Identify AMPK-Mediated Autophagy and Oxphos As a Novel Metabolic Vulnerability for Targeted Therapy in Mantle Cell Lymphoma

Introduction: Metabolic reprogramming promotes cancer cell growth, metastasis, and therapeutic resistance in a multitude of cancers. Mantle cell lymphoma (MCL) is a rare and aggressive hematopoietic malignancy that exhibits dramatic alterations in the cellular metabolism, especially in advanced stages and at the time of relapse. Unchecked pro-tumor growth signaling and multidrug therapy induce genomic instability and metabolic stress. To sustain tumor survival, MCL cancer cells upregulate multiple essential metabolic pathways that include mitochondria biogenesis, OXPHOS, glutaminolysis, cytoprotective autophagy, and fatty acid β-oxidation. While we recently have demonstrated that OXPHOS is upregulated and mediates resistance to ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, in MCL, the underlying mechanism remains to be elucidated. Cytoprotective autophagy is known to be induced in chronic hypoxic microenvironments such as bone marrow and secondary lymphoid organs, and it in turn contributes to drug resistance and promotes MCL tumor survival. AMPK, a central regulator of cellular metabolism, is activated when MCL cells are challenged with metabolic stress. Consistently, AMPKα1 is highly activated in a subset of MCL at the leukemic phase. AMPK dependence and metabolic reprogramming represent vulnerabilities that could be potentially targeted for MCL treatment.

Methods: Primary MCL biopsies, apheresis, or blood specimens, as well as MCL cell lines, were used for metabolic and functional analyses. Metabolomics profiling was carried out using Liquid Chromatography Mass Spectrometry (LC-MS). Mitochondrial membrane potential was determined by staining MCL cells with MitoStatus or TMRE and flow cytometry analysis. OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were determined by Seahorse metabolic flux analysis. Autophagy flux was monitored with tandem mRFP-EGFP-LC3 fluorescence reporter analysis. Other analyses included western blotting, real-time qPCR, and cell viability assay (Cell Titer-Glo). Pharmacological inhibitors were used for the down-modulation of ULK1, AMPK, autophagy, and OXPHOS.

Results: Metabolomics profiling of steady-state levels of TCA metabolites showed significant increases in levels of α-KG, glutamate, malate, and fumarate, indicating upregulated glutaminolysis in subsets of MCL that are recurrent or refractory to targeted therapeutics including ibrutinib. Functional analysis illustrates that glutamine deprivation leads to reduced ATP and cell viability in a subset of MCL, while pharmacological inhibition of glutamine metabolism leads to reduced mitochondria membrane potential and increased cellular apoptosis. Seahorse metabolic flux analysis revealed enhanced OXPHOS activity in ibrutinib-resistant MCL cells as indicated by increased OCR. Inhibition of OXPHOS or glutamine metabolism impairs OCR and subsequently induces dose-dependent cellular apoptosis. Further, autophagy flux is increased in the same MCL cells with mRFP-EGFP-LC3 as a reporter. IHC and western blot analysis demonstrated that AMPKα1 is highly activated in a subset of MCL at the leukemic phase, concomitant with an increased NADP:NADPH and AMP:ATP ratio, indicating energy stress. AMPK activation promotes cytoprotective autophagy, and AMPK blockade suppresses autophagy, mitochondria biogenesis, and activity (OCR and ATP production), ultimately inhibiting MCL proliferation under metabolic stress.

Conclusions: Our results demonstrate that AMPK activation promotes cytoprotective autophagy, glutaminolysis, and OXPHOS in established MCL, thus representing unique metabolic vulnerabilities that could be potentially targeted for MCL treatment. As AMPKα1 inhibitors are underexplored, it is critical to identify and develop more potent and specific inhibitors of the kinase and define the in vivo functions of its activation in certain advanced cancers. Similarly, pharmacological blockade of the dysregulated autophagy, glutaminolysis, and OXPHOS can be extensively exploited for treatment of MCL patients, especially those at advanced stages of the disease.