Genetic mutations in apoptosis-inducing factor (AIF) have a strong association with mitochondrial disorders; however, little is known about the aberrant splicing variants in affected patients and how these variants contribute to mitochondrial dysfunction and brain development defects. We identified pathologic AIF3/AIF3-like splicing variants in postmortem brain tissues of pediatric individuals with mitochondrial disorders. Mutations in AIFM1 exon-2/3 increase splicing risks. AIF3-splicing disrupts mitochondrial complexes, membrane potential, and respiration, causing brain development defects. Mechanistically, AIF is a mammalian NAD(P)H dehydrogenase and possesses glutathione reductase activity controlling respiratory chain functions and glutathione regeneration. Conversely, AIF3, lacking these activities, disassembles mitochondrial complexes, increases ROS generation, and simultaneously hinders antioxidant defense. Expression of NADH dehydrogenase NDI1 restores mitochondrial functions partially and protects neurons in AIF3-splicing mice. Our findings unveil an underrated role of AIF as a mammalian mitochondrial complex-I alternative NAD(P)H dehydrogenase and provide insights into pathologic AIF-variants in mitochondrial disorders and brain development.

Breast cancer stem cells (BCSCs) mitigate oxidative stress to maintain their viability and plasticity. However, the regulatory mechanism of oxidative stress in BCSCs remains unclear. We recently found that the histone reader ZMYND8 was upregulated in BCSCs. Here, we showed that ZMYND8 reduced ROS and iron to inhibit ferroptosis in aldehyde dehydrogenase-high (ALDHhi) BCSCs, leading to BCSC expansion and tumor initiation in mice. The underlying mechanism involved a two-fold posttranslational regulation of nuclear factor erythroid 2-related factor 2 (NRF2). ZMYND8 increased stability of NRF2 protein through KEAP1 silencing. On the other hand, ZMYND8 interacted with and recruited NRF2 to the promoters of antioxidant genes to enhance gene transcription in mammospheres. NRF2 phenocopied ZMYND8 to enhance BCSC stemness and tumor initiation by inhibiting ROS and ferroptosis. Loss of NRF2 counteracted ZMYND8's effects on antioxidant genes and ROS in mammospheres. Interestingly, ZMYND8 expression was directly controlled by NRF2 in mammospheres. Collectively, these findings uncover a positive feedback loop that amplifies the antioxidant defense mechanism sustaining BCSC survival and stemness.

Supplementary Table 1-2 and Figure 1-7

Supplementary Table 1. The shRNA and sgRNA Oligonucleotide Sequences Supplementary Table 2. 5'-and 3' RACE Primers Supplementary Table 3. qPCR Primers Supplementary Table 4. Hypoxia-induced lncRNAs in TNBC cells. Supplementary Table 5. Abundance of hypoxia-induced lncRNAs in both MDA-MB-231 and SUM159 cells. Supplementary Figure 1. The nucleotide sequence alignment of lncIHAT. Supplementary Figure 2. LncIHAT promotes TNBC growth and metastasis in mice. Supplementary Figure 3. LncIHATis required for expression of PDK1 and ITGA6 in TNBC cells. Supplementary Figure 4 .LncIHAT overexpression has no effect on PDK1 and ITGA6 expression. Supplementary Figure 5. Time-dependent induction of lncIHAT and PDK1 by hypoxia in TNBC cells. Supplementary Figure 6. PDK1 and ITGA6 mediate the effect of lncIHAT on TNBC cell growth in vitro.

Fig. S1-6. Supplementary Fig. S1. The nucleotide sequence of RAB11B-AS1 identified by 5'- and 3'- RACE in human MDA-MB-231 cells. The RAB11B-AS1 sequence from 5'- and 3'-RACE is shown in orange and the mutated nucleotides are marked in blue as compared with the reference sequence in the GenBank (NR_038237.1), which is shown in black. The nucleotide sequence of RAB11B-AS1 has been deposited in the GenBank with the accession number MK855053. Supplementary Fig. S2. Validation of HIF-2a protein levels in overexpressed and knockout cells. Supplementary Fig. S3. RAB11B-AS1 increases breast cancer cell migration and invasion in vitro. Supplementary Fig. S4. RAB11B-AS1 has no effect on caspase-3-dependent cell death in tumors. Supplementary Fig. S5. RAB11B-AS1 increases hypoxia-induced expression of angiogenic factors in breast cancer cells. Supplementary Fig. S6. RAB11B-AS1 fails to regulate HIF transcriptional activity.

