Protein Kinase C Epsilon Is a Key Regulator of Mitochondrial Redox Homeostasis in Acute Myeloid Leukemia

The intracellular redox environment is largely influenced by the production and clearance of reactive species such as reactive oxygen species (ROS). ROS encompass a heterogeneous class of small oxygen-containing reactive species that are produced by several intracellular processes such as aerobic glucose metabolism and enzymatic reactions (1) and contribute to physiologic functions such as innate and acquired immune defense. However, aberrantly elevated ROS levels, referred to as “oxidative stress,” can cause DNA lesions, organelle dysfunction, and metabolic alterations that contribute to tumorigenesis (2). Furthermore, extremely high ROS levels can promote cell death (2). Therefore, intracellular ROS levels are tightly regulated by an array of enzymatic and nonenzymatic systems (1).

Several human cancers, including AML, display perturbations in genes that encode regulators of intracellular ROS biology (3). For example, primary AML patient samples display decreased expression of the mitochondrial superoxide-neutralizing gene SOD2 and decreased glutathione metabolism (4). The activities of the ROS-generating enzymes NADPH-oxidases (NOX) are also elevated in primary human AML samples compared with healthy controls and this is associated with increased steady-state levels of intracellular superoxides (5). Moreover, the total antioxidant capacity of leukemic cells from AML patients at initial diagnosis and relapse is decreased compared with healthy controls (6). Several of the oncogenic signaling molecules that are either mutated (e.g., FLT3^ITD, KRAS^G12D or BCR-ABL1; refs. 7–12) or dysregulated (e.g., c-MYC; ref. 13) in myeloid malignancies are known to drive ROS production. Furthermore, small nucleotide polymorphisms (SNP) of several redox-regulatory enzymes have been identified to be associated with myeloid neoplasia susceptibility and prognosis (14). Despite these studies as well as the emerging evidence that elevated ROS levels promote the proliferation and survival of solid cancers (15, 16), the underlying molecular mechanisms that govern ROS biology in AML remain unresolved.

PKCϵ is a serine/threonine kinase that is expressed in numerous tissues and supports tumorigenesis in many solid cancers (17). In normal hematopoietic development, PKCϵ influences erythrocyte and megakaryocyte lineage commitments (18); however, in primary myelofibrosis, elevated PKCϵ expression antagonizes megakaryocytic differentiation (19). Furthermore, downmodulation of PKCϵ expression is a key event during phorbol ester–induced differentiation of primary human AML samples and increased PKCϵ expression protects AML cells from TRAIL-induced apoptosis (20). However, the molecular mechanism(s) by which PKCϵ influences AML cell fate has yet to be resolved.

Here, we show that PKCϵ inhibition obstructs disease progression in a GEMM of AML driven by MLL-AF9 and impairs cell survival in multiple patient-derived AML samples. Furthermore, we find that PKCϵ inhibition results in increased steady-state levels of multiple mitochondrial ROS and that chemical or genetic neutralization of mitochondrial ROS counteracts the antileukemia effects of PKCϵ. Furthermore, we report that overexpression of PKCϵ protects AML cells from otherwise lethal doses of mitochondrial ROS–inducing agents. Finally, direct inhibition of the mitochondrial ROS–neutralizing enzyme SOD2 phenocopies PKCϵ inhibition indicating that maintenance of mitochondrial ROS homeostasis is crucial to the maintenance of AML pathogenesis.

Human AML cell lines were obtained from the ATCC and the German Collection of Microorganism and Cell Cultures (DMSZ). Cells were cultured in the recommended media conditions. Murine AML cells were cultured in cytokine-enriched media (CEM): RPMI1640 supplemented with 10% FBS and penicillin/streptomycin, 10 ng/mL mSCF (Peprotech), 6 ng/mL mIL3 (Peprotech), and 5 ng/ml mIL6 (Peprotech).

Patient-derived AML samples were obtained from The Ohio State Comprehensive Cancer Center and used according to the approved Institutional Review Board protocol 16-9037. Cells were plated on irradiated monolayers of HS27 cells (21) and cultured in Stemspan (Stem Cell Technology) supplemented with 10% FBS, 100 ng/mL hSCF (Peprotech), 100ng/ml hFLT3 ligand (Peprotech), 20ng/ml hIL-3 (Peprotech), 20ng/mL hIL6 (Peprotech), 20 ng/mL G-CSF (Peprotech). Cells (5 × 10⁵) were transduced with pLKO.1 GFP lentiviral shRNA vectors and evaluated for GFP expression every 3 days for 12 days after staining with human CD45 APC-Cy7 (BD Biosciences) and propidium iodide (PI) by flow cytometry.

