Inhibition Of DNA Damage Repair Research Articles

Long noncoding RNA SNHG8 promotes chemoresistance in gastric cancer via binding with hnRNPA1 and stabilizing TROY expression.

To determine SNHG8's function and potential mechanisms in gastric cancer (GC) chemoresistance. We assessed SNHG8 expression in GC cell lines, GC/CDDP cell lines (cell lines treated with cisplatin), and 42 GC tissues and SNHG8 levels in the lncRNA microarray analysis of AGS/CDDP and AGS cell lines. We also examined GC cell viability in vivo and in vitro and its apoptosis level with Flow cytometry assays. SNHG8 was localized in subcells using fluorescence in situ hybridization (FISH) and cell fraction assays, hnRNPA1's link to SNHG8 was determined utilizing RNA immunoprecipitation (RIP) and FISH assays, gene expression profiles were assessed employing RNA transcriptome sequencing, and hnRNPA1's relationship with TROY was ascertained with the RIP assay. SNHG8 increased significantly in GC cell lines and GC tissues. However, a decrease in its expression promoted sensitivity to chemotherapy and inhibited DNA damage repair in vitro and in vivo. SNHG8 appeared to regulate TROY expression via linking with hnRNPA1. Reducing TROY levels considerably stimulated GC cell chemosensitivity, whereas heightening them partially rescued the rate of chemoresistance caused by downregulating SNHG8. In summary, the "SNHG8/hnRNPA1-TROY" axis is crucial to GC chemoresistance.

Metformin modified chitosan as a multi-functional adjuvant to enhance cisplatin-based tumor chemotherapy efficacy

Recently, it was newly revealed that the DNA damage induced by cis‑platinum (Cis-Pt) mediated chemotherapy was significantly impaired by the highly expressed programmed death ligand-1 (PD-L1) in tumor cells. Besides, the efficacy of Cis-Pt was also limited due to its severe side effects, especially enhanced drug efflux induced by multidrug resistance protein 1 (MDR-1) and increased tumor metastasis. Up to now, few drugs or carbohydrates could simultaneously solve these defects of Cis-Pt mediated chemotherapy. Here, we newly found that metformin-modified chitosan (Ch-Met) possessed ideal selective mitochondria accumulation capacity, leading to the further disrupted mitochondrial function, which then effectively inhibited the upregulated PD-L1 expression to inhibit DNA damage repair in tumor cells, as well as impaired drug efflux and lowered tumor metastasis. Therefore, it was demonstrated that Ch-Met could sensitize the chemotherapy efficacy of Cis-Pt.

Preclinical Evaluation of Tumor Treating Fields Combined with Personalized Ultra-Fractionated Stereotactic Adaptive Radiotherapy (PULSAR)

Purpose/Objective(s)The PULSAR paradigm, treats patients using a few large dose “pulses,” delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination.Materials/MethodsThe Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined.ResultsIn the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported.ConclusionThe addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI. The PULSAR paradigm, treats patients using a few large dose “pulses,” delivered at least a week apart. These split treatments represent a radical break from conventional radiation treatments lasting six to nine weeks. The expectation is that therapy will be less toxic and will allow treatments to personalized based upon noted changes in tumor morphology, location and radiation response. Early pre-clinical experiments identified an optimal timing between radiation pulses with the use of immune checkpoint inhibitors (ICI). Other therapeutic combinations are being explored. Tumor Treating Fields (TTFields) is a novel noninvasive physical modality of cancer therapy that disrupts mitosis, causes replication stress, inhibits DNA damage repair, and sets up a conditional vulnerability to DNA damaging agents. This study combined the conditional vulnerabilities caused by TTFields exposure with a preclinical model of PULSAR to determine treatment efficacy for this combination. The Inovivo system was used to generate Tumor Treating Fields across subcutaneously implanted tumors. Three distinct syngeneic mouse tumor models, LLC (Murine Lewis Lung Carcinoma), KPC63 (Pancreatic Cancer) and MC38 (Colon Cancer). Tumor growth delay (TGD) was the endpoint determined. In the three mice models tested in vitro, TTFields exposure alone modestly decreased tumor volume. A difference in tumor growth rate between the heat (sham) and TTFields group was not statistically significant. TTFields combined with 8 or 10 Gy radiation pulses, depending on tumor model radiosensitivity, produced a significant TGD effect on the MC38 mouse model after one round. The second round of TTFields and radiation treatment further delayed TGD, in a synergistic manner, confirming that TTFields induces a conditional vulnerability that is particularly suited to the PULSAR concept. The combination of TTFields, PULSAR and an ICI is being tested and will be reported. The addition of TTFields with a pre-clinical model of PULSAR resulted in synergistic delays in tumor growth compared to the PULSAR strategy alone when tested using three different subcutaneous tumor models. This combination is well suited for the addition of an ICI.

Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair

Developing an effective binder for a specific ubiquitin (Ub) chain is a promising approach for modulating various biological processes with potential applications in drug discovery. Here, we combine the Random Non-standard Peptides Integrated Discovery (RaPID) method and chemical protein synthesis to screen an extended library of macrocyclic peptides against synthetic Lys63-linked Di-Ub to discover a specific binder for this Ub chain. Furthermore, next-generation binders are generated by chemical modifications. We show that our potent cyclic peptide is cell-permeable, and inhibits DNA damage repair, leading to apoptotic cell death. Concordantly, a pulldown experiment with the biotinylated analog of our lead cyclic peptide supports our findings. Collectively, we establish a powerful strategy for selective inhibition of protein-protein interactions associated with Lys63-linked Di-Ub using cyclic peptides. This study offers an advancement in modulating central Ub pathways and provides opportunities in drug discovery areas associated with Ub signaling.

Immune Checkpoint Inhibitors and Other Immune Therapies in Breast Cancer: A New Paradigm for Prolonged Adjuvant Immunotherapy.

Background: Breast cancer is the most common form of cancer in women worldwide. Advances in the early diagnosis and treatment of cancer in the last decade have progressively decreased the cancer mortality rate, and in recent years, immunotherapy has emerged as a relevant tool against cancer. HER2+ and triple-negative breast cancers (TNBCs) are considered more immunogenic and suitable for this kind of treatment due to the higher rate of tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) expression. In TNBC, genetic aberrations further favor immunogenicity due to more neo-antigens in cancer cells. Methods: This review summarizes the principal ongoing conventional and investigational immunotherapies in breast cancer. Particularly, immune checkpoint inhibitors (ICIs) and their use alone or combined with DNA damage repair inhibitors (DDRis) are described. Then, the issue on immunotherapy with monoclonal antibodies against HER-2 family receptors is updated. Other investigational immunotherapies include a new schedule based on the interferon beta-interleukin-2 sequence that was given in ER+ metastatic breast cancer patients concomitant with anti-estrogen therapy, which surprisingly showed promising results. Results: Based on the scientific literature and our own findings, the current evaluation of tumor immunogenicity and the conventional model of adjuvant chemotherapy (CT) are questioned. Conclusions: A novel strategy based on additional prolonged adjuvant immunotherapy combined with hormone therapy or alternated with CT is proposed.

Compartmentalized regulation of NAD+ by Di (2-ethyl-hexyl) phthalate induces DNA damage in placental trophoblast

Di (2-ethyl-hexyl) phthalate (DEHP) is a wildly used plasticizer. Maternal exposure to DEHP during pregnancy blocks the placental cell cycle at the G2/M phase by reducing the efficiency of the DNA repair pathways and affects the health of offsprings. However, the mechanism by which DEHP inhibits the repair of DNA damage remains unclear. In this study, we demonstrated that DEHP inhibits DNA damage repair by reducing the activity of the DNA repair factor recruitment molecule PARP1. NAD+ and ATP are two substrates necessary for PARP1 activity. DEHP abated NAD+ in the nucleus by reducing the level of NAD+ synthase NMNAT1 and elevated NAD+ in the mitochondrial by promoting synthesis. Furthermore, DEHP destroyed the mitochondrial respiratory chain, affected the structure and quantity of mitochondria, and decreased ATP production. Therefore, DEHP inhibits PARP1 activity by reducing the amount of NAD+ and ATP, which hinders the DNA damage repair pathways. The supplement of NAD+ precursor NAM can partially rescue the DNA and mitochondria damage. It provides a new idea for the prevention of health problems of offsprings caused by DEHP injury to the placenta.

In vitro radiosensitization of breast cancer with hypoxia-activated prodrugs.