Supplementary Table S1-S3 and Figure S1-S3

Table S1-3. Supplementary Table S1. Oligonucleotide sequence used for qPCR primers. Supplementary Table S2. Oligonucleotide sequence used for HIF-1b sgRNA and RAB11B-AS1 shRNA, HRE, and RACE cloning primers. Supplementary Table S3. Gene ontology analysis of LncRAB11B-AS1-induced genes in hypoxic MDA-MB-231 cells.

SAP30 is a core subunit of the transcriptional corepressor SIN3 complex, but little is known about its role in gene regulation and human cancer. Here, we show that SAP30 was a nonmutational oncoprotein upregulated in more than 50% of human breast tumors and correlated with unfavorable outcomes in patients with breast cancer. In various breast cancer mouse models, we found that SAP30 promoted tumor growth and metastasis through its interaction with SIN3A/3B. Surprisingly, the canonical gene silencing role was not essential for SAP30's tumor-promoting actions. SAP30 enhanced chromatin accessibility and RNA polymerase II occupancy at promoters in breast cancer cells, acting as a coactivator for genes involved in cell motility, angiogenesis, and lymphangiogenesis, thereby driving tumor progression. Notably, SAP30 formed a homodimer with 1 subunit binding to SIN3A and another subunit recruiting MLL1 through specific Phe186/200 residues within its transactivation domain. MLL1 was required for SAP30-mediated transcriptional coactivation and breast tumor progression. Collectively, our findings reveal that SAP30 represents a transcriptional dependency in breast cancer.

27-Hydroxycholesterol (27-HC) is the most abundant oxysterol that increases the risk of breast cancer progression. However, little is known about epigenetic regulation of 27-HC metabolism and its role in breast tumor initiation. Using genetic mouse mammary tumor and human breast cancer models, we showed here that the histone reader ZMYND8 was selectively expressed in breast cancer stem cells (BCSCs) and promoted epithelial-mesenchymal transition (EMT), BCSC maintenance and self-renewal, and oncogenic transformation through its epigenetic functions, leading to breast tumor initiation. Mechanistically, ZMYND8 was a master transcriptional regulator of 27-HC metabolism. It increased cholesterol biosynthesis and oxidation but blocked cholesterol efflux and 27-HC catabolism, leading to accumulation of 27-HC in BCSCs. Consequently, 27-HC promoted EMT, oncogenic transformation, and tumor initiation through activation of liver X receptor. These findings reveal that ZMYND8 is an epigenetic booster that drives breast tumor initiation through metabolic reprogramming.

Abstract Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by reexpression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKA), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, cotreatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable. Significance: Metabolic synthetic lethal screening in IDHWT glioblastoma defines a vulnerability to ΑΚG following BCAT1 loss, uncovering a therapeutic strategy to improve glioblastoma treatment. See related commentary by Meurs and Nagrath, p. 2354

The excitatory neurotransmitter glutamate shapes learning and memory, but the underlying epigenetic mechanism of glutamate regulation in neuron remains poorly understood. Here, we showed that lysine demethylase KDM6B was expressed in excitatory neurons and declined in hippocampus with age. Conditional knockout of KDM6B in excitatory neurons reduced spine density, synaptic vesicle number and synaptic activity, and impaired learning and memory without obvious effect on brain morphology in mice. Mechanistically, KDM6B upregulated vesicular glutamate transporter 1 and 2 (VGLUT1/2) in neurons through demethylating H3K27me3 at their promoters. Tau interacted and recruited KDM6B to the promoters of Slc17a7 and Slc17a6, leading to a decrease in local H3K27me3 levels and induction of VGLUT1/2 expression in neurons, which could be prevented by loss of Tau. Ectopic expression of KDM6B, VGLUT1, or VGLUT2 restored spine density and synaptic activity in KDM6B-deficient cortical neurons. Collectively, these findings unravel a fundamental mechanism underlying epigenetic regulation of synaptic plasticity and cognition.