A total of 5 × 10⁵ human AML cells were transduced for 30 hours with recombinant pLKO.1 lentiviruses coexpressing either GFP or a puromycin-resistant cassette with shRNAs from the TRC shRNA library (shRNA Pkcϵ: TRCN0000022759; shRNA PKCϵ_1: TRCN0000000848 and shRNA PKCϵ_2: TRCN0000000846: shRNA SOD2: TRCN0000350349; shRNA Sod2_1: TRCN0000123392; shRNA Sod2_2: TRCN0000123390). Cells transduced with recombinant lentiviruses expressing the puromycin-resistant cassette were initially selected with 2 μg/mL puromycin for 48 or 72 hours (MOI = 0.5–0.9) and thereafter maintained in 0.5 μg/mL puromycin. To generate pLKO.1 GFP vectors, the puromycin-resistant cassette was replaced with eGFP. Cells transduced with lentiviruses expressing GFP (MOI = 0.2–0.5) were purified by FACS at the indicated times following transduction.

A total of 5 × 10⁵ human or murine AML cells were subjected to spin transduction with recombinant retroviruses (MOI = 0.2–0.3). The MSCV.PKCϵ.GFP construct was obtained by cloning murine Pkcϵ into the BglII and XhoI sites of the MSCV.IRES.GFP vector. Cells transduced with vectors expressing GFP were purified by FACS 96 hours after transduction. SF91-IRES-eGFP and SF91-SOD2/Catalase-IRES-eGFP vectors, were described previously (22) and provided by M. Milsom. SF91-Grx1-roGFP2, SF91-Mito-Grx1-roGFP2, SF91-roGFP2-Orp1, and SF91-Mito-roGFP2-Orp1 were described previously (23) and provided by Dr. T. Dick (German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany).

Cell pellets were lysed and resolved on 4%–12% Bis Tris gels (Thermo Fisher Scientific; Life Technologies). Following protein transfer and blocking with 5% nonfat milk, blots were incubated with primary antibody overnight at 4°C. Secondary antibody was incubated at room temperature for 1 hour and blots were developed with ECL prime (GE Healthcare). The following antibodies were used: anti-PKCϵ (catalog no.: 2683S: lot no.: 4) and anti-Catalase (catalog no.: 12980S, lot no.: 1) from Cell Signaling Technology with the dilution 1:1,000, anti-SOD2 (catalog no.: ADI-SOD-111, lot no.: 08021202) from Enzo Life Sciences with the dilution 1:1,000, anti-α-tubulin (catalog no.: T9026, lot no.: 083M4847V) from Sigma-Aldrich with the dilution 1:5,000. Secondary antibodies anti-rabbit-HRP (catalog no.: 7074S, lot no.: 26) and anti-mouse-HRP (catalog no.: 7076S, lot no.: 31) were from Cell Signaling Technology with the dilution 1:4,000.

Cells were plated at the density of 1–2 × 10⁵ cells/mL at either 72 or 96 hours posttransduction (day 0). At days 0, 2, and 4, 100 μL of cell suspension was incubated with 20 μL of CellTiter Aqueous One solution (Promega) for 90 minutes. Plates were read at the wavelength of 495 nm. Culture media was used as blank.

Cells were washed in PBS and stained with Annexin V and Propidium Iodide (PI) or 7AAD according to the manufacturer's instruction (BD Biosciences). Cells were acquired and analyzed using an LSRII flow cytometer (Beckton Dickinson). All the data were analyzed using FlowJo software.

Human AML cell lines were washed and incubated with PBS + 5 μmol/L of CellROX DeepRed reagent (Life Technologies; catalog no.: C10422) at 37°C for 30 minutes, or with PBS + 5 μmol/L of MitoSOX Red reagent (Life Technologies; catalog no.: M36008) at 37°C for 10 minutes. After incubation, cells were washed two times in PBS and stained with Annexin V-APC according to the manufacturer's instructions (BD Biosciences). Cells were analyzed using an LSRII flow cytometer (Beckton Dickinson).

