Homologous Tumor Cells Research Articles

The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin

Poly (ADP-ribose) polymerase (PARP) inhibitors elicit antitumour activity in homologous recombination-defective cancers by trapping PARP1 in a chromatin-bound state. How cells process trapped PARP1 remains unclear. Using wild-type and a trapping-deficient PARP1 mutant combined with rapid immunoprecipitation mass spectrometry of endogenous proteins and Apex2 proximity labelling, we delineated mass spectrometry-based interactomes of trapped and non-trapped PARP1. These analyses identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase/segregase. We found that following trapping, PARP1 is SUMOylated by PIAS4 and subsequently ubiquitylated by the SUMO-targeted E3 ubiquitin ligase RNF4, events that promote recruitment of p97 and removal of trapped PARP1 from chromatin. Small-molecule p97-complex inhibitors, including a metabolite of the clinically used drug disulfiram (CuET), prolonged PARP1 trapping and enhanced PARP inhibitor-induced cytotoxicity in homologous recombination-defective tumour cells and patient-derived tumour organoids. Together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 and the response of tumour cells to PARP inhibitors.

Near-Infrared Responsive Membrane Nanovesicles Amplify Homologous Targeting Delivery of Anti-PD Immunotherapy against Metastatic Tumors.

The major obstacles of anti-PD therapy in metastatic tumors are limited drug delivery in primary tumors and metastatic foci, and the lack of tumor-infiltrating lymphocytes (TILs). Here, the authors constructed a novel cellular membrane nanovesicles platform (M/IR NPs) based on homologous targeting and near-infrared (NIR) responsive release strategy to potentiate PD-1/PD-L1 blockade therapy against metastatic tumors. In tumor-bearing mice, biomimetic M/IR NPs targeted both primary tumors and their lung metastases. Upon laser irradiation, M/IR NPs reduced cancer-associated fibroblasts (CAFs) in tumor microenvironment, thus increasing the penetration of TILs. When shed from homologous tumor cell membranes, positively charged nanoparticles (IR NPs) core can capture released tumor-associated antigens, thereby enhancing the antigen-presenting ability of DCs to activate cytotoxic T lymphocytes. When the photothermal conversion temperature under NIR-laser is higher than 42°C, M/IR NPs initiated the rupture of cell membranes and the responsive release of PD-1/PD-L1 inhibitor BMS, which significantly attenuated tumor-associated immunosuppression and synergistically induced T cellular immunity to inhibit the tumor growth and metastasis. Overall, biomimetic M/IR NPs can improve the targeting and therapeutic efficacy of anti-PD therapy in primary tumors and metastases, opening up a new avenue for the diagnosis and treatment of metastatic tumors in the future.

Cancer-Erythrocyte Hybrid Membrane-Camouflaged Magnetic Nanoparticles with Enhanced Photothermal-Immunotherapy for Ovarian Cancer

Cell-membrane-coated nanoparticles are widely studied due to their inherent cellular properties, such as immune escape and homologous homing. A cell membrane coating can also maintain the relative stability of nanoparticles during circulation in a complex blood environment through cell membrane encapsulation technology. In this study, we fused a murine-derived ID8 ovarian cancer cell membrane with a red blood cell (RBC) membrane to create a hybrid biomimetic coating (IRM), and hybrid IRM camouflaged indocyanine green (ICG)-loaded magnetic nanoparticles (Fe3O4-ICG@IRM) were fabricated for combination therapy of ovarian cancer. Fe3O4-ICG@IRM retained both ID8 and RBC cell membrane proteins and exhibited highly specific self-recognition of ID8 cells in vitro and in vivo as well as a prolonged circulation lifetime in blood. Interestingly, in the bilateral flank tumor model, the IRM-coated nanoparticles also activated specific immunity, which killed homologous ID8 tumor cells but had no effect on B16-F10 tumor cells. Furthermore, Fe3O4-ICG@IRM showed synergistic photothermal therapy, resulting in the release of whole-cell tumor antigens by photothermal-induced tumor necrosis, which further enhanced antitumor immunotherapy for primary tumor and metastatic tumor by activating CD8+ cytotoxic T cells and reducing regulatory Foxp3+ T cells. Together, the biomimetic Fe3O4-ICG@IRM nanoparticles showed synergistic photothermal-immunotherapy for ovarian cancer.