Traumatic brain injury (TBI), often induced by sports, car accidents, falls, or other daily occurrences, is a primary non-genetically related risk factor for the development of subsequent neurodegeneration and neuronal cell death. However, the molecular mechanisms underlying neurodegeneration, cell death, and neurobehavioral dysfunction following TBI remain unclear. Here, we found that poly(ADP-ribose) polymerase-1 (PARP-1) was hyperactivated following TBI and its inhibition reduced TBI-induced brain injury. Macrophage migration inhibitory factor (MIF), a newly identified nuclease involved in PARP-1-dependent cell death, was translocated from the cytosol to the nucleus in cortical neurons following TBI and promoted neuronal cell death in vivo. Genetic deletion of MIF protected neurons from TBI-induced dendritic spine loss, morphological complexity degeneration, and subsequent neuronal cell death in mice. Moreover, MIF knockout reduced the brain injury volume and improved long-term animal behavioral rehabilitation. These neuroprotective effects in MIF knockout mice were reversed by the expression of wild-type MIF but not nuclease-deficient MIF mutant. In contrast, genetic deletion of MIF did not alter TBI-induced neuroinflammation. These findings reveal that MIF mediates TBI-induced neurodegeneration, neuronal cell death and neurobehavioral dysfunction through its nuclease activity, but not its pro-inflammatory role. Targeting MIF's nuclease activity may offer a novel strategy to protect neurons from TBI.

Abstract Emerging studies indicate that DNA damage in cancer cells triggers antitumor immunity, but its intrinsic regulatory mechanism in breast cancer cells remains poorly understood. Here, we show that ZMYND8 is upregulated and inhibits micronucleus formation and DNA damage in breast cancer cells. Loss of ZMYND8 triggered activation of the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase in micronuclei, leading to further activation of the downstream signaling effectors stimulator of IFN genes and NF-κB, but not TANK-binding kinase 1 and IFN regulatory factor 3, thereby inducing the expression of IFNβ and IFN-stimulated genes (ISG) in breast cancer cells in vitro and tumors in vivo. ZMYND8 knockout (KO) in breast cancer cells promoted infiltration of CD4+ and CD8+ T cells, leading to tumor inhibition in syngeneic mouse models, which was significantly attenuated by treatment of anti-CD4/CD8–depleting antibodies or anti-IFNAR1 antibody and in immunodeficient Rag1 KO mice. In human breast tumors, ZMYND8 was negatively correlated with ISGs, CD4, CD8A, CD8B, and the tumor-lymphocyte infiltration phenotype. Collectively, these findings demonstrate that maintenance of genome stability by ZMYND8 causes breast cancer cells to evade cytotoxic T-lymphocyte surveillance, which leads to tumor growth. Significance: These findings show that ZMYND8 is a new negative and intrinsic regulator of the innate immune response in breast tumor cells, and ZMYND8 may be a possible target for antitumor immunotherapy.

Abstract Hypoxia induces thousands of mRNAs and miRNAs to mediate tumor malignancy. However, hypoxia-induced long noncoding RNA (lncRNA) transcriptome and their role in triple-negative breast cancer (TNBC) have not been defined. Here we identified hypoxia-induced lncRNA transcriptome in two human TNBC cell lines by whole transcriptome sequencing. AC093818.1 was one of 26 validated lncRNAs and abundantly expressed in TNBC in vitro and in vivo. 5′- and 3′-rapid amplification of cDNA ends assays revealed that the isoform 2 was a dominant AC093818.1 transcript in TNBC cells and thus referred to as lncIHAT (lncRNA induced by hypoxia and abundant in TNBC). Hypoxia-inducible factor 1 (HIF1) but not HIF2 bound to the hypoxia response element at the promoter of lncIHAT to activate its transcription in hypoxic TNBC cells. LncIHAT promoted TNBC cell survival in vitro and tumor growth and lung metastasis in mice. Mechanistically, lncIHAT was required for the expression of its proximal neighboring oncogenic genes PDK1 and ITGA6 in TNBC cells and tumors. Reexpression of PDK1 and ITGA6 rescued survival and growth of lncIHAT knockdown TNBC cells in vitro. Collectively, these findings uncovered lncIHAT as a new hypoxia-induced oncogenic cis-acting lncRNA in TNBC. Implications: This study systematically identified hypoxia-induced lncRNA transcriptome in TNBC and sheds light on multiple layers of regulatory mechanisms of gene expression under hypoxia.

