Development and crystal structures of a potent second-generation dual degrader of BCL-2 and BCL-xL.

Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determine crystal structures of VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/753b-linker/target interfaces. The importance of these interfacial contacts is validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This results in the design of a degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we are able to explain the enhanced target degradation of BCL-xL/BCL-2 in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.

MiR-223-3p promotes genomic stability of hematopoietic progenitors after radiation.

When hematopoietic cells are overwhelmed with ionizing radiation (IR) DNA damage, the alternative non-homologous end-joining (aNHEJ) repair pathway is activated to repair stressed replication forks. While aNHEJ can rescue cells overwhelmed with DNA damage, it can also mediate chromosomal deletions and fusions, which can cause mis-segregation in mitosis and resultant aneuploidy. We previously reported that a hematopoietic microRNA, miR-223-3p, normally represses aNHEJ. We found that miR-223-/- mice have increased survival of hematopoietic stem and progenitor cells (HSPCs) after sublethal IR. However, this came at the cost of significantly more genomic aberrancies, with miR-223-/- hematopoietic progenitors having increased metaphase aberrancies, including chromothripsis, and increased sequence abnormalities, especially deletions, which is consistent with aNHEJ. These data imply that when an HSPC is faced with substantial DNA damage, it may trade genomic damage for its own survival by choosing the aNHEJ repair pathway, and this choice is regulated in part by miR-223-3p.

Cellular Responses to Widespread DNA Replication Stress.

Replicative DNA polymerases are blocked by nearly all types of DNA damage. The resulting DNA replication stress threatens genome stability. DNA replication stress is also caused by depletion of nucleotide pools, DNA polymerase inhibitors, and DNA sequences or structures that are difficult to replicate. Replication stress triggers complex cellular responses that include cell cycle arrest, replication fork collapse to one-ended DNA double-strand breaks, induction of DNA repair, and programmed cell death after excessive damage. Replication stress caused by specific structures (e.g., G-rich sequences that form G-quadruplexes) is localized but occurs during the S phase of every cell division. This review focuses on cellular responses to widespread stress such as that caused by random DNA damage, DNA polymerase inhibition/nucleotide pool depletion, and R-loops. Another form of global replication stress is seen in cancer cells and is termed oncogenic stress, reflecting dysregulated replication origin firing and/or replication fork progression. Replication stress responses are often dysregulated in cancer cells, and this too contributes to ongoing genome instability that can drive cancer progression. Nucleases play critical roles in replication stress responses, including MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, FEN1, and TATDN2. Several of these nucleases cleave branched DNA structures at stressed replication forks to promote repair and restart of these forks. We recently defined roles for EEPD1 in restarting stressed replication forks after oxidative DNA damage, and for TATDN2 in mitigating replication stress caused by R-loop accumulation in BRCA1-defective cells. We also discuss how insights into biological responses to genome-wide replication stress can inform novel cancer treatment strategies that exploit synthetic lethal relationships among replication stress response factors.

TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

In Vitro Reconstitutive Base Excision Repair (BER) Assay.

The mammalian cell genome is continuously exposed to endogenous and exogenous insults that modify its DNA. These modifications can be single-base lesions, bulky DNA adducts, base dimers, base alkylation, cytosine deamination, nitrosation, or other types of base alteration which interfere with DNA replication. Mammalian cells have evolved with a robust defense mechanism to repair these base modifications (damages) to preserve genomic stability. Base excision repair (BER) is the major defense mechanism for cells to remove these oxidative or alkylated single-base modifications. The base excision repair process involves replacement of a single-nucleotide residue by two sub-pathways, the single-nucleotide (SN) and the multi-nucleotide or long-patch (LP) base excision repair pathways. These reactions have been reproduced in vitro using cell free extracts or purified recombinant proteins involved in the base excision repair pathway. In the present chapter, we describe the detailed methodology to reconstitute base excision repair assay systems. These reconstitutive BER assay systems use artificially synthesized and modified DNA. These reconstitutive assay system will be a true representation of biologically occurring damages and their repair.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

The RNA Demethylase ALKBH5 Maintains Endoplasmic Reticulum Homeostasis by Regulating UPR, Autophagy, and Mitochondrial Function.

