Increased Tumor Cell Death Research Articles

Abstract B009: Sub-oxidant ascorbate sensitizes glioblastoma stem cells to DNA damaging agents by inducing DNA de-methylation and altering the chromatin landscape

Abstract Gliomas are the most common primary brain cancer and glioblastoma (GBM) represents the most aggressive and lethal subtype. The high GBM morbidity and mortality are due to profound genetic, epigenetic, and phenotypic heterogeneity within the tumor that contributes to an almost guaranteed development of therapeutic resistance and tumor recurrence. GBM stem-like cells (GSCs) are tumor cell sub-populations that disproportionately drive tumor propagation, therapy resistance, and tumor recurrence. Epigenetic deregulation is thought to play a fundamental role in the formation and expansion of GSCs and their contributions to oncogenesis. However, the epigenetic reprogramming events that contribute to GSC tumor propagation and therapy resistance remain elusive. DNA methylation (5mC) can be modified to generate hydroxymethylation (5hmC) or reversed through enzymatic reactions coordinated by the Ten-Eleven Translocation (TET) enzymes. Multiple studies show a decrease in both 5hmC levels and TET expression in gliomas and a correlation between loss of 5hmC and increasing glioma grade and poor prognosis. These observations suggest that re-establishing TET activity and 5hmC levels in tumor cells will have tumor suppressive effects. Ascorbate (vitamin C) has gained pre-clinical interest as an attractive antitumor agent for GBM due to its ability to regulate redox status, cross the blood-brain barrier, and enhance TET enzymatic activity; therefore, providing an innovative potential epigenetic therapeutic for GBM. We show that low-dose ascorbate (i.e. sub-pro-oxidant concentrations) enhances TET activity and elevates global 5hmC levels concurrent with decreases in 5mC in GSCs. These changes in DNA methylation correlate with a decrease in self-renewal capacity and increased tumor cell death induced by temozolomide (TMZ) and ionizing radiation (IR). We also present a novel mechanism whereby ascorbate increases the histone mark H3K36me3 by upregulating GSC expression of the histone methyltransferase NSD1 via demethylation of the gene promoter region. We propose that the enhanced susceptibility of ascorbate-treated tumor cells to DNA damage therapies results from increased H3K36me3-induced euchromatin states that increases genomic susceptibility to DNA damage. These findings support the need to better understand the epigenetic, transcriptomic, and chromatin architectural effects of ascorbate in cancer. These results also strongly indicate that ascorbate could serve as an adjunct to standard-of-care GBM treatment by restoring TET tumor-suppressor activity and re-sensitizing GSCs to conventional radio- and chemotherapeutics. Citation Format: Maureen Malloy, Sweta Sudhir, Harmon Khela, Maria Fagundo-Lugo, Bachchu Lal, Joseph Bressler, John Laterra, Hernando Lopez Bertoni. Sub-oxidant ascorbate sensitizes glioblastoma stem cells to DNA damaging agents by inducing DNA de-methylation and altering the chromatin landscape. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B009.

Phase 1b/2 Study of Autologous CD30.CAR-T Cells in Combination with Nivolumab in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma after Failure of Frontline Therapy (ACTION)

