Are DNA damage response kinases a target for the differentiation treatment of acute myeloid leukemia?

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by the expansion of hematopoietic stem/progenitor cells (HPCs) blocked at different stages of maturation/differentiation. The poor outcome of AMLs necessitates therapeutic improvement. In AML, genes encoding for myeloid transcription factors, signaling receptors regulating cell proliferation, and epigenetic modifiers can be mutated by somatically acquired genetic mutations or altered by chromosomal translocations. These mutations modify chromatin organization at genes sites regulating HPCs proliferation, terminal differentiation, and DNA repair, contributing to the development and progression of the disease. The reversibility of the epigenetic modifications by drug treatment makes epigenetic changes attractive targets for AML therapeutic intervention. Recent findings underline increased DNA damage and abnormalities in the DNA damage response (DDR) as a critical feature of AML blasts. The DDR preserves cell integrity and must be tightly coordinated with DNA methylation and chromatin remodeling to ensure the accessibility to the DNA of transcription factors and repair enzymes. A crucial role in these events is played by the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) kinases, which are hyperactive in AML. Based on these findings, we hypothesize the inhibition of DNA damage kinases as an alternative or complementary strategy for the differentiation treatment of AML as it leads to a reduced ability to repair the DNA damage, and to the inhibition of specific epigenetic modifiers whose function is altered in leukemic cells. © 2018 IUBMB Life, 70(11):1057-1066, 2018.

Molecular Evolution of Alternative Oxidase Proteins: A Phylogenetic and Structure Modeling Approach.

Alternative oxidases (AOXs) are mitochondrial cyanide-resistant membrane-bound metallo-proteins catalyzing the oxidation of ubiquinol and the reduction of oxygen to water bypassing two sites of proton pumping, thus dissipating a major part of redox energy into heat. Here, the structure of Arabidopsis thaliana AOX 1A has been modeled using the crystal structure of Trypanosoma brucei AOX as a template. Analysis of this model and multiple sequence alignment of members of the AOX family from all kingdoms of Life indicate that AOXs display a high degree of conservation of the catalytic core, which is formed by a four-α-helix bundle, hosting the di-iron catalytic site, and is flanked by two additional α-helices anchoring the protein to the membrane. Plant AOXs display a peculiar covalent dimerization mode due to the conservation in the N-terminal region of a Cys residue forming the inter-monomer disulfide bond. The multiple sequence alignment has also been used to infer a phylogenetic tree of AOXs whose analysis shows a polyphyletic origin for the AOXs found in Fungi and a monophyletic origin of the AOXs of Eubacteria, Mycetozoa, Euglenozoa, Metazoa, and Land Plants. This suggests that AOXs evolved from a common ancestral protein in each of these kingdoms. Within the Plant AOX clade, the AOXs of monocotyledon plants form two distinct clades which have unresolved relationships relative to the monophyletic clade of the AOXs of dicotyledonous plants. This reflects the sequence divergence of the N-terminal region, probably due to a low selective pressure for sequence conservation linked to the covalent homo-dimerization mode.

A PET-Surrogate Signature for the Interrogation of the Metabolic Status of Breast Cancers.

Metabolic alterations in cancers can be exploited for diagnostic, prognostic, and therapeutic purposes. This is exemplified by 18F-fluorodeoxyglucose (FDG)-positron emission tomography (FDG-PET), an imaging tool that relies on enhanced glucose uptake by tumors for diagnosis and staging. By performing transcriptomic analysis of breast cancer (BC) samples from patients stratified by FDG-PET, a 54-gene signature (PETsign) is identified that recapitulates FDG uptake. PETsign is independently prognostic of clinical outcome in luminal BCs, the most common and heterogeneous BC molecular subtype, which requires improved stratification criteria to guide therapeutic decision-making. The prognostic power of PETsign is stable across independent BC cohorts and disease stages including the earliest BC stage, arguing that PETsign is an ab initio metabolic signature. Transcriptomic and metabolomic analysis of BC cells reveals that PETsign predicts enhanced glycolytic dependence and reduced reliance on fatty acid oxidation. Moreover, coamplification of PETsign genes occurs frequently in BC arguing for their causal role in pathogenesis. CXCL8 and EGFR signaling pathways feature strongly in PETsign, and their activation in BC cells causes a shift toward a glycolytic phenotype. Thus, PETsign serves as a molecular surrogate for FDG-PET that could inform clinical management strategies for BC patients.

The clinically relevant CHK1 inhibitor MK-8776 induces the degradation of the oncogenic protein PML-RARα and overcomes ATRA resistance in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by the expression of the oncogenic fusion protein PML-RARα. The current treatment approach for APL involves differentiation therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the development of resistance to therapy, occurrence of differentiation syndrome, and relapses necessitate the exploration of new treatment options that induce differentiation of leukemic blasts with low toxicity. In this study, we investigated the cellular and molecular effects of MK-8776, a specific inhibitor of CHK1, in ATRA-resistant APL cells. Treatment of APL cells with MK-8776 resulted in a decrease in PML-RARα levels, increased expression of CD11b, and increased granulocytic activity consistent with differentiation. Interestingly, we showed that the MK-8776-induced differentiating effect resulted synergic with ATO. We found that the reduction of PML-RARα by MK-8776 was dependent on both proteasome and caspases. Specifically, both caspase-1 and caspase-3 were activated by CHK1 inhibition, with caspase-3 acting upstream of caspase-1. Activation of caspase-3 was necessary to activate caspase-1 and promote PML-RARα degradation. Transcriptomic analysis revealed significant modulation of pathways and upstream regulators involved in the inflammatory response and cell cycle control upon MK-8776 treatment. Overall, the ability of MK-8776 to induce PML-RARα degradation and stimulate differentiation of immature APL cancer cells into more mature forms recapitulates the concept of differentiation therapy. Considering the in vivo tolerability of MK-8776, it will be relevant to evaluate its potential clinical benefit in APL patients resistant to standard ATRA/ATO therapy, as well as in patients with other forms of acute leukemias.