Human AML cell lines stably expressing the roGFP2 probes (27) were transduced with recombinant lentiviruses for 30 hours, then selected with 2 μg/μL puromycin for 72 hours and subsequently analyzed by flow cytometry. RoGFP2 is excited at 400 nm (oxidized state) and 475–490 nm (reduced state) when fluorescence emission is monitored at 510 nm. Diamide (DIA; 1 mmol/L) or dithiothreitol (DTT; 1 mmol/L) were used as controls to induce a complete oxidized or reduced state of the probes, respectively. Propidium iodide staining was performed to exclude dead cells from the analysis.

Human AML cells stably expressing PKCϵ, PKCϵ-targeting shRNAs, or corresponding controls were seeded at the concentration of 2 × 10⁵ cells/mL in 24-well plates and treated with the indicated concentrations of Antimycin A (AA; Sigma Aldrich A8674) and Thenoyltrifluoroacetone (TTFA, Sigma Aldrich T27006) for 24 hours and then analyzed by flow cytometry after Annexin V and 7AAD staining.

All animal studies conducted were approved by the IUCAC of the Fox Chase Cancer Center. For recombinant viral transduction, bone marrow cells recovered from leukemia mice were cultured in CEM media overnight. The next day, cells were counted and then 5 × 10⁵ cells were subjected to spin transduction with recombinant lentiviruses (MOI = 0.4–0.6) or retroviruses (MOI = 0.2–0.3) supplemented with polybrene (5 μg/mL) in 12-well nonadherent plates. Plates were centrifuged at 2,400 rpm at 30 °C for 90 minutes. Transduced cells were then incubated overnight. The following day, viral supernatants were removed from cells and replenished with fresh CEM. For in vivo survival assays, sorted GFP⁺ cells (1 × 10⁶ cells/mouse) were transplanted into sublethally irradiated (450 rad) syngenic recipient mice 48 hours post-transduction. For Western blot analysis and colony formation assays, transduced cells were subjected to FACS to isolate GFP⁺ cells 48 hours post-transduction. For colony formation assays, 500 purified GFP⁺ leukemia cells were cultured in 1 mL of methylcellulose supplemented with cytokines (M3434, StemCell Technologies) for 5–7 days.

To evaluate the functional role of PKCϵ expression in AML biology, we employed an shRNA approach in a panel of genetically distinct AML cell lines in vitro (OCI-AML3, THP-1, NOMO1, and U937). We identified two shRNA constructs, PKCϵ shRNA_1 and PKCϵ shRNA_2, which target distinct regions of the PKCϵ mRNA and effectively deplete PKCϵ protein levels (Supplementary Fig. S1A). Each of these PKCϵ-targeting shRNAs significantly reduced the expansion of the four AML cell lines compared with nontargeting shRNA controls (CTRL shRNA; Supplementary Fig. S1B). This reduction in cell growth was accompanied by a significant increase in the percentage of Annexin V⁺ cells (Supplementary Fig. S1C), and CD11b expression (Supplementary Fig. S1D and S1E) indicating that PKCϵ inhibition is detrimental to survival and growth and may induce differentiation of these AML cell lines.

To assess the impact of PKCϵ inhibition in vivo, we utilized a GEMM of AML that is driven by the human leukemogenic fusion protein MLL-AF9 (24). Mouse MLL-AF9 leukemia cells were transduced with recombinant lentiviruses that coexpress GFP in combination with either control (CTRL shRNA) or murine Pkcϵ-targeting shRNAs (Pkcϵ shRNA). The transduced cells were subsequently purified by FACS to assess protein expression, cell growth in vitro and leukemia induction in vivo (Supplementary Figs. S2A and S2B). Depletion of Pkcϵ protein significantly reduced the growth of mouse MLL-AF9 leukemia cells in cytokine-enriched liquid culture (Fig. 1A and B; Supplementary Fig. S2C–S2E). Furthermore, mice transplanted with FACS-purified mouse MLL-AF9 leukemia cells expressing Pkcϵ shRNA exhibited a significantly longer onset of disease compared with CTRL shRNA–expressing cells (Fig. 1C). Furthermore, depletion of Pkcϵ protein significantly reduced the colony-forming capacity (CFC) of mouse MLL-AF9 leukemia cells in cytokine-enriched methylcellulose (Fig. 1D).