KP167 is a novel hypoxia‐activated prodrug (HAP), targeting cancer cells via DNA intercalating and alkylating properties. The single agent and radiosensitizing efficacy of KP167 and its parental comparator, AQ4N, were evaluated in 2D and 3D cultures of luminal and triple negative breast cancer (TNBC) cell lines and compared against DNA damage repair inhibitors. 2D normoxic treatment with the DNA repair inhibitors, Olaparib or KU‐55933 caused, as expected, substantial radiosensitization (sensitiser enhancement ratio, SER0.01 of 1.60–3.42). KP167 induced greater radiosensitization in TNBC (SER0.01 2.53 in MDAMB‐231, 2.28 in MDAMB‐468, 4.55 in MDAMB‐436) and luminal spheroids (SER0.01 1.46 in MCF‐7 and 1.76 in T47D cells) compared with AQ4N. Significant radiosensitization was also obtained using KP167 and AQ4N in 2D normoxia. Although hypoxia induced radioresistance, radiosensitization by KP167 was still greater under 2D hypoxia, yielding SER0.01 of 1.56–2.37 compared with AQ4N SER0.01 of 1.13–1.94. Such data show KP167 as a promising single agent and potent radiosensitiser of both normoxic and hypoxic breast cancer cells, with greater efficacy in TNBCs.

Discovery of CN0 as a novel proteolysis-targeting chimera (PROTAC) degrader of PARP1 that can activate the cGAS/STING immunity pathway combined with daunorubicin

Poly ADP-ribose polymerase 1 (PARP1) plays an essential role in DNA repair signaling, rendering it an attractive target for cancer treatment. Despite the success of PARP1 inhibitors (PARPis), only a few patients can currently benefit from PARPis. Moreover, drug resistance to PARPis occurs during clinical treatment. Natural and acquired resistance to PARPis has forced us to seek new therapeutic approaches that target PARP1. Here, we synthesized a series of compounds by proteolysis-targeting chimera (PROTAC) technology to directly degrade the PARP1 protein. We found that CN0 (compound 3) with no polyethylene glycol (PEG) linker can degrade the PARP1 protein through the proteasome pathway. More importantly, CN0 could inhibit DNA damage repair, resulting in highly efficient accumulation of cytosolic DNA fragments due to unresolved unrepaired DNA lesions when combined with daunorubicin (DNR). Therefore, CN0 can activate the cyclic GMP-AMP synthase/stimulator of the interferon gene (cGAS/STING) pathway of innate immunity and then spread the resulting inflammatory signals, thereby reshaping the tumor microenvironment, which may eventually enhance T cell killing of tumor cells.

Functional Immune Cell-Derived Exosomes Engineered for the Trilogy of Radiotherapy Sensitization.

The limited efficacy of radiotherapy leads to radio‐resistance and high rates of tumor recurrence and metastasis, which is caused by tumor hypoxia, rapid DNA damage repair, and especially the suppressive immune microenvironment of tumor. Lots of immune cell‐derived exosomes can regulate antitumor immunity, but their application in enhancing radiotherapy is rarely studied. Herein, as a model of concept, M1 macrophage‐derived exosomes (M1Exos) is engineered as effective radiotherapy sensitizers, realizing the trilogy of radiotherapy sensitization: 1) M1Exos is engineered to express catalases on the inside of membrane, which can effectively relieve tumor hypoxia, and enhance DNA damage. 2) The DNA damage repair inhibitor is loaded in M1Exos to effectively inhibit DNA damage repair. 3) M1Exos can polarize M2 macrophages into M1 phenotypes, and the anti‐PD‐L1 nanobody engineered on the outside of M1Exos can relieve the immunosuppression of T cells, both ultimately leading to the remodeling of the tumor suppressive microenvironment. The trilogy of radiotherapy sensitization achieves excellent antitumor efficacy, exhibiting the good utility of engineering immune cell‐derived exosomes as radiotherapy sensitizers, inspiring the future efforts to explore different kinds of immune cell‐derived exosomes for enhanced radiotherapy.

