DNA Damage-induced Cell Death Research Articles

Cooperative effects of RIG-I-like receptor signaling and IRF1 on DNA damage-induced cell death

Properly responding to DNA damage is vital for eukaryotic cells, including the induction of DNA repair, growth arrest and, as a last resort to prevent neoplastic transformation, cell death. Besides being crucial for ensuring homeostasis, the same pathways and mechanisms are at the basis of chemoradiotherapy in cancer treatment, which involves therapeutic induction of DNA damage by chemical or physical (radiological) measures. Apart from typical DNA damage response mediators, the relevance of cell-intrinsic antiviral signaling pathways in response to DNA breaks has recently emerged. Originally known for combatting viruses via expression of antiviral factors including interferons (IFNs) and establishing of an antiviral state, RIG-I-like receptors (RLRs) were found to be critical for adequate induction of cell death upon the introduction of DNA double-strand breaks. We here show that presence of IRF3 is crucial in this process, most likely through direct activation of pro-apoptotic factors rather than transcriptional induction of canonical downstream components, such as IFNs. Investigating genes reported to be involved in both DNA damage response and antiviral signaling, we demonstrate that IRF1 is an obligatory factor for DNA damage-induced cell death. Interestingly, its regulation does not require activation of RLR signaling, but rather sensing of DNA double-strand breaks by ATM and ATR. Hence, even though independently regulated, both RLR signaling and IRF1 are essential for full-fledged induction/execution of DNA damage-mediated cell death programs. Our results not only support more broadly developing IRF1 as a biomarker predictive for the effectiveness of chemoradiotherapy, but also suggest investigating a combined pharmacological stimulation of RLR and IRF1 signaling as a potential adjuvant regimen in tumor therapy.

Constitutive High Expression of NOXA Sensitizes Human Embryonic Stem Cells for Rapid Cell Death.

Human embryonic stem (hES) cells are highly sensitive to apoptotic stimuli such as DNA damage, which allows for the rapid elimination of mutated cells during development. However, the mechanisms that maintain hES cells in the primed apoptotic state are not completely known. Key activators of apoptosis, the BH3-only proteins, are present at low levels in most cell types. In contrast, hES cells have constitutive high levels of the BH3-only protein, NOXA. We examined the importance of NOXA for enabling apoptosis in hES cells. hES cells deleted for NOXA showed remarkable protection against multiple apoptotic stimuli. NOXA was constitutively localized to the mitochondria, where it interacted with MCL1. Strikingly, inhibition of MCL1 in NOXA knockout cells was sufficient to sensitize these cells to DNA damage-induced cell death. Our study demonstrates that an essential function of constitutive high levels of NOXA in hES cells is to effectively antagonize MCL1 to permit rapid apoptosis.

Mitochondrial glutamine metabolism regulates sensitivity of cancer cells after chemotherapy via amphiregulin

The DNA damage response is essential for sustaining genomic stability and preventing tumorigenesis. However, the fundamental question about the cellular metabolic response to DNA damage remains largely unknown, impeding the development of metabolic interventions that might prevent or treat cancer. Recently, it has been reported that there is a link between cell metabolism and DNA damage response, by repression of glutamine (Gln) entry into mitochondria to support cell cycle arrest and DNA repair. Here, we show that mitochondrial Gln metabolism is a crucial regulator of DNA damage-induced cell death. Mechanistically, inhibition of glutaminase (GLS), the first enzyme for Gln anaplerosis, sensitizes cancer cells to DNA damage by inducing amphiregulin (AREG) that promotes apoptotic cell death. GLS inhibition increases reactive oxygen species production, leading to transcriptional activation of AREG through Max-like protein X (MLX) transcription factor. Moreover, suppression of mitochondrial Gln metabolism results in markedly increased cell death after chemotherapy in vitro and in vivo. The essentiality of this molecular pathway in DNA damage-induced cell death may provide novel metabolic interventions for cancer therapy.