In addition to MLL-AF9, we observed that PKCϵ supports the growth of mouse hematopoietic stem and progenitor cells (HSPC) by expressing alterations in genes commonly mutated in AML. Briefly, leukemia cells coexpressing MLL-AF9 and an internal tandem duplication (ITD) of the murine Flt3 gene (MLL-AF9;Flt3^ITD) were transduced with lentiviruses expressing GFP in combination with either CTRL or Pkcϵ shRNAs. HSPCs null for the combination of Dnmt3a and Tet2 deletion (Dnmt3a^−/−;Tet2^−/−) as well as Dnmt3a^−/−;Tet2^−/− HSPCs coexpressing human FLT3-ITD and GFP (Dnmt3a^−/−;Tet2^−/−;FLT3^ITD) were transduced with lentiviruses expressing RFP in combination with either CTRL or Pkcϵ shRNAs. Following stable transduction, cells from each condition were purified by FACS and plated separately in cytokine-enriched methycellulose. In all of the models analyzed, shRNA-mediated inhibition of PKCϵ expression significantly hindered the CFC of leukemic cells compared with CTRL shRNA–expressing cells (Fig. 1E–G).

To investigate the functional role of PKCϵ in patient-derived AML samples, we employed an shRNA approach to inhibit the expression of PKCϵ in 10 subtype diverse patient-derived AML samples (Supplementary Table S1; Supplementary Fig. S3A). Briefly, cryopreserved patient-derived samples were thawed and plated on the HS-27 supportive stroma in cytokine-enriched media. After a short recovery period, cells were transduced with lentiviruses coexpressing GFP and either CTRL shRNA or PKCϵ shRNA_1 and subsequently replated on fresh HS-27 stroma cells. Three days after transduction, cells were assessed by flow cytometry to determine the percentage of live hCD45⁺, GFP⁺ cells (represented as time point day 0). Cells were cocultured for an additional 9 days and then assessed for the percentage of live hCD45⁺, GFP⁺ cells (time point day 9). We then determined the fold change in the percentage of hCD45⁺, GFP⁺ cells by dividing the day 9 percentages of hCD45⁺, GFP⁺ divided by the percentages of hCD45⁺, GFP⁺ at day 0. From this analysis, we observed that 8 of the 10 patient samples expressing PKCϵ-targeting shRNAs displayed a significantly lower fold change in the percentage of hCD45⁺, GFP⁺ cells compared with those samples expressing CTRL shRNAs (Fig. 2; Supplementary Fig. S3B). These data indicate that PKCϵ supports leukemia growth in a genetically diverse subset of patient-derived AML samples.

Given the emerging recognition that ROS influences both normal and malignant HSPCs (4–6) and the previous connections of PKCϵ to redox biology (25–28), we investigated the relationship between PKCϵ and ROS biology in AML cells. As an initial assessment of whether shRNA-mediated inhibition of PKCϵ impacted intracellular ROS levels, we stained CTRL and PKCϵ-shRNA expressing cells with the fluorogenic probe CellROX, which detects multiple types of ROS. From this analysis, we found that inhibition of PKCϵ resulted in an increase of CellROX staining in OCI-AML3, NOMO1, and THP-1 cells (Fig. 3A; Supplementary Figs. S3C and S3D).

To determine the localization and further define the specific types of ROS regulated by PKCϵ, OCI-AML3 cells were engineered to express the redox-sensitive roGFP2 protein genetically fused with glutaredoxin (Grx1) or the hydrogen peroxide (H₂O₂)-neutralizing yeast peroxidase Orp1. The Grx1-roGFP2 probe measures the redox potential of GSH:GSSG redox couples, where roGFP2-Orp1 probe measures changes in H₂O₂ levels. Both probes are expressed in the cytoplasm, however, we also engineered cells to express versions of each probe that are tagged with mitochondrial localization signals (Grx1-roGFP2–mito and roGFP2-Orp1–mito) to evaluate changes in mitochondria ROS biology (Supplementary Figs. S4A and S4B). roGFP2 has an emission of 510 nm as well as excitation peaks at 400 and 490 nm. Upon oxidation, the intensity of the 400-nm roGFP2 peak increases while the amplitude of the 490-nm peak decreases resulting in the ratio of peak intensities shifting toward 400 nm (23). OCI-AML3 cells were treated with either the oxidizing agent diamide (DIA) or the reducing agent dithiothreitol (DTT) to establish the oxidized and reduced gates (data not shown). Using these probes, we observed that the expression of PKCϵ shRNA_1 or shRNA_2 resulted in a significantly higher percentage of oxidized OCI-AML3 cells expressing either cytoplasmic or mitochondrial Grx1-roGFP2, compared with control shRNAs. We also observed that PKCϵ inhibition led to a significant increase in the percentage of cells expressing oxidized roGFP2-Orp1 in the mitochondria (Fig. 3B; Supplementary Fig. S4A and S4B).