Abstract 1866: Transcriptomics analysis for identification of pathways involved in the response to Tumor Treating Fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are alternating electric fields that disrupt the function of polarized molecules within cancer cells. Initial data showed an anti-mitotic effect on cancerous cells whilst more recent findings confirmed additional effects such as downregulation of DNA double strand break repair, replication stress, upregulation of autophagy, and immunogenic cell death. Identification of TTFields-driven alterations in pan-cancer and tumor specific pathway is needed to aid selection of therapeutic modalities to be applied concomitant with TTFields for improved treatment outcomes. The aim of this study was to identify common pathways involved in the response to TTFields by comparing transcriptomics of various tumor type. Methods: Control and TTFields-treated non-small cell lung carcinoma cell lines and animal model, glioblastoma (GBM) cell lines and patient-derived cell lines, and a hepatocellular carcinoma animal model were examined. Samples from GBM patients treated with concomitant TTFields and temozolomide (TMZ) were compared to samples from patients treated with TMZ alone. A list of differentially expressed genes (DEGs) was generated from transcriptomics analysis. Enrichment analysis was conducted according to the Gene Set Enrichment Analysis (GSEA) of MSigDB, Reactome, and Kegg pathway databases. Significantly overlapping pathways were identified using ActivePathways package according to the Reactome and Kegg gene sets, and an enrichment map was created according to the number of datasets supporting each pathway. Results: DEGs in response to TTFields application included genes of the cytoskeleton, immune system, and some secretion proteins. Common pathways downregulated by TTFields (negative GSEA) included DNA repair, DNA and RNA synthesis, and cell cycle regulation, specifically the G2M checkpoint, E2F targets, and Myc targets. Common pathways upregulated by TTFields (positive GSEA) included the immune response, specifically complement cascades, coagulation, and lysosome activity. ActivePathways enrichment map results agreed with the gene set enrichment results, and revealed additional pathways involved in the response to TTFields, such as SUMOylation, metabolism of carbohydrates, unfolded protein response, and signaling by interleukins. Conclusions: Transcriptomic analysis revealed common pathways involved in the responses to TTFields, regardless of the origin of the sample. Some identified pathways were in line with previously demonstrated effect of TTFields, such as mitotic interference, inhibition of DNA damage repair and upregulation of the innate immune response. New pathways revealed in this work support the examination of novel combination strategies with TTFields to increase the therapeutic effect in patients bearing various solid tumor types. Citation Format: Kerem Wainer-Katsir, Gitit Lavy-Shahaf, Shiri Davidi, Sara Jacobovitch, Tali Voloshin, Itai Tzchori, Yaara Porat, Lianghao Ding, Michael Story, Niv Pencovich, Ilan Volovitz, Joshua Branter, Stuart J. Smith, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Transcriptomics analysis for identification of pathways involved in the response to Tumor Treating Fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1866.

Abstract 5990: Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer

Abstract Background: Metastatic triple-negative breast cancer (TNBC), especially with BRCA1/2 mutations, is a deadly subtype of breast cancer with ~12% of 5-year survival. Despite their FDA approval, the therapeutic benefits of poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) are limited to BRCA1/2 mutated malignancies. Moreover, the PARPi olaparib has been shown to upregulate the expression of programmed death-ligand 1 (PD-L1) leading to immune suppression. The purpose of this study is to investigate if a novel nanoparticle formulation of oligomer hyaluronic acid (oHA) and doxorubicin (DOX) co-loaded in an iRGD-conjugated polymer-lipid nanocarrier (iRGD-DOX-oHA-PLN) can inhibit DNA damage repair and suppress PD-L1 in both mutant and non-mutant TNBC cells and reduce tumor progression and lung metastasis compared to the PARPi olaparib. We postulate that the co-administered oHA will block the signaling pathways of native hyaluronic acid (HA) receptors, the cluster of differentiation 44 (CD 44) and receptor for HA mediated motility (RHAMM), that regulate DNA damage repair and immunosuppression and enhance DOX efficacy. Methods: In vitro cellular uptake of various DOX formulations by TNBC MDA-MB-231-luc-D3H2LN cells and MDA-MB-436 (BRCA1 mutant) cells was examined using confocal laser scanning microscopy or spectrophotometer. The expression level of RHAMM, PD-L1, and PARP1 parylation (PAR) was evaluated after treatment with iRGD-DOX-oHA-PLN or olaparib in both cell lines using western blot or confocal laser scanning microscopy. The therapeutic efficacy of iRGD-DOX-oHA-PLN compared to olaparib was determined by monitoring the tumor progression and lung metastasis development in orthotopic breast tumor models of MDA-MB-231-luc-D3H2LN or MDA-MB-436 cell line. Results: The expression of native HA receptor RHAMM was profoundly reduced by ~5 folds in vivo and the DNA DSB was significantly increased by the iRGD-DOX-oHA-PLN treatment with 40% of γH2AX positive cells in tumor tissue sections. The iRGD-functionalized PLN enhanced DOX cellular uptake compared to DOX free drug or non-targeted NPs by ~3- and ~1.5-fold, in MDA-MB-231 and MDA-MB-436 cells, respectively. While olaparib upregulated PD-L1 and free DOX increased PAR level, the iRGD-DOX-oHA-PLN reduced the parylation and PD-L1 expression in both TNBC cell lines. The iRGD-DOX-oHA-PLN treatment (two dose of 10 mg/kg i.v. biweekly) outperformed olaparib (twenty doses of 50 mg/kg i.p 5x/week) in preventing tumor progression and lung metastasis in vivo over a 4-week period. Conclusion: The results suggest that the iRGD-DOX-oHA-PLN can effectively inhibit DNA damage repair and immunosuppression of cancer cells and could be a promising multitargeted nanomedicine for the treatment of both BRCA1-mutant and non-mutant metastatic TNBC. Citation Format: Ibrahim Alradwan, Pei Zhi, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Rauth, Xiao Yu Wu. Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5990.

