Excess endogenous L(S)-2HG in hypoxic cells

Similar to D-2HG (or R-2HG), excessive L-2HG (or S-2HG), an enantiomer of D-2HG, can also inhibit numerous α-KG-dependent enzymes^26,27. Recent studies showed that hypoxia dramatically induces L-2HG production in both normal and malignant cells^28,29. Under hypoxic or acidic conditions, L-2HG production was increased via promiscuous catalytic activities of lactate dehydrogenase A (LDHA) and malate dehydrogenase (MDH1 and 2) (Fig. 3). Purified LDH and MDH enzymes stereo-specifically reduced α-KG to L-2HG using NADH. Acidic conditions increased the levels of the protonated α-KG form, which more stably binds to the catalytic region of LDH-A, thereby increasing L-2HG production³⁰. Hypoxic condition in the core region of many solid tumors enhanced L-2HG production via HIF-1α-dependent induction of LDH-A expression. Similar to D-2HG, L-2HG inhibits the Jumonji family histone lysine demethylase KDM4C, resulting in aberrant accumulation of trimethylated histone 3 lysine 9 (H3K9me3)²⁸.

D- and L-2HG accumulation due to defects in 2-HG dehydrogenases

L-2HG is oxidized and converted to α-KG by L-2HG dehydrogenase in the mitochondria. Similarly, D-2HG is also oxidized by D2HGDH. These 2-HG oxidation reactions are coupled with the reduction of FAD or NAD+³¹. Oxidation of 2-HG increases the NADH and FADH levels in cells; thus, 2-HG may act as a reservoir of reducing equivalents^32–34. Consistent with this idea, the NADH/NAD ratio increases when mitochondrial respiration is impaired; limited 2-HG oxidation in hematopoietic stem cells increases the levels of 2-HG, which is accompanied by inhibition of DNA and histone demethylation, leading to increased hypermethylation of both DNA and histones³⁴. Homozygous germline loss-of-function mutations in D2HGDH or L-2HG dehydrogenase (L2HGDH) increase the D-2HG or L-2HG levels, respectively, in both urine and blood³⁵. Systemic L-2HG elevations arising from inherited L2HGDH mutations have been associated with brain tumors³⁶. L2hgdh knockout mice display an increased L-2HG level in the brain with progressive leukoencephalopathy and neurodegeneration³⁷.

JARID1C/KDM5C as a tumor suppressor

JARID1C was identified as an X-linked mental retardation-related gene that escapes X inactivation during embryogenesis. Thus, females harbor two active copies of JARID1C, whereas males possess a single copy⁵⁴. Loss of JARID1C in males contributes to sex bias phenotypes^55,56. Mutations in JARID1C are associated with short stature, hyperreflexia, and autism^57,58. Mutations in JARID1C have been identified in many cancers, such as clear cell renal cell carcinoma (ccRCC), pancreatic cancer, and human papillomavirus (HPV)-associated cancer (Table 1)^59–63. In ccRCC, von Hippel Lindau (VHL), a tumor suppressor, is dominantly inactivated. As VHL is an HIF-α subunit-specific E3 ubiquitin ligase, HIF-α is constitutively activated in VHL-inactive ccRCC. Whole-exome sequencing analyses of ccRCC revealed that in addition to VHL, JARID1C/KDM5C, UTX/KDM6A, and SETD2 (H3K36 methyltransferase) were new cancer genes in ccRCC⁶¹. In 786-O VHL−/− ccRCC cells, JARID1C knockdown significantly enhanced tumor growth in a xenograft mouse model, showing that JARID1C is a tumor suppressor and that its inactivating mutations in ccRCC promote tumors⁶⁰. Whole-exome sequencing of human pancreatic cancers identified truncating insertions and deletion mutations in JARID1C⁶³. Genome-wide siRNA screening has identified SMCX (another name of JARID1C) as an E2-dependent regulator of the HPV oncogenes E6 and E7⁶². The HPV protein E2 represses the expression of oncogenic E6 and E7 genes by inactivating p53 and Rb, respectively. JARID1C was shown to repress E6 and E7 by interacting with E2. JARID1C knockdown increased the expression levels of the E6 and E7 oncogenes, suggesting that JARID1C can function as a tumor suppressor in HPV-associated cancers.

