ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

mTOR inhibition acts as an unexpected checkpoint in p53-mediated tumor suppression.

Here, we showed that the acetylation-defective p53-4KR mice, lacking the ability of cell cycle arrest, senescence, apoptosis, and ferroptosis, were tumor prone but failed to develop early-onset tumors. By identifying a novel p53 acetylation site at lysine K136, we found that simultaneous mutations at all five acetylation sites (p53-5KR) diminished its remaining tumor suppression function. Moreover, the embryonic lethality caused by the deficiency of mdm2 was fully rescued in the background of p535KR/5KR , but not p534KR/4KR background. p53-4KR retained the ability to suppress mTOR function but this activity was abolished in p53-5KR cells. Notably, the early-onset tumor formation observed in p535KR/5KR and p53-null mice was suppressed upon the treatment of the mTOR inhibitor. These results suggest that p53-mediated mTOR regulation plays an important role in both embryonic development and tumor suppression, independent of cell cycle arrest, senescence, apoptosis, and ferroptosis.

Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.

Cell-cycle arrest, apoptosis, and senescence are widely accepted as the major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are the rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53(K117R)) or three (p53(3KR); K117R+K161R+K162R) of p53 acetylation sites. Although p53(K117R/K117R) cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p53(3KR/3KR) cells. Surprisingly, unlike p53 null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53(K117R/K117R) or p53(3KR/3KR) animals. Notably, p53(3KR) retains the ability to regulate energy metabolism and reactive oxygen species production. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.

Brown remodeling of white adipose tissue by SirT1-dependent deacetylation of Pparγ.

Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like features in white adipose (WAT) is an attractive, if elusive, therapeutic approach to staunch the current obesity epidemic. Here we report that gain of function of the NAD-dependent deacetylase SirT1 or loss of function of its endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote "browning" of WAT by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268 and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective induction of BAT genes and repression of visceral WAT genes associated with insulin resistance. An acetylation-defective Pparγ mutant induces a brown phenotype in white adipocytes, whereas an acetylated mimetic fails to induce "brown" genes but retains the ability to activate "white" genes. We propose that SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of potential therapeutic import.

Acetylation-regulated interaction between p53 and SET reveals a widespread regulatory mode.

Although lysine acetylation is now recognized as a general protein modification for both histones and non-histone proteins, the mechanisms of acetylation-mediated actions are not completely understood. Acetylation of the C-terminal domain (CTD) of p53 (also known as TP53) was an early example of non-histone protein acetylation and its precise role remains unclear. Lysine acetylation often creates binding sites for bromodomain-containing 'reader' proteins. Here we use a proteomic screen to identify the oncoprotein SET as a major cellular factor whose binding with p53 is dependent on CTD acetylation status. SET profoundly inhibits p53 transcriptional activity in unstressed cells, but SET-mediated repression is abolished by stress-induced acetylation of p53 CTD. Moreover, loss of the interaction with SET activates p53, resulting in tumour regression in mouse xenograft models. Notably, the acidic domain of SET acts as a 'reader' for the unacetylated CTD of p53 and this mechanism of acetylation-dependent regulation is widespread in nature. For example, acetylation of p53 also modulates its interactions with similar acidic domains found in other p53 regulators including VPRBP (also known as DCAF1), DAXX and PELP1 (refs. 7, 8, 9), and computational analysis of the proteome has identified numerous proteins with the potential to serve as acidic domain readers and lysine-rich ligands. Unlike bromodomain readers, which preferentially bind the acetylated forms of their cognate ligands, the acidic domain readers specifically recognize the unacetylated forms of their ligands. Finally, the acetylation-dependent regulation of p53 was further validated in vivo by using a knock-in mouse model expressing an acetylation-mimicking form of p53. These results reveal that acidic-domain-containing factors act as a class of acetylation-dependent regulators by targeting p53 and, potentially, other proteins.

Ferroptosis as a p53-mediated activity during tumour suppression.