Abstract 5298: Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models

Abstract Background: Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate composed of an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. T-DXd is approved for patients with HER2+ metastatic breast cancer who have received two or more anti-HER2 based regimens as well as HER2+ gastric cancer, and has demonstrated antitumor activity in both HER2+ and HER2-low cancers clinically. Trapping of Topo1cc by DXd in targeted cells, induces replication stress (RS) and double strand breaks (DSB), activating a DNA Damage Repair (DDR) response. Therefore we aimed to investigate if combination of T-DXd with DDR inhibitors enhances efficacy and durability of response. Methods: We evaluated the antiproliferative effect of the combination of T-DXd with AZD6738 (ATR inhibitor), AZD1775 (WEE1 inhibitor), AZD1390 (ATM inhibitor), in a panel of 27 breast cancer cell lines in an in vitro 7-day viability assay. Combinations were also evaluated in vivo in the HER2-high gastric NCI-N87 cell line xenograft. To evaluate the specificity of the combination activity in tumor cells (vs normal tissue), we further evaluated the combination in a 2D in vitro human bone marrow progenitor assay. Results: We found that the combination with all DDRi significantly enhanced in vitro cell killing activity over single agents in many of the models tested (12/27 for AZD6738, 9/27 for AZD1775, 6/27 for AZD1390). This was not limited to models with DNA damage repair mutations (e.g. BRCA1, BRCA2, or ATM). Mechanistically, T-DXd activated PARP and the ATR, Wee1, ATM pathways, inducing cell cycle arrest. Combination of T-DXd with AZD6738 and AZD1775 inhibited the RS response (pATR-T1989, pChk1-S345) and cell cycle arrest (pCdc2-Y15), while AZD1390 inhibited the DSB response pathway (pATM-S1981, pRad50-S685, pKap1-S824, pChk2-T68). All DDR inhibitors exacerbated DNA damage (pRPA-S4/8, gH2AX) and cell death (cCasp3). In the in vitro human bone marrow (BM) assay, T-DXd had no synergistic interaction with AZD6738 and AZD1775 inhibitors, but synergy with AZD1390. In vivo, combination therapy with all DDRi tested achieved superior Tumor Growth Inhibition (TGI) compared to monotherapy alone. At day 41 in NCI-N87, 3mg/kg of T-DXd provided TGI of 74% while monotherapy arms of 25mg/kg BID AZD6738, 60mg/kg (5-days on/2-days off) AZD1775 or 10mg/kg AZD1390 provided 68%, 47%, 31% TGI respectively. The combinations of T-DXd with AZD6738, AZD1775 and AZD1390 provided TGI of >100% (19% regression), 90%, and 99% respectively. Conclusions: These results suggest that AZD6738, AZD1775, and AZD1390 all potentiate T-DXd activity in vitro and in vivo, and the combination interactions with AZD6738 and AZD1775 were much stronger in some tumor models over bone marrow cells in vitro. These data suggest further investigation of these combinations in clinical studies may be warranted. Citation Format: Yann Wallez, Theresa Proia, Azadeh Cheraghchi-Bashi-Astaneh, Ankur Karmokar, Zena Wilson, Suzanne Randle, Mark Anderton, Stephen Durant, Elisabetta Leo, Alan Lau, Mark O'Connor, Jerome Mettetal. Activity and tolerability of combinations of trastuzumab deruxtecan (T-DXd) with inhibitors of the DNA damage response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5298.

