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Eukaryotic cells have two separate genomes; nuclear chromosomal DNA and circular mitochondrial DNA. Mitochondrial DNA lacks introns, and encodes 2 rRNAs, 22 t-RNAs and 13 of the 90 proteins that constitute the mitochondrial respiratory chain. To maintain homeostasis, mitochondrial RNA degradation machinery regulates RNA turnover. ATP-dependent helicase, SUV3 (gene SUPV3L1), and exonuclease PNPase (gene PNPT1) function as a complex to degrade mitochondrial dsRNA. By immunoblotting, PNPase and SUV3 proteins were increased in 7/7 AML patient samples and 13/13 of AML cell lines, compared to the normal hematopoietic cells. Analysis of the TARGET AML dataset revealed AML patients with increased expression of SUPV3L1 (p = 0.051, p= 0.045) and PNPT1 (p = 0.0013, p = 0.018) had decreased overall and event free survival. Genetic knockdown or knockout using shRNA or sgRNA against PNPT1 or SUPV3L1 decreased growth and viability of OCI-AML2, TEX, K562, U937, NB4 and OCI-AML 8227 cells. Furthermore, SUPV3L1 & PNPT1 ranked in the top 5.2% and 7.4% of essential genes in 26 leukemia cell lines in CRISPR screens and 2.7% and 4.9% in RNAi screens (depmap.org). Knockdown of PNPT1 & SUPV3L1 also reduced the clonogenic growth of OCI-AML2, TEX and U937 cells and significantly reduced engraftment of TEX cells into the marrow of immune deficient mice, demonstrating the functional importance on leukemia initiating cells in vivo. SUPV3L1 knockdown in primary AML cells reduced engraftment in marrow of immune deficient mice. Bioinformatics analysis to detect processes associated with PNPT1 and SUPV3L1, we identified associations with Response to exogenous dsRNA, Response to virus, and RNA catabolic process ontologies. Consistent with this, we observed knockdown of PNPT1 or SUPV3L1 increased expression of genes (INFgR1, ICAM, IRF7 & JAK/STAT) suggesting an interferon response. As PNPT1 and SUPV3L1 degrade mitochondrial dsRNA, we measured levels of dsRNA after knockdown of these genes. Knockdown of PNPT1 and SUPV3L1 in OCI-AML2 cells increased levels of cytoplasmic/mitochondrial dsRNA 3-4 fold compared to control. Knockdown of PNPT1 and SUPV3L1 also increased dsRNA in 143B cells, but not Rho (0) 143B cells that lack mitochondrial DNA. Upregulation of inflammatory genes leads to viral mimicry and can increase sensitivity to immune mediated killing. We observed enhanced sensitivity to Double Negative T (DNT) cells mediated killing in PNPT1 and SUPV3L1 knockdown OCI-AML2 cells compared to control cells. In summary, RNA degradosome complex proteins SUPV3L1 and PNPT1 are overexpressed in AML, and are essential for the survival of AML cells and AML stem/progenitors. These enzymes regulate levels of mitochondrial dsRNA, and their inhibition leads to increased cytoplasmic dsRNA triggering a viral mimicry response and enhanced sensitivity to immune-mediated killing. Citation Format: Geethu Emily Thomas, Kazem Nouri, Jong Bok Lee, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiao Ming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Li Zhang, Vito Spadavecchio, Aaron Schimmer. Inhibiting the mitochondrial RNA degradosome complex SUV3 and PNPase increases dsRNA in the cytoplasm, triggers a viral mimicry response and kills AML cells and progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4833.

Mitochondria possess unique proteases that localize to specific sub-compartments of the organelle. However, the functions of these proteases are largely ill-defined. Here, we used proximity-dependent biotinylation (BioID) to map the interactomes of seven proteases located in the intermembrane space of the mitochondria. The mitochondrial intermembrane space proteases HTRA2, OMA1, YME1L1, LACTB, IMMP1L, IMMP2L and PARL were cloned in-frame with the abortive E. coli biotin ligase BirA*, and expressed in 293 T-REx cells. Cell culture media was spiked with biotin for 24 hrs, the cells lysed, and biotinylated proteins were isolated and identified by mass spectrometry. In total, we identified 342 different proteins as high confidence interactors of the seven mitochondrial proteases. Of these, 272 are assigned a GO mitochondrial annotation, and 230 proteins interacted with only 1 or 2 proteases in our dataset. Validation efforts were focused on high temperature requirement peptidase A 2 (HTRA2). HTRA2 is a serine protease that is released into the cytoplasm during apoptosis where it binds Inhibitor of Apoptosis Proteins (IAPs). However, little is known about the function of HTRA2 in the mitochondria. HTRA2 interacted with 60 mitochondrial, 11 nuclear and 4 cytoplasmic proteins, including its known interactor XIAP, and consistent with its known localization to these cellular compartments. HTRA2 interacted with 8 out of 13 components of the MIB complex, a multiprotein assembly that is essential for proper mitochondrial cristae formation. Knockdown of HTRA2 with shRNA in 293T-REx cells disrupted cristae formation and this phenotype was rescued by expression of an shRNA-resistant HTRA2 cDNA. Compared to normal