The most well-defined ROS generated by mitochondria are superoxides, which are either neutralized by glutathione or converted to H₂O₂ by superoxide-dimutase (SOD) enzymes. Given that PKCϵ inhibition consistently altered mitochondrial levels of glutathione and H₂O₂, we next examined how PKCϵ inhibition impacted mitochondrial superoxide levels using the fluorogenic probe MitoSOX. From this analysis, we observed that shRNA-mediated inhibition of PKCϵ led to a significant increase in MitoSOX staining in both human [OCI-AML3, Fig. 3C; Supplementary Figs. S5A (THP-1) and S5B] and mouse (MLL-AF9, Fig. 3D) AML cells. We also observed that shRNA-mediated inhibition of PKCϵ in patient-derived AML cells expressing MLL-AF9 also display elevated MitoSOX levels (Supplementary Fig. S5C). Collectively, these findings reveal that PKCϵ regulates steady-state levels of mitochondrial ROS and possibly certain cytoplasmic ROS in human and mouse AML cells.

To determine whether changes in the intracellular redox biology contribute to the antileukemia effects of PKCϵ inhibition, we assessed how various chemical antioxidants impacted the growth and survival of AML cells expressing CTRL and PKCϵ-targeting shRNAs. Twice daily treatment with either N-acetyl-l-cysteine (NAC) or glutathione was unable to reverse the antileukemia effects of PKCϵ inhibition in both human and mouse cells despite being able to effectively block the cytotoxicity of the glutathione-depleting pro-oxidant, Menadione (Supplementary Fig. S6A and S6B and data not shown). However, administration of either butylated hydroxyanisole (BHA) or MitoTEMPO, both compounds that neutralize mitochondrial ROS, was able to significantly reduce the cell death of human and mouse AML cells mediated by PKCϵ inhibition (Fig. 4A and B; Supplementary Fig. S6C).

To examine how specific neutralization of mitochondrial ROS impacts the antileukemia effects of PKCϵ inhibition, we engineered OCI-AML3 cells to constitutively coexpress SOD2 and Catalase (SOD2-Catalase), which neutralize mitochondrial superoxides and H₂O₂, respectively. Control and SOD2-Catalase–expressing OCI-AML3 cells were then transduced with CTRL or PKCϵ shRNAs and subsequently analyzed for MitoSOX levels and cell death. SOD2-Catalase expression significantly blocked the induction of MitoSOX staining mediated by PKCϵ inhibition (Supplementary Fig. S6D) and significantly reduced cell death induced by PKCϵ-targeting shRNAs (Fig. 4C). We also observed that SOD2-Catalase expression restored the CFC of mouse MLL-AF9 leukemia cells expressing Pkcϵ shRNA (Fig. 4D) further indicating that AML cells rely on PKCϵ to maintain mitochondrial ROS levels and survival.

To examine how a specific induction of mitochondrial superoxides impacts AML cell growth, we evaluated how shRNAs directly targeting SOD2 impacted human AML cell biology (Supplementary Fig. S7A). Similar to PKCϵ inhibition, shRNA-mediated depletion of SOD2 led to increased steady-state levels of MitoSOX and reduced AML cell growth and survival (Supplementary Fig. S7B–S7D). Furthermore, direct inhibition of SOD2 in mouse MLL-AF9 leukemia cells (Fig. 5A) led to increased levels of MitoSOX staining (Fig. 5B), reduced CFC (Supplementary Fig. S7E) and significantly delayed the time of disease onset in vivo (Fig. 5C). These results show that similar to the antileukemia effects of PKCϵ inhibition, SOD2 inhibition leads to an accumulation of mitochondrial ROS and diminishes AML cell growth and survival.

