Marko Skrtic

INHIBITION OF MITOCHONDRIAL TRANSLATION AS A THERAPEUTIC STRATEGY FOR HUMAN ACUTE MYELOID LEUKEMIA

To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.

The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma

The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.

Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML.

AML cells have low spare reserve capacity in their respiratory chain that renders them susceptible to oxidative metabolic stress

Mitochondrial respiration is a crucial component of cellular metabolism that can become dysregulated in cancer. Compared with normal hematopoietic cells, acute myeloid leukemia (AML) cells and patient samples have higher mitochondrial mass, without a concomitant increase in respiratory chain complex activity. Hence these cells have a lower spare reserve capacity in the respiratory chain and are more susceptible to oxidative stress. We therefore tested the effects of increasing the electron flux through the respiratory chain as a strategy to induce oxidative stress and cell death preferentially in AML cells. Treatment with the fatty acid palmitate induced oxidative stress and cell death in AML cells, and it suppressed tumor burden in leukemic cell lines and primary patient sample xenografts in the absence of overt toxicity to normal cells and organs. These data highlight a unique metabolic vulnerability in AML, and identify a new therapeutic strategy that targets abnormal oxidative metabolism in this malignancy.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

Therapeutic potential of mitochondrial translation inhibition for treatment of acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and is the most common form of acute leukemia in adults [1]. While there have been advances in the treatment of certain hematological malignancies, the prognosis of AML remains grim. For example, patients older than 60 years have a 2-year survival of less than 20% despite aggressive treatment. Thus, further research is warranted into developing novel targeted AML therapies with greater efficacy and less toxicity than standard chemotherapy regimens. A challenging aspect of cancer therapy is selectively eliminating the cancer stem cell. Much of the evidence for the cancer stem cell hypothesis has come from studies on hematologic malignancies. Dick and colleagues found leukemia stem cells in a small compartment of the peripheral blood in AML patients [2]. These quiescent malignant cells are capable of self-renewal and differentiation. They are often chemoresistant and are a likely cause of disease relapse. Therefore, therapeutic strategies that target the AML stem cell as well as the bulk AML cell population preferentially over normal stem cells could improve the outcome of this disease. A custom library of the US FDAapproved drugs for agents cytotoxic to TEX and M9-ENL1 cells was recently screened to identify such therapies. These leukemic cell lines have features of leukemia stem cells, including hierarchal differentiation and marrow repopulation [3,4]. This screen identified tigecycline [5], a recently characterized antimicrobial agent of the novel glycylcycline class that is active against a range of Gram-positive and Gram-negative bacteria, particularly drug-resistant pathogens [6]. Subsequent validation studies demonstrated that tigecycline targeted AML cells and AML stem cells preferentially over normal hematopoietic stem cells in vitro and in vivo. To understand the antileukemic mechanism of action of tigecycline, the authors performed genome-wide druginduced haploinsufficiency profiling in Saccharomyces cerevisiae and identified mitochondrial translation inhibition as the mechanism of tigecycline cell death in eukaryotic cells. This finding was validated in AML cell lines and primary AML patient samples. Moreover, it was shown that mitochondrial translation inhibition is functionally important for cell death of leukemia progenitors and stem cells using both chemical and genetic approaches. Finally, it was demonstrated that the heightened sensitivity of AML cells to the inhibition of mitochondrial translation was a derivative of increased mitochondrial mass in these cells [5]. In addition to nuclear DNA, eukaryotic cells have circular mitochondrial DNA located within the mitochondria that is 16.6 kB in length and consists only of exons [7]. Mitochondrial DNA encodes two rRNAs, 22 tRNAs and 13 of the 90 proteins that comprise the mitochondrial respiratory chain and oxidative Expert Rev. Hematol. 5(2), 117–119 (2012)

Hyperfiltration, urinary albumin excretion, and ambulatory blood pressure in adolescents with Type 1 diabetes mellitus

Adolescents with Type 1 diabetes mellitus (T1DM) are at risk for hyperfiltration and elevated urinary albumin-to-creatinine ratio (ACR), which are early indicators of diabetic nephropathy. Adolescents with T1DM also develop early changes in blood pressure, cardiovascular structure, and function. Our aims were to define the relationships between hyperfiltration, ACR, and 24-h ambulatory blood pressure over time in adolescents with T1DM. Normotensive, normoalbuminuric adolescents ( n = 98) with T1DM underwent baseline and 2-yr 24-h ambulatory blood pressure monitoring, glomerular filtration rate (eGFR) estimated by cystatin C (Larsson equation), and ACR measurements. Linear regression models adjusted for diabetes duration, sex, and HbA1c were used to determine associations. Hyperfiltration (eGFR ≥ 133 ml/min) was present in 31% at baseline and 21% at 2-yr follow-up. Hyperfiltration was associated with greater odds of rapid GFR decline (>3 ml·min-1·yr-1) [OR: 5.33, 95%; CI: 1.87-15.17; P = 0.002] over 2 yr. Natural log of ACR at baseline was associated with greater odds of hyperfiltration (OR: 1.71, 95% CI: 1.00-2.92; P = 0.049) and 2-yr follow-up (OR: 2.14, 95%; CI: 1.09-4.19; P = 0.03). One SD increase in eGFR, but not ln ACR, at 2-yr follow-up conferred greater odds of nighttime nondipping pattern (OR: 1.96, 95% CI: 1.06-3.63; P = 0.03). Hyperfiltration was prevalent at baseline and at 2-yr follow-up, predicted rapid decline in GFR, and was related to ACR. Elevated GFR at 2-yr follow-up was associated with nighttime nondipping pattern. More work is needed to better understand early relationships between renal hemodynamic and systemic hemodynamic changes in adolescents with T1DM to reduce future cardiorenal complications.