hematopoietic cells, HTRA2 mRNA expression levels are increased in a subgroup of primary AML cells. HTRA2 knockdown in OCI-AML2 leukemia cells led to a similar disruption of mitochondrial cristae. Knockdown of HTRA2 in OCI-AML2 cells led to increased levels of the MIB subunit IMMT, but not two other MIB complex subunits, SAMM50 and CHCHD3. Finally, in cell-free assays, we demonstrate that recombinant HTRA2 can degrade recombinant IMMT, but not SAMM50 or CHCHD3.Thus, we have mapped the interactomes of the proteases of the mitochondrial intermembrane space. Through this effort, we discovered that HTRA2 regulates protein levels of the MIB complex subunit IMMT and that disruption of this process affects mitochondrial cristae formation. Citation Format: Aaron D. Botham, Etienne Coyaud, Sanjit Nirmalanandhan, Marcela Gronda, Rose Hurren, Neil Maclean, Jonathan St. Germain, Sara Mirali, Estelle Laurent, Brian Raught, Aaron Schimmer. Mapping the protein interactome of mitochondrial intermembrane space proteases identifies a novel function for HTRA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4529.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational conjugation of NEDD8 onto target proteins. Cullin proteins are major substrates of the neddylation pathway, and are involved in diverse cellular processes. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924 in leukemic cells. MLN4924 shows potent activity in K562 human leukemia cells (EC50 = 100 nM). In contrast, by exposing K562 cells to increasing concentrations of MLN4924 over 6 months, we selected a population of cells resistant to MLN4924 (R-K562MLN; EC50 > 2 µM). R-K562MLN cells proliferated similarly to parental K562 cells, and remained resistant to MLN4924 even after culturing in the absence of MLN4924 for 5 weeks. Compared to parental cells, R-K562MLN cells were similarly sensitive to a broad spectrum of chemotherapeutic drugs, including daunorubicin, a classical substrate of the multidrug efflux transporter P-glycoprotein. Thus, the mechanism of resistance to MLN4924 did not appear to be related to generalized multidrug efflux. While basal levels of NEDD8-conjugated cullin proteins were equivalent in parental and resistant cells, NEDD8-cullins were diminished in parental cells treated with 25 nM MLN4924. In contrast, no appreciable reduction in NEDD8-cullins was observed in R-K562MLN cells after treatment with up to 250 nM MLN4924. However, knockdown of NEDD8 by shRNA was cytotoxic to R-K562MLNcells, suggesting that NEDD8 conjugation remains essential for the survival of resistant cells. To further probe mechanisms of MLN4924 resistance in these cells, we sequenced the coding region of the NAE catalytic subunit UBA3 from K562 and R-K562MLN cells, and identified a mutation in codon 310 [ATT (Isoleucine, I) > AAT (Asparagine, N)] in R-K562MLN cells. Biochemical analyses using recombinant wild-type and I310N NAE complexes indicated that the I310N mutation alters biochemical properties of the NAE, increasing the enzyme’s affinity for ATP while decreasing its affinity for NEDD8. The I310N NAE complex was ∼7-fold less sensitive to MLN4924 (IC50 = 225 nM) compared to wild-type NAE (IC50 = 32 nM), providing a mechanistic basis for MLN4924 resistance in R-K562MLNcells through the UBA3 I310N mutation. To investigate whether another malignant leukemic cell line employs a similar on-target resistance mechanism, we similarly selected a population of U937 cells resistant to MLN4924 (R-U937MLN). Parental U937 cells were sensitive to MLN4924 (EC50 = 25 nM), while R-U937MLN cells were more than 40-fold more resistant (EC50 > 1 µM). Sequencing of the coding region of UBA3 in U937 and R-U937MLN cells revealed a point mutation in codon 352 [TAT (Tyrosine, Y) > CAT (Histidine, H)] only in R-U937MLN cells. As for the UBA3 I301N mutation, the Y352H mutation conferred a ∼10-fold decrease in the sensitivity of the NAE to MLN4924 (IC50= 138.5 nM and 13 nM for Y352H and wild-type, respectively) through decreased NEDD8 and increased ATP affinities. These findings suggest that the Y352H mutation, like I310N, is sufficient to provide MLN4924 resistance in leukemia cells while sustaining adequate NAE function for leukemic cell survival. We next found that R-K562MLN cells were cross-resistant to other NAE-selective inhibitors derived from Compound 1, a pan-E1 inhibitor. Nevertheless, compared to parental cells, R-K562MLN cells were not refractory to Compound 1 (EC50 = 27 nM and 81 nM, respectively). The cytotoxicity of Compound 1 towards MLN4924-resistant cells might be explained by its inhibition of other E1 enzymes. Indeed, Compound 1 diminished the abundance of ubiquitinated proteins in R-K562MLNcells similarly to its effects in K562 cells, while NEDD8-cullins in the MLN4924-resistant cells were unaffected. Overall, as MLN4924 continues to be evaluated clinically, our work provides insight into the mechanisms of MLN4924 resistance, which may facilitate the development of more effective second-generation NAE inhibitors. Disclosures: Petroski: Allostere, Inc.: Consultancy, Equity Ownership.