ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

The Synergistic Anticancer Effect of a Combination of Chidamide and Etoposide Against NK/T Cell Lymphoma

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive hematological malignancy. However, there is currently no consensus on first-line therapies for refractory/relapsed patients. Chidamide is a self-researched and developed HDACs inhibitor, and when combined with DNA-damaging agents, exhibited a clinical synergistic effect for the treatment of some solid tumors and hematological malignancies. Thus in this study, a series of in vitro and in vivo experiments were conducted to explore the efficacy and potential mechanisms of combined chidamide and etoposide treatment in NKTCL. We demonstrated that chidamide or etoposide alone dose- and time-dependently inhibited the cell viability of NKTCL cell lines, YT, NKYS and KHYG-1. Functional experiments suggested that combined chidamide and etoposide treatment exerted synergistic antiproliferation effect and enhanced cell apoptotic death both in vitro and in vivo. Furthermore, the expression of DNA damage related proteins was detected and we also examined the alternations in histone acetylation, cell cycle progression, and mitochondrial membrane potential (MMP). The results suggested that increased histone acetylation, cell cycle arrest at the G2/M phase and loss of MMP, converging to greater DNA damage, might account for the synergism of the combination of chidamide and etoposide in NKTCL. Taken together, our study supplements the clinical application of combining HDACs inhibitors and DNA-damaging agents on treating hematological malignancies but also provide an experimental basis for improved therapeutic efficacy and decreased complications for patients with NKTCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Cullin-5 neddylation-mediated NOXA degradation is enhanced by PRDX1 oligomers in colorectal cancer

NOXA, a BH3-only proapoptotic protein involved in regulating cell death decisions, is highly expressed but short-lived in colorectal cancer (CRC). Neddylated cullin-5 (CUL5)-mediated ubiquitination and degradation of NOXA is crucial to prevent its overaccumulation and maintain an appropriate action time. However, how this process is manipulated by CRC cells commonly exposed to oxidative stress remain unknown. The peroxiredoxin PRDX1, a conceivable antioxidant overexpressed in CRC tissues, has been shown to inhibit apoptosis and TRAF6 ubiquitin-ligase activity. In this study, we found that PRDX1 inhibits CRC cell apoptosis by downregulating NOXA. Mechanistically, PRDX1 promotes NOXA ubiquitination and degradation, which completely depend on CUL5 neddylation. Further studies have demonstrated that PRDX1 oligomers bind with both the Nedd8-conjugating enzyme UBE2F and CUL5 and that this tricomplex is critical for CUL5 neddylation, since silencing PRDX1 or inhibiting PRDX1 oligomerization greatly dampens CUL5 neddylation and NOXA degradation. An increase in reactive oxygen species (ROS) is not only a hallmark of cancer cells but also the leading driving force for PRDX1 oligomerization. As shown in our study, although ROS play a role in upregulating NOXA mRNA transcription, ROS scavenging in CRC cells by N-acetyl-L-cysteine (NAC) can significantly reduce CUL5 neddylation and extend the NOXA protein half-life. Therefore, in CRC, PRDX1 plays a key role in maintaining intracellular homeostasis under conditions of high metabolic activity by reinforcing UBE2F-CUL5-mediated degradation of NOXA, which is also evidenced in the resistance of CRC cells to etoposide treatment. Based on these findings, targeting PRDX1 could be an effective strategy to overcome the resistance of CRC to DNA damage-inducing chemotherapeutics.