CBIO-19. GENOME SHREDDING ENABLES CRISPR-MEDIATED GLIOBLASTOMA ONCOLYSIS

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults 1. Despite multimodal treatment regimens including surgical resection, radio- and chemotherapy, the growth of residual tumor often results in therapy resistance and ultimately death. GBMs are highly diffuse and exhibit extensive intratumoral heterogeneity 2,3, confounding diagnostic efforts and presenting opportunities for therapy evasion. Therefore, innovative treatment paradigms that can efficiently eliminate GBM cells irrespective of their mutational and epigenetic profile are urgently needed. CRISPR technologies have revolutionized medicine by enabling targeted genome editing through RNA-guided introduction of DNA double-strand breaks 4,5. Here, we show that CRISPR-Cas9 mediated genome fragmentation through targeting of highly repetitive loci, termed “genome shredding”, enables rapid and robust elimination of GBM cells. We characterized genome shredding across mammalian and vertebrate cells, and identified optimal repetitive pan-vertebrate and species-specific loci. Genome shredding is equally effective in temozolomide (TMZ)-sensitive and -resistant GBM cells, and multi-cycle treatment regimens are feasible. Importantly, when deployed in intracerebral GBM xenografts through local delivery, CRISPR-Cas9 genome shredding efficiently eliminated all targeted cells. Together, genome shredding enables the rapid and efficient fragmentation of a target cell’s genome and subsequent DNA damage-induced cell death. This provides an innovative treatment paradigm that is independent of a tumor’s mutational and epigenetic profile and leverages CRISPR-Cas9 as a breakthrough therapeutic modality for GBM.

Cell Death and Survival Pathways Involving ATM Protein Kinase

Cell death is the ultimate form of cellular dysfunction, and is induced by a wide range of stresses including genotoxic stresses. During genotoxic stress, two opposite cellular reactions, cellular protection through DNA repair and elimination of damaged cells by the induction of cell death, can occur in both separate and simultaneous manners. ATM (ataxia telangiectasia mutated) kinase (hereafter referred to as ATM) is a protein kinase that plays central roles in the induction of cell death during genotoxic stresses. It has long been considered that ATM mediates DNA damage-induced cell death through inducing apoptosis. However, recent research progress in cell death modality is now revealing ATM-dependent cell death pathways that consist of not only apoptosis but also necroptosis, ferroptosis, and dysfunction of autophagy, a cellular survival mechanism. In this short review, we intend to provide a brief outline of cell death mechanisms in which ATM is involved, with emphasis on pathways other than apoptosis.

Beyond the Double-Strand Breaks: The Role of DNA Repair Proteins in Cancer Stem-Cell Regulation.

Simple SummaryCancer stem cells (CSCs) are a tumor cell population maintaining tumor growth and promoting tumor relapse if not wholly eradicated during treatment. CSCs are often equipped with molecular mechanisms making them resistant to conventional anti-cancer therapies whose curative potential depends on DNA damage-induced cell death. An elevated expression of some key DNA repair proteins is one of such defense mechanisms. However, new research reveals that the role of critical DNA repair proteins is extending far beyond the DNA repair mechanisms. This review discusses the diverse biological functions of DNA repair proteins in CSC maintenance and the adaptation to replication and oxidative stress, anti-cancer immune response, epigenetic reprogramming, and intracellular signaling mechanisms. It also provides an overview of their potential therapeutic targeting.Cancer stem cells (CSCs) are pluripotent and highly tumorigenic cells that can re-populate a tumor and cause relapses even after initially successful therapy. As with tissue stem cells, CSCs possess enhanced DNA repair mechanisms. An active DNA damage response alleviates the increased oxidative and replicative stress and leads to therapy resistance. On the other hand, mutations in DNA repair genes cause genomic instability, therefore driving tumor evolution and developing highly aggressive CSC phenotypes. However, the role of DNA repair proteins in CSCs extends beyond the level of DNA damage. In recent years, more and more studies have reported the unexpected role of DNA repair proteins in the regulation of transcription, CSC signaling pathways, intracellular levels of reactive oxygen species (ROS), and epithelial–mesenchymal transition (EMT). Moreover, DNA damage signaling plays an essential role in the immune response towards tumor cells. Due to its high importance for the CSC phenotype and treatment resistance, the DNA damage response is a promising target for individualized therapies. Furthermore, understanding the dependence of CSC on DNA repair pathways can be therapeutically exploited to induce synthetic lethality and sensitize CSCs to anti-cancer therapies. This review discusses the different roles of DNA repair proteins in CSC maintenance and their potential as therapeutic targets.

The PI3K/mTOR Inhibitor Ompalisib Suppresses Nonhomologous End Joining and Sensitizes Cancer Cells to Radio- and Chemotherapy.

