Ibrutinib-Resistant Mantle Cell Lymphoma Undergoes Metabolic Reprogramming Towards Oxphos
Authors: Nomie Krystle , Zhang Liang , Yao Yixin , Liu Yang , Zhang Shaojun , Zhang Hui , Guo Hui , Ahmed Makhdum , Badillo Maria , Wang Linghua , Wang Michael 
Published: October 2019

Abstract
Introduction
Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma subtype and constitutive activation of the B-cell receptor pathway is a hallmark of B-cell lymphomas. Bruton's tyrosine kinase (BTK) is a critical component of the B-cell receptor pathway, and ibrutinib, a first-in-class, once-daily, and oral covalent inhibitor of BTK, was developed to reduce/silence B-cell receptor pathway activity, leading to clinically remarkable anti-tumor activity. In our prior multiple-center Phase II clinical trial, the overall response rate in relapsed/refractory MCL patients was 68% (Wang et al., NEJM, 2013), surpassing the effectiveness of other therapies. Although ibrutinib is extremely efficacious in patients with relapsed/refractory MCL, the one-year overall survival rate of ibrutinib-exposed patients who relapse is only 22%.
Methods
Patient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with reverse phase protein array (RPPA) analysis of ibrutinib-sensitive and -resistant MCL cell lines. Metabolic assays including the measurement of mitochondria respiration rates with the Seahorse analyzer and reactive oxygen species (ROS) levels, targeted metabolomics, and ATP analysis. Functional studies targeting this molecular pathway were conducted, including in vitro cell viability and apoptosis assays, as well as in vivo efficacy studies in an ibrutinib-resistant MCL patient-derived xenograft mouse model.
Results
WES data analysis identified frequent inactivating somatic alterations in ATM, KMT2D, and TP53 in both the ibrutinib-sensitive and -resistant tumors. CDKN2A (5/7, 71%) was frequently deleted, and the deletion was only observed in the ibrutinib-resistant tumors (p = 0.010). The RNA-seq analysis identified a total of 63 protein-coding genes as the most differentially expressed genes (DEGs) between the ibrutinib-resistant and -sensitive groups, with a fold change of ≥ 2 or ≤ -2 and the false discovery rate (FDR q-value) ≤ 0.01. Among the DEGs, 26 genes were upregulated in ibrutinib-resistant tumors. In addition, gene set enrichment analysis (GSEA) revealed the marked upregulation of oncogenic pathways including c-MYC, mTOR (mTORC1), Wnt, and NF-ĸb signaling, followed by cell cycle, apoptosis, BCR signaling and DNA repair in the ibrutinib-resistant tumors. Notably, in addition to these oncogenic pathways, the metabolic pathways, including oxidative phosphorylation (OXPHOS), were significantly enriched in the ibrutinib-resistant tumors (normalized enrichment score > 3 and FDR q-value < 1e-5). In further support of this finding, metabolomics analysis and the measurement of ATP production and mitochondrial respiration indicated that the OXPHOS pathway is the predominant metabolic pathway employed by ibrutinib-resistant MCL cells. To determine the effects of targeting these pathways, OXPHOS was inhibited with a novel electron transport complex I inhibitor (IACS-010759, developed by MD Anderson Cancer Center) in both MCL cell lines and ibrutinib-resistant MCL patient-derived xenograft (PDX) models. Single agent IACS-010759 treatment at 10 mg/kg oral gavage for 5 consecutive days/week completely prevented tumor growth compared with the vehicle control as shown by measuring tumor volume (n = 5, p < 0.0001) and human β2M levels (n = 5; p < 0.0001) throughout treatment. No apparent toxicities were observed in the IACS-010759-treated MCL PDX mice.
Conclusion
This current study warrants the exploitation of active cancer metabolic pathways, especially OXPHOS, to improve the clinical outcomes of MCL and additional lymphoma, which is actively being investigated in a Phase I lymphoma clinical trial (NCT03291938).

Disclosures
Wang: AstraZeneca: Consultancy, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; Novartis: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MoreHealth: Consultancy; Acerta Pharma: Honoraria, Research Funding; Kite Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Dava Oncology: Honoraria.