DNA Damage Signaling Research Articles

Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers

Abstract Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities. Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B173.

Abstract C039: Cancer labeling should be retained for prostate cancer with a Gleason score of 3+3=6 - based on genomic, transcriptomic, and proteomic aspects

Abstract Purpose: The decision to remove the cancer label for prostate cancer with a Gleason score (GS) of 3+3 relies on pathology and clinical manifestations. However, evidence at the molecular level, especially for comparing the Excluding 3 group (GS4+4, GS4+5, GS5+4 and GS5+5) and the Gleason Grade Group 1 (GG1) (GS3+3), is lacking. To address this gap, we aimed to explore the differences between these two groups at the genomic, transcriptomic, and proteomic levels. Methods: We compared the GG1 group (n=82) and Excluding 3 group (n=148) using data from the TCGA in terms of genomic alterations, including gene mutations, chromosomal structure variants, and copy number variants. Pathways were revealed by using GSEA enrichment analysis at the transcriptome level. We also conducted a quantitative whole protein analysis of prostate cancer and paraneoplastic tissues in 6 pairs of GG1 and 11 pairs of Excluding 3 groups to compare the differences at the protein level and explored the enriched pathways between them by GSEA enrichment analysis. Finally, we validated the accuracy of DIA quantification by performing immunohistochemical experiments on radical resection pathology specimens from 29 patients diagnosed with GG1 and 29 patients diagnosed with Excluding 3 group. Results: At the genomic level, we found that the mutation frequencies of GG1 and Excluding 3 group were comparable. But, the mutation frequencies of TP53, FOXA1 and PTEN were significantly higher in the Excluding 3 group than in the GG1 group , while the TMPRSS2-ERG fusion was not significantly different between the two groups. And the incidence of the p53 signaling pathway, WNT pathway, PI3K pathway, and DNA damage was significantly higher in the Excluding 3 group than in the GG1 group. For the transcriptomic level, GSEA enrichment analysis revealed that both groups were enriched for multiple tumor-related pathways. At the proteome level, with protein quantitative testing by mass spectrometry, we identified that androgen-response pathway was inhibited in the GG1 group, while DNA repair and PPAR signaling pathway was activated in the Excluding 3 group. Six differential proteins were identified which had important roles in cancer development. And immunohistochemical experiments confirmed the accuracy of quantification analysis for 3 representative proteins (DDX21, NOLC1, and TIMP1). Conclusion: The GG1 and Excluding 3 groups showed similar oncogene mutation frequencies and chromosomal structural variations. However, shared and distinct pathway alterations were observed in genomic and transcriptomic analyses, indicating common driving factors and differential influences of biological factors. Proteomic analysis identified differentially expressed proteins and enriched cancer-related pathways associated with tumor characteristics. These findings support the malignancy features of GG1 prostate cancer. Citation Format: Sijie Wen, Yinzhao Wang, Lin Qi, Hongling Yin, Xiaomei Gao, Minfeng Chen, Yi Cai. Cancer labeling should be retained for prostate cancer with a Gleason score of 3+3=6 - based on genomic, transcriptomic, and proteomic aspects [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C039.

The MRE11–ATM–SOG1 DNA damage signaling pathway confers rice immunity to Xanthomonas oryzae

Plants are constantly exposed to microbial pathogens in the environment. One branch of innate plant immunity is mediated by cell-membrane-localized receptors, but less is known about associations between DNA damage and plant immune responses. Here, we show that rice (Oryza sativa) mesophyll cells are prone to DNA double-stranded breaks (DSBs) in response to ZJ173, a strain of Xanthomonas oryzae pv. oryzae (Xoo). The DSB signal transducer ataxia telangiectasia mutated (ATM), but not the ATM and Rad3-related branch, confers resistance against Xoo. Mechanistically, the MRE11–ATM module phosphorylates suppressor of gamma response 1 (SOG1), which activates several phenylpropanoid pathway genes and prompts downstream phytoalexin biosynthesis during Xoo infection. Intriguingly, overexpression of the topoisomerase gene TOP6A3 causes a switch from the classic non-homologous end joining (NHEJ) pathway to the alternative NHEJ and homologous recombination pathways at Xoo-induced DSBs. The enhanced ATM signaling of the alternative NHEJ pathway strengthens the SOG1-regulated phenylpropanoid pathway and thereby boosts Xoo-induced phytoalexin biosynthesis in TOP6A3-OE1 overexpression lines. Overall, the MRE11–ATM–SOG1 pathway serves as a prime example of plant–pathogen interactions that occur via host non-specific recognition. The function of TOP6-facilitated ATM signaling in the defense response makes it a promising target for breeding of rice germplasm that exhibits resistance to bacterial blight disease without a growth penalty.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