As the predominant pathway for the repair of DNA double-strand breaks (DSB), non-homologous end joining (NHEJ) attenuates the efficacy of cancer treatment which relies on the introduction of DSBs, such as radiotherapy and genotoxic drugs. Identifying novel NHEJ inhibitors is of great importance for improving the therapeutic efficiency of radio- or chemotherapy. Here we miniaturized our recently developed NHEJ detecting system into a 96-well plate-based format and interrogated an FDA approved drug library containing 1732 compounds. A collection of novel hits were considered to be potential DSB repair inhibitors at the noncytotoxic concentration. We identified omipalisib as an efficient sensitizer for DNA damage-induced cell death in vitro Furthermore, in vitro analysis uncovered the repressive effect of omipalisib on the phosphorylation of DNA-dependent protein kinase catalytic subunit induced by ionizing radiation and doxorubicin, which led to the suppression of NHEJ pathway. IMPLICATIONS: In summary, our findings suggested the possibility for repurposing these candidates as radio- or chemosensitizers, which might extend their clinical application in cancer therapy.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death

SummaryBRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.

Uncovering molecular mechanisms of regulated cell death in the naked mole rat.

The naked mole rat (NMR), Heterocephalus glaber, is the longest-living rodent species, and is extraordinarily resistant to cancer and aging-related diseases. The molecular basis for these unique phenotypic traits of the NMR is under extensive research. However, the role of regulated cell death (RCD) in the longevity and the protection from cancer in the NMR is still largely unknown. RCD is a mechanism restricting the proliferation of damaged or premalignant cells, which counteracts aging and oncotransformation. In this study, DNA damage-induced cell death in NMR fibroblasts was investigated in comparison to RCD in fibroblasts from Mus musculus. The effects of methyl methanesulfonate, 5-fluorouracil, and etoposide in both cell types were examined using contemporary cell death analyses. Skin fibroblasts from Heterocephalus glaber were found to be more resistant to the action of DNA damaging agents compared to fibroblasts from Mus musculus. Strikingly, our results revealed that NMR cells also exhibit a limited apoptotic response and seem to undergo regulated necrosis. Taken together, this study provides new insights into the mechanisms of cell death in NMR expanding our understanding of longevity, and it paves the way towards the development of innovative therapeutic approaches.

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

Polyploidy in the adult Drosophila brain.

Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal's lifespan. How long-lived cells deal with ageing-related damage is poorly understood. Here we show that polyploid cells accumulate in the adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid in the adult Drosophila brain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. Our results suggest polyploidy may serve a protective role for neurons and glia in adult Drosophila melanogaster brains.

Breast-Specific Epigenetic Regulation of DeltaNp73 and Its Role in DNA-Damage-Response of BRCA1-Mutated Human Mammary Epithelial Cells.

The function of BRCA1/2 proteins is essential for maintaining genomic integrity in all cell types. However, why women who carry deleterious germline mutations in BRCA face an extremely high risk of developing breast and ovarian cancers specifically has remained an enigma. We propose that breast-specific epigenetic modifications, which regulate tissue differentiation, could team up with BRCA deficiency and affect tissue susceptibility to cancer. In earlier work, we compared genome-wide methylation profiles of various normal epithelial tissues and identified breast-specific methylated gene promoter regions. Here, we focused on deltaNp73, the truncated isoform of p73, which possesses antiapoptotic and pro-oncogenic functions. We showed that the promoter of deltaNp73 is unmethylated in normal human breast epithelium and methylated in various other normal epithelial tissues and cell types. Accordingly, deltaNp73 was markedly induced by DNA damage in human mammary epithelial cells (HMECs) but not in other epithelial cell types. Moreover, the induction of deltaNp73 protected HMECs from DNA damage-induced cell death, and this effect was more substantial in HMECs from BRCA1 mutation carriers. Notably, when BRCA1 was knocked down in MCF10A, a non-malignant breast epithelial cell line, both deltaNp73 induction and its protective effect from cell death were augmented upon DNA damage. Interestingly, deltaNp73 induction also resulted in inhibition of BRCA1 and BRCA2 expression following DNA damage. In conclusion, breast-specific induction of deltaNp73 promotes survival of BRCA1-deficient mammary epithelial cells upon DNA damage. This might result in the accumulation of genomic alterations and allow the outgrowth of breast cancers. These findings indicate deltaNp73 as a potential modifier of breast cancer susceptibility in BRCA1 mutation carriers and may stimulate novel strategies of prevention and treatment for these high-risk women.

