Gene Expression Profile Reveals a Unique Pattern of Protein Kinases in Chronic Lymphocytic Leukemia (CLL): Potential Role of the Second Generation Protein Kinase Inhibitors.

Purpose. CLL is a malignancy of mature B cells with an heterogeneous clinical course. In order to identify novel therapeutic targets and given the role of protein kinase (PK) deregulation in cancer development and the availability of specific PK inhibitors, oligonucleotide arrays were used to determine if CLL patients exhibit a specific PK pattern. The results were validated by Q-PCR, and the in vitro efficacy of a dual specific inhibitor, dasatinib (Bristol-Myers Squibb, Princeton, NJ), was tested.

Methods. The gene expression profile of 44 CLL and 137 acute lymphocytic leukemia (ALL) patients was evaluated using the HG U133 Plus 2.0 Affymetrix arrays. Two additional sets of CLL (49 cases) were utilized as test sets: 505 PK genes were used for all analyses, which included unsupervised clustering, Analysis of Variance (ANOVA) and t-test analysis. To validate the gene expression data, a Q-PCR approach was used on 14 CLL, 6 B-lineage ALL, 3 T-ALL and CD19+ enriched normal B cells, isolated from peripheral blood of 3 healthy donors. Finally, to evaluate the in vitro effects of dasatinib on primary CLL cells, the MTT assay was performed on 21 untreated CLL samples following 72 and 96 hours of drug exposure.

Results. Unsupervised analysis on CLL samples and different ALL subgroups showed a very homogeneous PK gene profile in CLL. ANOVA corroborated these results. Among the PK that proved overexpressed in CLL, we identified 16 PK genes highly expressed in all 3 CLL sets analyzed. For these genes, Q-PCR analysis showed that CLL cases display a significantly higher expression level, when compared to B-lineage ALL, T-ALL and, more importantly, to CD19+ cells from healthy controls. PK expression was also analyzed within different CLL subclasses, subdivided on the basis of the IgV_H mutational status, as well as CD38 and ZAP-70 expression. The comparison among these groups did not show a specific PK signature associated with biologic features; similar results were obtained by Q-PCR analysis. To validate the gene expression profiling and Q-PCR findings, and to test the efficacy of dasatinib, functional in vitro experiments were performed on primary CLL cells. Treatment with 1μM dasatinib induced an overall viability reduction of 40% after 96 hours in the CLL samples analyzed; no significant differences were associated with the IgV_H mutational status. These findings indicate that this inhibitor is functionally active on CLL cells, without however reaching marked levels of cytotoxicity when used as a single agent.

Conclusions. Our results highlight a very homogeneous PK signature in CLL, independently of specific biologically-defined prognostic subgroups. A set of these PKs is more highly expressed in CLL cells than in CD19+ cells from healthy donors. Dasatinib treatment induces a ∼40% viability reduction after 96 hours exposure. Overall, these results suggest that PK inhibitors, namely dual kinase inhibitors, may have a role in the management of CLL patients particularly in combination with other therapeutic agents. * ST and SC equally contributed to the study