Identification of Genes Cooperating with Mpl Signaling Using Retroviral Insertional Mutagenesis.

Abstract 4558

The thrombopoietin receptor (Mpl) regulates the proliferation of hematopoietic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. Mutations in the Mpl gene have been shown to be associated with myeloproliferative neoplasms (MPN). We attempted to identify genes cooperating with Mpl signaling that could provide cell survival and proliferative advantage using MSCV based retroviral insertional mutagenesis coupled with an inducible Mpl signaling system. The retroviral Mpl vectors used in our screening system were based on the well-described retroviral vector MGIFM. That vector carried a bacterial shuttle plasmid coding for the Neo gene and a bacterial origin of replication in its 3′ untranslated region, and also coding sequences of a drug dependent, dimerizable, fusion protein containing the cytoplasmic domain of Mpl. This vector can interrupt gene structure through insertion, and its intact long terminal repeats can activate adjacent genes. The shuttle plasmid allows nonbiased recovery of proviral genomic integration sites (RIS). The retroviral Mpl vectors were used to transduce the human leukemia cell line K562. Selection for Mpl signaling dependent cells required blocking of the endogenous transforming Bcr-Abl kinase with imatinib and supplying the dimerizer drug, AP20187. Similar selection approach was applied in another leukemia cell line, a cytokine dependent UT7 cells, after transduction depriving them of exogenous cytokines and supplying the AP20187. In multiple independent transductions only a few percent of the transduced cells survived in the presence of AP20187, as assessed by the GFP expression, which was co-expressed with the Mpl fusion gene through an internal ribosome entry site. The surviving cells in long term culture were dependent on Mpl signaling and were sensitive to a Jak2 inhibitor, AG490. A wide variation in the mean fluorescent intensity of the surviving cells (from 37.5 to 238.8) in independent transductions indicated that a high level of Mpl expression was not the critical factor of selection. Transduction conditions were designed so that each experiment started with 30,000 independent insertion events from which selection of an Mpl-dependent cell population could be derived. Thirty six independent transductions have been performed with a presumed ∼ 1.89 × 10⁶ independent insertions in the initial non-selected, cell population. After selection, 668 retroviral insertion sites (RIS) have been recovered that represent 203 independent insertion events. Within each independent transduction, a small number of RISs were identified suggesting selection of insertion events that result in a proliferative advantage. This was not observed in an independent experiment in K562 cells, in which the cells were not switched to Mpl dependent growth. Insertion events that occurred in close proximity are considered to indicate a potential cooperating mutation that results in a proliferative and/or survival advantage to the cell. Insertional events in several instances fell in regions of known frequent chromosomal aberrations in MPN. The distribution of insertions appears to be non-random. Many sites are adjacent to genes that have been identified as regulators of apoptosis, proliferation and myeloid development (data not shown). We are validating this conclusion by performing a parallel screening of RIS in multiple (40) populations of independent transductions of the K562 and UT7 cells transduced with retrovirus/Mpl vector without selection on AP20187. This screening will provide sufficient data to statistically compare the outcome of the two sets of data, AP20187- selected versus non selected cell populations. To date, we have a number of candidate genes. The Discs large homolog-associated protein 1 (DLGAP1) gene is implicated secondary to three independent insertion events at 18p11.3. All insertions are in the fourth intron. This protein interacts with a known tumor suppressor and is being investigated as a possible novel hematopoietic regulator.