Klin Padiatr 2016; 228 - A12
DOI: 10.1055/s-0036-1582489

Aberrant transcriptional pathways in t(12;21) Acute Lymphoblastic Leukemia

A Sundaresh 1, L Gasparoli 1, M Mangolini 1, D Edwards 1, 2, M Hubank 3, T Brooks 3, J Bartram 2, N Goulden 2, P Ancliff 2, J de Boer 1, O Williams 1
  • 1Developmental Biology and Cancer Section Programme, UCL Institute of Child Health
  • 2Great Ormond Street Hospital
  • 3Centre for Translational Omics, UCL Institute of Child Health, London, UK

Introduction: The single most frequent chromosomal translocation associated with childhood Acute Lymphoblastic Leukemia is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy. Recent data from our lab has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Studies have shown that STAT3 regulates SMAD7 gene expression in other human cancers. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways.

Methods: Lentiviral-mediated shRNA knockdown of SMAD7 in ALL cell lines was verified by qRT-PCR and Western blot analysis. The functional effects of the knockdown were analysed using cell cycle, colony forming ability and apoptosis assays. RNA isolated from knockdown cells were subjected global gene expression analysis using Ilumina NextSeq 500 and results analysed using Strand NGS software and Ingenuity Pathway Analysis. The role of SMAD7 in leukaemia progression in vivo was studied using bioluminescence xenograft models.

Results: We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. Furthermore, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. We also observed growth arrest following loss of SMAD7 in other ALL and AML subtypes. Silencing of SMAD7 in TEL-AML1+ ALL cells transplanted into immunodeficient mice significantly impaired disease progression in vivo, resulting in prolonged disease latency. Global gene expression analysis revealed SMAD7 to be a regulator of cholesterol biosynthesis, a pathway critical for lymphoblastic leukaemia cell survival. Further experiments validated the mechanism for transcriptional regulation of this pathway and identified pharmacological inhibitors capable of blocking this pathway.

Conclusion: Our data indicate although STAT3 regulation of SMAD7 is exclusive to TEL-AML1+ ALL, SMAD7 has a more general, critical role in leukaemia survival. RNA-seq analysis indicates that SMAD7 regulates cholesterol biosynthesis through a transcriptional mediator that is pharmacologically targetable. Therefore, we have established a novel transcriptional pathway operating specifically in t(12;21) ALL but regulating downstream pathways essential for ALL in general.