Targeting Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia with a Novel Transcriptional Inhibitor

Philadelphia chromosome positive acute lymphoblastic leukemia (Ph⁺ALL) is caused by reciprocal translocation between chromosome 9 (region q34) and chromosome 22 (region q11). This translocation results in oncogenic BCR-ABL fusion gene that encodes a chimeric BCR-ABL protein, continuously activated tyrosine kinase which causes unregulated cell division and leukemia. Although tyrosine kinase inhibitors (TKIs) are the most useful treatment against Ph⁺ALL patients, a part of them steadily become refractory to current therapy mainly through acquired mutations in ATP binding sites of BCR-ABL, necessitating a novel strategy to treat TKI resistant Ph⁺ALL. Here we show that our novel small molecule which targets specific transcriptional factor induces Ph⁺ALL cell death through directly controlling the expression of BCR-ABL gene. We have previously reported the fundamental requirement of transcription factor A (TFA) in the development of acute myeloid leukemia (AML), another form of acute leukemia originating from myeloid progenitor cells. Since previous reports suggest the involvement of TFA family genes in the development of lymphoid cells also, we first addressed the role of TFA in Ph⁺ALL cells using tetracycline-responsive short hairpin RNA (shRNA)-mediated gene knockdown system. Upon doxycycline treatment, shRNAs targeting TFA (sh_TFA)-transduced SU-Ph2 (Ph⁺ALL) and SU/SR (SU-Ph2 with BCR-ABL T315I mutation, therefore resistant to Imatinib) cells showed decreased proliferation rate with cell cycle arrest at G₀/G₁ phase and profound apoptosis. Intriguingly, TFA knockdown suppressed the expression of BCR-ABL fusion gene both at mRNA and protein levels. Concomitant down-regulation of BCR-ABL cytoplasmic gene targets such as mTOR-AKT, STAT and MEK-ERK pathways were also observed in sh_TFA-transduced ALL cells. These results indicate the possible interaction between BCR-ABL and TFA.

Since expression of BCR-ABL fusion gene is tightly regulated by gene promoter of BCR andthere exists a TFA-consensus binding site in the proximal promoter region of BCR, we next addressed the TFA-mediated transcriptional regulation of BCR-ABL. In silico data analysis of gene expression array sets extracted from ALL patients revealed the positive correlation of BCR and TFA expressions. Analysis of chromatin immunoprecipitation and sequencing (ChIP-Seq) in mouse ES cells disclosed the potential TFA binding in the proximal regulatory region of BCR, which we have confirmed by ChIP assay in SU/SR cells. These findings suggest that TFA positively and directly controls BCR-ABLexpression through BCR promoter binding in ALL cells.

We next sought to explore the effect of TFA inhibition in ALL cells in vivo. Immunodeficient NOG mice transplanted with SU/SR cells that are transduced with sh_TFA or control vector were followed for overall survival and chimerism of ALL cells in the peripheral blood. As expected, TFA inhibition decelerated the dissemination of leukemia and prolonged overall survival period in these mice.

We next challenged this ALL-xenografted mice with newly synthesized small compound which irreversibly attenuates the function of TFA as a transcription factor. Existence of T315I mutation in BCR-ABL gene in SU/SR cells naturally provides TKI-therapy resistance to these mice, and oral Imatinib treatment at 100 mg/kg body weight did not prolonged their survivals actually. In contrast, our novel compound was highly effective in these mice at 375 microgram/kg body weight and significantly prolonged the overall survival period without serious side effects, overcoming TKIs resistance. These findings collectively underscore the importance of TFA in the maintenance of ALL cells with resistance to TKIs and is a druggable target in the leukemia therapy.