Fig 2: Loss of Phf6 results in decreased dependence on the driving oncogene BCR–ABL1 in vivo. (A) GSEA plots depicting significant changes in the targets of the BCR–ABL1 fusion signature (P = 0.0129) and dasatinib resistance signature (P = 0.0056) upon Phf6 deletion, as compared with Phf6WT cells. (NES) Normalized enrichment score. (B) Kaplan-Meier survival analysis of mice injected with 106 B-ALL cells of the indicated genotypes with subsequent mock or ponatinib treatment (30 mg/kg daily for four consecutive days). The number (n) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P-value is shown for the comparison. (C) Kaplan-Meier survival analysis of mice injected with 106 B-ALL cells of the indicated genotypes with mock or 10 mg/kg doxorubicin (single immunoprecipitation dose) treatment. The number (n) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in the indicated comparisons. P-value is shown for the comparison.

Fig 3: PHF6 exerts transcriptional regulation by interacting with histones rather than binding sequence-specific DNA sites. (A) Pie chart showing the distribution of 77,749 PHF6-binding sites across genomic regions in B-ALL cells. (TTS) Transcription termination site; (UTR) untranslated region. (B) De novo DNA sequence motifs identified in PHF6-bound regions at promoters of differentially expressed genes with their associated P-values. Shown are sequence logos of de novo position-weight matrices found by the MEME motif discovery tool (left) or those of known transcription factors whose motifs are found to be most similar to the de novo motif discovery results by Tomtom software (right). (C) Endogenous coimmunoprecipitation (co-IP) assay of TCF12, NF-κB, and PHF6 in the absence (top) or presence (bottom) of ethidium bromide (EtBr). Input is 7% of immunoprecipitation lysate. (D, left) Metagene tracks of PHF6 ChIP-seq signal averaged over all promoter–TSS tracks grouped by relative expression levels. (Red) High; (green) genomic; (purple) low. (Right) Metagene track of H3K27ac ChIP-seq signal averaged over all promoter–TSS regions. Shaded regions around average tracks denote estimates of 95% confidence interval (CI) of the metagene average signals based on resampling. (E) Metagene tracks of PHF6 ChIP-seq signal and correlation with histone marks: H3K27ac (blue), H3K4me3 (yellow; GSE66234), H3K27me3 (green). Pearson correlation of PHF6 and histone ChIP-seq signals across 10-kb regions spanning the TSS. Differentially expressed genes (solid line) and genome-wide genes (dotted line) are shown. (F) Endogenous co-IP assay of histone H3 and PHF6 in the absence (top) or presence (bottom) of EtBr. Input is 7% of immunoprecipitation lysate.

Fig 4: Model of PHF6 as a chromatin state regulator, permitting transcription factor binding through chromatin accessibility. (A) In wild-type cells, PHF6 binds to the ±1 nucleosome flanking the open TSSs of genes, allowing B-cell-specific transcription factors to bind, drive gene expression, and maintain B-cell identity. Conversely, PHF6 binds nucleosomes surrounding the TSSs of T-cell-specific genes, coordinating chromatin compaction and thus blocking the binding of T-cell-specific transcription factors. (B) In the absence of PHF6, chromatin is no longer maintained in an open state, B-cell transcription factors cannot bind, and expression of B-cell identity genes is down-regulated. However, T-cell identity genes are no longer inaccessible, allowing T-cell-specific transcription factors to bind and activate aberrant transcriptional programs.

Fig 5: Phf6 loss decreases the leukemogenic potential of cells in vivo and triggers a change in disease presentation. (A) PHF6 is a lineage-specific regulator of tumor growth in B-ALL and T-cell acute lymphoblastic leukemia (T-ALL). (B). Kaplan-Meier survival analysis of mice injected with either 103 (dotted) or 106 (solid) Phf6WT (blue) and Phf6KO (red) B-ALL cells. The number (n) of mice per genotype analyzed is shown. Statistical analysis (log-rank test, Mantel-Cox) was performed for the different groups in comparison with mice injected with Phf6WT cells. P-values are shown for the comparisons. (C) Representative hematoxylin and eosin (H&E) (top) and immunohistochemistry (bottom) staining of serial sections from lymph nodes (LNs) and lymphoma (mass) of recipient mice injected with Phf6WT, shPhf6, and Phf6KO cells. mCherry immunochemistry demarcates tumor cells. Bars, 600 µm. (D, top) Size comparison of representative LNs from Phf6WT (left) and Phf6KO (right) recipient mice. (Bottom) Quantification of combined LN weight of Phf6WT (blue; n = 5) and Phf6KO (red; n = 5) recipients. (E) Tumor burden in the blood of Phf6WT (blue; n = 7) and Phf6KO (red; n = 8) recipient mice. mCherry demarcates tumor cells. (F) Bar graphs showing the percentage of the CD4+ fraction among mCherry+ cells isolated from Phf6WT (blue; n = 9) and Phf6KO (red; n = 5) tumors in bone marrow (left) and LNs (right). Data represent the mean ± standard deviation (SD) in D–F. Statistics were calculated with two-sided Student's t-test. (***) P < 0.001; (****) P < 0.0001.