Background Autologous CD30-directed chimeric antigen receptor T-cell (CD30.CAR-T) therapy is well tolerated, with significant clinical efficacy in relapsed or refractory (r/r) classical Hodgkin Lymphoma (cHL) patients, as demonstrated in phase 1/2 clinical studies [LCCC 1532-ATL (NCT02690545) and H-37966 (RELY-30, NCT02917083); Ramos et al., 2020] and the pilot part of CHARIOT phase 2 trial (TESSCAR001, NCT0535288; Ahmed et al., 2021). cHL is characterized by the presence of malignant Hodgkin Reed-Sternberg (HRS) cells, which frequently harbor amplifications in chromosome 9p24.1, leading to overexpression of programmed death-ligand 1 (PD-L1) (Chen et al., 2017). The efficacy and safety profile of programmed death (PD)-1 checkpoint inhibitors, nivolumab and pembrolizumab, in cHL have been well demonstrated (Chen et al., 2017; Kuruvilla et al., 2021; Younes et al., 2016), with FDA approval for r/r cHL. PD-1 checkpoint inhibitors, in combination with CD30-directed antibody therapy (brentuximab vedotin) or other chemotherapies, have also shown high efficacy in this setting (Advani et al., 2021; Mei et al., 2022; Moskowitz et al., 2021). Preclinical studies demonstrated potential synergism between PD-1 checkpoint inhibitors and CAR-T cell therapy, where PD-1 or PD-L1 antibodies boost CAR-T cell therapy and promote increased tumor cell death in vivo (Cherkassky et al., 2016; Gargett et al., 2016; Moon et al., 2014). Despite multiple treatment options available in the second line therapy for cHL, 10-40% of patients do not achieve response to salvage chemotherapy (von Keudell and Younes, 2019). Additionally, salvage chemotherapies are associated with short-term toxicity, long-term morbidity and non-lymphoma-related mortality (de Vries et al., 2021; Dores et al., 2020). We have designed a phase 1b/2 trial (ACTION) to evaluate the safety and antitumor activity of CD30.CAR-T cells combined with nivolumab in advanced cHL patients in whom standard frontline chemotherapy has failed. Study Design and Methods Approximately 15 adult and pediatric patients (≥12 years old) with r/r cHL following progressive disease after standard frontline therapy will be enrolled in the present single-arm, phase 1b/2, multicenter study (ClinicalTrials.gov NCT05352828). Following a leukapheresis procedure for manufacture of CD30.CAR-T cells, patients will be treated with 4 cycles of nivolumab every 4 weeks (Q4W), and a single infusion of CD30.CAR-T cells (given between nivolumab Cycles 2 and 3, and after lymphodepletion with bendamustine and fludarabine). At the end of treatment (EOT) visit (4 weeks after nivolumab Cycle 4) and at the Investigators' discretion, patients with complete response (CR), partial response (PR), or stable disease (SD) will undergo either standard treatment with autologous stem cell transplant (ASCT group) or continue nivolumab Q4W for up to 6 additional cycles, unless patients experience either progressive disease (PD) or unacceptable toxicity, whichever occurs earlier (non-ASCT group). Patients will be followed with periodic response assessments and safety monitoring until end of study (EOS), approximately 3 years after leukapheresis. Long-term follow-up will continue for up to 15 years. The primary objective of this study is to assess the safety of autologous CD30.CAR-T in combination with nivolumab. The key secondary objective is to evaluate potential anti-tumor activity, as assessed by the CR rate of autologous CD30.CAR-T in combination with nivolumab at EOT in 14 evaluable patients, as per Lugano Classification Revised Response System for malignant lymphoma (Cheson et al., 2014). Other secondary objectives are to assess the overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS) of patients in the ASCT and non-ASCT groups. This study is ongoing and one patient has been enrolled to date. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dosimetric Evaluation of Physical Radiation Delivery Limits of a Weak Magnetic Field Generator on GBM Dose Distributions

<h3>Purpose/Objective(s)</h3> To demonstrate the feasibility of meeting dose prescription criteria when using a weak magnetic field generator to support glioblastoma multiforme (GBM) radiation therapy. <h3>Materials/Methods</h3> Weak magnetic fields extend free radical life, and recent experimental results have shown that adding weak magnetic fields (20G-40G) to radiation therapy delivery enhances free radical production, increases tumor cell death, and slows tumor growth. Based on these results, we have built the first clinical unit, consisting of two 63 cm ID Helmholtz coils, spaced 35 cm apart. The coil system is placed such that the patient and Linac couch are within the coils, and the Linac isocenter is coincident with the center of the coil system. We propose to use GBM for our first clinical trial. The limitation imposed by the coil system is a fixed Linac couch angle, constraining the gantry rotation to slightly more than 180°, from lateral to lateral. The impact on treatment plan quality of the corresponding reduction in available beam angles was unknown, so we conducted a treatment planning study to determine if we could provide clinically equivalent treatment plans with the coil in place. We evaluated the treatment plans for 10 GBM patients that had been treated with arc therapy. The prescription was 60 Gy (2 Gy x 30 fractions) for 6 patients, 55.8 Gy (1.8 Gy x 31 fractions) for 2 patients, 54 Gy (1.8 Gy x 30 fractions) for 1 patient, and 40.05 Gy (2.67 Gy x 15 fractions) for 1 patient. The dose was delivered to 95% of the planning target volume (PTV). The PTV margin was 1.3-2 cm surrounding the gross tumor volume. Critical structures, such as the brainstem, optic nerves, and optic chiasm, were contoured and assigned constraints of 62 Gy, 54 Gy, and 54 Gy, respectively. We repeated the treatment planning session using only coplanar arcs that spanned from 90° to 270° (half-arcs). <h3>Results</h3> All the treatment plans generated with the half-arcs met the clinical criteria. The minimum PTV doses for the half arc and clinical plans were 50.0 ± 6.1 Gy and 51.4 ± 7.1 Gy, and the maximum PTV doses were 61.9 ± 7.1 Gy and 62.7 ± 8.6 Gy, respectively. Table 1 summarizes the results of four reported critical structures. All the critical structures met the criteria, and the half-arc plans were neither better nor worse than the clinical plans. <h3>Conclusion</h3> The physical limits imposed by the magnetic field generator on the Linac beam angles do not degrade treatment planning for GBM radiation therapy. The prescribed dose can be adequately delivered while avoiding critical structures. With this groundwork, we can develop clinical trials to evaluate the impact of utilizing weak magnetic fields in radiation therapy.