To identify potential downstream effectors of PKCϵ in AML, we performed nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC/MS-MS) on OCI-AML3 cells expressing CTRL shRNA, PKCϵ shRNA_1, or PKCϵ shRNA_2. From this analysis, 3,192 peptides were captured and using a statistical cutoff of P < 0.05, we found that 707 peptides were differently expressed between CTRL shRNA and PKCϵ shRNA_1–expressing cells and 641 peptides were differently expressed between CTRL shRNA and PKCϵ shRNA_2–expressing cells. Furthermore, we observed that 226 peptides were similarly differentially expressed between OCI-AML3 cells expressing CTRL shRNA versus PKCϵ shRNA_1 and CTRL shRNA versus PKCϵ shRNA_2. Integrated pathway analysis did not reveal any significant enrichment of particular molecular pathways or processes however, we did observe that approximately 15% (34 proteins) of these differentially expressed proteins between CTRL shRNA and either PKCϵ shRNA_1 or PKCϵ shRNA_2 were related to mitochondrial biology. Aberrant mitochondrial ROS arise from multiple disruptions in mitochondrial biology such as reduced expression of antioxidant systems, perturbations in the activities of the complexes that regulate electron flux through the electron transport chain (ETC), as well as alterations in outer mitochondrial membrane (OMM) potential and transport. Many of the proteins whose expression is altered by PKCϵ inhibition are components of ETC complexes involved in the regulation of OMM potential (UQCR10, ATPC1, ATP5H, COX6C, SDHAF2, and NDUFB10) or proteins involved in mitochondrial membrane transport (VDAC1, VDAC3, TOMM22, SLC25A1, SLC25A11, and SLC25A12; Fig. 6A). We also observed that two antioxidant proteins, GSS and TXN, were significantly reduced by PKCϵ inhibition as well as a variety of other antioxidant proteins that were reduced but not statistically significant (Supplementary Fig. S7F).

To assess the role PKCϵ in superoxide-induced oxidative stress, we examined how modulating PKCϵ expression impacted the survival of AML cells challenged with either of the mitochondrial superoxide inducing agents, 2-thenoyltrifluoroacetone (TTFA) or antimycin A (AA). To examine whether increased expression of PKCϵ is able to protect AML cells from the cytotoxic effects of TTFA or AA, we generated OCI-AML3, THP-1, and mouse MLL-AF9–expressing AML cells that constitutively express PKCϵ (Supplementary Fig. S8A and data not shown). Constitutive PKCϵ expression did not impact OCI-AML3 growth or the CFC of mouse MLL-AF9 leukemia cells (Supplementary Fig. S8B and S*C). However, PKCϵ-expressing OCI-AML3 or THP-1 cells treated with TTFA or AA displayed significantly lower percentages of cell death compared with similarly treated control cells (Fig. 6B and C; Supplementary Fig. S8E and S8F). In addition, constitutive PKCϵ expression significantly improved the CFC of mouse MLL-AF9 leukemia cells challenged with TTFA or AA compared with similarly treated control cells (Fig. 6D). Of all the human AML cell lines we tested, U937 cells were the least impacted by PKCϵ inhibition, cell viability–wise (Supplementary Fig. S1B, bottom). Therefore, we evaluated whether PKCϵ inhibition rendered U937 cells more sensitive to TTFA and/or AA treatment. From this analysis, we found that shRNA-mediated inhibition of PKCϵ exacerbated the cytotoxic effects of TTFA and AA in comparison with PKCϵ inhibition or pro-oxidant treatment alone (Supplementary Fig. S8G and S8H). Collectively, these results suggest that PKCϵ protects AML cells from agents that perturb mitochondrial function and induce oxidative stress.

PKCϵ has been implicated as an oncogenic kinase in several human cancers including prostate, breast, colon, lung, and certain forms of squamous cell carcinoma (29–34). In AML, downmodulation of PKCϵ is necessary for the prodifferentiating actions of phorbol esters (20, 35), insinuating that PKCϵ supports leukemia growth and expansion. However, the impact of blocking PKCϵ expression on AML growth and survival has not been comprehensively investigated.

Here, we demonstrate that shRNA-mediated reduction of PKCϵ significantly reduces the survival of human AML cells in vitro and significantly impedes disease progression in a GEMM of AML driven by MLL-AF9. In addition, inhibition of PKCϵ reduced the in vitro growth properties of multiple, genetically diverse patient-derived AML samples confirming that the proleukemia roles of PKCϵ is not restricted to a particular genetic subtype of AML.

