SOX4 enables Oncogenic Survival Signals in Acute Lymphoblastic Leukemia
Authors: Ramezani-Rad Parham , Geng Huimin , Chan Lai N , Hurtz Christian , Jumaa Hassan , Melnick Ari , Paietta Elisabeth , Carroll William L. , Willman Cheryl L , Lefebvre Véronique , Muschen Markus 
Published: October 2019

Abstract
Abstract 863

Background:
The SOX4 (SRY-related HMG-box) transcription factor is expressed in early B- and T- cell development. In the absence of SOX4, B cell development is arrested at the pro-B to pre-B cell transition. In Sox4−/− mice, pro-B cells fail to proliferate in response to IL7 and to expand and differentiate past the pre-B cell receptor checkpoint. Interestingly, SOX4 functions a transcription factor yet closely interacts with membrane-proximal cytokine receptor signaling. The PDZ domain-containing adaptor protein syntenin (SDCBP) recruits the Sox4 protein directly to the cytoplasmic tail of the cytokine receptors. With regard to its receptor-proximal activation, Sox4 resembles the SMAD and STAT transcription factors. A role for Sox4 in acute myeloid leukemia was proposed based on the identification of viral insertions, and Sox4-overexpression causes myeloid leukemia. In this study, we found SOX4 was strongly upregulated upon tyrosine kinase inhibitor (TKI) treatment in Ph+ ALL and high expression of Sox4 correlates with poor clinical outcome of patients with ALL.

Results:
To elucidate the potential contribution of Sox4 to BCR-ABL1-driven B cell lineage leukemia, we studied a conditional Sox4 knockout mouse model. To this end, we transformed bone marrow B cell precursors from Sox4fl/fl mice with BCR-ABL1 to test its function in a mouse-model for Ph+ ALL. Tamoxifen (4-OHT)-inducible activation of Cre and subsequent deletion of loxP-flanked Sox4 alleles in B cell precursor ALL resulted in reduced mRNA levels of multiple genes that play a known role in survival signaling in Ph+ ALL cells including BCLXL (BCL2l1), Mapk8, Map2k6, PI3K/AKT (Pik3r2, Pik3r3, Rps6ka2, Mtor). Overexpression of Sox4 had the opposite effect and resulted in increased mRNA levels of BCLXL (Bcl2l1), Mapk8, PI3K/AKT (Pik3r2, Pik3r3). Single-locus quantitative ChIP confirmed binding of SOX4 to BCL2l1, PIK3R3 and MAPK8 promoters in human Ph+ ALL cells via its putative DNA binding motif. Transcriptional regulation of components within the PI3K/AKT pathway is indeed functionally relevant since Cre-mediated deletion of Sox4 caused multiple de-phosphorylation events including AKTS473, SRCY416 and RPS6S235/6. In addition, inducible deletion of Sox4 resulted in strong upregulation of both Arf and p53 protein, suggesting that Sox4 mediates survival signaling both by activation of BCLXL (Bcl2l1) and repression of Arf/p53. Consistent with Sox4-mediated negative regulation of Arf/p53, deletion of Sox4 resulted in G0/G1 arrest of BCR-ABL1 ALL cells and loss of self-renewal capacity, failure to form colonies in methylcellulose replating assays and delayed the onset of disease and substantially prolonged overall survival of recipient mice. In agreement with reduced expression of BCLXL (Bcl2l1) and increased levels of Arf/p53, the overall outcome of Sox4 deletion is rapid loss of viability and apoptosis in BCR-ABL1-transformed ALL but not normal pre-B cells. Loss of viability upon Sox4 deletion in the leukemia cells was rescued by transduction with constitutively active (myristoylated; CA) mutants of AKTCA and the p110 catalytic subunit of PI3K (p110αCA) and as well with BCLXL.

Clinical relevance:
These findings are also relevant to human disease since promoter CpG methylation analysis (HELP assay) revealed lower levels SOX4 promoter methylation in human Ph+ALL cells compared to their normal pre-B cell counterparts. In addition, samples from patients with high risk ALL (n=31) show a trend towards higher SOX4 mRNA levels compared to patients with intermediate risk ALL (n=23, p=0.07; ALL REZ BFM 2002). Analyzing data from a larger study based on 207 patients with ALL (COG P9906) showed a statistically significant association between high mRNA levels of SOX4 and poor clinical outcome (shorter overall survival).

Conclusion:
Collectively, these findings identify SOX4 as a critical upstream regulator of survival signaling in Ph+ ALL. Pathways affected by SOX4 include PI3K/AKT signaling downstream of BCR-ABL1, activation of BCLXL (BCL2L1) and negative regulation of Arf and p53.

Disclosures:
No relevant conflicts of interest to declare.