Evaluating the Combined Anticancer Response of Checkpoint Inhibitor Immunotherapy and FAP-Targeted Molecular Radiotherapy in Murine Models of Melanoma and Lung Cancer.

Simple SummaryAlthough newer cancer medicines that help the immune system recognize and attack cancer cells have improved responses to therapy, most patients ultimately have cancer recurrence. Additional therapies and therapy combinations are needed so that responses can last longer or indefinitely. Molecular targeted radiotherapy is another kind of therapy that targets radioactive particles directly to cancer in the hopes of killing cancer cells to stop tumor growth with limited side effects. Prior studies have shown that targeted radiotherapies activate the immune system and can work together with immunotherapy to improve response. Here, we tested a promising new therapy targeting fibroblast activation protein (FAP) with a therapeutic radionuclide 177Lu alone and with immunotherapy in mouse models of melanoma and lung cancer. The FAP-targeted radiotherapy reduced tumor growth in both models and melanoma, resulting in tumor regression. We saw increased tumor cell death in dual-treated tumors. We also found that myeloid cells were affected by the combined therapy to a greater degree than the additive effect of either therapy. These results demonstrate that this is a promising new therapy regimen and requires further preclinical and clinical study to better understand the molecular mechanisms underpinning response. Immunotherapy has dramatically improved outcomes for some cancer patients; however, novel treatments are needed for more patients to achieve a long-lasting response. FAP-targeted molecular radiotherapy has shown efficacy in both preclinical and clinical models and has immunomodulatory effects. Here, we studied if combined immunotherapy and radiotherapy could increase antitumor efficacy in murine models of lung cancer and melanoma and interrogated the mechanisms by which these treatments attenuate tumor growth. Using LLC1 and B16F10 murine models of lung cancer and melanoma, respectively, we tested the efficacy of 177Lu-FAPI-04 alone and in combination with immunotherapy. Alone, 177Lu-FAPI-04 significantly reduced tumor growth in both models. In animals with melanoma, combined therapy resulted in tumor regression while lung tumor growth was attenuated, but tumors did not regress. Combined therapy significantly increased caspase-3 and decreased Ki67 compared with immunotherapy alone. Flow cytometry demonstrated that tumor-associated macrophages responded in a tumor-dependent manner which was distinct in animals treated with both therapies compared with either therapy alone. These data demonstrate that 177Lu-FAPI-04 is an effective anticancer therapy for melanoma and lung cancer which mediates effects at least partially through induction of apoptosis and modulation of the immune response. Translational studies with immunotherapy and 177Lu-FAPI-04 are needed to demonstrate the clinical efficacy of this combined regimen.

Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment

Systemic delivery of oncolytic viruses has been widely regarded as an impractical option for antitumor treatment. Here, we selected two target genes as leading components, and significant therapeutic effects were obtained by simultaneously reducing the expression of transforming growth factor β 1 (TGF-β1) and heat shock protein 27 (HSP27) in various cancer cell types. Downregulation of HSP27 reduced the cellular levels of tumor progression-related proteins, and the simultaneous downregulation of HSP27 and TGF-β1 increased tumor cell death beyond that observed with TGF-β1 downregulation alone. To increase the potential for systemic administration, we generated modified mesenchymal stem cells (MSCs) to act as oncolytic adenovirus factories and carriers and assessed bioavailability in tumors after MSC injection. The MSCs were modified to express 78-kDa glucose-regulated protein (GRP78) and adenovirus early-region 1B 55 kDa (E1B55K). The tightly controlled inducible system permitted selective timing of viral release from carrier MSCs within the tumor. This approach significantly improved viral production, tumor targeting, timely viral release at the tumor site, and antitumor efficacy of the oncolytic adenovirus. These combined results demonstrate that engineered MSCs can significantly enhance the antitumor effects of oncolytic viruses without adverse safety issues, which may greatly extend the clinical applicability of oncolytic adenoviruses.

Nanomicelles of Radium Dichloride [223Ra]RaCl2 Co-Loaded with Radioactive Gold [198Au]Au Nanoparticles for Targeted Alpha-Beta Radionuclide Therapy of Osteosarcoma.

Alpha and beta particulate radiation are used for non-treated neoplasia, due to their ability to reach and remain in tumor sites. Radium-223 (223Ra), an alpha emitter, promotes localized cytotoxic effects, while radioactive gold (198Au), beta-type energy, reduces radiation in the surrounding tissues. Nanotechnology, including several radioactive nanoparticles, can be safely and effectively used in cancer treatment. In this context, this study aims to analyze the antitumoral effects of [223Ra]Ra nanomicelles co-loaded with radioactive gold nanoparticles ([198Au]AuNPs). For this, we synthesize and characterize nanomicelles, as well as analyze some parameters, such as particle size, radioactivity emission, dynamic light scattering, and microscopic atomic force. [223Ra]Ra nanomicelles co-loaded with [198Au]AuNPs, with simultaneous alpha and beta emission, showed no instability, a mean particle size of 296 nm, and a PDI of 0.201 (±0.096). Furthermore, nanomicelles were tested in an in vitro cytotoxicity assay. We observed a significant increase in tumor cell death using combined alpha and beta therapy in the same formulation, compared with these components used alone. Together, these results show, for the first time, an efficient association between alpha and beta therapies, which could become a promising tool in the control of tumor progression.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathway, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

UV emitting nanoparticles enhance the effect of ionizing radiation in 3D lung cancer spheroids

Purpose Radiation therapy for cancer is limited by damage to surrounding normal tissues, and failure to completely eradicate a tumor. This study investigated a novel radiosensitizer, composed of lutetium phosphate nanoparticles doped with 1% praseodymium and 1.5% neodymium cations (LuPO4:Pr3+,Nd3+). During X-ray exposure, the particles emit UVC photons (200–280 nm), resulting in increased tumor cell death, by oxygen-independent UVC-induced damage. Methods and Materials Specially designed LuPO4:Pr3+,Nd3+ nanoscintillator particles were characterized by dynamic light scattering, TEM and emission spectroscopy upon excitation. Cell death was determined by reduction in tumor spheroid growth over a 3-week period using a 3 D A549 lung cancer model. Cell cycle was evaluated by flow cytometry and cell death pathways were assessed by Annexin V/PI stain as well as quantify apoptotic bodies. Results Lung cancer cells expressed no long-term or nonspecific toxicity when incubated with LuPO4:Pr3+,Nd3+ nanoscintillators. In contrast, there was significant growth inhibition of cell spheres treated with 2.5 mg/ml LuPO4:Pr3+,Nd3+ in combination with ionizing radiation (4 or 8 Gy X-ray), compared to radiation alone. Homogeneous distribution of small NPs throughout the entire sphere resulted in more pronounced lethality and growth inhibition, compared to particle distribution limited to the outer cell layers. Growth inhibition after the combined treatment was caused by necrosis, apoptosis and G2/M cell cycle arrest. Conclusions Newly designed UVC-emitting nanoscintillators (LuPO4:Pr3+,Nd3+) in combination with ionizing radiation cause tumorsphere growth inhibition by inducing cell cycle arrest, apoptosis and necrosis. UVC-emitting nanoparticles offer a promising new strategy for enhancing local tumor response to ionizing radiation treatment.