Abstract Background Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. Methods AIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. Results We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. Conclusions We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

How cancer cells cope with high levels of replication stress during rapid proliferation is currently unclear. Here, we show that macrophage migration inhibitory factor (MIF) is a 3’ flap nuclease that translocates to the nucleus in S phase. Poly(ADP-ribose) polymerase 1 co-localizes with MIF to the DNA replication fork, where MIF nuclease activity is required to resolve replication stress and facilitates tumor growth. MIF loss in cancer cells leads to mutation frequency increases, cell cycle delays and DNA synthesis and cell growth inhibition, which can be rescued by restoring MIF, but not nuclease-deficient MIF mutant. MIF is significantly upregulated in breast tumors and correlates with poor overall survival in patients. We propose that MIF is a unique 3’ nuclease, excises flaps at the immediate 3’ end during DNA synthesis and favors cancer cells evading replication stress-induced threat for their growth.

AbstractRecruitment of RNA polymerase II to hypoxia-inducible factor (HIF) target genes under normoxia is a prerequisite for HIF-mediated transactivation. However, the underlying mechanism of this recruitment remains unknown. Here we report that chromodomain helicase DNA-binding protein 4 (CHD4) physically interacts with α and β subunits of HIF1 and HIF2 and enhances HIF-driven transcriptional programs to promote breast cancer progression. Loss of HIF1/2α abolished CHD4-mediated breast tumor growth in mice. In breast cancer cells under normoxia, CHD4 enrichment at HIF target gene promoters increased RNA polymerase II loading through p300. Hypoxia further promoted CHD4 binding to the chromatin via HIF1/2α, where CHD4 in turn enhanced recruitment of HIF1α, leading to HIF target gene transcription. CHD4 was upregulated and correlated with HIF target gene expression in human breast tumors; upregulation of CHD4 and other known HIF coactivators in human breast tumors was mutually exclusive. Furthermore, CHD4 was associated with poor overall survival of patients with breast cancer. Collectively, these findings reveal a new fundamental mechanism of HIF regulation in breast cancer, which has clinical relevance.Significance:This study identifies CHD4 as a HIF coactivator and elucidates the fundamental mechanism underlying CHD4-mediated HIF transactivation in breast tumors.

Abstract Hypoxia induces a vast array of long noncoding RNAs (lncRNA) in breast cancer cells, but their biological functions remain largely unknown. Here, we identified a hitherto uncharacterized hypoxia-induced lncRNA RAB11B-AS1 in breast cancer cells. RAB11B-AS1 is a natural lncRNA upregulated in human breast cancer and its expression is induced by hypoxia-inducible factor 2 (HIF2), but not HIF1, in response to hypoxia. RAB11B-AS1 enhanced the expression of angiogenic factors including VEGFA and ANGPTL4 in hypoxic breast cancer cells by increasing recruitment of RNA polymerase II. In line with increased angiogenic factors, conditioned media from RAB11B-AS1-overexpressing breast cancer cells promoted tube formation of human umbilical vein endothelial cells in vitro. Gain- and loss-of-function studies revealed that RAB11B-AS1 increased breast cancer cell migration and invasion in vitro and promoted tumor angiogenesis and breast cancer distant metastasis without affecting primary tumor growth in mice. Taken together, these findings uncover a fundamental mechanism of hypoxia-induced tumor angiogenesis and breast cancer metastasis. Significance: This study reveals the molecular mechanism by which the lncRNA RAB11B-AS1 regulates hypoxia-induced angiogenesis and breast cancer metastasis, and provides new insights into the functional interaction between a lncRNA and tumor microenvironment.