PHF6 is a novel regulator of B-cell identity in acute lymphoblastic leukemia

Abstract

Mutations in the zinc finger gene PHF6 are seen in approximately 20% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. The notable absence of PHF6 mutations in B-cell lineage malignancies has led to the hypothesis that PHF6 may act as a lineage-specific tumor suppressor gene. Recent work from our group described a role for PHF6 as a positive regulator of growth in B-cell acute lymphoblastic leukemia (B-ALL). To identify the mechanisms by which PHF6 acts to promote B-ALL growth in vivo, we utilized CRISPR-Cas9 to delete Phf6 in murine B-ALL cells. Transplantation of Phf6 knockout cells (Phf6KO) into immunocompetent recipients significantly extended disease latency and survival. Strikingly, these mice developed lymphomas, characterized by significantly enlarged lymph nodes, decreased disease burden in the spleen and increased expression of the canonical T-cell marker CD4, suggesting that Phf6KO B-ALL cells adopt alternate lineage programs in vivo. To dissect the molecular mechanisms by which Phf6 regulates this lineage decision, we carried out a combination of RNA and chromatin immunoprecipitation (ChIP-Seq) sequencing analyses in Phf6WT and Phf6KO cells. RNA sequencing analysis revealed many differentially expressed genes in Phf6KO B-ALL cells. Notably, genes and gene sets that were significantly down-regulated in Phf6KO cells included those involved in pathways important for B-cell development and function. ChIP-Seq analysis of PHF6 and several histone marks revealed that PHF6 and H3K27ac signals co-localize close to the transcription start site and enhancer regions of a significant proportion of differentially expressed genes. Transcription factor binding motif analysis revealed significant enrichment for several well-described transcriptional regulators of B-cell development. Importantly, we demonstrated that the transcription factors TCF12 and NF-kB co-immunoprecipitated with PHF6 in Phf6WT B-ALL cells. These findings discovered a novel role for PHF6 in the maintenance of B-cell identity in B-ALL, by activating genes that are crucial for B-cell lineage maintenance. Collectively, these results indicate that loss-of-function of Phf6 in B-ALL leads to an unstable cell identity state, in which cells need to acquire alternate developmental programs in order to survive. These findings could potentially explain the absence of PHF6 mutations in human B-cell lineage malignancies.