Hudec, Roman

The mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PRKN (Parkin) together mediate the selective degradation of damaged mitochondria via the autophagy‐lysosome system (mitophagy). Complete loss of either gene function results in failure of this mitochondrial quality control and thus accumulation of damaged organelles. While homozygous or compound heterozygous mutations in PINK1 or PRKN are the most common causes of early‐onset Parkinson's disease (PD), the contribution of heterozygous variants to PD is debated. However, recently we identified a partial dominant‐negative effect of a heterozygous PINK1 missense mutations that is associated with a significant increased risk and an earlier onset compared to late‐onset, sporadic PD. This underscores that not all missense mutations are alike and suggests critical threshold functions of this stress‐activated, neuroprotective pathway. Here, we analyzed the underlying mechanisms in greater detail and found that the pathogenic PINK1 mutant, which can dimerize with wild‐type PINK1 protein, is aberrantly auto‐phosphorylated, impacting its enzymatic activity towards the substrates Ub and PRKN. Phosphorylation of both PINK1 targets however, is critical for PRKN recruitment as well as efficient labeling of mitochondria with phosphorylated poly‐Ub chains that serve as the mitophagy tag. In addition to cells from PD patients, we confirmed the altered phosphorylation pattern of mutant PINK1 under endogenous conditions in isogenic CRISPR/Cas9 genome engineered cells. Excitingly, while studying the pathogenic mechanisms in detail with a structure‐function approach, we identified a novel PINK1 variant that exerts even greater kinase activity than wild type PINK1. Our results further suggest that this hyperactive form of PINK1 likely modulates substrate receptivity thereby resulting in enhanced kinase activity and greater mitophagy rates. Going forward, it will be interesting to study if enhanced PINK1 signaling translates into greater neuroprotection. An in‐depth understanding of the regulation and enzyme functions of PINK1 may provide a rationale for a structure‐function based drug design in the future. Support or Funding Information This work was partially supported by funding from the Mayo Clinic Foundation, the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) [R01#NS085070] and the Michael J. Fox Foundation for Parkinson's Research to W.S. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models. Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000. Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S). Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.