Control of replication stress and mitosis in colorectal cancer stem cells through the interplay of PARP1, MRE11 and RAD51.

Cancer stem cells (CSCs) are tumor subpopulations driving disease development, progression, relapse and therapy resistance, and their targeting ensures tumor eradication. CSCs display heterogeneous replication stress (RS), but the functionality/relevance of the RS response (RSR) centered on the ATR-CHK1 axis is debated. Here, we show that the RSR is efficient in primary CSCs from colorectal cancer (CRC-SCs), and describe unique roles for PARP1 and MRE11/RAD51. First, we demonstrated that PARP1 is upregulated in CRC-SCs resistant to several replication poisons and RSR inhibitors (RSRi). In these cells, PARP1 modulates replication fork speed resulting in low constitutive RS. Second, we showed that MRE11 and RAD51 cooperate in the genoprotection and mitosis execution of PARP1-upregulated CRC-SCs. These roles represent therapeutic vulnerabilities for CSCs. Indeed, PARP1i sensitized CRC-SCs to ATRi/CHK1i, inducing replication catastrophe, and prevented the development of resistance to CHK1i. Also, MRE11i + RAD51i selectively killed PARP1-upregulated CRC-SCs via mitotic catastrophe. These results provide the rationale for biomarker-driven clinical trials in CRC using distinct RSRi combinations.

NBS1 interacts with HP1 to ensure genome integrity.

Heterochromatin Protein 1 (HP1) and the Mre11-Rad50-Nbs1 (MRN) complex are conserved factors that play crucial role in genome stability and integrity. Despite their involvement in overlapping cellular functions, ranging from chromatin organization, telomere maintenance to DNA replication and repair, a tight functional relationship between HP1 and the MRN complex has never been elucidated. Here we show that the Drosophila HP1a protein binds to the MRN complex through its chromoshadow domain (CSD). In addition, loss of any of the MRN members reduces HP1a levels indicating that the MRN complex acts as regulator of HP1a stability. Moreover, overexpression of HP1a in nbs (but not in rad50 or mre11) mutant cells drastically reduces DNA damage associated with the loss of Nbs suggesting that HP1a and Nbs work in concert to maintain chromosome integrity in flies. We have also found that human HP1α and NBS1 interact with each other and that, similarly to Drosophila, siRNA-mediated inhibition of NBS1 reduces HP1α levels in human cultured cells. Surprisingly, fibroblasts from Nijmegen Breakage Syndrome (NBS) patients, carrying the 657del5 hypomorphic mutation in NBS1 and expressing the p26 and p70 NBS1 fragments, accumulate HP1α indicating that, differently from NBS1 knockout cells, the presence of truncated NBS1 extends HP1α turnover and/or promotes its stability. Remarkably, an siRNA-mediated reduction of HP1α in NBS fibroblasts decreases the hypersensitivity to irradiation, a characteristic of the NBS syndrome. Overall, our data provide an unanticipated evidence of a close interaction between HP1 and NBS1 that is essential for genome stability and point up HP1α as a potential target to counteract chromosome instability in NBS patient cells.

Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair.

Correction: Pennisi, R., et al. Hsp90: A New Player in DNA Repair? Biomolecules 2015, 5, 2589-2618.

n/a.

Hsp90: A New Player in DNA Repair?

Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone that, together with Hsp70 and co-chaperones makes up the Hsp90 chaperone machinery, stabilizing and activating more than 200 proteins, involved in protein homeostasis (i.e., proteostasis), transcriptional regulation, chromatin remodeling, and DNA repair. Cells respond to DNA damage by activating complex DNA damage response (DDR) pathways that include: (i) cell cycle arrest; (ii) transcriptional and post-translational activation of a subset of genes, including those associated with DNA repair; and (iii) triggering of programmed cell death. The efficacy of the DDR pathways is influenced by the nuclear levels of DNA repair proteins, which are regulated by balancing between protein synthesis and degradation as well as by nuclear import and export. The inability to respond properly to either DNA damage or to DNA repair leads to genetic instability, which in turn may enhance the rate of cancer development. Multiple components of the DNA double strand breaks repair machinery, including BRCA1, BRCA2, CHK1, DNA-PKcs, FANCA, and the MRE11/RAD50/NBN complex, have been described to be client proteins of Hsp90, which acts as a regulator of the diverse DDR pathways. Inhibition of Hsp90 actions leads to the altered localization and stabilization of DDR proteins after DNA damage and may represent a cell-specific and tumor-selective radiosensibilizer. Here, the role of Hsp90-dependent molecular mechanisms involved in cancer onset and in the maintenance of the genome integrity is discussed and highlighted.

Identification of the interactors of human nibrin (NBN) and of its 26 kDa and 70 kDa fragments arising from the NBN 657del5 founder mutation.

Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.