Curcumin and Radiotherapy Exert Synergistic Anti-Glioma Effect In Vitro.

Curcumin, a bioactive polyphenol, is known to have anticancer properties. In this study, the effectiveness of curcumin pretreatment as a strategy for radio-sensitizing glioblastoma cell lines was explored. For this, U87 and T98 cells were treated with curcumin, exposed to 2 Gy or 4 Gy of irradiation, and the combined effect was compared to the antiproliferative effect of each agent when given individually. Cell viability and proliferation were evaluated with the trypan blue exclusion assay and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The synergistic effects of the combination treatment were analyzed with CompuSyn software. To examine how the co-treatment affected different phases of cell-cycle progression, a cell-cycle analysis via flow cytometry was performed. Treatment with curcumin and radiation significantly reduced cell viability in both U87 and T98 cell lines. The combination treatment arrested both cell lines at the G2/M phase to a higher extent than radiation or curcumin treatment alone. The synergistic effect of curcumin when combined with temozolomide resulted in increased tumor cell death. Our results demonstrate for the first time that low doses of curcumin and irradiation exhibit a strong synergistic anti-proliferative effect on glioblastoma cells in vitro. Therefore, this combination may represent an innovative and promising strategy for the treatment of glioblastoma, and further studies are needed to fully understand the molecular mechanism underlying this effect.

Phase I Study of Novel Radioenhancer NBTXR3 Activated by Radiotherapy in Cisplatin-Ineligible Locally Advanced HNSCC Patients

<h3>Purpose/Objective(s)</h3> Concurrent radiotherapy (RT) with high-dose cisplatin, or cetuximab in case of intolerance to cisplatin, are the non-surgical standard treatment for locally advanced head and neck squamous cell carcinoma (LA HNSCC). However, elderly patients, patients with poor performance status, comorbidities, and/or intolerance may not benefit from these treatments and represent a high unmet medical need. New approaches are thus needed to improve the patient clinical outcomes without adding toxicity. NBTXR3, composed of functionalized hafnium oxide nanoparticles, is injected once intratumorally and activated by RT. NBTXR3 increases the RT energy deposit inside tumor cells and subsequently increases tumor cell death compared to RT alone, without adding toxicity to healthy tissues. We present here current results of the dose expansion part of the phase I study evaluating NBTXR3 plus intensity modulated radiation therapy (IMRT) in this population (ClinicalTrials.gov: NCT01946867). <h3>Materials/Methods</h3> Patients with stage III-VA or T3/T4 (AJCC/UICC TNM staging system 8th ed.) HNSCC of the oropharynx or oral cavity, ineligible to cisplatin or cetuximab and amenable for RT, received a single intratumoral injection of NBTXR3 and IMRT (70 Gy in 35 fractions /7 weeks). A classical 3 + 3 dose escalation design has tested four doses of NBTXR3, equivalent to 5, 10, 15, and 22% of baseline theoretical tumor volume. The RP2D established as 22% of baseline tumor volume is further tested in the dose expansion part of the study. The primary endpoints of the dose expansion part are objective response rate (ORR) and complete response rate (CRR) of the primary tumor, by imaging according to RECIST v1.1. Safety is also evaluated. <h3>Results</h3> As of August 13, 2020, 43 patients have been treated in the dose expansion part of the study. The median age was 70.7 years old (range: 50.7- 89.9), 70% of patients had cardiac disorder risk, 44% had gastrointestinal disorder risk and 44% metabolic and nutrition disorder risk. The median tumor volume was 42.8 mL (range: 1.3 - 222.3). In the evaluable population for efficacy (N = 31), the ORR of the primary lesion was 83.9% and the CRR 67.7% at a median time of 7.8 months after NBTXR3 injection. Three patients (7%) experienced at least one serious adverse event (AE) related to the injection procedure and/or NBTXR3 which represented less than 1% of all reported AEs. RT-related toxicity was as expected with IMRT. Three deaths due to AEs related to RT and other causes were reported. The recruitment is ongoing and updated efficacy and safety results will be presented. <h3>Conclusion</h3> NBTXR3 intratumoral administration followed by IMRT may represent an option in elderly in elderly patients or patients with multiple comorbidities with LA-HNSCC who have limited therapeutic options. NBTXR3 activated by RT showed promising anti-tumor efficacy, supporting further evaluation in a phase III randomized trial.