Lipid-Mediated Modulation of DNA Damage Signaling As a Prognostic and Therapeutic Strategy Against Multiple Myeloma

Multiple myeloma (MM), characterized by the accumulation of tumor plasma cells in the bone marrow, is the second most common hematologic cancer. Occurrence of chemoresistance invariably leads patients to relapse and results in loss of clinical control over the disease. Cancer cells show a deregulation of different pathways, including lipid metabolism alterations and high genomic instability. Yet, no functional relationship between these two features has been identified. We have shown in a previous work that the metabolism of lipids impacts in how DNA damage is detected, signaled and repaired (Ovejero et al 2023 EMBO J). We reported that breaks in the DNA trigger the birth of lipid droplets (LD), a central lipid-storage organelle, and that this event is key for cells to tolerate this specific DNA damage. Given that MM cells are particularly dependent on DNA repair pathways for survival, we postulated that new therapeutic perspectives would emerge by targeting the formation of LD. Analysis of the LD formation gene expression profile of a cohort of newly diagnosed MM patients (UAMS-TT2 MM cohort, n = 345) allowed the establishment of a LD score comprising 13 genes associated with a significant prognostic value after multiple testing correction. The LD score gene expression-based risk score enabled the identification of MM patients with a poor outcome. The prognostic value of the LD score was validated in two other independent cohorts of newly diagnosed patients (HM cohort, n = 206; UAMS-TT3 MM cohort, n = 158) and in a cohort of patients at relapse treated with anti-CD38 MoAb (Mtp-Dara, n = 49). We subsequently studied the presence of LD in primary MM cells obtained from 34 patients. Importantly, we observed a significant positive correlation between the percentage of LD-positive cells and the value of the LD score (p = 0,02). This prompted us to investigate the therapeutic interest of interfering with LD formation to target MM cells. To this end, we treated 26 Human Myeloma Cell Lines (HMCLs) with drugs selected on an educated-guess basis and identified positive hits. These results were validated employing a shRNA strategy in selected HCMLs. Flow cytometry analyses revealed that this regime triggered a cell cycle halt, where cells accumulated in the G 1 phase at the expense of S and G 2. This was accompanied by a decrease in c-MYC expression, as well as an induction of cell proliferation arrest markers such as the G 1-checkpoint CDK inhibitors p21 and p27. Mechanistically, we observed a significant increase in Chk1 and Chk2 phosphorylations, two downstream events of DNA damage signaling despite that basal DNA damage levels remained unchanged. Together, these data convey the notion that limiting LD formation in MM cell lines boosts the detection of endogenously present DNA damage, thus enforcing a more robust DNA damage signaling which culminates in a drastic loss of proliferative capacity and viability. Of major importance, we validated the therapeutic interest of this strategy in primary MM cells from patients (n = 19). A significant higher toxicity in MM cells was found compared to normal cells from the microenvironment (tumoral: ****, p < 0.0001; normal: ***, p = 0,0006). Moreover, we tested the therapeutic interest of combining this approach with conventional treatments used in MM. Interestingly, we identified a synergistic effect when combined with melphalan, a DNA-alkylating agent used in MM treatment. This synergy was observed in HCMLs and in primary MM cells of patients (n = 7). Furthermore, significant synergistic effects were found in HMCLs by combining with the proteasome inhibitors bortezomib and carfilzomib. Altogether, these data unveil both the LD score and LD-bearing cell quantification as new prognostic factors in MM, and open new therapeutic perspectives to improve the treatment of MM patients by targeting LD metabolism.

