RNA INTERFERENCE BASED THERAPEUTIC INTERVENTIONS FOR LEUKEMIAS

Abstract

Leukemia, a heterogeneous group of hematological malignancies, continues to cause significant morbidity and mortality despite decades of research and development. Chromosomal aberrations are the main cause of leukemia and lead to the generation of fusion/chimeric genes, resulting in activation of proto-oncogenes and suppression of tumor-suppressor genes. Incidence of different aberrations associated with different leukemias varies in different regions of the world and the data from population based studies in South Asia, including Pakistan, are lacking. The expression of chimeric/fusion genes can be detected using sensitive molecular methods like reverse transcriptase polymerase chain reaction (RT-PCR) and dot blot hybridization. In this study, classic BCR-ABL t(9;22) variants (e13a2 and e14a2) were detected in 96% of CML patients, while one of the patient possessed a unique e13-1a BCR-ABL variant. A total of 68 patients of paediatric ALL, were screened by RT-PCR to determine the relative frequency of t(9;22), t(12;21), t(1;19), and t(4;11,). Translocation (9;22) was seen in 2/68 (3%) and t(1;19) in 2/68 (3) children. Seven children showed t(12;21) while 8 showed t(4;11) translocations. In AML patients, t(8;21) was found in 4/21 patients while t(1;19) was seen in only one of the patient out of 21 screened. Thus, there appears to be a significant under representation of the fusion transcripts for TEL-AML, a good prognostic marker, in this study, unlike in the West, where it is seen in 35% of children with ALL. This, together with the generally increased leukemic burden seen in Pakistani patients, may explain in part, the poor treatment outcome reported. Conventional therapeutic approaches for leukemias include chemotherapy, radiation therapy, interferon therapy, stem-cell transplantation and surgery but their application is limited due to their side effects. The advent of RNA interference (RNAi) technology has opened the door to previously unrefined methods of therapeutic interventions. Gene targeting of the chimeric genes by small interfering RNA is an ideal way to kill tumor cells specifically, while leaving the normal cells unaffected. In case of CML, the over- expressed protein, tyrosine kinase, from BCR-ABL fusion genes, trigger malignant transformation and abnormal proliferation of the cells. Although targeting the BCR-ABL tyrosine kinase activity by imatinib mesylate has rapidly become first-line therapy, drug resistance suggests that combination therapy directed to a complementing target may significantly improve treatment results. To identify such potential targets, we used lentivirus-mediated RNA interference (RNAi) as a tool for functional genomics in human leukemic as well as murine hematopoietic cell lines. Expression of STAT1 and STAT3 proteins was successfully knocked down using specific shRNAs targeting STAT1 and STAT3 mRNA. RNAi-mediated reduction of STAT1 and STAT3 protein expression inhibited BCR-ABL–dependent (K562) but not cytokine-dependent (32D) cell proliferation. The data indicate that BCR-ABL expression may affect the function of normal signaling molecules. Targeting these molecules may harbor significant therapeutic potential for the treatment of patients with CML.