Although p53-mediated cell-cycle arrest, senescence and apoptosis serve as critical barriers to cancer development, emerging evidence suggests that the metabolic activities of p53 are also important. Here we show that p53 inhibits cystine uptake and sensitizes cells to ferroptosis, a non-apoptotic form of cell death, by repressing expression of SLC7A11, a key component of the cystine/glutamate antiporter. Notably, p53(3KR), an acetylation-defective mutant that fails to induce cell-cycle arrest, senescence and apoptosis, fully retains the ability to regulate SLC7A11 expression and induce ferroptosis upon reactive oxygen species (ROS)-induced stress. Analysis of mutant mice shows that these non-canonical p53 activities contribute to embryonic development and the lethality associated with loss of Mdm2. Moreover, SLC7A11 is highly expressed in human tumours, and its overexpression inhibits ROS-induced ferroptosis and abrogates p53(3KR)-mediated tumour growth suppression in xenograft models. Our findings uncover a new mode of tumour suppression based on p53 regulation of cystine metabolism, ROS responses and ferroptosis.

Differential effects on ARF stability by normal versus oncogenic levels of c-Myc expression.

ARF suppresses aberrant cell growth upon c-Myc overexpression by activating p53 responses. Nevertheless, the precise mechanism by which ARF specifically restrains the oncogenic potential of c-Myc without affecting its normal physiological function is not well understood. Here, we show that low levels of c-Myc expression stimulate cell proliferation, whereas high levels inhibit by activating the ARF/p53 response. Although the mRNA levels of ARF are induced in both scenarios, the accumulation of ARF protein occurs only when ULF-mediated degradation of ARF is inhibited by c-Myc overexpression. Moreover, the levels of ARF are reduced through ULF-mediated ubiquitination upon DNA damage. Blocking ARF degradation by c-Myc overexpression dramatically stimulates the apoptotic responses. Our study reveals that ARF stability control is crucial for differentiating normal (low) versus oncogenic (high) levels of c-Myc expression and suggests that differential effects on ULF- mediated ARF ubiquitination by c-Myc levels act as a barrier in oncogene-induced stress responses.

FBXO11 inactivation leads to abnormal germinal-center formation and lymphoproliferative disease.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)-mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor-mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice.

PHLDA2-mediated phosphatidic acid peroxidation triggers a distinct ferroptotic response during tumor suppression.

Although the role of ferroptosis in killing tumor cells is well established, recent studies indicate that ferroptosis inducers also sabotage anti-tumor immunity by killing neutrophils and thus unexpectedly stimulate tumor growth, raising a serious issue about whether ferroptosis effectively suppresses tumor development in vivo. Through genome-wide CRISPR-Cas9 screenings, we discover a pleckstrin homology-like domain family A member 2 (PHLDA2)-mediated ferroptosis pathway that is neither ACSL4-dependent nor requires common ferroptosis inducers. PHLDA2-mediated ferroptosis acts through the peroxidation of phosphatidic acid (PA) upon high levels of reactive oxygen species (ROS). ROS-induced ferroptosis is critical for tumor growth in the absence of common ferroptosis inducers; strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth but has no obvious effect in normal tissues in both immunodeficient and immunocompetent mouse tumor models. These data demonstrate that PHLDA2-mediated PA peroxidation triggers a distinct ferroptosis response critical for tumor suppression and reveal that PHLDA2-mediated ferroptosis occurs naturally in vivo without any treatment from ferroptosis inducers.

Inactivation of arf-bp1 induces p53 activation and diabetic phenotypes in mice.

It is well accepted that the Mdm2 ubiquitin ligase acts as a major factor in controlling p53 stability and activity in vivo. Although several E3 ligases have been reported to be involved in Mdm2-independent p53 degradation, the roles of these ligases in p53 regulation in vivo remain largely unknown. To elucidate the physiological role of the ubiquitin ligase ARF-BP1, we generated arf-bp1 mutant mice. We found that inactivation of arf-bp1 during embryonic development in mice resulted in p53 activation and embryonic lethality, but the mice with arf-bp1 deletion specifically in the pancreatic ß-cells (arf-bp1(FL/Y)/RIP-cre) were viable and displayed no obvious abnormality after birth. Interestingly, these mice showed dramatic loss of ß-cells as mice aged, and >50% of these mice died of severe diabetic symptoms before reaching 1 year of age. Notably, the diabetic phenotype of these mice was largely reversed by concomitant deletion of p53, and the life span of the mice was significantly extended (p53(LFL/FL)/arf-bp1(FL/Y)/RIP-cre). These findings underscore an important role of ARF-BP1 in maintaining ß-cell homeostasis in aging mice and reveal that the stability of p53 is critically regulated by ARF-BP1 in vivo.

Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators.