Abstract 4871: The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation

Abstract Children with high-risk neuroblastoma have poor survival rates and need novel treatment strategies. UBE4B is an E3/E4 ubiquitin ligase whose gene is found in the chromosome 1p36 region commonly deleted in high-risk neuroblastoma tumors and whose expression is associated with neuroblastoma patient outcomes. However, the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We hypothesized that UBE4B would interact with mediators of DNA damage and repair pathways, leading to the observed associations between UBE4B and patient outcomes. Using cell lines with stable UBE4B depletion and over-expression, we evaluated the interaction of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression levels on p53 and Ku70 ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in neuroblastoma differentiation, cell death, and responses to chemotherapy and HDAC inhibition using continuous live cell imaging and Western blots. We determined that UBE4B directly interacts with ITCH/AIP4 in the last WW domain of ITCH and we also determined that Ku70 directly interacts with ITCH in the three first WW domain of ITCH. We also determined that UBE4B depletion leads to reduced polyubiquitination and degradation of both p53 and Ku70 in neuroblastoma cells. UBE4B depletion also led to reduced apoptosis induced by both HDAC inhibition with vorinostat and chemotherapy treatment with carboplatin and doxorubicin via stabilizaton of the ITCH target protein c-FLIPL leading inhibition of caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role of the UBE4B-ITCH complex in HDAC inhibitor and DNA-damage induced cell death, and suggest that the association of UBE4B expression with neuroblastoma patient outcomes may be mediated by effects on responses of patient tumors to chemotherapy. Citation Format: Christophe Le Clorennec, Jacqueline Lesperance, Yuchen Huo, Peter E. Zage. The roles of E3/E4 ubiquitin ligase UBE4B and its targets p53 and Ku70 in neuroblastoma cell death and differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4871.

LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells

AbstractThe linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage–induced cell death and is a suitable therapeutic target for B-cell lymphomas.

Grape skin extract prevents UV irradiation induced DNA damage of normal human epidermal keratinocytes cells

BACKGROUND: There is concern that DNA damage may occur in skin cells due to UV irradiation. In this study, we investigated whether extracts from grape skin, which should be discarded, can suppress DNA damage caused by UV radiation. OBJECTIVE: This study aims to investigate the effectivity of GSE in diminishing UV-induced cytotoxicity in normal human epidermal keratinocytes (NHEK) cells. METHODS: The polyphenol content in GSE was carried out using the HPLC instrument. UV dose was selected by measurement of cell viability, and ELISA results of DNA photoproduct. Optimum extracts condition of grape skin selected by DNA photoproduct content as well as at this condition; Bax/Bcl-2 ratio and cytochrome c gene expressions were evaluated by Western blotting. RESULTS: In this study, we confirmed that GSE protect against DNA damage-induced cell death. Condition for 80% EtOH for 24 h at 60 °C was suitable to extract for grape skin. Red grape skin (Zweigelt) was more effective than white grape skin (Niagara) in preventing (before irradiation) and repairing (after irradiation). UV-induced upregulation of Bax/Bcl-2 ratio and cytochrome c expression were reduced by GSE treatment. CONCLUSIONS: The study demonstrated a promising potential of GSEs in skin therapeutics application.

Stem cell programs in cancer initiation, progression, and therapy resistance

Over the past few decades, substantial evidence has convincingly revealed the existence of cancer stem cells (CSCs) as a minor subpopulation in cancers, contributing to an aberrantly high degree of cellular heterogeneity within the tumor. CSCs are functionally defined by their abilities of self-renewal and differentiation, often in response to cues from their microenvironment. Biological phenotypes of CSCs are regulated by the integrated transcriptional, post-transcriptional, metabolic, and epigenetic regulatory networks. CSCs contribute to tumor progression, therapeutic resistance, and disease recurrence through their sustained proliferation, invasion into normal tissue, promotion of angiogenesis, evasion of the immune system, and resistance to conventional anticancer therapies. Therefore, elucidation of the molecular mechanisms that drive cancer stem cell maintenance, plasticity, and therapeutic resistance will enhance our ability to improve the effectiveness of targeted therapies for CSCs. In this review, we highlight the key features and mechanisms that regulate CSC function in tumor initiation, progression, and therapy resistance. We discuss factors for CSC therapeutic resistance, such as quiescence, induction of epithelial-to-mesenchymal transition (EMT), and resistance to DNA damage-induced cell death. We evaluate therapeutic approaches for eliminating therapy-resistant CSC subpopulations, including anticancer drugs that target key CSC signaling pathways and cell surface markers, viral therapies, the awakening of quiescent CSCs, and immunotherapy. We also assess the impact of new technologies, such as single-cell sequencing and CRISPR-Cas9 screening, on the investigation of the biological properties of CSCs. Moreover, challenges remain to be addressed in the coming years, including experimental approaches for investigating CSCs and obstacles in therapeutic targeting of CSCs.