Abstract P057: The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin

Abstract Poly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumour activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, “trapped” PARP1. How cells process trapped PARP1 remains unclear. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with either a rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME)-based approach or PARP1 Apex2-proximity labelling linked to mass-spectrometry, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. This combined approach identified an interaction between trapped PARP1 and the ubiquitin-regulated p97 ATPase (aka VCP). Subsequent experiments demonstrated that upon trapping, PARP1 is SUMOylated by the SUMO-ligase PIAS4 and subsequently ubiquitinated by the SUMO-targeted E3-ubiquitin ligase, RNF4, events that promote p97 recruitment and p97 ATPase-mediated removal of trapped-PARP1 from chromatin. Consistent with this, small molecule p97 complex inhibitors, including a metabolite of the clinically-used drug disulfiram, CuET that acts as a p97 sequestration agent, prolong PARP1 trapping and thus enhance PARPi-induced cytotoxicity in homologous recombination defective tumour cells and patient-derived tumour organoids. Taken together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 from chromatin and the response of homologous recombination defective tumour cells to PARPi. Citation Format: Dragomir Krastev, Shudong Li, Yilun Sun, Yves Pommier, Kristijan Ramadan, Christopher J. Lord. The ubiquitin-dependent ATPase p97 removes cytotoxic trapped PARP1 from chromatin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P057.

Cancer-specific calcium nanoregulator suppressing the generation and circulation of circulating tumor cell clusters for enhanced anti-metastasis combinational chemotherapy

Tumor metastasis is responsible for chemotherapeutic failure and cancer-related death. Moreover, circulating tumor cell (CTC) clusters play a pivotal role in tumor metastasis. Herein, we develop cancer-specific calcium nanoregulators to suppress the generation and circulation of CTC clusters by cancer membrane-coated digoxin (DIG) and doxorubicin (DOX) co-encapsulated PLGA nanoparticles (CPDDs). CPDDs could precisely target the homologous primary tumor cells and CTC clusters in blood and lymphatic circulation. Intriguingly, CPDDs induce the accumulation of intracellular Ca2+ by inhibiting Na+/K+-ATPase, which help restrain cell–cell junctions to disaggregate CTC clusters. Meanwhile, CPDDs suppress the epithelial–mesenchymal transition (EMT) process, resulting in inhibiting tumor cells escape from the primary site. Moreover, the combination of DOX and DIG at a mass ratio of 5:1 synergistically induces the apoptosis of tumor cells. In vitro and in vivo results demonstrate that CPDDs not only effectively inhibit the generation and circulation of CTC clusters, but also precisely target and eliminate primary tumors. Our findings present a novel approach for anti-metastasis combinational chemotherapy.

In Vitro Experimental and Numerical Studies on the Preferential Ablation of Chemo-Resistant Tumor Cells Induced by High-Voltage Nanosecond Pulsed Electric Fields.

Here, we introduce an emerging physical feature-based therapeutic approach based on nanosecond pulsed electric fields (nsPEFs). The goal of this study is to investigate the effect of nsPEFs on CRTCs. The cell viability, ablation effects on a 3D-cultured scaffold, and lethal thresholds of nsPEFs were evaluated according to fluorescence staining assays. nsPEF treatment preferentially affected chemoresistant cells (A549/CDDP) with a higher cell viability inhibition ability/cell death rate, larger ablation area, and lower ablation threshold compared to their respective homologous tumor cells (A549). The experimental and theoretical studies suggested that nsPEFs displayed selective behavior toward intracellular structures. With this selective character, nsPEFs can induce higher electroporation effects (e.g., higher pore number, larger electroporation area, and faster fluorescence dissipation on the nuclear envelope) on CRTCs due to their larger nuclear size and cell membrane capacitance. These findings demonstrated that nsPEFs induced preferential ablation of CRTCs over their respective homologous tumor cells. This study provides an experimental and theoretical basis for the study of killing CRTCs by electrical treatments and suggests potential applications in the optimization of novel anti-chemoresistance methods.

