Bi-allelic hydroxymethylbilane synthase inactivation defines a homogenous clinico-molecular subtype of hepatocellular carcinoma

Abstract

Background & aims: Acute intermittent porphyria (AIP), caused by heterozygous germline mutations of the heme synthesis pathway enzyme HMBS (hydroxymethylbilane synthase), confers a high risk of hepatocellular carcinoma (HCC) development. Yet, the role of HMBS in liver tumorigenesis remains unclear.

Methods: Herein, we explore HMBS alterations in a large series of 758 HCC cases, including 4 patients with AIP. We quantify the impact of HMBS mutations on heme biosynthesis pathway intermediates and we investigate the molecular and clinical features of HMBS-mutated tumors.

Results: We identify recurrent bi-allelic HMBS inactivation, both in patients with AIP acquiring a second somatic HMBS mutation and in sporadic HCC with 2 somatic hits. HMBS alterations are enriched in truncating mutations, in particular in splice regions, leading to abnormal transcript structures. Bi-allelic HMBS inactivation results in a massive accumulation of its toxic substrate porphobilinogen and synergizes with CTNNB1-activating mutations, leading to the development of well-differentiated tumors with a transcriptomic signature of Wnt/β-catenin pathway activation and a DNA methylation signature related to ageing. HMBS-inactivated HCC mostly affects females, in the absence of fibrosis and classical HCC risk factors.

Conclusions: These data identify HMBS as a tumor suppressor gene whose bi-allelic inactivation defines a homogenous clinical and molecular HCC subtype.

Lay summary: Heme (the precursor to hemoglobin, which plays a key role in oxygen transport around the body) synthesis occurs in the liver and involves several enzymes including hydroxymethylbilane synthase (HMBS). HMBS mutations cause acute intermittent porphyria, a disease caused by the accumulation of toxic porphyrin precursors. Herein, we show that HMBS inactivation is also involved in the development of liver cancers with distinct clinical and molecular characteristics.

Keywords: Acute intermittent porphyria; Cancer Genomics; Heme biosynthesis pathway; Hepatocellular carcinoma.

Fig. 1.. Molecular history of HCC developed in patients with AIP.

Whole-genome sequencing of HCC and matched non-tumor liver samples from 3 patients with AIP revealed germline HMBS mutations and somatic alterations acquired by cancer cells. Alterations in known liver cancer driver genes are annotated. Patient #4027 developed 2 independent synchronous HCCs. AIP, acute intermittent porphyria; HCC, hepatocellular carcinoma; SVs, structural variants.

Fig. 2.. Recurrent inactivating HMBS mutations in HCC.

(A) Lollipop plot showing the type and distribution of HMBS mutations along the protein sequence. Amino acid and exon numbering is given with respect to the NCBI Refseq NM_000190 isoform. (B) Impact of splice mutations on RNA structure. RNA-sequencing coverage is shown in grey. Arcs represent split reads indicating splicing events. Mutations are highlighted in each plot. HCC, hepatocellular carcinoma.

Fig. 3.. Massive porphobilinogen accumulation in HMBS-mutated HCC.

Three heme biosynthesis pathway intermediates (porphobilinogen, protoporphyrin IX and hemin) were quantified by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Porphobilinogen is the substrate of HMBS, protoporphyrin IX is the last intermediate before heme, and hemin results from the spontaneous oxidation of heme in vitro. These metabolites were quantified in 10 HCC/non-tumor liver pairs from 1 patient with AIP, 3 patients with HCC and somatic HMBS inactivation, and 6 patients with HCC without HMBS inactivation used as controls. AIP, acute intermittent porphyria; HCC, hepatocellular carcinoma.

Fig. 4.. Clinical and molecular features of HMBS-inactivated HCC.

Forest plots showing odds ratios for enrichment of clinical features (A) and driver alterations (B) among HMBS-inactivated HCC. P values were obtained using Fisher’s exact test. (C) t-distributed stochastic neighbor embedding plots showing the unsupervised classification of gene expression (RNA-sequencing) and DNA methylation (Illumina Infinium HumanMethylation450 BeadChip) profiles in the LICA-FR series. Previously defined transcriptomic (G1-G6) and DNA methylation (M1-M7) subgroups are indicated with a color code, and HMBS-mutated HCCs are highlighted with stars. (D) Gene expression signatures showing differential activity in HCC with or without HMBS mutations. (E) DNA methylation components showing differential activity in HCC with or without HMBS mutations. HCC, hepatocellular carcinoma.