Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

The arsenic-based cure of acute promyelocytic leukemia promotes cytoplasmic sequestration of PML and PML/RARA through inhibition of PML body recycling.

Arsenic in the form of arsenic trioxide (ATO) is used as a therapeutic drug for treatment of acute promyelocytic leukemia (APL). The mechanism by which this agent cures this disease was previously shown to involve direct interactions between ATO and the promyelocytic leukemia protein (PML), as well as accelerated degradation of the APL-associated fusion oncoprotein PML/retinoic acid receptor α (RARA). Here we investigated the fate of PML-generated nuclear structures called PML bodies in ATO-treated cells. We found that ATO inhibits formation of progeny PML bodies while it stabilizes cytoplasmic precursor compartments, referred to as cytoplasmic assemblies of PML and nucleoporins (CyPNs), after cell division. This block in PML body recycling is readily detected at pharmacologic relevant ATO concentrations (0.02-0.5µM) that do not cause detectable cell-cycle defects, and it does not require modification of PML by SUMOylation. In addition, PML and PML/RARA carrying mutations previously identified in ATO-resistant APL patients are impeded in their ability to become sequestered within CyPNs. Thus, ATO may inhibit nuclear activities of PML and PML/RARA in postmitotic cells through CyPN-dependent cytoplasmic sequestration.

Subcellular distribution of nuclear import-defective isoforms of the promyelocytic leukemia protein.

BACKGROUND: The promyelocytic leukemia (PML) protein participates in a number of cellular processes, including transcription regulation, apoptosis, differentiation, virus defense and genome maintenance. This protein is structurally organized into a tripartite motif (TRIM) at its N-terminus, a nuclear localization signal (NLS) at its central region and a C-terminus that varies between alternatively spliced isoforms. Most PML splice variants target the nucleus where they define sub-nuclear compartments termed PML nuclear bodies (PML NBs). However, PML variants that lack the NLS are also expressed, suggesting the existence of PML isoforms with cytoplasmic functions. In the present study we expressed PML isoforms with a mutated NLS in U2OS cells to identify potential cytoplasmic compartments targeted by this protein. RESULTS: Expression of NLS mutated PML isoforms in U2OS cells revealed that PML I targets early endosomes, PML II targets the inner nuclear membrane (partially due to an extra NLS at its C-terminus), and PML III, IV and V target late endosomes/lysosomes. Clustering of PML at all of these subcellular locations depended on a functional TRIM domain. CONCLUSIONS: This study demonstrates the capacity of PML to form macromolecular protein assemblies at several different subcellular sites. Further, it emphasizes a role of the variable C-terminus in subcellular target selection and a general role of the N-terminal TRIM domain in promoting protein clustering.

Replication protein A prevents accumulation of single-stranded telomeric DNA in cells that use alternative lengthening of telomeres.

The activation of a telomere maintenance mechanism is required for cancer development in humans. While most tumors achieve this by expressing the enzyme telomerase, a fraction (5-15%) employs a recombination-based mechanism termed alternative lengthening of telomeres (ALT). Here we show that loss of the single-stranded DNA-binding protein replication protein A (RPA) in human ALT cells, but not in telomerase-positive cells, causes increased exposure of single-stranded G-rich telomeric DNA, cell cycle arrest in G2/M phase, accumulation of single-stranded telomeric DNA within ALT-associated PML bodies (APBs), and formation of telomeric aggregates at the ends of metaphase chromosomes. This study demonstrates differences between ALT cells and telomerase-positive cells in the requirement for RPA in telomere processing and implicates the ALT mechanism in tumor cells as a possible therapeutic target.

The DNA repair protein ALKBH2 mediates temozolomide resistance in human glioblastoma cells.