Abstract 1123: AML cells have low reserve capacity in their respiratory chain complexes leading to increased sensitivity to palmitate-induced cell death

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Mitochondria contain their own DNA that encodes 13 proteins that comprise the respiratory chain. Recently we demonstrated that inhibiting mitochondrial protein synthesis is preferentially cytotoxic to AML cells and stem cells over normal hematopoietic stem cells due to dysregulated mitochondrial biogenesis in AML. Given the dysregulated mitochondrial biogenesis in primary AML cells, we examined the activity of the respiratory chain complexes in primary AML and normal PBSCs. The enzymatic activity of the respiratory chain complexes were equivalent between primary AML cell and normal hematopoietic cells. We then evaluated the reserve capacity of the respiratory chain complexes. Primary AML cells (n=11) and PBSCs (n =5) were treated with increasing concentrations of chemical respiratory complex inhibitors and the effects on oxygen consumption were measured using the 96-well Seahorse XF Extracellular Flux (XF) Analyzer. AML cells with increased mitochondrial mass displayed heightened sensitivity to the complex inhibitors and less reserve capacity in the respiratory complex compared to normal hematopoietic cells. For example, the mean concentration of the complex III inhibitor antimycin required to reduce oxygen consumption by 50% in primary AML cells was 13.7 ± 1.6 nM vs 29.0 ± 2.4 nM (p=0.0007) for normal hematopoietic cells. Thus, small reductions in the enzyme activity of the respiratory complexes produced greater reductions in oxygen consumption in AML cells compared to normal hematopoietic cells. The reduced capacity of the respiratory chain in primary AML cells highlighted a potential therapeutic approach. We speculated that increasing electron flux through the respiratory chain would preferentially overwhelm the capacity of the complexes in AML cells. To test this strategy, AML cell lines and primary AML samples were treated with increasing concentrations of the fatty acid substrate palmitate to increase the production of Acetyl-CoA and increase flux of electrons through the respiratory chain. Treatment of AML cells with palmitate transiently increased oxygen consumption. However, by 4 hours after treatment, reactive oxygen species increased, oxygen consumption decreased and cell death ensued. In contrast, cell lines with greater reserve capacity including normal hematopoietic cells displayed no change in oxygen consumption, reactive oxygen species or cell viability after palmitate treatment. Thus, we have demonstrated that AML cells have reduced reserve capacity in their respiratory chain leading to heightened sensitivity to reductions in respiratory complexes by inhibiting mitochondrial protein synthesis. The reduced reserve capacity also heightens their sensitivity to increased electron flux through the respiratory chain. As such, targeting the aberrant metabolism of AML may be a novel therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1123. doi:1538-7445.AM2012-1123

Abstract 4538: Inhibition of mitochondrial protein synthesis with antimicrobial tigecycline preferentially induces cell death in leukemia cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To identify known drugs with unrecognized anti-leukemia activity, we compiled a library of 500 on patent and off patent compounds, and screened it to identify compounds cytotoxic to leukemia cell lines. From this screen we identified Tigecycline, an antibiotic approved for the treatment of cutaneous and intra-abdominal infections. Tigecycline induced cell death in leukemia cell lines (LD50 3 to 8 μM, n = 6 cell lines) and primary Acute Myeloid Leukemia (AML) patient samples (LD50 5-10 μM, n = 7), preferentially over normal hematopoietic cells (10% cell death at 20 μM, n = 4) by MTS assays and Annexin V staining. Likewise, in colony formation assays, Tigecycline (5μM) reduced the clonogenic growth of primary AML patient samples (n = 4) by 95±1.5 %, demonstrating an effect on leukemia progenitor cells. In contrast, 5 μM of Tigecycline reduced the clonogenic growth of normal hematopoetic cells by 34± 5% (n = 5). Although Tigecycline is structurally related to tetracycline and minocycline, these drugs were not cytotoxic towards AML cell lines up to 25 μM. Thus, Tigecycline appears preferentially cytotoxic to leukemia cells at pharmacologically achievable concentrations. Given the anti-leukemic effects of Tigecycline in vitro, we evaluated the efficacy of Tigecycline as a potential anti-leukemic agent in vivo. Mice injected subcutaneously with OCI-AML2 leukemia cells were treated with Tigecycline 50 mg/kg i.p. daily. Compared to control, Tigecycline decreased tumour mass and volume without toxicity. We also assessed the effect of Tigecycline on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD-SCID mice were injected intra-femorally with primary AML cells. Two weeks after injection, mice were treated with Tigecycline 50 mg/kg i.p. daily for two weeks. After treatment, engraftment of human AML cells was measured by flow cytometry. Compared to control, Tigecycline decreased engraftment of AML cells without toxicity. Tigecycline binds and inhibits the bacterial 30S ribosome. Bacterial ribosomes are more homologous to mitochondrial ribosomes than cytosolic ribosomes, so we compared the effects of Tigecycline on mitochondrial and cytosolic protein synthesis. At times preceding the onset of cell death, Tigecycline decreased levels of the mitochondrial protein Cox-1. In contrast, it did not decrease the expression of cytosolic short half-life proteins Bcl-XL and XIAP, suggesting a preferential effect on mitochondrial protein synthesis. Thus, Tigecycline demonstrated preclinical activity through a mechanism related to mitochondrial protein synthesis inhibition. Moreover, Tigecycline appeared cytotoxic to leukemia stem cells over normal hematopoetic stem cells. Given its prior pharmacology and toxicology testing, Tigecycline could be rapidly repositioned for a new anti-leukemic indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4538.