Abstract 2599: Targeted delivery of a colchicine analogue provides synergy with ATR inhibition

Abstract Introduction: Vascular disrupting agents (VDA) are an important class of therapeutic that have yet to reach their clinical potential. Colchicine is representative of the class and is an effective disruptor of microtubule dynamics. Microtubules play an important role in the maintenance of vascular integrity, hence the ability of these agents to collapse tumour blood vessels, and consequently cause tumour necrosis. Colchicine, in addition to its vascular disrupting properties, is known to induce G2/M arrest and trigger apoptosis. ICT2588 is a non-toxic, tumour targeted prodrug of a potent colchicine analogue with reduced potential for toxicity, (cardiotoxicity in particular) due to its metabolic stability in plasma and normal tissues. The preclinical evaluation of ICT2588 to-date has been focused solely on its selective tumour vascular disrupting ability and subsequent antitumour activity. The aim of this current study was to investigate the cellular metabolism of ICT2588, examine its G2/M cell cycle arrest potential, and to explore mechanisms of synergy between ICT2588 and ATR inhibition. Method: The cellular mechanism of uptake and MT1-MMP-selective metabolism of ICT2588 was studied in MT1-MMP positive (HT1080) and negative (MCF-7) cell lines using LCMS based metabolism assays. Flow cytometry was used to analyse the effect of ICT2588 and active azademethylcolchicine (the ‘warhead’) on G2/M cell cycle arrest in both MT1-MMP positive and negative cell lines. The growth inhibitory effects of ICT2588 and its warhead either alone or in combination with ceralasertib (AZD6738, ATR inhibitor) were performed using an MTT assay in MT1-MMP positive cell lines (HT1080, MDA-MB-231, and SH-SY5Y). Synergy Finder (https://synergyfinder.fimm.fi) was used to evaluate combinations. Results: The expression of MT1-MMP correlates with selective metabolism of ICT2588 and subsequent selective release of its warhead in HT1080 cells, but not MCF-7 cells. This metabolism is inhibited by an MT1-MMP inhibitor. Metabolism of ICT2588 induced G2/M arrest and triggered apoptosis in HT1080 cells, but not in MCF-7 cells, though the warhead induced G2/M arrest and triggered apoptosis in both cell lines. ICT2588 was synergistic with AZD6738 in all MT1-MMP positive cell lines, contrary to the antagonism observed with the warhead and AZD6738. These findings will be explored. Conclusion: ICT2588 is metabolised by only MT1-MMP expressing cells, releasing the potent colchicine analogue in a targeted-manner, which synergised with ATR inhibition in a way not observed when the warhead was administered alone. This in vitro data has important potential implications for possible combinations of ICT2588 and DNA damage repair inhibitors in the clinic. Citation Format: Francis M. Barnieh, Goreti Ribeiro Morais, Paul M. Loadman, Robert A. Falconer. Targeted delivery of a colchicine analogue provides synergy with ATR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2599.

Abstract 5399: Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. Despite the early response to chemotherapy, high incidence of recurrence leads to short overall survival and poor prognosis. Native hyaluronic acid (HA) interacts with CD44 and receptors for hyaluronan mediated motility (RHAMM) overexpressed in TNBC mediating chemo-resistance and metastasis. In contrast, oligomeric HA (oHA) can disrupt HA-CD44/RHAMM interactions and attenuate oncogenic and pro-metastatic pathways, such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway, reducing downstream DNA repair and drug resistance markers. Our previous work demonstrated that oHA can exert synergistic anti-tumor and anti-metastatic effects when combined with doxorubicin (DOX) and co-loaded in an integrin-targeted iRGD-modified nanoparticle system (iRGD-DOX-oHA-PLNs). This study aims to investigate the inhibitory effect of iRGD-DOX-oHA-PLNs on both DNA single-strand break (SSB) and double-strand break (DSB) repair proteins and drug efflux pumps responsible for multidrug resistance to enhance DOX efficacy in breast cancer gene 1 (BRCA1) mutant and non-mutant TNBC. Methods: The cytotoxicity of DOX, oHA and their combinations in free solution or in nanoparticles was evaluated by clonogenic assay in human MDA-MB-231-luc-D3H2LN and MDA-MB-436 (BRCA1 mutant) TNBC cells. The in vitro expression of a DNA DSB marker, DNA repair markers, and drug efflux pump P-glycoprotein (P-gp) were measured by Western blot. The in vivo expression level of BRCA1 and Rad51 in an orthotopic TNBC mouse model was determined by immunohistochemical staining. Results: The combination of DOX-oHA showed synergism against both BRCA1 mutant and non-mutant TNBC cells. The iRGD-DOX-oHA-PLNs induced great increases in DNA DSBs demonstrated by the highest γH2AX level compared to other treatment groups. These nanoparticles also showed inhibitory effects on the expression of both DNA SSB repair protein (poly (ADP-ribose) polymerase) and DNA DSB repair proteins (Rad50 and Rad51), contributing to the enhanced efficacy of chemotherapy. The immunohistochemical staining of tumor tissues indicated lower levels of BRCA1 and Rad51 after iRGD-DOX-oHA-PLN treatment than the formulation without oHA, attributable to the effect of intracellularly delivered oHA on limiting the MAPK signaling. Additionally, iRGD-DOX-oHA-PLNs reduced the expression of the drug efflux pump P-gp as compared to DOX treatment groups without oHA. Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently blocked DNA damage repair and down-regulated the drug efflux pump P-gp, thus improving the efficacy of DOX. Collectively, this nanoparticle system could be a promising option for metastatic TNBC treatment. Citation Format: Pei Zhi, Ibrahim Alradwan, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Michael Rauth, Xiao Yu Wu. Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5399.