Methyltransferase Set7/9 controls PARP1 expression and regulates cisplatin response of breast cancer cells

The protein-specific methyltransferase Set7/9 is known for its ability to add methyl groups to lysine residues on many targets, including as histones H1.4, H2A, H2B, H3, and non-histone proteins such as p53, NFκB, E2F1, pRb, Hif1α, β-catenin, STAT3, and YY1 transcription factors. Set7/9 affects both the landscape of histone modifications and the functionality of the aforementioned TFs, and acts as an essential mediator of vital cellular functions, regulating tumor growth and the neoplastic transformation of normal cells.The number of studies demonstrating the determining role of Set7/9 in cancer is growing. Importantly, the effect of Set7/9 on tumor progression is ambivalent and cancer-type dependent. In this study we analyzed the potential participation of Set7/9 in the essential cellular processes in breast cancer cells and revealed that Set7/9 may be involved in DNA damage signaling and DNA repair processes. We further demonstrated that Set7/9 expression is downregulated in cancerous breast tissues and inversely correlated to PARP1 expression level.Using breast cancer cell lines of HER2-positive and triple negative subtypes we have shown that the attenuation of Set7/9 led to the stabilization of PARP1 on both mRNA and protein levels that in turn resulted in cisplatin resistance acquiring.Finally, we demonstrated that the combination of cisplatin with FDA approved PARP1 inhibitor niraparib (Zejula) has a synergistic effect with cisplatin and thereby allows to overcome cisplatin resistance of Set7/9 deficient breast cancer cells.

Pan-cancer analyses of senescence-related genes in extracellular matrix characterization in cancer

PurposeThe aged microenvironment plays a crucial role in tumor onset and progression. However, it remains unclear whether and how the aging of the extracellular matrix (ECM) influences cancer onset and progression. Furthermore, the mechanisms and implications of extracellular matrix senescence-related genes (ECM-SRGs) in pan-cancer have not been investigated.MethodsWe collected profiling data from over 10,000 individuals, covering 33 cancer types, 750 small molecule drugs, and 24 immune cell types, for a thorough and systematic analysis of ECM-SRGs in cancer.ResultsWe observed a significant correlation between immune cell infiltrates and Gene Set Variation Analysis enrichment scores of ECM-SRGs in 33 cancer types. Moreover, our results revealed significant differences in immune cell infiltration among patients with copy number variations (CNV) and single nucleotide variations (SNV) in ECM-SRGs across various malignancies. Aberrant hypomethylation led to increased ECM-SRGs expression, and in specific malignancies, a connection between ECM-SRGs hypomethylation and adverse patient survival was established. The frequency of CNV and SNV in ECM-SRGs was elevated. We observed a positive correlation between CNV, SNV, and ECM-SRGs expression. Furthermore, a correlation was found between the high frequency of CNV and SNV in ECM-SRGs and poor patient survival in several cancer types. Additionally, the results demonstrated that ECM-SRGs expression could serve as a predictor of patient survival in diverse cancers. Pathway analysis unveiled the role of ECM-SRGs in activating EMT, apoptosis, and the RAS/MAPK signaling pathway while suppressing the cell cycle, hormone AR, and the response to DNA damage signaling pathway. Finally, we conducted searches in the “Genomics of Drug Sensitivity in Cancer” and “Genomics of Therapeutics Response Portal” databases, identifying several drugs that target ECM-SRGs.ConclusionsWe conducted a comprehensive evaluation of the genomes and immunogenomics of ECM-SRGs, along with their clinical features in 33 solid tumors. This may provide insights into the relationship between ECM-SRGs and tumorigenesis. Consequently, targeting these ECM-SRGs holds promise as a clinical approach for cancer treatment.

The entanglement of DNA damage and pattern recognition receptor signaling

Cells are under constant pressure to suppress DNA damage originating from both exogenous and endogenous sources. Cellular responses to DNA damage help to prevent mutagenesis and cell death that arises when DNA damage is either left unrepaired or repaired inaccurately. During the “acute phase” of DNA damage signaling, lesions are recognized, processed, and repaired to restore the primary DNA sequence whilst cell cycle checkpoints delay mitotic progression, cell death and the propagation of errors to daughter cells. Increasingly, there is recognition of a “chronic phase” of DNA damage signaling, exemplified by the secretion of dozens of cytokines days after the inciting damage event. In this review, we focus on the cellular origin of these chronic responses, the molecular pathways that control them and the increasing appreciation for the interconnection between acute and chronic DNA damage responses.

