PHF6 mutations in T-cell acute lymphoblastic leukemia

Abstract

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.

Figure 1

Next generation sequencing and array CGH analysis of the X chromosome identifies PHF6 mutations in human T-ALL. (a) Overview of mutation screening approach of the human X chromosome exome in a panel of tumor DNA samples from 12 male T-ALL cases using oligonucleotide sequence capture and next generation sequencing with SOLiD3. After filtering and confirmation of high throughput sequencing data, analysis of corresponding remission DNA samples led to the identification of three somatically acquired changes in the PHF6 gene. (b) Schematic overview of the recurrent genomic deletions involving chromosomal band Xq26.3 in 8 human T-ALL samples. Specific genes located in Xq26.3 are shown. (c) Detailed view of a representative oligo array-CGH plot of leukemia DNA/control DNA ratios (blue tracing) versus the dye-swap experiment (red tracing) in a patient harboring an Xq26.3 deletion. (d) DNA quantitative PCR analysis of PHF6 copy number dose in female and male reference genomic DNAs and 2 primary samples from male T-ALL cases harboring Xq26.3 deletions.

Figure 2

PHF6 mutations and expression in T-ALL lymphoblasts. (a) Structure of the PHF6 protein including four nuclear localization signals and two imperfect PHD zinc finger domains. Overview of all PHF6 mutations identified in primary T-ALL samples and T-ALL cell lines. Filled circles represent nonsense and frameshift mutations, whereas missense mutations are depicted as open circles. Circles filled in gray indicate mutations identified in female T-ALL cases. (b) Representative DNA sequencing chromatograms of paired diagnosis and remission genomic T-ALL DNA samples showing a somatic mutation in exon 7 of PHF6. (c) Western blot analysis of T-ALL cell lines revealed complete loss of PHF6 protein expression in the PHF6 mutated T-ALL cell lines. (d) PHF6 immunostaining in the Jurkat and HPB-ALL, wild-type and mutant T-ALL cell lines, respectively. (e) Western blot analysis of PHF6 and gamma-H2AX expression in HEK293T cells upon PHF6 shRNA knockdown. Actin levels are shown as loading control.

Figure 3

PHF6 expression in T-ALL lymphoblasts. (a) Sequence analysis of paired genomic DNA and cDNA samples shows monoallelic expression of PHF6 SNP rs17317724 in lymphoblasts from a wild-type PHF6 female T-ALL case. (b) Differential distribution of PHF6 mutations in T-ALL samples from male and female cases. (c) Immunohistochemical analysis of PHF6 expression in wild type and mutant T-ALL lymphoblasts.

Figure 4

Clinical and biological characteristics associated with PHF6 mutations in T-ALL. (a) Frequencies of PHF6 mutations in pediatric and adult T-ALL samples. (b) Differential distribution of PHF6 mutations in TLX1/TLX3 positive and negative T-ALL samples. (c) Kaplan-Meier curve of overall survival in pediatric T-ALL patients from DCOG trials ALL7, ALL8 and ALL9 with and without PHF6 mutations. (d) Kaplan-Meier survival curve in adult T-ALL patients with and without mutations in PHF6 treated in ECOG clinical trial ECOG2993.