Eukaryotic cells maintain cellular fitness by employing well-coordinated and evolutionarily conserved processes that negotiate stress induced by internal or external environments. These processes include the unfolded protein response, autophagy, endoplasmic reticulum-associated degradation (ERAD) of unfolded proteins and altered mitochondrial functions that together constitute the ER stress response. Here, we show that the RNA demethylase ALKBH5 regulates the crosstalk among these processes to maintain normal ER function. We demonstrate that ALKBH5 regulates ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain the calcium flux between the ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently, mitochondrial biogenesis. Thus, these findings reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness.

EEPD1 promotes repair of oxidatively-stressed replication forks.

Unrepaired oxidatively-stressed replication forks can lead to chromosomal instability and neoplastic transformation or cell death. To meet these challenges cells have evolved a robust mechanism to repair oxidative genomic DNA damage through the base excision repair (BER) pathway, but less is known about repair of oxidative damage at replication forks. We found that depletion or genetic deletion of EEPD1 decreases clonogenic cell survival after oxidative DNA damage. We demonstrate that EEPD1 is recruited to replication forks stressed by oxidative damage induced by H2O2 and that EEPD1 promotes replication fork repair and restart and decreases chromosomal abnormalities after such damage. EEPD1 binds to abasic DNA structures and promotes resolution of genomic abasic sites after oxidative stress. We further observed that restoration of expression of EEPD1 via expression vector transfection restores cell survival and suppresses chromosomal abnormalities induced by oxidative stress in EEPD1-depleted cells. Consistent with this, we found that EEPD1 preserves replication fork integrity by preventing oxidatively-stressed unrepaired fork fusion, thereby decreasing chromosome instability and mitotic abnormalities. Our results indicate a novel role for EEPD1 in replication fork preservation and maintenance of chromosomal stability during oxidative stress.

Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibit small-cell lung cancer growth without causing on-target toxicities in mice.

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation.

The tumor suppressor BRCA2 participates in DNA double-strand break repair by RAD51-dependent homologous recombination and protects stressed DNA replication forks from nucleolytic attack. We demonstrate that the C-terminal Recombinase Binding (CTRB) region of BRCA2, encoded by gene exon 27, harbors a DNA binding activity. CTRB alone stimulates the DNA strand exchange activity of RAD51 and permits the utilization of RPA-coated ssDNA by RAD51 for strand exchange. Moreover, CTRB functionally synergizes with the Oligonucleotide Binding fold containing DNA binding domain and BRC4 repeat of BRCA2 in RPA-RAD51 exchange on ssDNA. Importantly, we show that the DNA binding and RAD51 interaction attributes of the CTRB are crucial for homologous recombination and protection of replication forks against MRE11-mediated attrition. Our findings shed light on the role of the CTRB region in genome repair, reveal remarkable functional plasticity of BRCA2, and help explain why deletion of Brca2 exon 27 impacts upon embryonic lethality.

Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice.

Hem1 (hematopoietic protein 1), a hematopoietic cell-specific member of the Hem family of cytoplasmic adaptor proteins, is essential for lymphopoiesis and innate immunity as well as for the transition of hematopoiesis from the fetal liver to the bone marrow. However, the role of Hem1 in bone cell differentiation and bone remodeling is unknown. Here, we show that deletion of Hem1 resulted in a markedly increase in bone mass because of defective bone resorption in mice of both sexes. Hem1-deficient osteoclast progenitors were able to differentiate into osteoclasts, but the osteoclasts exhibited impaired osteoclast fusion and decreased bone-resorption activity, potentially because of decreased mitogen-activated protein kinase and tyrosine kinase c-Abl activity. Transplantation of bone marrow hematopoietic stem and progenitor cells from wildtype into Hem1 knockout mice increased bone resorption and normalized bone mass. These findings indicate that Hem1 plays a pivotal role in the maintenance of normal bone mass.

Resistance to the BCL-XL degrader DT2216 in T-cell acute lymphoblastic leukemia is rare and correlates with decreased BCL-XL proteolysis.