Rosmarinic acid enhances CHO cell productivity and proliferation through activation of the unfolded protein response and the mTOR pathway.

Rosmarinic acid (RA) has gained attraction in bioprocessing as a media supplement to improve cellular proliferation and protein production. Here, we observe up to a two-fold increase in antibody production with RA-supplementation, and a concentration-dependent effect of RA on cell proliferation for fed-batch Chinese hamster ovary (CHO) cell cultures. Contrary to previously reported antioxidant activity, RA increased the reactive oxygen species (ROS) levels, stimulated endoplasmic reticulum (ER) stress, activated the unfolded protein response (UPR), and elicited DNA damage. Despite such stressful events, RA appeared to maintained cell health via mammalian target of rapamycin (mTOR) pathway activation; both mTOR complex 1 (mTORC1) and mTOR complex 2(mTORC2) were stimulated in RA-supplemented cultures. By reversing such mTOR pathway activity through either chemical inhibitor addition or siRNA knockdown of genes regulating the mTORC1 and mTORC2 complexes, antibody production, UPR signaling, and stress-induced DNA damage were reduced. Further, the proliferative effect of RA appeared to be regulated selectively by mTORC2 activation and have reproduced this observation by using the mTORC2 stimulator SC-79. Analogously, knockdown of mTORC2 strongly reduced X-box binding protein 1 (XBP1) splicing, which would be expected to reduce antibody folding and secretion, sugging that reduced mTORC2 would correlate with reduced antibody levels. The crosstalk between mTOR activation and UPR upregulation may thus be related directly to the enhanced productivity. Our results show the importance of the mTOR and UPR pathways in increasing antibody productivity, and suggest that RA supplementation may obviate the need for labor-intensive genetic engineering by directly activating pathways favorable to cell culture performance.

DNAR-01. PAR-MEDIATED DNA DAMAGE SIGNALING IN IDH MUTANT GLIOMAS: COMBINING PARG INHIBITION AND RADIATION FOR SELECTIVE CYTOTOXICITY

Abstract Isocitrate dehydrogenase (IDH) 1 or 2 mutant gliomas have unique metabolic characteristics and DNA damage response. Radiotherapy and chemotherapy have been proven to be effective treatments for IDH mutant gliomas. Poly (ADP-ribose) (PAR) mediates DNA damage response and is regulated by opposing enzymes: poly (ADP-ribose) polymerases (PARPs) synthesize PAR, while poly (ADP-ribose) glycohydrolase (PARG) breaks it down. Combining PARG inhibitor (PARGi) and Temozolomide (TMZ) in IDH mutant cells leads to imbalanced NAD sequestration into PAR, causing metabolic lethality. This study characterizes the combination of PARGi and radiation. PARGi monotherapy showed no difference between IDH mutant and wild-type cells. However, we found that combining PARGi with radiation significantly increased cytotoxicity in IDH mutant glioma models, similar to PARPi and radiation. (Wang et al, ACi.Adv. 2020) Our findings showed that unlike TMZ+PARGi, PARGi and radiation did not consume and deplete NAD. Cytotoxicity was partially reversed by IDH inhibitor, highlighting the contribution of PAR-mediated DNA damage signaling in IDH mutant contexts. This convergence of vulnerabilities in IDH mutant cells identifies PAR-mediated DNA repair mechanisms that operate independently of NAD metabolism. IDH mutant enzyme generates 2 hydroxyglutarate (2HG), which is critical early in tumor development. However, the effect of 2HG on radiation sensitivity varies across cellular contexts. In this study, we found that IDH1 mutation and 2HG production enhance radiosensitivity in glioma cells both in vivo and in in vitro models, facilitated by PAR-dependent mechanisms. This augmentation of radiosensitivity is associated with S-phase blockage of the cell cycle, unveiling a vulnerability in the replication process of these cancer cells. In summary, this study demonstrates that the combination of PARGi and radiation regulates DNA repair pathways, augmenting cytotoxicity specifically in IDH mutant gliomas. Targeting PAR during radiation holds promise for treating IDH mutant gliomas, tailoring therapy to maximize durability while minimizing impact on normal cells.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Inhibiting Drp1 Dependent Mitochondrial Fission

