in self-renewal of CML LSCs. Yap1/Yorkie is the terminal target of the Hippo signaling pathway. Hippo signaling pathway regulates organ size in Drosophila. Over expression of Yap1 can transform cells and Yap1 expression is required for ES cell self renewal. We observed LSC growth inhibition in Yap1shRNA knockdown studies whereas conditions leading to induced expression of Yap1 lead to increased LSC self renewal. Based on the results we have developed a new model for LSC self-renewal, where HH signaling promotes self-renewal by inhibiting p53 activity. Down regulation of p53 results in expression of YAP1, this results in inhibition of genes that promote differentiation and induces self-renewal. Furthermore we show that expression of BCR-ABL inYap1 knock down cells fails to induce CML in mice. Taken together our data suggest that Yap1 could be a potential target for new therapies to eradicate CML Leukemic stem cells. This discovery of the HH-p53-Yap1 gene regulatory network and its role in LSC self renewal will help to find new target for therapeutic invention to treat CML.