PURPOSE: The BCL-2 family of anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1, can mediate survival of some types of cancer. DT2216 is a PROteolysis-TArgeting Chimera (PROTAC) that degrades BCL-XL specifically and is in phase 1 trials. We sought to define the frequency and mechanism of resistance to DT2216 in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. METHODS: We measured cell survival and protein levels of BCL-XL, BCL-2, MCL-1 and the pro-apoptotic BIM in 13 distinct T-ALL cell lines after exposure to varying concentrations of DT2216. RESULTS: We identified concentrations of DT2216 which were cytotoxic to each T-ALL cell line. These concentrations have no correlation with the initial protein levels of BCL-XL, BCL-2, MCL-1 or BIM in each cell line. However, there was a correlation between survival to DT2216 and the efficiency of degradation of BCL-XL by DT2216. Only one cell line, SUP-T1, had significant resistance to DT2216, defined as an IC50 above what is achievable in murine tumors in vivo. CONCLUSION: Resistance to DT2216 is rare in a wide variety of T-ALL cells but when it occurs is correlated with decreased BCL-XL degradation. Resistance to DT2216 in T-ALL is not predicted by initial BCL-XL or BIM protein levels, or BCL-2 or MCL-1 levels before or after treatment. These data imply that a phase 2 clinical trial of DT2216 in T-ALL should be widely available and not limited to a subset of patients.

Roles of homologous recombination in response to ionizing radiation-induced DNA damage.

PURPOSE: Ionizing radiation induces a vast array of DNA lesions including base damage, and single- and double-strand breaks (SSB, DSB). DSBs are among the most cytotoxic lesions, and mis-repair causes small- and large-scale genome alterations that can contribute to carcinogenesis. Indeed, ionizing radiation is a 'complete' carcinogen. DSBs arise immediately after irradiation, termed 'frank DSBs,' as well as several hours later in a replication-dependent manner, termed 'secondary' or 'replication-dependent DSBs. DSBs resulting from replication fork collapse are single-ended and thus pose a distinct problem from two-ended, frank DSBs. DSBs are repaired by error-prone nonhomologous end-joining (NHEJ), or generally error-free homologous recombination (HR), each with sub-pathways. Clarifying how these pathways operate in normal and tumor cells is critical to increasing tumor control and minimizing side effects during radiotherapy. CONCLUSIONS: The choice between NHEJ and HR is regulated during the cell cycle and by other factors. DSB repair pathways are major contributors to cell survival after ionizing radiation, including tumor-resistance to radiotherapy. Several nucleases are important for HR-mediated repair of replication-dependent DSBs and thus replication fork restart. These include three structure-specific nucleases, the 3' MUS81 nuclease, and two 5' nucleases, EEPD1 and Metnase, as well as three end-resection nucleases, MRE11, EXO1, and DNA2. The three structure-specific nucleases evolved at very different times, suggesting incremental acceleration of replication fork restart to limit toxic HR intermediates and genome instability as genomes increased in size during evolution, including the gain of large numbers of HR-prone repetitive elements. Ionizing radiation also induces delayed effects, observed days to weeks after exposure, including delayed cell death and delayed HR. In this review we highlight the roles of HR in cellular responses to ionizing radiation, and discuss the importance of HR as an exploitable target for cancer radiotherapy.

BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1.

Tumors with BRCA1 mutations have poor prognoses due to genomic instability. Yet this genomic instability has risks and BRCA1-deficient (def) cancer cells must develop pathways to mitigate these risks. One such risk is the accumulation of unfolded proteins in BRCA1-def cancers from increased mutations due to their loss of genomic integrity. Little is known about how BRCA1-def cancers survive their genomic instability. Here we show that BRCA1 is an E3 ligase in the endoplasmic reticulum (ER) that targets the unfolded protein response (UPR) stress sensors, Eukaryotic Translation Initiation Factor 2-alpha Kinase 3 (PERK) and Serine/Threonine-Protein Kinase/Endoribonuclease Inositol-Requiring Enzyme 1 (IRE1) for ubiquitination and subsequent proteasome-mediated degradation. When BRCA1 is mutated or depleted, both PERK and IRE1 protein levels are increased, resulting in a constitutively activated UPR. Furthermore, the inhibition of protein folding or UPR signaling markedly decreases the overall survival of BRCA1-def cancer cells. Our findings define a mechanism used by the BRCA1-def cancer cells to survive their increased unfolded protein burden which can be used to develop new therapeutic strategies to treat these cancers.