Ferroptosis, an iron-dependent programmed cell death triggered by excessive lipid peroxidation, has shown promising therapeutic potentials in human diseases. Here, we describe a protocol of a CRISPR-Cas9 loss-of-function screen to identify regulators in response to different inducers of ferroptosis. We emphasize the steps of library amplification, drug treatment, high-throughput sequencing preparation, and bioinformatics analysis using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK). We also present a method to discover the regulators of ferroptosis and verify the potential targets efficiently. For complete details on use and execution of this protocol, please refer to Yang et al. (2023).1.

Regulation of VKORC1L1 is critical for p53-mediated tumor suppression through vitamin K metabolism.

Here, we identified vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1) as a potent ferroptosis repressor. VKORC1L1 protects cells from ferroptosis by generating the reduced form of vitamin K, a potent radical-trapping antioxidant, to counteract phospholipid peroxides independent of the canonical GSH/GPX4 mechanism. Notably, we found that VKORC1L1 is also a direct transcriptional target of p53. Activation of p53 induces downregulation of VKORC1L1 expression, thus sensitizing cells to ferroptosis for tumor suppression. Interestingly, a small molecular inhibitor of VKORC1L1, warfarin, is widely prescribed as an FDA-approved anticoagulant drug. Moreover, warfarin represses tumor growth by promoting ferroptosis in both immunodeficient and immunocompetent mouse models. Thus, by downregulating VKORC1L1, p53 executes the tumor suppression function by activating an important ferroptosis pathway involved in vitamin K metabolism. Our study also reveals that warfarin is a potential repurposing drug in cancer therapy, particularly for tumors with high levels of VKORC1L1 expression.

Inhibition of USP7 induces p53-independent tumor growth suppression in triple-negative breast cancers by destabilizing FOXM1.

Although numerous studies indicate that inhibition of USP7 suppresses tumor growth by activating p53, the precise mechanism by which USP7 contributes to tumor growth through the p53-independent manner is not well understood. p53 is frequently mutated in most triple-negative breast cancers (TNBC), characterized as the very aggressive form of breast cancers with limited treatment options and poor patient outcomes. Here, we found that the oncoprotein Forkhead Box M1 (FOXM1) acts as a potential driver for tumor growth in TNBC and, surprisingly, through a proteomic screen, we identified USP7 as a major regulator of FOXM1 in TNBC cells. USP7 interacts with FOXM1 both in vitro and in vivo. USP7 stabilizes FOXM1 through deubiquitination. Conversely, RNAi-mediated USP7 knockdown in TNBC cells, dramatically reduced the levels of FOXM1. Moreover, based upon the proteolysis targeting chimera (PROTAC) technology, we generated PU7-1 (protein degrader for USP7-1), as a USP7 specific degrader. PU7-1 induces rapid USP7 degradation at low nanomolar concentrations in cells but shows no obvious effect on other USP family proteins. Strikingly, the treatment of TNBC cells with PU7-1 significantly abrogates FOXM1 functions and effectively suppresses cell growth in vitro. By using xenograft mouse models, we found that PU7-1 markedly represses tumor growth in vivo. Notably, ectopic overexpression of FOXM1 can reverse the tumor growth suppressive effects induced by PU7-1, underscored the specific effect on FOXM1 induced by USP7 inactivation. Together, our findings indicate that FOXM1 is a major target of USP7 in modulating tumor growth in a p53-independent manner and reveals the USP7 degrader as a potential therapeutic tool for the treatment of triple-negative breast cancers.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

Hepatic Huwe1 loss protects mice from non-alcoholic fatty liver disease through lipid metabolic rewiring.

Non-alcoholic fatty liver disease (NAFLD) is the most pervasive liver pathology worldwide. Here, we demonstrate that the ubiquitin E3 ligase Huwe1 is vital in NAFLD pathogenesis. Using mass spectrometry and RNA sequencing, we reveal that liver-specific deletion of Huwe1 (Huwe1LKO) in 1-year-old mice (approximately middle age in humans) elicits extensive lipid metabolic reprogramming that involves downregulation of de novo lipogenesis and fatty acid uptake, upregulation of fatty acid ß-oxidation, and increased oxidative phosphorylation. ChEA transcription factor prediction analysis inferred these changes result from attenuated PPARɑ, LXR, and RXR activity in Huwe1LKO livers. Consequently, Huwe1LKO mice fed chow diet exhibited significantly reduced hepatic steatosis and superior glucose tolerance compared to wild-type mice. Huwe1LKO also conferred protection from high-fat diet-induced hepatic steatosis by 6-months of age, with increasingly robust differences observed as mice reached middle age. Together, we present evidence that Huwe1 plays a critical role in the development of age- and diet-induced NAFLD.

