The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that ...hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.
•Activation of the mitochondrial ClpP induces p53-independent cancer cell lethality•Imipridones are allosteric agonists of ClpP, being tested in human clinical trials•ClpP activation increases proteolysis of mitochondrial proteins•ClpP-mediated mitochondrial proteolysis impairs mitochondrial respiratory function
Ishizawa et al. report that hyperactivating the mitochondrial caseinolytic protease P (ClpP) degrades respiratory chain proteins, disrupts mitochondrial function, and selectively kills cancer cells, regardless of p53 status. They identify imipridones as hyperactivators of ClpP and show their anti-tumor activity.
AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants ...of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin β family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.
From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has ...structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.
•ClpP is a mitochondrial protease overexpressed in a subset of AML and stem cells•Inhibition of ClpP decreases the viability of AML cells with high ClpP expression•ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes•Genetic knockdown of ClpP impairs oxidative phosphorylation and complex II
Cole et al. show that ClpP, a mitochondrial protease, is overexpressed in a large fraction of human acute myeloid leukemias (AMLs) and that inactivation of ClpP selectively kills these AML cells via inhibition of oxidative phosphorylation and mitochondrial metabolism.
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small ...molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like ...modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive ...chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.
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.
On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we ...conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.
AML (acute myeloid leukemia) cells have a unique reliance on mitochondrial metabolism and fatty acid oxidation (FAO). Thus, blocking FAO is a potential therapeutic strategy to target these malignant ...cells. In the current study, we assessed plasma membrane carnitine transporters as novel therapeutic targets for AML. We examined the expression of the known plasma membrane carnitine transporters, OCTN1, OCTN2, and CT2 in AML cell lines and primary AML samples and compared expression to normal hematopoietic cells. Of the three carnitine transporters, CT2 demonstrated the greatest differential expression between AML and normal cells. Using shRNA, we knocked down CT2 and demonstrated that target knockdown impaired the function of the transporter. In addition, knockdown of CT2 reduced the growth and viability of AML cells with high expression of CT2 (OCI-AML2 and HL60), but not low expression. CT2 knockdown reduced basal oxygen consumption without a concomitant increase in glycolysis. Thus, CT2 may be a novel target for a subset of AML.
Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole ...mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine
, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.
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