Senotherapeutic peptide reduces skin biological age and improves skin health markers

Skin aging has been primarily related to aesthetics and beauty. Therefore, interventions have focused on reestablishing skin appearance, but not necessarily skin health, function, and resilience. Recently, cellular senescence was shown to play a role in age-related skin function deterioration and influence organismal health and, potentially, longevity. In the present study, a two-step screening was performed to identify peptides capable of reducing cellular senescence in human dermal fibroblasts (HDF) from Hutchinson-Gilford Progeria (HGPS) patients. From the top four peptides of the first round of screening, we built a 764-peptide library using amino acid scanning, of which the second screen led to the identification of peptide 14. Peptide 14 effectively decreased HDF senescence induced by HGPS, chronological aging, ultraviolet-B radiation, and etoposide treatment, without inducing significant cell death, and likely by modulating longevity and senescence pathways. We further validated the effectiveness of peptide 14 using human skin equivalents and skin biopsies, where peptide 14 promoted skin health and reduced senescent cell markers, as well as the biological age of samples, according to the Skin-Specific DNA methylation clock, MolClock. Topical application of peptide 14 outperformed Retinol treatment, the current gold-standard in “anti-aging” skin care. Finally, we determined that peptide 14 is safe for long-term applications and also significantly extends both the lifespan and healthspan of C. elegans worms tested in two independent testings. This highlights the potential for geroprotective applications of the senotherapeutic compounds identified using our screening platform beyond the skin.

Phosphorylation of SMURF2 by ATM exerts a negative feedback control of DNA damage response

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser384 by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser384 constitutes a negative feedback loop that regulates DSB repair.

Nonhistone Proteins HMGB1 and HMGB2 Differentially Modulate the Response of Human Embryonic Stem Cells and the Progenitor Cells to the Anticancer Drug Etoposide

HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells(hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functionalroles in pluripotency and the mechanisms underlying their dierentiation in response to the anticancerdrug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown(KD) by shRNA in hESCs did not aect the cell stemness/pluripotency regardless of etoposidetreatments, while in hESC-derived neuroectodermal cells, treatment resulted in dierential eects oncell survival and the generation of rosette structures. The objective of this work was to determinewhether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitorcells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent,HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 ontelomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the otherhand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs(but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres.Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, withthe highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activitywere found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedlydecreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treatedHMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and couldalready be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1or HMGB2 dierentially modulate the impact of etoposide treatment on human embryonic stem cellsand their progenitor cells, suggesting possible strategies for the enhancement of the ecacy of thisanticancer drug.

Curing genetic skin disease through altered replication stress response

SummaryRevertant mosaicism, or ‘natural gene therapy’, refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell1. Only ∼50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation2. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes3. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.

Lamotrigine-induced hemophagocytic lymphohistiocytosis with Takotsubo cardiomyopathy: a case report

Background Hemophagocytic lymphohistiocytosis is a rare hematological syndrome characterized by excessive and uncontrolled activation of the immune system. The often nonspecific nature of early symptoms and the potential for progression to multiorgan failure and death if appropriate therapy is not started promptly, highlight the importance of heightened recognition for this uncommon disease. Although there are well-described associations of hemophagocytic lymphohistiocytosis with infectious, malignant, and autoimmune diseases and an established treatment protocol for these cases, the link between medications and hemophagocytic lymphohistiocytosis is less clearly established and the optimal treatment of these cases less well defined. Case presentation Here we describe the case of a 45-year-old caucasian woman presenting with signs and symptoms consistent with hemophagocytic lymphohistiocytosis, induced by recent exposure to lamotrigine. She had a rapidly progressive clinical course, complicated by multiorgan failure including stress-induced Takotsubo cardiomyopathy and cardiac arrest. With dexamethasone and etoposide therapy, she made a full and sustained recovery. Conclusions This case highlights that medication-induced hemophagocytic lymphohistiocytosis appears to respond similarly to the same dexamethasone and etoposide treatment regimen developed for other non-drug-induced forms of hemophagocytic lymphohistiocytosis. With the continued cessation of the offending agent there has not been need for maintenance therapy and no relapse to date. In addition, given the risk for cardiomyopathy, a clinical complication not classically associated with hemophagocytic lymphohistiocytosis, echocardiogram and telemetry monitoring should be considered in the initial workup of suspected hemophagocytic lymphohistiocytosis.