Deciphering the acetylation code of p53 in transcription regulation and tumor suppression.

Although it is well-established that p53-mediated tumor suppression mainly acts through its ability in transcriptional regulation, the molecular mechanisms of this regulation are not completely understood. Among a number of regulatory modes, acetylation of p53 attracts great interests. p53 was one of the first non-histone proteins found to be functionally regulated by acetylation and deacetylation, and subsequent work has established that reversible acetylation is a general mechanism for regulation of non-histone proteins. Unlike other types of posttranslational modifications occurred during stress responses, the role of p53 acetylation has been recently validated in vivo by using the knock-in mice with both acetylation-defective and acetylation-mimicking p53 mutants. Here, we review the role of acetylation in p53-mediated activities, with a focus on which specific acetylation sites are critical for p53-dependent transcription regulation during tumor suppression and how acetylation of p53 recruits specific "readers" to execute its promoter-specific regulation of different targets. We also discuss the role of p53 acetylation in differentially regulating its classic activities in cell cycle arrest, senescence and apoptosis as well as newly identified unconventional functions such as cell metabolism and ferroptosis.

Robust p53 Stabilization Is Dispensable for Its Activation and Tumor Suppressor Function.

p53 is a short-lived protein with low basal levels under normal homeostasis conditions. However, upon DNA damage, levels of p53 dramatically increase for its activation. Although robust stabilization of p53 serves as a "trademark" for DNA damage responses, the requirement for such dramatic protein stabilization in tumor suppression has not been well addressed. Here we generated a mutant p53KQ mouse where all the C-terminal domain lysine residues were mutated to glutamines (K to Q mutations at K367, K369, K370, K378, K379, K383, and K384) to mimic constitutive acetylation of the p53 C-terminus. Because of p53 activation, p53KQ/KQ mice were perinatal lethal, yet this lethality was averted in p53KQ/- mice, which displayed normal postnatal development. Nevertheless, p53KQ/- mice died prematurely due to anemia and hematopoiesis failure. Further analyses indicated that expression of the acetylation-mimicking p53 mutant in vivo induces activation of p53 targets in various tissues without obviously increasing p53 levels. In the well-established pancreatic ductal adenocarcinoma (PDAC) mouse model, expression of the acetylation-mimicking p53-mutant protein effectively suppressed K-Ras-induced PDAC development in the absence of robust p53 stabilization. Together, our results provide proof-of-principle evidence that p53-mediated transcriptional function and tumor suppression can be achieved independently of its robust stabilization and reveal an alternative approach to activate p53 function for therapeutic purposes. SIGNIFICANCE: Although robust p53 stabilization is critical for acute p53 responses such as DNA damage, this study underscores the important role of low basal p53 protein levels in p53 activation and tumor suppression.

iPLA2ß-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

Here, we identify iPLA2ß as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2ß is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2ß-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2ß is overexpressed in human cancers; inhibition of endogenous iPLA2ß sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2ß acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2ß has no obvious effect on normal development or cell viability in normal tissues but iPLA2ß plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2ß is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns.

An unexpected role for p53 in regulating cancer cell-intrinsic PD-1 by acetylation.

Cancer cell-intrinsic programmed cell death protein-1 (PD-1) has emerged as a tumor regulator in an immunity-independent manner, but its precise role in modulating tumor behaviors is complex, and how PD-1 is regulated in cancer cells is largely unknown. Here, we identified PD-1 as a direct target of tumor suppressor p53. Notably, p53 acetylation at K120/164 played a critical role in p53-mediated PD-1 transcription. Acetylated p53 preferentially recruited acetyltransferase cofactors onto PD-1 promoter, selectively facilitating PD-1 transcription by enhancing local chromatin acetylation. Reexpression of PD-1 in cancer cells inhibited tumor growth, whereas depletion of cancer cell-intrinsic PD-1 compromised p53-dependent tumor suppression. Moreover, histone deacetylase inhibitor (HDACi) activated PD-1 in an acetylated p53-dependent manner, supporting a synergistic effect by HDACi and p53 on tumor suppression via stimulating cancer cell-intrinsic PD-1. Our study reveals a mechanism for activating cancer cell-intrinsic PD-1 and indicates that p53-mediated PD-1 activation is critically involved in tumor suppression in an immunity-independent manner.