Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

Clinical impact and mechanistic contributions to leukemogenesis are difficult to assign to less common somatic mutations. However, the genetics of inherited syndromes can often be helpful in discerning the biological functions and mechanistic consequences of genes in other diseases. PHF6 (Xq26.2) encodes a protein consisting of two PHD-type zinc finger domains with activity in transcriptional regulation. PHF6 translocations were originally described in T-ALL and its mutations were later observed also in CML and adult AML. Germline (GL) PHF6^MT cause Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked disorder characterized by intellectual disability and, to date, only a few BFLS cases were found to develop lymphoma or T-ALL. While regularly encountered in myeloid neoplasia (MN), the impact and functional meaning of PHF6 are not well established.

To determine the incidence, distribution and molecular context of PHF6^MT we studied a large cohort of patients with MN (n=8617) collected from our institution and public series. ¹ Overall, 73% of patients were AML (pAML 69%; sAML 4%), MDS (22%) and MDS/MPN (5%) with a median age at diagnosis of 67 ys (18-100).

We detected 149 patients (2%) carrying at least 1 PHF6^MT with 11 harboring more than 1 hit. Four patients carried -X in addition to PHF6^MT (2 males; 2 females). Majority of patients (68%) carried frameshift del/ins and nonsense. Mutations were scattered across all coding region with a slightly enrichment (47%) in the second PHD domain (239-330 aa) including the frequent R274Q/X (n=17). Common hits mainly affected arginine residues essential for DNA binding capacity (R129X n=9, R116X=7, R319X=5, R225X=3) followed by other hits (I314T=6, Y301X and C20fs=4 each). Of note, R116X, R225X, R274X, C280Y, H329R and Y303* lesions overlapped with the T-ALL PHF6^MT spectrum while no overlap was found with GL mutations found in BFLS.

Overall, 75% of all PHF6^MT carriers were males and carried mostly (80%) truncating lesions. Compared to mutational frequencies observed in other X-linked genes, truncating PHF6^MT behaved similarly to those in ZRSR2 (78%), STAG2 (73%) and BCOR (62%). Conversely, BCORL1^MT, KDM6A^MT and PIGA^MT were evenly distributed between genders. When evaluating mutational characteristics in males and females, no differences were found in sex-adjusted median variant allelic burden of PHF6^MT (54.8 vs 51%) nor its mRNA expression levels suggesting locus inactivation. PHF6^MT tended to be older than PHF6^WT patients (72 vs 68 ys; P= .05) and had mostly (63%) AML followed by MDS (23%) and MDS/MPN (14%). OS was similar between PHF6^MT and PHF6^WT patients (P= .16).

Expression analyses showed that PHF6 loss leads to deregulation of chromatin and transcriptional factor genes. Indeed, in our cohort the most comutated genes were transcriptional factors and chromatin modifiers genes such as RUNX1 (26/149, 17%), ASXL1 (23/149, 15%) and TET2 (17/149, 11%). Of note, this group characterized by the triple ASXL1, RUNX1, TET2 mutational configuration clustered in one of the genomic groups previously identified (GC-3) ¹ but the presence of these lesions did not worsen the OS as compared to PHF6^MT without this mutational constellation. A low frequency of SF3B1^MT (4%) was also noted confirming the enrichment of PHF6^MT in AML rather than in low risk MDS. Further, 12% (14 males; 4 females) of PHF6^MT patients had X-mutation mosaicism as shown by concomitant hits in BCOR (n=8), ZRSR2 (4), STAG2 (5), KDM6A (1).

PHF6^MT were equally founder lesions (30%; 44/149) and subclonal (34%; 50/149) whereas the rest was indistinguishable by VAF discrimination (co-dominant). The most common subclonal mutations were U2AF1 (14%, 6/44), IDH1/2 (9%, 4/44) and RUNX1 (7%, 3/44). When PHF6^MT were subclonal, the founder hits were in TET2 (14%, 7/50), DNMT3A and RUNX1 (12%, each 6/50) genes. Given the high frequency of RUNX1^MT in PHF6^MT we investigated whether RUNX1 and PHF6 might be correlated. Transcriptomic analysis of 6246 patients (from 9 public studies) ² showed a direct linear correlation (AdjR2= .03, P=5.55e-05) between the expression of the two genes.

Our study is the largest to date to investigate the genetic landscape of PHF6^MT in MN and highlights a strong connection of PHF6 with transcriptional regulation and chromatin genes. Ongoing scDNA-seq will clarify whether these mutations were acquired in distinct clones helping in dissecting the clonal hierarchy of PHF6^MT cases.