Targeting mitochondrial RNA polymerase in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.

Keywords: acute myeloid leukemia; electron transport chain; mitochondria; mitochondrial RNA polymerase; oxidative phosphorylation.

Figure 1. Effects of shRNA against POLRMT on target knockdown

A. Western blot of POLRMT levels after treatment of OCI-AML2 cells with shGFP, shPOLRMT or sh spRNAP-IV B. Western blot of POLRMT levels in OCI-AML2 cells treated with doxycycline-inducible shNonTargeting or shPOLRMT in the absence or presence of doxycycline C. Relative expression of spRNAP-IV transcript, normalized to 18S, in OCI-AML2 cells treated with shGFP, shPOLRMT or sh spRNAP-IV.

Figure 2. Consequences of POLRMT knockdown on mitochondrial function

A. Effect of POLRMT knockdown on expression of mitochondrial genes ND6, ND3, CO2 and ATP normalized to 18S, expressed relative to shGFP controls at 4 or 6 days post-infection. B. Relative amount of assembled ETC complexes I, II, III, IV and V in OCI-AML2 cells treated with shGFP or shPOLRMT. C. Western blotting of individual ETC subunits in lysates from OCI-AML2 cells infected with shGFP or shPOLRMT. D. Relative viability of OCI-AML2 cells treated with shGFP or shPOLRMT and the complex II inhibitor TTFA or vehicle. E. Basal respiration (oxygen consumption rate, OCR) in OCI-AML2 cells infected with shRNA against a non-targeting control or shPOLRMT. F. Basal respiration rate of OCI-AML2 cells treated with inducible shRNA against a non-targeting control or POLRMT, and treated with doxycycline. G. Basal respiration rate of K562 cells treated with shRNA against POLRMT or GFP H. The proportion of cells with a depolarized inner mitochondrial membrane, in OCI-AML2 cells treated with shGFP or shPOLRMT I. Frequency histogram of TMRM- (left peak) and TMRM + (right peak) OCI-AML2 cells treated with shGFP (solid line) or shPOLRMT#1 (dashed bars).

Figure 3. POLRMT knockdown inhibits growth and viability of OCI-AML2 cells

A. Proliferation curve of OCI-AML2 cells treated with shGFP or shPOLRMT. B. The proportion of Annexin V positive/PI negative cells in OCI-AML2 cells treated with shGFP or shPOLRMT C. The proportion of Annexin V positive/PI negative cells in cells infected with doxycycline-inducible shRNA against a non-targeting control or POLRMT, and treated with doxycycline or vehicle for 6 days. D. Cumulative cell number of OCI-AML2 cells infected with inducible shRNA against a non-targeting control or POLRMT. Cells were treated with doxycycline or vehicle for 10 days E. Histograms of propidium iodide staining intensity in OCI-AML2 cells treated with POLRMT shRNA or GFP shRNA for 6 days.

Figure 4. Effects of 2-C-methyladenosine on leukemia cells

A. Expression of mitochondrial gene cytochrome b normalized to 18S in OCI-AML2 and K562 cells treated with 2-CM for 24 hours. B. Basal respiration (oxygen consumption rate) of OCI-AML2 cells treated with 2-CM for 48 hours, normalized to vehicle controls. The basal respiration (oxygen consumption rate) of K562 cells with POLRMT knockdown or shGFP controls treated with 2-CM for 48 hours, values are normalized to shGFP vehicle controls. C. The proportion of cells with a depolarized inner mitochondrial membrane, in OCI-AML2 cells treated with 2-CM D. Proportion of Annexin V positive/PI negative OCI-AML2 cells treated with 2-CM E. Normal human hematopoietic cells (n = 3) were treated with 0, 10, 50 or 100 μM 2-CM and plated in clonogenic growth assays. Colonies were counted, including CFU-G, CFU-M, CFU-Eo and BFU-E colony-forming units. Similarly, clonogenic growth of OCI-AML2 cells treated with 0, 10, 20 or 50 μM 2-CM was measured. F. Proliferation curve of OCI-AML2 cells treated with 2-C-methyladenosine (2-CM). G. Dose-response curves of growth and viability after 2-CM in five leukemia and lymphoma cell lines. H. Histograms of propidium iodide staining intensity in OCI-AML2 cells treated with 2-CM for 24 hours.

Figure 5. 2-C-methyladenosine decreases tumour growth in OCI-AML2 xenografts

OCI-AML2 human leukemia cells (5 × 10⁵) were injected subcutaneously into the flanks of male SCID mice. At 11 days after injection, once tumours were palpable, mice were treated with 70 mg/kg 2-C-methyladenosine i.p. (n = 10) or vehicle (n = 10). After 8 days of drug treatment, mice were euthanized, tumours and organs were excised, and tumour mass was measured. A. Tumour volume was measured over time. B. At the completion of the experiment, tumour mass of excised tumours was measured. C. Expression of mitochondrial genes CO2 and ND6 in excised tumours was measured and normalized to 18S. D. Plasma levels of asparate transaminase, alkaline phosphatase, creatine, creatine kinase, and bilirubin were measured at the end of the experiment (n = 4 per group). E. Hematoxylin and eosin staining of sections of liver, kidney, heart, skeletal muscle and lung from tumours of mice treated with 2-CM or vehicle.