The molecular mechanism through which JARID1C suppresses tumor growth remains unclear. One explanation is that JARID1C represses important oncogenic target genes by reducing the active H3K4me3 marks at several promoters, where it is recruited via interactions with DNA-binding proteins⁶⁴; furthermore, JARID1C is recruited to the promoters of IGFBP1, DNAJC12, COL6A1, and GDF15 by interacting with HIF in 786-O VHL−/− ccRCC cells⁶⁰, and at the long control region promoter of the viral E6 and E7 genes via interaction with E2⁶². Rondinelli et al. suggested that in addition to transcriptional regulation, the H3K4me3 demethylase activity of JARID1C is also associated with heterochromatin assembly and genomic instability after replication in ccRCC cells⁶⁵. JARID1C binds to heterochromatic regions where transcription is tightly suppressed. Demethylation of H3K4me3 by JARID1C is required for Suv39H1-mediated methylation of H3K9 for postreplication heterochromatin reconstruction. In ccRCC cells, JARID1C inactivation failed to reduce H3K4me3 at heterochromatic regions, preventing Suv38H1 H3K9 methyl transferase and HP1a from binding to heterochromatin. Under this condition, heterochromatin assembly is hindered, leading to unrestrained expression of aberrant noncoding RNAs, which trigger genomic rearrangement. Therefore, JARID1C prevents tumor progression by guarding genomic stability via maintenance of heterochromatic regions. JARID1C is also required for proper DNA replication by removing tri-methyl groups from H3K4 at replication origins, which drives origin binding by the preinitiation proteins CDC45 and PCNA⁶⁶. Whether promotion of DNA replication is related to the tumor suppression function of JARID1C is unclear.

Other KDM5 isoforms, JARID1A, 1B, and 1D, as tumor suppressors

JARID1A and 1B are not cancer drivers; however, they are the most intensively studied isoforms. Several studies provided evidence that they are involved in tumor suppression. JARID1A/KDM5A/RBP2/RBBP2 was identified as an Rb-interacting protein, while JARID1B/KDM5B/PLU-1 was identified as a gene that is upregulated by overexpression of c-ErbB2^67,68. The H3K4 demethylation activities of both JARID1A and 1B mediate Rb’s function of repressing E2F target genes, suggesting that JARID1A and 1B may be involved in tumor suppression by enhancing cellular senescence⁶⁹. Consistent with this idea, Nijwening et al. demonstrated that JARID1B depletion led to phenotypes similar to those observed after Rb1 knockdown and that JARID1B associates with E2F target genes during cellular senescence⁷⁰. The genome-wide epigenetic state of leukemic stem cells in MLL-associated leukemia revealed that leukemic stem cells maintained a high level of H3K4me3 and a low level of H3K79me2. JARID1B negatively regulates self-renewal and the oncogenic potential of leukemic stem cells by reducing H3K4me3 abundance⁷¹. A genome-wide transcriptional analysis revealed that the JARID1B-LSD1-NuRD1 complex targeted the cell migration- and angiogenesis-related CCL14 chemokine pathway⁵². Experiments using MDA-MB-231 and MCF7 breast cancer cells showed that JARID1B suppressed cell invasion and angiogenesis by repressing CCL14 expression, suggesting that JARID1B suppresses tumor progression. JARID1D was originally reported as a minor histocompatibility antigen on the Y chromosome⁷². JARID1D was considered a tumor suppressor because of its downregulation, mutation, or loss in prostate cancer and ccRCC^73,74. JARID1D directly interacts with the androgen receptor (AR) to attenuate the transcriptional activation of AR target genes in hormone-sensitive prostate cancers, whereas JARID1D expression is downregulated during the progression to hormone-resistant tumors.

These diverse roles for the JARID1 isoforms in multiple cancer types highlight the roles of JARID1A, 1B, and 1D as tumor suppressers; however, unlike JARID1C, the functions of JARID1A, 1B, and 1D depend on the cellular context, which underscores their potential as cancer biomarkers and drug targets. In contrast, several studies showed that JARID1A and 1B are involved in tumor progression rather than in suppression.

