The Identification of Genes Cooperating with Mpl Signaling Using Insertional Mutagenesis

In addition to its role in megakaryopoiesis and platelet production, thrombopoietin activation of its receptor, myeloproliferative leukemia virus protooncogene (Mpl), results in hematopoietic stem cell (HSC) homeostasis and self-renewal. Mutations in the Mpl gene have been shown to be associated with myeloproliferative disorders. In an attempt to identify genes that could cooperate with Mpl signaling leading to an increase in cell survival and proliferation, we have used retroviral expression of a drug dependent, dimerizable, fusion protein which contains the cytoplasmic domain of Mpl. In previous experiments the expression of this protein in both mouse and human blood progenitors resulted in drug dependent hematopoietic cell expansion. We cloned a bacterial shuttle plasmid carrying the Neo gene and a bacterial origin of replication into the 3′ untranslated region of the well-described MSCV based retroviral vector MGIFM to produce the MGIFMNO vector. The shuttle plasmid allows nonbiased recovery of provirus genomic integration sites. This vector can interrupt gene structure through insertion, and has an intact long terminal repeat which can activate adjacent genes. We transduced the human leukemia cell line K562 with the MGIFMNO vector carrying inducible Mpl fusion construct and bacterial shuttle plasmid. The transduction efficiency and relative Mpl expression were assayed by GFP expression since GFP and the Mpl fusion gene are co-expressed through an internal ribosome entry site (IRES). After transduction, the endogenous, transforming Bcr-Abl kinase was blocked using imatinib, and cells dependent on Mpl signaling were selected by the addition of the dimerizer drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. A small proportion of the transduced cells (3%) survived in the presence of AP20187. The mean fluorescent intensity of the surviving cells was increased relative to non-selected, transduced cells indicating that selection required a high level of Mpl expression. The surviving cells in long term culture were dependent on Mpl signaling and were sensitive to a Jak2 inhibitor, AG490. We cloned retroviral integration sites from the established Mpl dependent population using the plasmid rescue method. A scoring system to assign relevance to isolated integration sites was developed, based on:

1. multiple independent recoveries,

2. relative distance from a target gene, and

3. proximity to a target gene that encodes a product involved in cell cycle, apoptosis, senescence, or gene expression regulation.

Cells that have a proliferative advantage and are dependent on Mpl signaling should contain synergistic mutations. Insertion sites in genomic regions appearing repeatedly in multiple experiments are considered highly significant. A preliminary screen of 46 positive integration clones revealed 17 independent integration sites, confirming feasibility of the screening method. Within the rescued integrations we identified possible candidate genes including:

1. the RSL1D1 gene, also labeled PBK1 – nucleostemin interacting protein (nucleostemin was suggested to regulate HSC proliferation),

2. the acidic nuclear phosphoprotein 32 family member B – cell cycle and survival factor,

3. the SH3-domain kinase binding protein 1 – Cbl interacting protein (Cbl was indicated in a repression of HSC self renewal),

4. and Angiotensin II receptor-associated protein isoform E.

Large scale screening of integration sites and experimental analysis of candidate genes are under way.