Background: Primary or secondary resistance to venetoclax is frequently associated with mutational/non-mutational activation of MAPK pathways. ERK1/2 are terminal kinases in MAPK pathway and may be an appropriate target regardless of the upstream mechanisms that activate the pathway. ERK1/2 mediated phosphorylation of Drp1 promotes mitochondrial fission and MAPK-driven tumor growth in RAS driven solid cancers (Kashatus et al., Mol Cell. 2015). However, the role of Drp1 dependent mitochondrial dynamics in therapeutic resistance in AML is unexplored. Methods: ERK1/2 was inhibited using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029 (Munck et al., Mol Cancer Ther. 2021) in vitro and using ASTX029 in vivo. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition (ERKi). In addition, a comprehensive analysis of alteration of signaling pathways, apoptotic signatures and DNA damage responses in response to ERKi+/-venetoclax were analyzed by mass cytometry based proteomic analysis (CyTOF) and immunoblotting. The potential clinical relevance of ERK1/2 inhibition to overcome venetoclax resistance was confirmed in a PDX model of AML (established from an AML patient who relapsed after venetoclax/decitabine treatment). Mitochondrial images were acquired using super-resolution imaging with OMX-Blaze followed by quantification with Imaris. Results : We previously reported the synergy of ERK1/2 inhibition using Compound 27 with venetoclax at inducing apoptosis in RAS mutated and/or venetoclax resistant AML cells including venetoclax resistant isogenic lines (Sharma et al., Blood 140; Supplement 1, 2022). Venetoclax+ERKi depleted leukemia progenitor cells in primary AML samples (CI:0.03-0.23) and impaired clonogenic growth of NRAS mutant PDX cells. In a PDX mouse model of post venetoclax/decitabine-relapsed AML, ASTX029+venetoclax treatment improved survival compared to vehicle (median survival 76.5 days vs. 50 days, p=0.0006) and venetoclax alone (median survival 76.5 days vs. 51.5 days, p=0.0065) (Figure 1) with corresponding reduction in leukemia burden in bone marrow (p<0.0001) and spleen (p<0.0001). CyTOF analysis using PDX bone marrow showed decreased expression of Mcl-1 and pMcl-1-T163 and an increased expression of BIM in response to ERKi+/-venetoclax (Figure 1). To maintain stemness in AML, mitochondrial ROS mitigation and Drp1-mediated mitochondrial fission are crucial (Schimmer et al., Cell Stem Cell. 2018). The inhibition of ERK1/2 resulted in decreased pDrp1-Ser616, along with an increase in mitochondrial length (p<0.001) suggesting impaired mitochondrial fission. This was accompanied by a decrease in mitochondrial membrane potential (p<0.0001) and an increase in mitochondrial ROS (p<0.0001). Overexpression (OE) of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala led to shorter mitochondrial length (Figure 2), suggesting enhanced fission, a distinct metabolic phenotype with decreased ROS production and decreased mitochondrial depolarization with venetoclax+/- ERKi. Finally, Drp1 phospho-mimetic OE reversed apoptosis induction by venetoclax +/- ERKi (Figure 2) as compared to the wild-type and phospho-null (Ser616Ala) Drp1 (p<0.001) expressing cells, supporting the role of mitochondrial fission in resistance to venetoclax. Conclusion: The increased mitochondrial fission driven by ERK1/2 mediated phosphorylation of Drp1 contributes to venetoclax resistance in AML and inhibiting ERK1/2/Drp1 axis overcomes resistance to venetoclax by inhibiting mitochondrial fission (Figure 2). These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in the treatment of AML.

Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

Regulation of DNA damage-induced HLA class I presentation

Immune checkpoint inhibitors (ICI) are cancer therapies that restore anti-tumor immunity; however, only a small percentage of patients have been completely cured by ICI alone. Multiple approaches in combination with other modalities have been used to improve the efficacy of ICI therapy. Among conventional cancer treatments, radiotherapy or DNA damage-based chemotherapy is a promising candidate as a partner of ICI because DNA damage signaling potentially stimulates immune activities turning the tumor’s immune environment into hot tumors. Programmed death-ligand 1 (PD-L1) and human leukocyte antigen class I (HLA-I), which are immune ligands, regulate the balance of anti-tumor immunity in the tumor microenvironment. PD-L1 functions as a brake to suppress cytotoxic T cell activity, whereas HLA-I is an immune accelerator that promotes the downstream of the T cell signaling. Accumulating evidence has demonstrated that DNA damage enhances the presentation of HLA-I on the surface of damaged cells. However, it is unclear how signal transduction in DNA-damaged cells upregulates the presentation of HLA-I with antigens. Our recent study uncovered the mechanism underlying DNA damage-induced HLA-I presentation, which requires polypeptide synthesis through a pioneer round of translation. In this review, we summarize the latest overview of how DNA damage stimulates antigen production presented by HLA-I.

Bone marrow stromal cells reduce low-dose cytarabine-induced differentiation of acute myeloid leukemia.

Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-β1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.

Systematic in vivo candidate evaluation uncovers therapeutic targets for LMNA dilated cardiomyopathy and risk of Lamin A toxicity

BackgroundDilated cardiomyopathy (DCM) is a severe, non-ischemic heart disease which ultimately results in heart failure (HF). Decades of research on DCM have revealed diverse aetiologies. Among them, familial DCM is the major form of DCM, with pathogenic variants in LMNA being the second most common form of autosomal dominant DCM. LMNA DCM is a multifactorial and complex disease with no specific treatment thus far. Many studies have demonstrated that perturbing candidates related to various dysregulated pathways ameliorate LMNA DCM. However, it is unknown whether these candidates could serve as potential therapeutic targets especially in long term efficacy.MethodsWe evaluated 14 potential candidates including Lmna gene products (Lamin A and Lamin C), key signaling pathways (Tgfβ/Smad, mTor and Fgf/Mapk), calcium handling, proliferation regulators and modifiers of LINC complex function in a cardiac specific Lmna DCM model. Positive candidates for improved cardiac function were further assessed by survival analysis. Suppressive roles and mechanisms of these candidates in ameliorating Lmna DCM were dissected by comparing marker gene expression, Tgfβ signaling pathway activation, fibrosis, inflammation, proliferation and DNA damage. Furthermore, transcriptome profiling compared the differences between Lamin A and Lamin C treatment.ResultsCardiac function was restored by several positive candidates (Smad3, Yy1, Bmp7, Ctgf, aYAP1, Sun1, Lamin A, and Lamin C), which significantly correlated with suppression of HF/fibrosis marker expression and cardiac fibrosis in Lmna DCM. Lamin C or Sun1 shRNA administration achieved consistent, prolonged survival which highly correlated with reduced heart inflammation and DNA damage. Importantly, Lamin A treatment improved but could not reproduce long term survival, and Lamin A administration to healthy hearts itself induced DCM. Mechanistically, we identified this lapse as caused by a dose-dependent toxicity of Lamin A, which was independent from its maturation.ConclusionsIn vivo candidate evaluation revealed that supplementation of Lamin C or knockdown of Sun1 significantly suppressed Lmna DCM and achieve prolonged survival. Conversely, Lamin A supplementation did not rescue long term survival and may impart detrimental cardiotoxicity risk. This study highlights a potential of advancing Lamin C and Sun1 as therapeutic targets for the treatment of LMNA DCM.Graphical

Epigenetic regulation of TP53 is involved in prostate cancer radioresistance and DNA damage response signaling