At the molecular level, we have discovered that PKCϵ is a key regulator of intracellular redox homeostasis in several mouse and human AML models. Using multiple ROS detection strategies we have found that shRNA-mediated inhibition of PKCϵ increases the steady-state levels of multiple ROS, including several mitochondrial ROS. On the basis of these observations, we postulated that increased/excess production of mitochondrial ROS antagonizes leukemia cell viability and that management of mitochondrial ROS levels is a key proleukemia function of PKCϵ. We have made three central observations that support these hypotheses. First, chemical antioxidants that specifically neutralize mitochondrial ROS, such as BHA (36) and MitoTEMPO (37) are able to significantly and consistently blunt cell death mediated by PKCϵ inhibition, whereas indiscriminant antioxidants such as NAC and glutathione are not. Moreover, reconstitution of mitochondrial ROS-neutralizing enzymes SOD2 and Catalase, partially reverses mitochondrial superoxide induction and cell death mediated by PKCϵ inhibition. Second, similar to PKCϵ inhibition, shRNA-mediated inhibition of SOD2 increased mitochondrial ROS, limited disease progression in vivo and suppressed the growth of patient-derived AML samples. Third, elevated expression of PKCϵ is able to largely protect AML cells from otherwise toxic doses of agents that drive mitochondrial ROS production. Moreover, impeding PKCϵ expression rendered leukemia cells more sensitive to these pro-oxidants. Collectively, these results support that certain sub-types of AML rely on PKCϵ for proper management of mitochondrial ROS biology and survival.

Our observations that the anti-leukemia effects of PKCϵ inhibition could not by fully rescued by SOD2-Catalase expression, MitoTEMPO or BHA suggest that PKCϵ may regulate additional molecular processes to support AML cell survival. Our proteomic analysis shows that, in addition to multiple key ROS-buffering enzymes, such as TXN and GSS, several proteins related to mitochondrial biology and function are impacted by PKCϵ inhibition. Therefore, it remains possible that the increase in mitochondrial ROS mediated by PKCϵ inhibition is due to mitochondrial dysfunction either in addition to or in place of the observed decrease in ROS-regulating enzymes. Consistent with this idea, several studies have shown that the mitochondrial redox state of AML cells is directly related to their metabolic needs. Specifically, compared to bulk AML cells, leukemia-initiating cells from multiple AML patients display a lower oxidized redox environment that is associated with lower rates of oxidative phosphorylation (OXPHOS; ref. 38). Furthermore, cytarabine-treated AML cells display high levels of mitochondrial ROS and OXPHOS and this altered mitochondrial state may contribute to cytarabine resistance (39). Also, multiple AML patients display increased mitochondrial mass compared with healthy HSPCs and as a result are more sensitive to OXPHOS-induced oxidative stress (40).

Although our results indicate that a key proleukemia function of PKCϵ is to manage mitochondrial ROS biology, they do not exclude the possibility that PKCϵ regulates additional redox-related (that are mitochondrial-independent) and/or redox-independent mechanisms to support AML cell survival. For example, our proteomic analysis shows that PKCϵ inhibition, although not statistically significant, impacts multiple redox-regulatory systems. For example, protein levels of both PRDX2 and PRDX4, which have been implicated as growth suppressors in AML and acute promyelocytic leukemia (APL), respectively, increase upon PKCϵ inhibition (41, 42). PKCϵ inhibition also altered the expression of various glutathione-regulatory components and Pei and colleagues (4) have shown that pharmacologic inhibition of glutathione metabolic enzymes, such as GPX1 and GCLC, antagonize primitive human leukemia cell survival. In fact, the relationship of PKCϵ and redox biology varies among distinct biological settings. PKCϵ activation in neuronal and cardiac tissues correlates with the induction of ROS mediated by ischemia, hypoxia or pro-oxidants such as buthionine sulfoximine (BSO) or AA. However, other studies have shown that PKCϵ activation promotes ROS generation in smooth muscle and immortalized epithelial cells and that hepatocytes void of PKCϵ display enhanced stress-induced ROS formation (28, 43). These divergent observations may result from distinct tissue- or ROS-specific roles of PKCϵ; however, in the context of AML, our results establish that PKCϵ works to suppress mitochondrial ROS and possibly other types of ROS.