UTX/KDM6A as a tumor suppressor

Genomic analyses of several tumors revealed that loss of UTX occurs in various cancers, including B-cell lymphoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, pancreatic adenocarcinoma, lung squamous cell carcinoma, and kidney renal papillary cell carcinoma (Table 1)^88–93. However, the molecular mechanism through which UTX suppresses tumor progression remains unclear. Depending on the cancer type, UTX is involved in tumor suppression, not only via its H3K27me3 demethylase activity but also via interactions with other epigenetic complexes.

Due to its H3K27me3, me2, and me1 demethylase activities, loss of UTX increases the H3K27me3 level at its target genes. Several studies showed that loss of UTX represses a certain subset of genes by increasing the levels of the repressive H3K27me3 mark, ultimately promoting proliferation, clonogenicity, adhesion, and tumorigenicity in myeloid, bladder, and lung transformation^94–97. Upon loss of the UTX demethylases, H3K27 methyl transferases, such as PRC2/EZH2, determine the levels of H3K27me3 on the UTX-EZH2 target genes. Consistently, loss of UTX increases cellular sensitivity to EZH2 inhibition. In UTX-mutated multiple myeloma cells, such altered sensitivity was related to changes in gene expression triggered by a rebalancing of the H3K27me3 levels at specific genes, such as IRF4 and c-MYC⁹⁴. In UTX/KDM6A-mutated urothelial bladder carcinoma, PRC2/EZH2 target genes, such as IGFBP3, are deregulated due to H3K27me3 enrichment. EZH2 inhibition upregulated UTX-EZH2 target genes and contributed to the EZH2-dependent growth suppression in KDM6A-null bladder tumors in both patient-derived and cell line xenograft models⁹⁵. PRC2/EZH2 inhibition has emerged as a potential precision therapeutic strategy for genetically defined UTX/KDM6A null cancers that acts by rebalancing the H3K27me3 levels at specific genes^98–100. EZH2 inhibition also restored the normal gene expression patterns and impaired the proliferation of tumor cells harboring mutations in an H3K4 methyltransferase, MLL3/KMT2C, or a tumor suppressor, BAP1, by rebalancing the H3K27me3 levels at MLL3/BAP1 target genes¹⁰¹. In a range of human tumor types, a cancer-associated mutational hotspot was detected in the PHD domain of MLL3, which mediates association with BAP1. Cancer cells that harbored mutations in the PHD domain of MLL3 or lacked BAP1 showed reduced recruitment of UTX/KDM6A to gene enhancers, with no reduction in the levels of the repressive H3K27me3 mark. Thus, in cancer cells with MLL3 or BAP1 mutations, reduction of H3K27me3 via EZH2 inhibitors can restore the balance of H3K27me3 at enhancers where UTX is not properly recruited. Thus, UTX and PRC2/EZH2 inhibition contribute to enhancer restoration by preventing the deposition of excess H3K27me3¹⁰². Several studies showed that PRC2/EZH2 inhibition compensates for the loss of UTX function, suggesting that the catalytic activity of UTX plays an important role in tumor suppression. However, other studies showed that in certain types of cancer, an inactive UTX paralog is required for tumor development in males concomitant with loss of UTY and that the UTX-UTY double-knockout cells exhibited higher proliferation than the single-knockout cells, suggesting demethylase-independent tumor suppressor functions of UTX/KDM6A and UTY¹⁰³.