Suppression of Extracellular Vesicle VEGF-C-mediated Lymphangiogenesis and Pancreatic Cancer Early Dissemination By a Selective HDAC1/2 Inhibitor.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by early dissemination and poor drug response. Therefore, it is an unmet medical need to develop new strategies for treatment. As aberrant activation of ERK due to KRAS activating mutation is a driving force for PDAC, a brake system that can terminate ERK signaling represents an ideal druggable target. Herein, we demonstrate that forced expression of dual specificity phosphatase-2 (DUSP2), a specific ERK phosphatase, abrogated tumor formation and loss of Dusp2 facilitated Kras-driven PDAC progression. We report that a selective HDAC1/2 inhibitor (B390) has multifaceted therapeutic potential in PDAC by restoring the expression and function of DUSP2. In vitro study showed that treatment with B390 inhibited growth and migration abilities of PDAC cells, decreased extracellular vesicle-associated VEGF-C expression, and suppressed lymphatic endothelial cell proliferation. In vivo, B390 not only suppressed tumor growth by increasing tumor cell death, it also inhibited lymphangiogenesis and lymphovascular invasion. Taken together, our data demonstrate that B390 was able to alleviate loss of DUSP2-mediated pathologic processes, which provides the proof-of-concept evidence to demonstrate the potential of using selective HDAC1/2 inhibitors in PDAC treatment and suggests reinstating DUSP2 expression may be a strategy to subside PDAC progression.

Abstract 2329: ONC212-induced impairment of oxidative phosphorylation is synergistic with glycolysis inhibition in treatment of pancreatic cancer in vitro and in vivo

Abstract ONC212 is a fluorinated imipridone shown to have preclinical efficacy against multiple cancers, including pancreatic cancer. One of the binding targets of ONC212 is the mitochondrial peptidase, ClpP, but its role in the anticancer activity of ONC212 is incompletely understood. Understanding the effects of ONC212 on the bioenergetics of pancreatic cancer could facilitate understanding and amplification of its anti-tumor effects. On extracellular flux analysis, ONC212 impairs mitochondrial ATP production and oxidative phosphorylation in multiple pancreatic cancer lines. However, pancreatic adenocarcinoma uses both glycolysis and mitochondrial function to meet its energy demands. Some pancreatic cancer cell lines are resistant to ONC212 inhibition of oxidative phosphorylation alone, but co-treatment with glycolytic inhibitor 2-deoxy-D-glucose (2-DG) synergizes with ONC212 and increases tumor cell death in vitro. Suppression of ClpX, the regulatory binding protein of ClpP, occurs with ONC212 treatment and was used as a biomarker of ONC212 activity. This suppression was demonstrated in multiple pancreatic cancer lines in vitro and in vivo. In a xenograft mouse tumor model of pancreatic cancer using both the ONC212 resistant Bxpc3 pancreatic cancer cell line and the more sensitive HPAF-II cell line, the combination of ONC212 and 2-DG treatment significantly decreased tumor growth and reduced the intertumoral variability. Immunohistochemistry also revealed increased apoptotic markers in these tumors. There was no significant toxicity from dual treatment in mice, which suggests that tumor energy deprivation from dual inhibition of oxidative phosphorylation and glycolysis via ONC212 and 2-DG has a therapeutic window and should be explored further as a potential combination therapy. In addition, if this effect holds true across other models and cancer types, baseline mitoATP percentage could be used as a functional biomarker of sensitivity to ONC212. Citation Format: Anna Donovan Louie, Isacco Ferrarini, Lanlan Zhou, Wafik S. El-Deiry. ONC212-induced impairment of oxidative phosphorylation is synergistic with glycolysis inhibition in treatment of pancreatic cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2329.