Tumor cell membrane-derived nano-Trojan horses encapsulating phototherapy and chemotherapy are accepted by homologous tumor cells

Tumor cell membrane-derived nanostructures targeting homologous tumors are promising biomimetic drugs. Herein, curcumin (Cur) and chlorin e6 (Ce6) were co-loaded into PLGA nanoparticles (NPs), and then the NPs were coated with MCF-7 cell membranes (MCNPs). Cell membrane coating sharply increased the uptake of MCNPs by homologous cells, as compared to that with naked NPs. The NPs co-loaded with Cur and Ce6 (Cur/Ce6-NPs) showed a stronger proliferation-inhibitory effect on MCF-7 cells than the NP groups loaded with Cur and Ce6 alone. Cytotoxicity and apoptosis rates of MCF-7 cells in the Cur/Ce6-MCNPs group were significantly higher than those in the uncoated Cur/Ce6-NPs group. Both Cur/Ce6-NPs and Cur/Ce6-MCNPs significantly inhibited the migration of MCF-7cells, although Cur/Ce6-MCNPs showed a stronger effect. Compared to that of Cur/Ce6-NPs, the elimination of Cur/Ce6-MCNPs was both decreased and retarded, prolonging their in vivo systemic circulation and resulting in improved bioavailability. After intravenous administration for 24 h, the fluorescence intensity of drugs in the liver and spleen of the Cur/Ce6-MCNPs group was significantly weaker than that in the Cur/Ce6-NPs group, but that in tumor tissue was enhanced. Further, Cur/Ce6-MCNPs treatment achieved significantly better tumor-suppressive effects in vivo than Cur/Ce6-NPs, resulting in smaller tumor weights, increased apoptosis rates, and the down regulation of Ki67 protein in the tumor tissue. Thus, the tumor cell membrane-camouflaged nanocomposites have potential for homologous tumor-targeted therapy. Furthermore, photodynamic therapy combined with chemotherapy has promising future prospects.

Endogenous DNA 3′ Blocks Are Vulnerabilities for BRCA1 and BRCA2 Deficiency and Are Reversed by the APE2 Nuclease

The APEX2 gene encodes APE2, a nuclease related to APE1, the apurinic/apyrimidinic endonuclease acting in base excision repair. Loss of APE2 is lethal in cells with mutated BRCA1 or BRCA2, making APE2 a prime target for homologous recombination-defective cancers. However, because the function of APE2 in DNA repair is poorly understood, it is unclear why BRCA-deficient cells require APE2 for viability. Here we present the genetic interaction profiles of APE2, APE1, and TDP1 deficiency coupled to biochemical and structural dissection of APE2. We conclude that the main role of APE2 is to reverse blocked 3' DNA ends, problematic lesions that preclude DNA synthesis. Our work also suggests that TOP1 processing of genomic ribonucleotides is the main source of 3'-blocking lesions relevant to APEX2-BRCA1/2 synthetic lethality. The exquisite sensitivity of BRCA-deficient cells to 3' blocks indicates that they represent a tractable vulnerability in homologous recombination-deficient tumor cells.

Application of bioimpedance spectroscopy to characterize chemoresistant tumor cell selectivity of nanosecond pulse stimulation

The discriminating effects of nanosecond pulsed electric fields (nsPEFs) between chemoresistant tumor cells (CRTCs) and their respective homologous chemosensitive tumor cells (CSTCs) were investigated based on bioimpedance spectroscopy (BIS). The electrical properties of individual untreated cells were determined by fitting the impedance spectra to an equivalent circuit model and then using aided simulations to calculate the nuclear envelope transmembrane potential (nTMP) and electroporation area on the nuclear envelope. Additionally, fluorescence staining assays of cell monolayers after nanopulse stimulation (80 pulses, 200 ns, 3 kV) were conducted to validate the simulation results. The staining results indicated that CRTCs showed a larger ablation area and lower lethal threshold compared to CSTCs after exposure to the same nsPEF energy, which was in accordance with the higher nTMP and larger electroporation area calculated for CRTCs. The increase in the lethal effects of nsPEFs on CRTCs compared to CSTCs mainly resulted from the superposition of the changes in the electrical properties and nuclear size. The work shows that BIS can distinguish CRTCs and CSTCs and the corresponding nsPEF effects, suggesting potential applications for the optimization of novel anti-chemoresistance methods, including nsPEF-treatments.