Several studies showed that UTX is essential for the establishment of the active enhancer histone marks H3K4me1 and H3K27ac in a demethylase activity-independent manner via recruitment and coupling of an H3K4 methyltransferase complex (named COMPASS) and the histone acetyl transferase p300. In embryonic stem cells, loss of UTX reduced the levels of H3K4me1/H3K27ac at enhancers and transcription. An interaction between UTX and MLL4 (a COMPASS component) enhances p300-dependent H3K27 acetylation not only via UTX-dependent recruitment of p300 but also via MLL4-mediated H3K4 monomethylation. The UTX-MLL4-p300 crosstalk coordinately establishes an active enhancer for transcription¹⁰⁴. Similarly, in many cancers and other diseases, enhancer-associated chromatin-modifying components, such as UTX and members of H3K4 methyltransferase complexes, are frequently mutated, leading to enhancer malfunction. In pancreatic cancer, loss of UTX causes aberrant activation of superenhancers that regulate oncogenes (such as Delta-Np63, MYC, and RUNX3) to drive an aggressive subtype of squamous-like pancreatic cancer¹⁰³. To investigate the tumor-suppressive function of UTX in vivo, Gozdecka et al. developed myeloid-specific Utx knockout mice, most of which (63%) developed AML¹⁰⁵. To mimic human leukemia, they expressed the AML1-ETO fusion gene in Utx−/− hematopoietic stem and progenitor cells. Global genomic and proteomic analyses using an Utx-null mouse leukemia model revealed that UTX suppresses myeloid leukemogenesis via noncatalytic functions. Loss of UTX/KDM6A leads to only minor changes in the level H3K27me3 modification, but significant changes in the levels H3K4me1/H3K27ac modifications and alterations in the binding of pioneering enhancer transcription factors, ETS and GATA. These findings suggest that loss of UTX contributes to drive tumor progression via repositioning of histone modification enzymes.

JMJD3, a KDM6 isoform, as a tumor suppressor

JMJD3 can be considered a tumor suppressor based on the finding that it increases the expression levels of the tumor suppressors p16^INK4a, p14^ARF, and p15^INK4B and the activities of the tumor suppressors p53 and Rb. During oncogene-induced senescence (OIS) in human fibroblasts, JMJD3 demethylates the repressive H3K27me3 mark in the INK locus, which contains the genes encoding p16^INK4a, p14^ARF, and p15^{INK4B 106}. Oncogenes such as B-RAF or RAS induce JMJD3 expression. JMJD3 is recruited by a long noncoding-RNA, ANRIL, to the INK locus¹⁰⁷. Cyclin-dependent kinase inhibitors, p16^INK4a and p15^INK4B, maintain the activity of the Rb protein by inhibiting CDK4/6 (which normally phosphorylates and inactivates the Rb tumor suppressor), whereas p14^ARF increases p53 stability by inhibiting the p53-specific E3 ligase, MDM2¹⁰⁸. JMJD3 also directly regulates Rb function during cellular senescence. Specific methylation of the K810 residue of Rb facilitates its interaction with CDK4 protein kinase. JMJD3 demethylates the K810 residue of Rb, which prevents CDK4 from phosphorylating the S807 and S811 residues of Rb in senescent cells¹⁰⁹. Both JMJD3 and Rb are together linked to the formation of senescence-associated heterochromatin foci (SAHF). SAHF is essential for silencing genes involved in cell cycle progression. JMJD3 and p53 mutually increase their activities. p53 increases the nuclear localization of JMJD3 via protein−protein interaction and recruits JMJD3 to its target genes, such as that encoding p21, where it demethylates the H3K27me3 repressive mark^110,111. The demethylase activity of JMJD3 eventually increases the activities of potent tumor suppressors, such as Rb and p53, leading to cellular senescence, a process that blocks tumor initiation, thereby supporting the role of JMJD3 as a tumor suppressor.

Several studies have shown potential tumor-suppressive roles for JMJD3 in different types of cancers. In glioblastoma, JMJD3 overexpression reduced the growth of glioma stem cells by increasing the p16 level and stabilizing p53 in the nucleus¹¹². JMJD3 expression correlates with the p15^INK4B expression level in clinical colorectal cancer samples. Low JMJD3 expression correlates with poor prognosis in patients with surgically resected colorectal cancer¹¹³. Loss of JMJD3 heterozygosity at chromosome 17p13.1 increased the aggressiveness of pancreatic ductal adenocarcinoma. JMJD3 expression is lower in patient samples of hepatocellular carcinoma, lung adenocarcinoma, non-Hodgkin’s lymphoma, and hematopoietic malignancies compared with that in normal tissues¹⁰⁶.

We thank Prof. Sung Soo Kim (School of Medicine, Kyung Hee University) for his critical comments and Ramhee Moon for assistance in organizing the references. This work was supported by grants NRF-2016R1A2B4012840 and NRF-2018R1A4A1025985 from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT). Sujin Yim was supported by NRF and the Center for Women In Science, Engineering and Technology (WISET) Grant funded by the Ministry of Science and ICT under the Program for Returners into R&D.