Histone H1 eviction by the histone chaperone SET reduces cell survival following DNA damage.

Many chromatin remodeling and modifying proteins are involved in the DNA damage response, where they stimulate repair or induce DNA damage signaling. Interestingly, we identified that downregulation of the histone H1 (H1)-interacting protein SET results in increased resistance to a wide variety of DNA damaging agents. We found that this increased resistance does not result from alleviation of an inhibitory effect of SET on DNA repair but, rather, is the consequence of a suppressed apoptotic response to DNA damage. Furthermore, we provide evidence that the histone chaperone SET is responsible for the eviction of H1 from chromatin. Knockdown of H1 in SET-depleted cells resulted in re-sensitization of cells to DNA damage, suggesting that the increased DNA damage resistance in SET-depleted cells is the result of enhanced retention of H1 on chromatin. Finally, clonogenic survival assays showed that SET and p53 act epistatically in the attenuation of DNA damage-induced cell death. Taken together, our data indicate a role for SET in the DNA damage response as a regulator of cell survival following genotoxic stress.This article has an associated First Person interview with the first author of the paper.

Anti-Bcma PBD MEDI2228 Combats Drug Resistance and Synergizes with Bortezomib and Inhibitors to DNA Damage Response in Multiple Myeloma

A novel anti-BCMA antibody drug conjugate (ADC) MEDI2228 preferentially binds to membrane-bound vs soluble BCMA (sBCMA) to effectively deliver the pyrrolobenzodiazepine (PBD) payload tesirine to multiple myeloma (MM) cells (Leukemia. 2019;33: 766). In preclinical models, this ADC targets both MM cells in bulk and CD19+CD138- patient MM progenitor cells. We here study the potency of MEDI2228 in MM cells in the bone marrow (BM) microenvironment and examine molecular mechanisms whereby MEDI2228 overcomes drug resistance. First, MEDI2228, more effectively than its microtubule-binding monomethyl auristatin-F (MMAF) ADC homolog, inhibits proliferation (>1-2-log) and survival of all MM cell lines and MM cells from patients with multiple relapsed and refractory diseases, regardless of BCMA levels, p53 status, and the protection conferred by BM stromal cells and IL-6. Significantly, MM cells with lower BCMA expression and resistance to bortezomib or immunomodulatory drugs (IMiDs, i.e., lenalidomide, pomalidomide) are more susceptible to MEDI2228 vs its MMAF ADC homolog. MEDI2228, but not its MMAF ADC homolog, activates critical DNA damage responses (DDR) via phosphorylation of ATM/ATR kinases, checkpoint kinases (CHK)1/2, and H2AX, associated with induction of multiple DDR pathway-associated genes. Low doses of MEDI2228 and bortezomib (btz) synergistically induce apoptosis of drug-sensitive and -resistant MM cells, at least in part, through modulation of RAD51, a DNA damage and repair protein. Importantly, MEDI2228 further triggers the ATM/ATR-CHK1/2 signaling cascade, associated with increased gH2AX, p21, and apoptosis molecules in MM1S-xenografted tumors in mice. In vivo, a single sub-optimal dose of 0.4 mg/kg MEDI2228 induces superior anti-MM activity than btz, indicating that MEDI2228 is significantly more effective and selective than btz as single agent therapy in vivo. Furthermore, combined treatments with MEDI2228 and btz result in potent tumor depletion and significantly prolonged host survival via increased nuclear gH2AX-expressing microfoci, DNA damage-induced growth arrest and cell death. Significant tumor necrosis is observed earlier in mice receiving both drugs than either agent alone. At 177d, 15% mice in the combination treatment group remain alive and without any tumor. Importantly, no weight loss is noted in all groups, indicating a favorable safety profile of MEDI2228, alone or with btz, in vivo. Moreover, DDR checkpoint inhibitors, i.e., AZD0156 (ATMi), AZD6738 (ATRi), AZD1775 (WEE1i), synergize with MEDI2228 to enhance MM cell cytotoxicity (combination index < 1). This study therefore further supports clinical development of MEDI2228 (NCT03489525) as an important next-generation immunotherapy to improve outcome of MM patients. Disclosures Kinneer: AstraZeneca: Employment. Munshi:Amgen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Celgene: Consultancy. Anderson:Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board.