Abstract OT2-03-02: A pilot trial of hyperthermia in combination with olaparib in breast cancer patients with chest wall recurrences

Abstract Background: Chest wall recurrences represent a significant source of morbidity for breast cancer patients causing symptoms including bleeding, ulceration, infection and pain. Approximately 5 to 10% of breast cancer patients undergoing a mastectomy will have a chest wall or nodal recurrence within ten years after surgery. Loco-regional recurrence isolated to the chest wall occurs in 50 to 70% of these patients and only one third of these patients having synchronous metastases. After radiation, patients with unresectable isolated loco-regional breast cancer have very few treatment options. Hyperthermia, temperature elevation to 40° to 45°C for 60 minutes, is a potent radiotherapy and chemotherapy sensitizer without significant toxicity. Additionally, superficial hyperthermia can improve the complete response and local control in breast cancer patients with chest wall disease. Chest wall hyperthermia induces degradation of the BRCA2 protein, thereby leading to defective homologous recombination DNA repair, and sensitization to PARP-1 inhibitors. Krawczyk and colleagues (2017) showed that mild hyperthermia innately sensitizes homologous recombination proficient tumor cells to treatment with a PARP-1 inhibitor in pre-clinical models. Olaparib, is an oral targeted therapy that is an inhibitor of the poly ADP ribose polymerase (PARP) and is currently approved for treatment for patients with metastatic ovarian cancer and metastatic breast cancer with BRCA1 or BRCA2 mutation. Our hypothesis is that the combination of olaparib and chest wall hyperthermia will be a safe and tolerable combination for breast cancer patients with chest wall metastases. Trial Design: In this novel dose-escalation pilot trial, breast cancer patients with chest wall recurrences will be treated with olaparib at the appropriate dose level, concurrently with chest wall hyperthermia involving heating the skin to 43 ° Celsius for 1 hour. We plan to treat patients first with olaparib given twice a day in tablet form at the appropriate dose level for one week and then add hyperthermia twice a week for 3 weeks, for a total duration of 4 weeks of treatment. Eligibility criteria: Female breast cancer patients regardless of ER/PR/HER2 status who have chest wall recurrences greater than 2 cm are eligible for this study. Patients are eligible after any line of therapy and if they have wild-type germ-line BRCA1 or BRCA2 status. Specific Aims: We will evaluate the safety and determine the maximum tolerated dose of the combination of olaparib and chest wall hyperthermia. We will assess the local progression free survival, one-year progression free survival, overall response and quality of life scores for these patients. We plan to obtain skin biopsies to evaluate biomarkers of homologous recombination deficiency including BRCA1, BRCA2, RAD51 as well as γH2AX as an indicator of DNA damage. Statistical Methods: A 3+3 dose-escalation design will be used to determine the MTD of olaparib in combination with hyperthermia. We will initially enroll 3 subjects at each dose level. If one of the three experiences that level of toxicity, we will accrue 3 more subjects at that dose. If at any time there are two or more dose-limiting toxicities (in the 3-6 subjects) on a given dose, we will terminate accrual to the trial. No patient will be treated at a higher dose until the 3 or 6 patients have completed their 30 day toxicity evaluation period at the current dose. The survival endpoints of PFS and local PFS will be analyzed using log-rank test with Kaplan-Meier curves, as well as Cox proportional hazard models. Present Accrual and target accrual: Target accrual is 12 patients in the next two years. This study opened in May of 2019 at Thomas Jefferson University in Philadelphia, PA with full support from AstraZeneca. Clinical trial information: NCT03955640 Citation Format: Saveri Bhattacharya, Matthew Schiewer, Rita C. Murphy, Pramila Rani Anne, Nicole Simone, Voichita Bar Ad, Maysa Abu Khalaf, Daniel P. Silver, Rebecca Jaslow, Frederick M. Fellin, Allison M. Zibelli, Ana Maria Lopez, Adam Berger, Theodore N. Tsangaris, Melissa A. Lazar, Alliric I. Willis, Paul Stauffer, Mark D. Hurwitz. A pilot trial of hyperthermia in combination with olaparib in breast cancer patients with chest wall recurrences [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT2-03-02.