Abstract 1133: Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers

Abstract Recently developed inhibitors of polymerase theta have demonstrated synthetic lethality in pre-clinical tumor models of BRCA-associated cancers. Given that other DNA damage repair inhibitor therapies, including PARP inhibitors, induce changes in the tumor immune microenvironment, we asked whether polymerase theta inhibition would also alter the tumor microenvironment in BRCA-associated cancers. We have demonstrated that novobiocin, a first-in-class inhibitor of the polymerase theta ATPase domain, induces both innate and adaptive immune responses through activation of the cGAS/STING pathway in BRCA-deficient tumor cells. Novobiocin-mediated polymerase theta inhibition generates micronuclei, activating the cGAS/STING pathway in both BRCA1-deficient MDA-MB-436 triple-negative breast cancer and BRCA2-deficient CAPAN1 pancreatic cancer models in vitro. Activation of the STING pathway upregulated the expression of interferon response genes, which was abrogated upon siRNA-mediated STING depletion. As an adaptive response, novobiocin also increased expression of PD-L1 in BRCA-deficient tumor cells. These effects were not observed in isogenic cell lines in which wild-type BRCA expression was corrected by add-back. Using a genetically engineered syngeneic mouse model of BRCA1-deficient breast cancer, we confirmed novobiocin-mediated activation of the cGAS/STING pathway in vivo, resulting in the induction of pro-inflammatory signaling and ultimately robust CD8+ T-cell infiltration and activation. Taken together, our findings demonstrate that novobiocin-mediated polymerase theta inhibition induces local and systemic immune responses in a cGAS/STING-dependent manner and provide a rationale for combining polymerase theta inhibition with immunotherapies for the treatment of BRCA-associated cancers. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Tin Phan, Alan D'Andrea, Geoffrey I. Shapiro. Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1133.

Protective role of electrophile-reactive glutathione for DNA damage repair inhibitory effect of dibromoacetonitrile

Dibromoacetonitrile (DBAN) is a disinfection byproduct (DBP) and linked with cancer in rodents, but the mechanism of its carcinogenicity has not been fully elucidated. We recently reported that DBAN induced inhibition of nucleotide excision repair (NER). In this study, we investigated if glutathione (GSH) is involved in the DBAN-induced inhibition of NER. Human keratinocytes HaCaT were pretreated with L-buthionine-(S,R)-sulfoximine (BSO) to deplete intracellular GSH. BSO treatment markedly potentiated the DBAN-induced NER inhibition as well as intracellular oxidation. The recruitment of NER proteins (transcription factor IIH, and xeroderma pigmentosum complementation group G) to DNA damage sites was inhibited by DBAN, which was further exacerbated by BSO treatment. Our results suggest that intracellular GSH protects cells from DBAN-induced genotoxicity including inhibition of DNA damage repair.

Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma

BackgroundAberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM.MethodsWe applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy.ResultsHDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM.ConclusionsOur study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM.Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018.