External beam radiotherapy (RT) is a leading first-line therapy for prostate cancer (PCa), and, in recent years, significant advances have been accomplished. However, RT resistance can arise and result in long-term recurrence or disease progression in the worst-case scenario. Thus, making crucial the discovery of new targets for PCa radiosensitization. Herein, we generated a radioresistant PCa cell line, and found p53 to be highly expressed in radioresistant PCa cells, as well as in PCa patients with recurrent/disease progression submitted to RT. Mechanism dissection revealed that RT could promote p53 expression via epigenetic modulation. Specifically, a decrease of H3K27me3 occupancy at TP53 gene promoter, due to increased KDM6B activity, was observed in radioresistant PCa cells. Furthermore, p53 is essential for efficient DNA damage signaling response and cell recovery upon stress induction by prolonged fractionated irradiation. Remarkably, KDM6B inhibition by GSK-J4 significantly decreased p53 expression, consequently attenuating the radioresistant phenotype of PCa cells and hampering in vivo 3D tumor formation. Overall, this work contributes to improve the understanding of p53 as a mediator of signaling transduction in DNA damage repair, as well as the impact of epigenetic targeting for PCa radiosensitization.

HPV Infection Causes Dependence on Alternative DNA Damage Response Pathways Providing Cancer Specific Targets for Radiosensitization

Cancer-specific radiosensitization is an attractive approach to improving the efficacy of radiotherapy. However, drugs targeting ubiquitous DNA damage response pathways are not cancer specific and can increase radiation related side effects. Thus, there is an unmet need for tumor specific molecular targets. This approach requires an identification of DNA damage signaling pathways that are unique to cancer cells. We hypothesized that by expressing its genome in the host cells HPV infection can rewire DNA damage signaling making HPV-positive tumor cells dependent on alternative pathways to survive radiation, which can be exploited for radiosensitization of HPV-induced cancers. We have performed a CRISPR/Cas9 screen in HPV-positive SiHa cells that either express HPV16 proteins E6 and E7 or contain a selective knock-out of E6 or E7. We used next-generation sequencing to determine the abundance of gRNA in cells that were mock-treated or irradiated. We identified genes that were required for survival of radiation specifically in the context of E6 or E7 expression. Results of the screen were validated by generating knock-outs of the discovered genes using CRISPR/Cas9. By applying an array of molecular tools to analyze cell survival, cell cycle progression and mitotic progression we assessed the role of these genes in radiation response in HPV-positive cancer cells. We have identified genes that are required for survival of radiation-induced DNA damage in the context of either E6 or E7 expression. We demonstrate that targeting these genes results in hypersensitization of cervical cancer cells to radiation specifically in the presence of E6 or E7. We show that the gene product required for survival in E6-expressing cells is critical for mitotic progression after radiation exposure serving as a member of a protein complex stabilizing the attachment of mitotic spindle to centromeres. We have uncovered specific genes that are critical for DNA damage response and cell survival after radiation exposure in HPV-positive cells. Our findings suggest that expression of the HPV proteins E6 and E7 rewires DNA damage signaling causing dependence on alternative response pathways. We propose that these pathways can be targeted for a tumor-specific radiosensitization of HPV-induced cancers.

Sodium-Glucose Cotransporter 2 (SGLT2) Inhibition Induces DNA Damage and Radiosensitization in Glioblastoma Cell Lines

Glioblastomas (GBM) are highly dependent on glucose for energy to promote cell proliferation and tumor growth. Preclinical studies have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors, drugs that are FDA approved for diabetes, reduce the viability and invasiveness of GBM cells. We hypothesize that SGLT2 inhibitors decrease GBM cell growth and viability by promoting DNA damage. The SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin were used and cell studies were performed under low glucose (0.6 mM) media conditions. In the cell proliferation assays, cells were counted in triplicate after 24-, 48- and 72-hour treatment with SGLT2 inhibitors. Glucose transport assays were performed using a 6-fluorescent iodinated glucose analog (6FIGA) which is selectively transported by SGLTs and not by glucose transporters (GLUTs). Immunoblots were used to measure protein expression of SGLT2 and markers of DNA damage (phospho-ATM and gamma-H2AX). Cellular radiosensitization was evaluated by clonogenic survival assays. SGLT2 inhibition with canagliflozin, dapagliflozin and empagliflozin decreased cell proliferation in LN229, U118MG and LN18 GBM cells lines. In LN229 cells, 72-hour treatment with canagliflozin (30 µM) decreased cell proliferation by 60% compared to vehicle control cells (p<0.0001) whereas dapagliflozin (30 µM) and empagliflozin (30 µM) decreased cell proliferation compared to vehicle control cells by 17% (p<0.0001) and 19% (p<0.001), respectively. Glucose transport assays show that 6FIGA uptake is reduced by canagliflozin and dapagliflozin in LN229 and U87 GBM cells. Canagliflozin treatment (50 µM) for 24 hours in LN229 cells results in increased phosphorylated ATM protein and increased gamma-H2AX protein, markers of DNA damage, by immunoblot. SGLT2 protein expression was not affected by canagliflozin. Treatment with canagliflozin (50 µM) 48 hours prior to 2, 4 and 6 Gy of ionizing radiation reduced clonogenic survival of LN229, LN18 and U118MG GBM cells. SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin decrease GBM cell proliferation, with the highest inhibitory effect seen with canagliflozin. Canagliflozin treatment alone increases DNA damage signaling in GBM cells. Canagliflozin radiosensitizes GBM cells. This work provides preliminary preclinical rationale for canagliflozin as a radiosensitizer in GBM.