A pH/Ultrasound dual-response biomimetic nanoplatform for nitric oxide gas-sonodynamic combined therapy and repeated ultrasound for relieving hypoxia

Sonodynamic therapy (SDT) has rapidly developed as a powerful alternative to traditional photodynamic therapy due to its intrinsically deeper tissue-penetration. However, single SDT dose is incapable of radical cure because the long-term hypoxia of tumor limits its therapeutic effect. Herein, we developed a biomimetic nanoplatform with dual pH/ultrasound response, homologous targeting and low phototoxicity for combined nitric oxide (NO) gas therapy with SDT to solve the problem. This nanoplatform is composed of zeolite imidazole framework-8 material embedded with nitrosoglutathione (GSNO) and chlorin e6 (Ce6) by one-step encapsulation, and then wrapped by homologous tumor cell membrane. In vitro and in vivo experiments indicate that the biomimetic nanoplatform has excellent biocompatibility and shows higher retention in tumor by homologous targeting. Importantly, it can sustainably release the encapsulated drug in acidic tumor microenvironment and accelerate degradation by ultrasound (US). Furthermore, NO released from GSNO and reactive oxygen species generated by Ce6, which are both triggered by US, react with each other to produce highly reactive peroxynitrite to inhibit the growth of tumor. Moreover, by repeated US irradiation, the tumor hypoxia can be relieved for a much-longer term, resulting in an effective gas-sonodynamic combined treatment. This study fully utilizes the advantages of US, providing a new strategy for high-performance cancer therapy.

Trasposon Mediated Horizontal Gene Transfer (T-HGT) of Circulating Tumor DNA Reshapes MM Clonal Architecture

Spatial genetic heterogeneity is a characteristic phenomenon that influences multiple myeloma's (MM) phenotype and drug sensitivity (Rasche L. et al and Bolli N et al.). Hence, the branch model of tumor evolution is not sufficient to explain the disorganized architecture observed in MM. In this study, we investigated whether MM ctDNA horizontal gene transfer (HGT) affect tumor genetic architecture and drug sensitivity, resembling what is seen in prokaryotes, and elucidated the mechanisms involved in the mobilization of genetic material from one cell to another. We identified that plasma from patients with MM transmits drug sensitivity or resistance to cells in culture. This transmission of drug sensitivity is mediated by ctDNA transfer of oncogenes to a host cell. Importantly, in vitro and in vivo demonstrated that ctDNA mainly targets cells resembling the cell of origin (tropism). Karyotype spreads and whole genome sequencing demonstrated that once patients ctDNA encounters host cells, it migrates into the nucleus where it ultimately integrates into the cell's genome. Integration to the genome was confirmed to be targeted to myeloma cells. Further sequencing analysis of multiple MM samples identified ctDNA tropism and integration is dependent on the 5' and 3' end presence of transposable elements (TE), particularly of the MIR and ALUsq family. These results were further validated by TE mediated delivery of GFP into MM cells in vitro and HSVTK in tumors of mouse xenografts. In conclusion, this data indicates for the first time that TE mediates MM ctDNA HGT into homologous tumor cells shaping the hierarchical architecture of tumor clones and affecting tumor response to treatment. Therapeutically, this unique quality of ctDNA can be exploited for targeted gene therapeutic approaches in MM and potentially other cancers. Disclosures Bernal-Mizrachi: Kodikas Therapeutic Solutions, Inc: Equity Ownership; TAKEDA: Research Funding; Winship Cancer Institute: Employment, Patents & Royalties.

Current status and future prospects of PARP inhibitor clinical trials in ovarian cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a class of targeted agents for the treatment of solid tumors. Concurrent PARP inhibition in Breast Cancer Susceptibility Gene (BRCA)-mutated or homologous recombination-deficient tumor cells can induce “synthetic lethality”, which targets two DNA repair pathways and induces serious cytotoxicity to tumor cells without damaging normal cells. Currently, PARP inhibitors such as olaparib, rucaparib and niraparib, which improve progression-free survival, particularly in patients harboring BRCA mutations, are approved by the Food and Drug Administration (FDA) and European Medicine Agency (EMA) for the treatment of ovarian cancers. Based on the results of different clinical trials, the indications for these drugs are slightly different. PARP inhibitors have been studied both as single agents and in combination with chemotherapy, antiangiogenic agents, and ionizing radiation. This review summarizes the critical clinical trials of PARP inhibitors that have been completed, provides an overview of the ongoing trials, presents the confirmed conclusions and notes the issues that need to be addressed in future studies.

