Germline HFE mutations predict for durable response to arginine depletion with pegargiminase-based chemotherapy in pleural mesothelioma

Background: Pegargiminase (ADI-PEG20)-based chemotherapy quadrupled the overall survival of patients at 3 years (12% versus 3%) with non-epithelioid (biphasic and sarcomatoid) pleural mesothelioma in the randomized placebo-controlled ATOMIC-Meso phase 2-3 trial (AACR 2023, JAMA Oncol 2024). Here, we report a novel genetic biomarker, namely HFE -related hereditary haemochromatosis as a predictor for durable response to arginine deprivation therapy for cancer using pegargiminase. Methods: We scrutinised the Barts patient cohort which recruited 20.9% (n=52/249) of patients enrolled on the ATOMIC-Meso study to identify potential pharmacogenomic factors linked to a prolonged therapeutic response. This led to identification of an index patient with the HFE C282Y variant who completed 2 years of pegargiminase. Common HFE variants (C282Y and H63D) were generated in the MSTO mesothelioma cell line using CRISPR/Cas9-mediated mutagenesis, followed by characterisation of mutant and parental controls relating to in vitro drug sensitivity, apoptosis, single/double strand DNA breaks (SSBs/DSBs), performed RNAseq to dissect the mechanism of drug action, and analyzed in vivo tumorigenesis and ADI-PEG20 efficacy using xenograft murine studies. Results: The index patient was homozygous for C282Y and was diagnosed with sarcomatoid mesothelioma during venesection to manage his iron overload. He was enrolled on ATOMIC-Meso and unexpectedly completed 2 years of pegargiminase with a durable partial response. Only one other patient at the same institution completed 2 years of pegargiminase with a durable partial response and was heterozygous for the HFE H63D variant with a biphasic mesothelioma. Bed-to-benchside studies confirmed increased ADI-PEG20 sensitivity in MSTO H63D+/- compared to parental cells using a 3D tumor spheroid model as evidenced by caspase 3 activation, PARP cleavage, higher Annexin V staining and upregulation of SSBs and DSBs. To understand the mechanism of enhanced cytotoxicity to ADI-PEG20 we performed RNA-Seq in MSTO H63D+/- and MSTO WT cells. This identified regulation of AP-1 signalling and suppression of Bcl-2 specifically in ADI-PEG20 treated MSTO H63D+/- cells by 48hrs. Differential modulation of the Bcl-2 family triggering enhanced apoptosis was confirmed in the MSTO H63D+/- compared to MSTO WT cells. Finally, MSTO H63D+/- tumors displayed earlier sensitivity to ADI-PEG20 monotherapy compared to MSTO WT tumor parental controls (14 days vs. 28 days) using murine xenografts. There was no difference in survival between MSTO H63D+/- and MSTO WT tumors, although there was a statistically significant earlier growth advantage for MSTO WT cells. Conclusions: In the Barts ATOMIC-Meso patient cohort, our data reveal mutant HFE as a novel pharmacogenomic biomarker for enhanced sensitivity and prolonged remission to pegargiminase-based chemotherapy for mesothelioma. Further analyses of durable responders to pegargiminase are ongoing with prospective clinical trials planned using HFE as a selection biomarker.