Novel diketopyrrolopyrrole NIR-II fluorophores and DDR inhibitors for in vivo chemo-photodynamic therapy of osteosarcoma

DNA damage response (DDR) inhibitors potentiate the therapeutic efficacy in osteosarcoma (OS) treatment. However, drug resistance, severe collateral toxicity, and poor bioavailability of DDR inhibitors in vivo are the main challenges. The NIR-II fluorescence image-guided photodynamic therapy exhibits advantageous properties and shows great potential in oncology, representing an emerging class of non-invasive treatment methods. Herein, a unique, synergistic strategy is evaluated to improve the therapeutic efficacy of a DDR inhibitor (AZD-2461) and a diketopyrrolopyrrole-based NIR-II fluorescent photosensitizer DT. The novel NIR-II photosensitizer DT is synthesized based on diacyl-substituted diketopyrrolopyrrole, and co-encapsulated with AZD-2461 using DSPE-mPEG5000-FA to form DTA dots. DTA dots significantly enhanced anti-osteosarcoma effects by increasing the generation of reactive oxygen species (ROS) and inhibiting DNA damage repair. NIR-II image-guided synergistic enhancement with high spatiotemporal resolution and strong tumor uptake is demonstrated in vivo, resulting in improved tumor control and increased apoptosis. This work reports for the first time a novel NIR-II phototheranostic nanoplatform for OS combination therapy without severe side effects, providing a practical strategy for simultaneous cancer diagnostics, therapeutics, and postoperative real-time monitoring.

Venetoclax enhances DNA damage induced by XPO1 inhibitors: A novel mechanism underlying the synergistic antileukaemic effect in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a highly heterogeneous haematologic malignancy with poor prognosis. We previously showed synergistic antileukaemic interaction between exportin 1 (XPO1) inhibitor KPT‐330 (Selinexor) and Bcl‐2 inhibitor venetoclax (ABT‐199) in preclinical models of AML, which was partially meditated by Mcl‐1, although the full mechanism of action remains unknown. In this study, using real‐time RT‐PCR and Western blot analysis, we show that inhibition of XPO1 via KPT‐330 or KPT‐8602 (Eltanexor) decreases the mRNA and protein levels of c‐Myc, CHK1, WEE1, RAD51 and RRM2. KPT‐330 and KPT‐8602 induce DNA damage, as determined by alkaline comet assay. In addition, we demonstrate that venetoclax enhances KPT‐330‐ and KPT‐8602‐induced DNA damage, likely through inhibition of DNA damage repair. This study provides new insight into the molecular mechanism underlying the synergistic antileukaemic activity between venetoclax and XPO1 inhibitors against AML. Our data support the clinical evaluation of this promising combination therapy for the treatment of AML.

PH-Responsive Hyaluronic Acid Nanoparticles for Enhanced Triple Negative Breast Cancer Therapy.

PurposeThis study emphasized that dasatinib (DAS) and olaparib (OLA) have synergistic effects on triple negative breast cancer, by inducing DNA damage and inhibiting DNA damage repair. However, poor water solubility, short half-life of drugs, and low drug concentration in tumor tissue limit the clinical application.MethodsIn this research, acid-sensitive ester bonds were used to connect hydrophobic DAS and hydrophilic hyaluronic acid (HA) to form the amphiphilic polymer prodrug HA-DAS, and then OLA was added as the core, the HA-DAS was used as the carrier to form nanomicelles (HDO-NPs) in aqueous. The characterization and drug release of HDO-NPs were studied, and the cytotoxicity, targeting effect, and intracellular transport behavior of HDO-NPs were evaluated in MDA-MB-231. In addition, the pharmacokinetic and therapeutic effect of HDO-NPs were further verified in vivo.ResultsIn vitro characterizations showed that HDO-NPs were spherical with uniform particle size, good stability and anti-dilution ability, and displayed favorable pH-responsive drug release behavior. In addition, the cell experiments showed that HDO-NPs could be effectively taken up by binding to the overexpressed CD44 proteins of MDA-MB-231 cells, resulting in increased intracellular drug concentration. In vivo experiments showed that HDO-NPs can effectively target tumor tissues, have excellent therapeutic effects on tumor, significantly prolong the circulation time of drugs in vivo, and effectively improved the bioavailability of drugs.ConclusionDAS and OLA were designed into micelles, the efficacy of HDO-NPs was higher than that of free drugs. Therefore, HDO-NPs have good application prospects in the treatment of triple negative breast cancer.