Apoptotic Melanoma B16-F1 Cells Induced by Lidamycin Could Initiate the Antitumor Immune Response in BABL/c Mice.

In the process of tumor cell apoptosis induced by specific regents, calreticulin (CRT) was transferred from endoplasmic reticulum (ER) onto the cell membrane. These tumor cells, when used as the cellular vaccine to immunize experimental animals, could initiate effective antitumor immunoresponse against homologous tumor cells. This is referred to as immunogenic cell death. Lidamycin (LDM) is an enediyne antibiotic, which has extremely potent cytotoxicity to cancer cells. In this study, the mouse melanoma B16-F1 cancer cells were used to investigate the ability of LDM in promoting immunogenic cell death. Our data showed that LDM could induce apoptosis of B16-F1 cancer cells, accompanied by CRT translocation onto the cell membrane. These LDM-treated B16-F1 cells could be recognized and phagocytosed more efficiently by macrophage and dendritic cells. When the LDM-treated apoptotic B16-F1 cells were used as a whole-cell tumor vaccine to immune mice, the mice obtained resistance against rechallenged B16-F1 living cells. At the same time, the specific antitumor immune response was observed in these vaccinated mice. The splenocytes from the mice vaccinated with LDM-treated B16-F1 cells showed significantly enhanced NK lymphocyte activities and also faster growth rate and increased secretion of IFN-γ when encountering the cellular antigens from B16-F1 cells. All these results suggested that LDM could promote immunogenic cell death in B16-F1 cells, and these LDM-treated B16-F1 cells could be used as a sort of cell vaccine to initiate effective antitumor immunoresponse in mice.

Abstract SY42-02: Sensitization to chemoradiation by inhibition of cell cycle checkpoints

Abstract Chemoradiation is uniquely effective at generating DNA DSBs (double-strand breaks) in the environment of chemotherapy-induced DNA damage and replication stress producing complex, difficult-to-repair DNA DSBs. However, chemoradiation therapy also initiates robust cell cycle checkpoint responses in tumor cells which promote repair of DNA damage and thus contribute to therapy resistance. Thus, inhibition of cell cycle checkpoints by inhibitors of kinases such as Wee1 or Chk1 is an especially effective strategy for sensitizing tumor cells to chemoradiation. In addition to abrogating cell cycle checkpoints, inhibitors of Wee1 and Chk1 inhibit homologous recombination repair of DNA DSBs. Furthermore, the efficacy of PARP1/2 [poly (ADP- ribose) polymerase1/2] inhibitors in homologous recombination repair defective tumor cells provides a rationale for the combination of Wee1 or Chk1 inhibitors with PARP1/2 inhibitors and radiation. Clinical investigation of Wee1 inhibitors with chemoradiation has recently been initiated. Novel combinations of targeted agents such as Wee1 and PARP1/2 inhibitors with radiation are an important area of emphasis for future clinical studies. Preclinical studies to define optimal agent combinations and scheduling, mechanisms of tumor cell selectivity, and biomarkers will improve the likelihood of clinical success for these combinations. Citation Format: Meredith Ann Morgan. Sensitization to chemoradiation by inhibition of cell cycle checkpoints. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY42-02. doi:10.1158/1538-7445.AM2014-SY42-02

ATR inhibition preferentially targets homologous recombination-deficient tumor cells.

Homologous recombination (HR) is required for faithful repair of double-strand DNA breaks. Defects in HR repair cause severe genomic instability and challenge cellular viability. Paradoxically, various cancers are HR defective and have apparently acquired characteristics to survive genomic instability. We aimed to identify these characteristics to uncover therapeutic targets for HR-deficient cancers. Cytogenetic analysis of 1143 ovarian cancers showed that the degree of genomic instability was correlated to amplification of replication checkpoint genes ataxia telangiectasia and Rad3-related kinase (ATR) and CHEK1. To test whether genomic instability leads to increased reliance on replication checkpoint signaling, we inactivated Rad51 to model HR-related genomic instability. Rad51 inactivation caused defective HR repair and induced aberrant replication dynamics. Notably, inhibition of Rad51 led to increased ATR/checkpoint kinase-1 (Chk1)-mediated replication stress signaling. Importantly, inhibition of ATR or Chk1 preferentially killed HR-deficient cancer cells. Combined, our data show that defective HR caused by Rad51 inhibition results in differential sensitivity for ATR and Chk1 inhibitors, implicating replication checkpoint kinases as potential drug targets for HR-defective cancers.

