Transcriptosome Profiling of B-CLL Identifies WNT-3A and ROR-1 as an Autocrine Mechanism in Cell Survival.

Background: B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia afflicting the Western world. B-CLL accounts for 25% of all newly diagnosed leukemias. Despite many new therapeutic advances, B-CLL is still not a curable malignancy. The hallmark feature is the presence of an elevated number of circulating clonal leukemic B cells that typically express CD 5, CD 19, CD 23, and low levels of surface immunoglobulins.

Methods: Mononuclear cells from 5 patients were analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from normal peripheral blood (PB) B-cell (AllCell, CA). Immunohistochemistry (IHC) confirmed tumor lineage and quantitative real time RT-PCR was performed on selected genes to validate the microarray study. The GEP data was processed and analyzed utilizing Affymetrix MAS 5.0 and GeneSpring 5.0 software. Our data was analyzed in the light of published GEP of B-cell CLL. Fifteen B-CLL patients (retrospectively) were evaluated by RT-PCR for ROR-1 and WNT-3A with gene specific probes. As a potential therapy, thalidomide was evaluated on B-CLL cells grown in cell culture for 24 hours. GEP of the thalidomide treated B-CLL from the initial 5 patients was performed to look for gene expression changes that could drive the B-CLL toward apoptosis. A homology model of ROR-1 tyrosine kinase was built, ATP docked and in silico databases screened for potential lead molecules.

Results: Data are represented as “robust” increases or decreases of relative gene expression common in the 5 patients. However, ROR-1 and WNT-3A were consistently over-expressed together in these 5 patients. The average increase was 25-fold for ROR-1 and 7-fold by WNT-3A when compared to normal B-cell RNA. Of the 15 patients we evaluated for ROR-1 and WNT-3A with gene specific probes, the increase in gene expression correlated well with our initial gene expression profiling study. Thalidomide specific gene changes included several molecules involved in apoptosis. Of these gene changes, Bcl-G, p35, and Cdk-5 were up-regulated several fold. Data will be presented on the influence of the stage of disease on ROR-1 and WNT-3A expression.

Conclusions: GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel therapeutic targets for therapy based on a comparison to normal (B-cell) RNA. GEP has identified ROR-1 as a key component in an autocrine pathway that helps B-CLL elude apoptosis. The identification of this novel tyrosine kinase-like protein has led to the development of a molecular target for future therapeutic applications. Several lead compounds have been identified and are being evaluated as potential therapeutics in B-CLL.