PDTM-41. INHIBITION OF DNA DOUBLE STRAND BREAK REPAIR PATHWAYS AS A PROMISING THERAPEUTIC TARGET IN DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

Abstract New approaches to the treatment of diffuse intrinsic pontine gliomas (DIPGs) are desperately needed. DNA damage response is essential for cells to maintain genome integrity as DNA is damaged by both endogenous and exogenous stressors. Many cancer cells exhibit hyper-dependency on specific DNA repair pathways due to either defects in DNA repair mechanisms and/or high levels of endogenous stress leading to accumulation of DNA damage lesions. Identification of DIPG-specific DNA repair deficiencies and resultant dependencies may establish novel therapeutic strategies for DIPGs. METHODS To identify pathways critical for DIPG cell survival, genome wide CRISPR-Cas9 screen was performed on patient derived DIPG cell lines followed by gene set enrichment analyses. To monitor the effects of pathway inhibition on survival, apoptosis, DNA damage and repair, assays were performed to measure cell proliferation, cleaved-caspase3, gamma-H2AX and reporter based-DNA repair efficiency. RESULTS Our unbiased CRISPR approach to uncover vulnerabilities in DIPGs identified DNA double strand break (DSBs) repair pathways as essential for DIPG cell proliferation and survival. Further studies revealed high basal DSBs in DIPG cells compared to neural stem cells and primary astrocytes that suggest dependence of DIPG cell survival on specific DSB repair pathways. We confirmed the intrinsic reliance of DIPG cells on the specific DSB repair pathway of mutagenic end-joining, and defined a key role for DNA repair in suppressing endogenous DNA damage-induced apoptotic cell death. CONCLUSION DIPG cells have high endogenous DNA damage levels and escape catastrophic genomic instability and cell death by engaging DNA repair pathways, in particular the mutagenic end-joining DNA repair pathway. Inhibition of this specific DNA repair pathway represents a promising new avenue for the treatment of DIPGs.

The Chk1 inhibitor SAR-020106 sensitizes human glioblastoma cells to irradiation, to temozolomide, and to decitabine treatment

BackgroundGlioblastoma is the most common and aggressive brain tumour in adults with a median overall survival of only 14 months after standard therapy with radiation therapy (IR) and temozolomide (TMZ). In a novel multimodal treatment approach we combined the checkpoint kinase 1 (Chk1) inhibitor SAR-020106 (SAR), disrupting homologue recombination, with standard DNA damage inducers (IR, TMZ) and the epigenetic/cytotoxic drug decitabine (5-aza-2′-deoxycitidine, 5-aza-dC). Different in vitro glioblastoma models are monitored to evaluate if the impaired DNA damage repair may chemo/radiosensitize the tumour cells.MethodsHuman p53-mutated (p53-mut) and -wildtype (p53-wt) glioblastoma cell lines (p53-mut: LN405, T98G; p53-wt: A172, DBTRG) and primary glioblastoma cells (p53-mut: P0297; p53-wt: P0306) were treated with SAR combined with TMZ, 5-aza-dC, and/or IR and analysed for induction of apoptosis (AnnexinV and sub-G1 assay), cell cycle distribution (nuclear PI staining), DNA damage (alkaline comet or gH2A.X assay), proliferation inhibition (BrdU assay), reproductive survival (clonogenic assay), and potential tumour stem cells (nestinpos/GFAPneg fluorescence staining). Potential treatment-induced neurotoxicity was evaluated on nestin-positive neural progenitor cells in a murine entorhinal-hippocampal slice culture model.ResultsSAR showed radiosensitizing effects on the induction of apoptosis and on the reduction of long-term survival in p53-mut and p53-wt glioblastoma cell lines and primary cells. In p53-mut cells, this effect was accompanied by an abrogation of the IR-induced G2/M arrest and an enhancement of IR-induced DNA damage by SAR treatment. Also TMZ and 5-aza-dC acted radioadditively albeit to a lesser extent. The multimodal treatment achieved the most effective reduction of clonogenicity in all tested cell lines and did not affect the ratio of nestinpos/GFAPneg cells. No neurotoxic effects were detected when the number of nestin-positive neural progenitor cells remained unchanged after multimodal treatment.ConclusionThe Chk1 inhibitor SAR-020106 is a potent sensitizer for DNA damage-induced cell death in glioblastoma therapy strongly reducing clonogenicity of tumour cells. Selectively enhanced p53-mut cell death may provide stronger responses in tumours defective of non-homologous end joining (NHEJ). Our results suggest that a multimodal therapy involving DNA damage inducers and DNA repair inhibitors might be an effective anti-tumour strategy with a low risk of neurotoxicity.