Nucleases and Co-Factors in DNA Replication Stress Responses.

DNA replication stress is a constant threat that cells must manage to proliferate and maintain genome integrity. DNA replication stress responses, a subset of the broader DNA damage response (DDR), operate when the DNA replication machinery (replisome) is blocked or replication forks collapse during S phase. There are many sources of replication stress, such as DNA lesions caused by endogenous and exogenous agents including commonly used cancer therapeutics, and difficult-to-replicate DNA sequences comprising fragile sites, G-quadraplex DNA, hairpins at trinucleotide repeats, and telomeres. Replication stress is also a consequence of conflicts between opposing transcription and replication, and oncogenic stress which dysregulates replication origin firing and fork progression. Cells initially respond to replication stress by protecting blocked replisomes, but if the offending problem (e.g., DNA damage) is not bypassed or resolved in a timely manner, forks may be cleaved by nucleases, inducing a DNA double-strand break (DSB) and providing a means to accurately restart stalled forks via homologous recombination. However, DSBs pose their own risks to genome stability if left unrepaired or misrepaired. Here we focus on replication stress response systems, comprising DDR signaling, fork protection, and fork processing by nucleases that promote fork repair and restart. Replication stress nucleases include MUS81, EEPD1, Metnase, CtIP, MRE11, EXO1, DNA2-BLM, SLX1-SLX4, XPF-ERCC1-SLX4, Artemis, XPG, and FEN1. Replication stress factors are important in cancer etiology as suppressors of genome instability associated with oncogenic mutations, and as potential cancer therapy targets to enhance the efficacy of chemo- and radiotherapeutics.

Targeting aberrant replication and DNA repair events for treating breast cancers.

The major limitations of DNA-targeting chemotherapy drugs include life-threatening toxicity, acquired resistance and occurrence of secondary cancers. Here, we report a small molecule, Carbazole Blue (CB), that binds to DNA and inhibits cancer growth and metastasis by targeting DNA-related processes that tumor cells use but not the normal cells. We show that CB inhibits the expression of pro-tumorigenic genes that promote unchecked replication and aberrant DNA repair that cancer cells get addicted to survive. In contrast to chemotherapy drugs, systemic delivery of CB suppressed breast cancer growth and metastasis with no toxicity in pre-clinical mouse models. Using PDX and ex vivo explants from estrogen receptor (ER) positive, ER mutant and TNBC patients, we further demonstrated that CB effectively blocks therapy-sensitive and therapy-resistant breast cancer growth without affecting normal breast tissue. Our data provide a strong rationale to develop CB as a viable therapeutic for treating breast cancers.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased nonhomologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors sensitive to olaparib. In contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its cofactor BARD1 but instead coimmunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TANK-binding kinase 1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and immune-independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker. SIGNIFICANCE: PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression.

Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4-based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth. SIGNIFICANCE: RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair-associated genes, driving osteosarcoma progression.

Metnase and EEPD1: DNA Repair Functions and Potential Targets in Cancer Therapy.

Cells respond to DNA damage by activating signaling and DNA repair systems, described as the DNA damage response (DDR). Clarifying DDR pathways and their dysregulation in cancer are important for understanding cancer etiology, how cancer cells exploit the DDR to survive endogenous and treatment-related stress, and to identify DDR targets as therapeutic targets. Cancer is often treated with genotoxic chemicals and/or ionizing radiation. These agents are cytotoxic because they induce DNA double-strand breaks (DSBs) directly, or indirectly by inducing replication stress which causes replication fork collapse to DSBs. EEPD1 and Metnase are structure-specific nucleases, and Metnase is also a protein methyl transferase that methylates histone H3 and itself. EEPD1 and Metnase promote repair of frank, two-ended DSBs, and both promote the timely and accurate restart of replication forks that have collapsed to single-ended DSBs. In addition to its roles in HR, Metnase also promotes DSB repair by classical non-homologous recombination, and chromosome decatenation mediated by TopoIIα. Although mutations in Metnase and EEPD1 are not common in cancer, both proteins are frequently overexpressed, which may help tumor cells manage oncogenic stress or confer resistance to therapeutics. Here we focus on Metnase and EEPD1 DNA repair pathways, and discuss opportunities for targeting these pathways to enhance cancer therapy.