INTRODUCTION: Glioblastoma multiforme (GBM; World Health Organization astrocytoma grade IV) is the most frequent and most malignant primary brain tumor in adults. Despite multimodal therapy, all such tumors practically recur during the course of therapy, causing a median survival of only 14.6 months in patients with newly diagnosed GBM. The present study was aimed at examining the expression of the DNA repair protein AlkB homolog 2 (ALKBH2) in human GBM and determining whether it could promote resistance to temozolomide chemotherapy. METHODS: ALKBH2 expression in GBM cell lines and in human GBM was determined by quantitative real-time PCR (qRT-PCR) and gene expression analysis, respectively. Drug sensitivity was assessed in GBM cells overexpressing ALKBH2 and in cells in which ALKBH2 expression was silenced by small-interfering (si)RNA. ALKBH2 expression following activation of the p53 pathway was examined by western blotting and qRT-PCR. RESULTS: ALKBH2 was abundantly expressed in established GBM cell lines and human GBM, and temozolomide exposure increased cellular ALKBH2 expression levels. Overexpression of ALKBH2 in the U87 and U251 GBM cell lines enhanced resistance to the methylating agents temozolomide and methyl methanesulfonate but not to the nonmethylating agent doxorubicin. Conversely, siRNA-mediated knockdown of ALKBH2 increased sensitivity of GBM cells to temozolomide and methyl methanesulfonate but not to doxorubicin or cisplatin. Nongenotoxic activation of the p53 pathway by the selective murine double minute 2 antagonist nutlin-3 caused a significant decrease in cellular ALKBH2 transcription levels. CONCLUSION: Our findings identify ALKBH2 as a novel mediator of temozolomide resistance in human GBM cells. Furthermore, we place ALKBH2 into a new cellular context by showing its regulation by the p53 pathway.

Cell-cycle regulation and dynamics of cytoplasmic compartments containing the promyelocytic leukemia protein and nucleoporins.

Nucleoporins and the promyelocytic leukemia protein (PML) represent structural entities of nuclear pore complexes and PML nuclear bodies, respectively. In addition, these proteins might function in a common biological mechanism, because at least two different nucleoporins, Nup98 and Nup214, as well as PML, can become aberrantly expressed as oncogenic fusion proteins in acute myeloid leukemia (AML) cells. Here we show that PML and nucleoporins become directed to common cytoplasmic compartments during the mitosis-to-G1 transition of the cell cycle. These protein assemblies, which we have termed CyPNs (cytoplasmic assemblies of PML and nucleoporins), move on the microtubular network and become stably connected to the nuclear membrane once contact with the nucleus has been made. The ability of PML to target CyPNs depends on its nuclear localization signal, and loss of PML causes an increase in cytoplasmic-bound versus nuclear-membrane-bound nucleoporins. CyPNs are also targeted by the acute promyelocytic leukemia (APL) fusion protein PML-RARalpha and can be readily detected within the APL cell line NB4. These results provide insight into a dynamic pool of cytoplasmic nucleoporins that form a complex with the tumor suppressor protein PML during the G1 phase of the cell cycle.

Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA.

The promyelocytic leukemia protein (PML) participates in several cellular functions, including transcriptional regulation, apoptosis and maintenance of genomic stability. A key feature of this protein is its ability to induce the assembly of nuclear compartments termed PML-nuclear bodies (PML-NBs). Here we show that these nuclear structures recruit single-stranded DNA (ssDNA) molecules in response to exogenous DNA damage. ssDNA was readily detected in PML-NBs within 1 hour following exposure of cells to UV light. Confocal real-time imaging of cells expressing YFP-tagged PML did not reveal de novo formation of new PML-NBs following UV-irradiation, which shows that ssDNA focus formation occurred within pre-existing PML-NBs. Moreover, siRNA-mediated depletion of PML prevented ssDNA focus formation and sensitized cells to UV-induced apoptosis. PML-dependent ssDNA focus formation was found to be particularly efficient during S-phase of the cell cycle, and PML-depleted cells became retarded in S-phase upon growth in the presence of etoposide. In addition, we found that caffeine and the poly(ADP-ribose) polymerase (PARP) inhibitor NU1027 enhanced UV-induced recruitment of ssDNA to PML-NBs. Together, our results show that PML-NBs have the capacity to accommodate DNA metabolic activities that are associated with processing of damaged DNA.