Several studies suggest that tumor cells, including leukemia cells, maintain high levels of ROS to drive cell growth and survival and therefore targeting redox regulators may be a viable anticancer therapeutic strategy (2, 34, 44). In our models, reducing steady-state levels of mitochondrial superoxides through the combined over-expression of SOD2 and Catalase does not impede colony formation suggesting that elevated mitochondrial ROS are not a central driver of AML cell growth in this model. However, increasing mitochondrial ROS, by inhibiting PKCϵ or SOD2 or by administering chemical ROS-inducing agents diminishes AML cell survival. Collectively, these results suggest that strategies for increasing, rather than decreasing, mitochondrial ROS may carry a significant therapeutic potential. Consistent with this concept, the efficacy of arsenic trioxide, which is commonly used to treat APL, works primarily by inducing ROS (45). Moreover, high doses of vitamin C, which have been previously shown to induce toxic ROS levels in certain types of cancer cells (46), were recently reported to selectively eliminate AML cells carrying TET2 (47, 48) or IDH (49) mutations. However, it should be noted that the proposed mechanism of action of Vitamin C in AML is to activate other TET family members (47, 48).

The intracellular redox environment of AML cells is often distinct from their normal counterparts. Therefore, identifying and defining the molecular regulators of redox biology, such as PKCϵ and SOD2, may provide key insights into the etiology and pathogenesis of AML as well as possibly contribute to the design of more effective antileukemia therapies. However, AML encompasses a wide variety of genetic subtypes and individual tumors often display complex clonal heterogeneity (50) and it remains unclear which genetic subtypes are susceptible to redox imbalances. Our loss-of-function studies in patient-derived AML cells show that not all AML samples rely on PKCϵ for growth and survival. Thus, it is possible that certain AML cells utilize PKCϵ- and/or SOD2-independent mechanisms to regulate mitochondrial superoxide biology or that certain genetic subtypes or clones are insensitive to increases in mitochondrial superoxide levels. Therefore, future studies defining the genetic subtypes that are sensitive to changes in redox homeostasis or PKCϵ/SOD2 inhibition as well as the role of PKCϵ/SOD2 in healthy HSPC biology will be needed to fully gauge the therapeutic potential of targeting these pathways in AML.

No potential conflicts of interest were disclosed.

Conception and design: D.Di Marcantonio, E. Masselli, M.D. Milsom, G. Gobbi, S.M. Sykes

Development of methodology: J. Michael Meadows, G. Gobbi, S.M. Sykes

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.Di Marcantonio, E. Martinez, J. Vadaketh, A. Gupta, J. Michael Meadows, G.A. Challen, B. Garcia, R. Garzon, S.M. Sykes

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.Di Marcantonio, J. Vadaketh, A. Gupta, J. Michael Meadows, F. Ferraro, M.D. Milsom, S. Froehling, P. Mirandola, G. Gobbi, S.M. Sykes

Writing, review, and/or revision of the manuscript: D.Di Marcantonio, E. Masselli, M.D. Milsom, C. Scholl, S. Froehling, S. Balachandran, T. Skorski, R. Garzon, M. Vitale, S.M. Sykes

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Nieborowska-Skorska, T. Skorski, S.M. Sykes

Study supervision: P. Mirandola, M. Vitale, S.M. Sykes

Other (responsible for the design, execution and interpretation of mass spectrometry studies): S. Sidoli

Other (advised in research design): C. Scholl

This work was supported by the NIH Grant R00 CA158461, the ASH Junior Scholar Award, W.W. Smith and Bob and Jeanne Brennan (to S.M. Sykes); the Rotary Foundation, Grant GG1414529 and the Board of Directors of Fox Chase Cancer Center Fellowship (to D. Di Marcantonio); CURE supplement (CA06927; to J. Vadaketh and A. Gupta); Jeanne E. and Robert F. Ozols Undergraduate Summer Research Fellowship, Fox Chase Cancer Center (to J. Michael Meadows); NIH grant P01CA196539, DOD grant W81XWH-113-1-0426 and the Leukemia and Lymphoma Society Dr. Robert Arceci Scholar Award (to B. Garcia), The Dietmar Hopp Stiftung (to M.D. Milsom), the NIH Grant 1R01DK102428 (to G.A. Challen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.