PD-0426: Homologous recombination repair deficient tumor cells exhibit increased radiosensitization by PARP inhibition

PD-0426: Homologous recombination repair deficient tumor cells exhibit increased radiosensitization by PARP inhibition

Melanoma B16-F1 cells coated with fusion protein of mouse calreticulin and virus G-protein coupled receptor induced the antitumor immune response in Balb/C mice

In apoptotic progress of tumor cells stimulated by special agents, the calreticulin (CRT) was relocated from endoplasmic reticulum onto the cell surface. When used as cellular antigen to immunize experimental animals, these CRT-coated apoptotic tumor cells could initiate effective anti-tumor immunoresponse against homologous tumor cells, indicating the value of CRT in anti-tumor immunotherapy. In order to develop an universal technique that could make CRT-coating more efficiently in the tumor cells, in this study, a mouse CRT recombinant gene with virus G-protein coupled receptor (vGPCR) was constructed and cloned into vector pcDNA3.1(+). When resulted plasmid pcDNA3.1(+)-mCRT/ vGPCR was stably transfected into the mouse melanoma B16-F1 cells, the mCRT-vGPCR recombinant protein was synthesized. With the membrane-locating ability of vGPCR in the recombinant protein, mCRT-vGPCR was carried onto the surface of B16-F1 cells efficiently. Overexpression of mCRT-vGPCR on the cell surface could enhance the phagocytosis of B16-F1 by macrophages in vitro. When mCRT-vGPCR coated B16-F1 cells were used as a cell-antigen to immunize mice, the specific anti-tumor immune response against the homologous tumor cells was initiated efficiently. Our data in this study may provide a new possibility for CRT-mediated tumor immune prevention and treatment. See commentary: Augmentation of tumor-specific immunity by upregulation of apoptotic melanoma cell calreticulin expression

Apoptotic B16-F1 cells coated with recombinant calreticulin mediated anti-tumor immune response in mice

To investigate the recombinant calreticulin (rCRT) mediated antitumor immune response. Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expression and cell localization were assayed by western blotting, QT-RT-PCR and immunofluorescence assays. The mouse melanoma cell line B16-F1 was treated with polyamine analogue BENS to induce apoptosis and incubated with rCRT to get rCRT coated on the membrane, and then the cells were used to immune BALB/c mice as a cell-antigen. Immunized animals were rechallenged by live B16-F1 cells and then tumor generation ratio and the lactate dehydrogenase release assay were used to evaluate antitumor effects of rCRT-mediated immunity. BENS induced apoptosis of B16-F1 cells without the redistribution of CRT within the cells. When B16-F1 cells coated with rCRT were used as cell-antigen to inoculate the animals, the mice obtained the ability in inhibiting proliferation of homologous tumor cells in vivo. Comparing with the positive control group, the splenocytes from those inoculated mice have an obvious enhancement on their cytolytic effects specifically against B16-F1 cells. rCRT coated on the cell surface can enhance immunogenicity of apoptotic tumor cells and mediated effective anti-tumor immunoresponse in mice.

Mitoxantrone-Mediated Apoptotic B16-F1 Cells Induce Specific Anti-tumor Immune Response

In the process of cell apoptosis induced by specific reagents, calreticulin (CRT) in endoplasmic reticulum is transferred and coated onto the cell membrane. As a sort of specific ligand, the CRT on the surface of apoptotic cells could mediate recognition and clearance of apoptotic cells by phagocytes. In this research we discovered that mitoxantrone could induce apoptosis of mouse melonoma B16-F1 tumor cells, accompanied by the membrane translocation and coating of CRT. When mitoxantrone-treated B16-F1 cells were used as antigen to inoculate mice, the mice acquired an ability to suppress proliferation of homologous tumor cells. Splenocytes from these mice showed an increased cytolytic effect on homologous B16-F1 cells but no such effect on non-homologous H22 tumor cells. All these results suggested that mitoxantrone-treated apoptotic B16-F1 cells could be used as a sort of cell vaccine to initiate effective anti-tumor immunoresponse in mice.