Telomeric DNA breaks in human induced pluripotent stem cells trigger ATR-mediated arrest and telomerase-independent telomere damage repair.

Although mechanisms of telomere protection are well-defined in differentiated cells, how stem cells sense and respond to telomere dysfunction, in particular telomeric double-strand breaks (DSBs), is poorly characterized. Here, we report the DNA damage signaling, cell cycle, and transcriptome changes in human induced pluripotent stem cells (iPSCs) in response to telomere-internal DSBs. We engineer human iPSCs with an inducible TRF1-FokI fusion protein to acutely induce DSBs at telomeres. Using this model, we demonstrate that TRF1-FokI DSBs activate an ATR-dependent DNA damage response, which leads to p53-independent cell cycle arrest in G2. Using CRISPR-Cas9 to cripple the catalytic domain of telomerase reverse transcriptase, we show that telomerase is largely dispensable for survival and lengthening of TRF1-FokI-cleaved telomeres, which instead are effectively repaired by robust homologous recombination (HR). In contrast to HR-based telomere maintenance in mouse embryonic stem cells, where HR causes ZSCAN4-dependent extension of telomeres beyond their initial lengths, HR-based repair of telomeric breaks is sufficient to maintain iPSC telomeres at a normal length, which is compatible with sustained survival of the cells over several days of TRF1-FokI induction. Our findings suggest a previously unappreciated role for HR in telomere maintenance in telomerase-positive iPSCs and reveal distinct iPSC-specific responses to targeted telomeric DNA damage.

Protective effect of Cornuside on OGD/R injury in SH-SY5Y cells and its underlying mechanism

Apoptosis induced by oxygen-glucose deprivation/reperfusion (OGD/R) injury is the main cause of neuronal damage. Cornuside, a small-molecule cyclic enol ether terpene glycoside extracted from the dried fruit of mature Cornus officinalis Sieb. et Zucc., has vigorous anti-apoptotic and antioxidant effects. Previous studies have shown that Cornuside can reduce apoptosis and improve mitochondrial energy metabolism in cortical neurons of rats by inhibiting caspase-3 and calcium release. In this study, we treated SH-SY5Y cells with OGD/R to simulated ischemia/reperfusion (I/R) injury. Using high-throughput transcriptome sequencing, differentially expressed genes were analyzed in the OGD/R group versus the OGD/R + Cornuside (10 μmol/L) group to explore the neuroprotective mechanisms of Cornuside. The differentially expressed genes were mainly enriched in apoptosis signaling pathway, cell cycle, DNA damage and repair, and p38/JNK MAPK and p53 signaling pathways. The results showed that OGD/R significantly reduced the survival of SH-SY5Y cells, induced apoptosis, disrupted the nucleus, promoted the release of ROS, and led to cell cycle arrest. Cornuside reversed OGD/R-induced damage. By upregulating MAPK8IP1 and downregulating MAPK14, TP53INP1, and signaling pathway-related proteins (p-p38, p-JNK, and p-p53), Cornuside ameliorated cell damage induced by p38/JNK MAPK and p53 signaling pathways. Cornuside also downregulated apoptosis regulatory proteins (Bax, Bcl-2, caspase-3, caspase-9, and cytochrome c) and cell cycle regulatory proteins (cyclin B1, cyclin E, and p21).