Regulatory mechanisms contributing to the homeostasis of normal and malignant hematopoietic cells

Abstract

Bone marrow is the site of origin of diverse population of hematological cells with lineage specific function. The different lineages of cells originate from a common progenitor that has a strong self-renewable capability giving rise to myeloid and lymphoid progenitor cells. The myeloid progenitor cells terminally differentiate to monocytes, granulocytes, erythrocytes and thrombocytes, while the lymphoid progenitor cells terminally differentiate to B, T and NK cells. A plethora of extracellular and intracellular factors in a complex yet tightly regulated orchestra controls the genesis and function of each cell type. However, breakdown of one or more regulatory processes can steer cells towards becoming cancerous, characterized by the loss of normal cellular functions and/or morphology. This thesis analyses; The contribution of p53 in the regulation of normal T cell homeostasis; A mechanism evolved by EBV for its sustenance in B cells and, a mechanism contributing to the development/survival of malignant hematopoietic cells.

The first half of the thesis describes the contribution of the tumor suppressor p53 in the regulation of T cell homeostasis. Paper I demonstrates the regulation of pro-apoptotic SAP by p53 in activated T cells. p53 and SAP were upregulated in activated but not in unstimulated T cells. To determine the effect of p53 on SAP expression, p53 was selectively induced by nutlin-3 in unstimulated T cells. This induced SAP mRNA and protein expression. Further, chromatin immuno-precipitation confirmed the binding of p53 to the SAP promoter in activated T cells. Paper II demonstrated the cMyc-p53 feedback mechanism contributing to T cell homeostasis. cMyc, p53 and p14ARF expression was induced in activated T cells. Further elevation of p53 in activated T cells by nutlin-3 treatment decreased cell proliferation, cMyc and p14ARF expression. At the other end of the feedback loop, inhibition of Notch (DAPT) signaling or cMyc (10058-F4) in activated primary T cells decreased proliferation accompanied by decreased cMyc, p14ARF and p53 expression. Thus, inhibition of cMyc expression had a negative outcome on p53 expression, and induction of p53 had a negative consequence on cMyc expression in activated T cells. Nutlin-3 induced p53 and 10058-F4 mediated cMyc inhibition in activated T cells had a combined effect of apoptosis and cell cycle arrest. On the contrary, activated T cells treated with nutlin-3 or cMyc inhibitor (10058-F4) retained the cytotoxic function despite the diminished proliferation.

The second half of the thesis explores mechanisms that virus infected and tumor cells exploit for their manifestation. In paper III we studied the role of CD4+T cells in the establishment and regulation of EBV latency. Our results showed that activated CD4+T cells from healthy donors downregulated the latency III protein EBNA2 (and its transcript, Cp) in LCLs, thus indicating that they induce a shift in latency towards II. By exploiting the transwell system we demonstrated that cell-cell contact is not essential to induce the latency shift, and that soluble factors produced by activated CD4+ T cells contributed to this effect. We also identified two soluble factors, IL21 and soluble CD40L that facilitates the latency shift.

In paper IV we analyzed the effects of IFNγ on BCL6 expression in chronic myeloid leukemia and multiple myeloma cells. IFNγ or imatinib treatment alone marginally induced BCL6 expression in CML cell lines and primary CML stem cells. On the contrary, combined treatment with IFNγ and imatinib induced remarkable BCL6 induction in a direct, STAT1 dependent manner. We also demonstrated that the sustained STAT5 activation is responsible for the low BCL6 expression in IFNγ only treated CML cells. IFNγ treatment alone in MM cells strongly induced BCL6 expression through STAT1 signaling. Interestingly, IFNα only marginally upregulated BCL6 in MM cells in spite of the strong STAT1 and STAT3 activation. We show that this effect is consequence of the concomitant IFNα induced phosphorylation of STAT5. Our finding regarding BCL6 upregulation, elucidates the adverse effect of IFNγ on the outcome of tyrosine kinase inhibitor therapy in CML. Furthermore, this may partially explain the inefficient therapeutic effects of IFNγ in MM.