Phip Is a Potent Leukemia Oncogene Identified in Acute Myeloid Leukemia (AML) Patients of African Ancestry

In the United States, AML patients of African Ancestry have shorter overall survival compared to patients of European descent, even if treated similarly on clinical frontline trials. To uncover genetic drivers of poor outcomes, we recently characterized the genomic landscape of African American AML. Tumor-normal whole-exome and total transcriptome sequencing was performed on samples from 100 patients, and uncovered several thus-far-unrecognized, recurrent mutations in the context of AML. We focused on Pleckstrin homology domain interacting protein (PHIP) because it was mutated in 7% of African ancestry patients, making it the 8^th most mutated gene. Variants of PHIP are found in congenital genetic disorders (i.e. Chung-Jansen syndrome) and solid malignancies like glioblastoma. PHIP is a putative chromatin regulator that contains two H3K14ac and H4K12ac binding bromodomains and is hypothesized to regulate gene expression. Our bulk RNA-seq analysis from 85 Black AML patients identified a distinct PHIP-mutant gene expression signature. Moreover, we noted reports of PHIP variants in both clonal hematopoiesis of indeterminate potential (CHIP) and myelodysplastic neoplasm (MDS) patients, supporting the potential role of PHIP mutation in hematologic disease initiation or transformation. We therefore sought to validate the oncogenic function of PHIP mutations.

To firmly delineate leukemogenic potential, we utilized CRISPR knock-in murine models in tandem with comprehensive, multi-omic efforts on primary human specimens. First, we generated a Phip^Y1316X/+ knock-in allele; a bromodomain stop gain variant observed in both Chung-Jansen syndrome patients and our AML cohort. In Phip^Y1316X/+ mice, we noted Chung-Jansen syndrome phenotypes including dysmorphic cranial features, abnormal brain histology, and neurological phenotypes like adult hydrocephaly. Transplantation of Phip^Y1316X/+ bone marrow led to myeloid skewing and a modest engraftment advantage compared to Phip^+/+ controls. However, combining Nras^G12D and/or Flt3^ITD sensitizing-mutant alleles with Phip^Y1316X/+ resulted in significantly increased CBC counts, splenomegaly, monocytic blasts, and decreased survival compared to Phip^+/+ controls. Phip^Y1316X/+Flt3^ITD and Phip^Y1316X/+Nras^G12D splenic tumor cells obtained from moribund primary animals generated a lethal AML in secondary recipient mice, unlike the Phip^+/+Flt3^ITD and Phip^+/+Nras^G12D controls. Thus, Phip^Y1316X/+ is an AML oncogene.

Single cell profiling of these murine AML models revealed that the shared differentially expressed genes (DEGs) are mostly downregulated and enriched for transcription coregulator activity, chromatin binding, and histone modifying activity. To validate our murine models, we next performed single cell profiling of 7 PHIP-mutated (2F, 5M; median age: 52) and 29 PHIP^+/+ human AML patient specimens; identifying the predominant populations as early progenitor or monocytic progenitor cells (Multilin-GMP1 and Intermediate-Mono-1 clusters¹). Strikingly, a significant number of DEGs were conserved in direction and amplitude between the Phip^Y1316X/+ mouse models and human PHIP-mutant AML; suggesting that an intersection of these DEG would reveal proximal targets of the PHIP oncoprotein. To understand the prognostic relevance of these targets we used the DEGs (shared mouse model/human AML) to derive a prognostic signature for survival using LASSO models in an established cohort of 876 adult AML patients. The optimal derived signature incorporated 61 genes and was independently associated with complete remission rates, disease-free and overall survival. This impact was independent of other established molecular features, and use of the signature refined all three risk assignments of the European LeukemiaNet 2022 genetic risk classification. Of note, many of the genes in the prognostic signature play a role in chromatin regulation, including KANSL1, MAML2, BTAF1, and PHIP itself.

Assigning oncogenic function to novel mutations like PHIP^Y1316X is requisite for their inclusion in cancer DNA-resequencing panels and to trigger the identification of new pathways for clinical intervention and drug development for all AML patients.

1. Zhang, X. et al. An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors. Nat Immunol25, 703-715, doi:10.1038/s41590-024-01782-4 (2024).

Walker:Karyopharm Therapeutics: Current Employment. Eisfeld:GTC: Honoraria; VJ HemeOnc: Honoraria; Karyopharm Therapeutics: Other: Spouse employment; OncLive: Honoraria; Dava Oncology: Honoraria; AstraZeneca US: Membership on an entity's Board of Directors or advisory committees.