Abstract 1043: Enhancing cytotoxic chemotherapy effects by nintedanib in gastric cancer preclinical models

Abstract Background: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer mortality in the world. Systemic chemotherapy is a preferred treatment option for advanced and recurrent GAC, but response rates and survival extension remain limited. Tumor angiogenesis plays a critical role in GAC growth, invasion and metastasis. In this study, we determined the antitumor efficacy of nintedanib, a potent triple angiokinase inhibitor for VEGFR-1/2/3, PDGFR-α/β and FGFR-1/2/3, alone or in combination with chemotherapy, in preclinical models of GAC. Methods: Animal survival and tumor growth studies were performed in 4-6 week-old female NOD/SCID mice with human GAC cell xenografts (MKN-45, KATO-III, SNU-5). The mechanistic evaluation involved Immunohistochemistry and Immunoblot analyses of subcutaneous tumors. In vitro cell viability assays were performed using colorimetric WST-1 reagent. Results: In MKN-45 cell-derived peritoneal dissemination xenografts, animal survival was improved by nintedanib (33%), docetaxel (100%) or irinotecan (181%), while oxaliplatin, 5-FU and epirubicin had no effect. The addition of nintedanib to docetaxel (157%) or irinotecan (214%) led to a further extension in animal survival. In KATO-III cell-derived xenografts carrying FGFR2 gene amplification, nintedanib monotherapy extended survival much more (209%). Docetaxel and irinotecan effects were again further enhanced by nintedanib. In MKN-45 subcutaneous xenografts, nintedanib, epirubicin, docetaxel or irinotecan reduced tumor growth (range: 68-87%), while 5-FU and oxaliplatin had a smaller effect (40%). The addition of nintedanib to all chemotherapy groups demonstrated a further reduction in tumor growth. Subcutaneous tumor analysis revealed that nintedanib attenuated tumor cell proliferation, reduced tumor vasculature and increased tumor cell death. Nintedanib (10 μM) inhibited in vitro cell proliferation of mutationally different GAC cells by 25% (MKN-45), 75% (KATO-III) or 82% (SNU-5), and confirmed additive inhibitory effects in combinations with chemotherapy agents. Conclusion: Nintedanib showed notable antitumor efficacy and significantly improved taxane or irinotecan chemotherapy responses. Combination regimens with nintedanib have the potential for improving clinical GAC therapy. Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Frank Hilberg, Roderich E. Schwarz. Enhancing cytotoxic chemotherapy effects by nintedanib in gastric cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1043.

Kinase Inhibitors of DNA-PK, ATM and ATR in Combination with Ionizing Radiation Can Increase Tumor Cell Death in HNSCC Cells While Sparing Normal Tissue Cells.

(1) Kinase inhibitors (KI) targeting components of the DNA damage repair pathway are a promising new type of drug. Combining them with ionizing radiation therapy (IR), which is commonly used for treatment of head and neck tumors, could improve tumor control, but could also increase negative side effects on surrounding normal tissue. (2) The effect of KI of the DDR (ATMi: AZD0156; ATRi: VE-822, dual DNA-PKi/mTORi: CC-115) in combination with IR on HPV-positive and HPV-negative HNSCC and healthy skin cells was analyzed. Cell death and cell cycle arrest were determined using flow cytometry. Additionally, clonogenic survival and migration were analyzed. (3) Studied HNSCC cell lines reacted differently to DDRi. An increase in cell death for all of the malignant cells could be observed when combining IR and KI. Healthy fibroblasts were not affected by simultaneous treatment. Migration was partially impaired. Influence on the cell cycle varied between the cell lines and inhibitors; (4) In conclusion, a combination of DDRi with IR could be feasible for patients with HNSCC. Side effects on healthy cells are expected to be limited to normal radiation-induced response. Formation of metastases could be decreased because cell migration is impaired partially. The treatment outcome for HPV-negative tumors tends to be improved by combined treatment.

Combination of chemotherapy with BRAF inhibitors results in effective eradication of malignant melanoma by preventing ATM-dependent DNA repair.

Invasive malignant melanoma (MM) is an aggressive tumor with no curative therapy in advanced stages. Chemotherapy has not demonstrated its efficacy in MM and current treatment for tumors carrying the most frequent BRAFV600E mutation consists of BRAF inhibitors alone or in combination with MAPK pathway inhibitors. We previously found that BRAF inhibition prevents activation of the DNA-damage repair (DDR) pathway in colorectal cancer thus potentiating the effect of chemotherapy. We now show that different chemotherapy agents inflict DNA damage in MM cells, which is efficiently repaired, associated with activation of the ATM-dependent DDR machinery. Pharmacologic inhibition of BRAF impairs ATM and DDR activation in these cells, leading to sustained DNA damage. Combination treatments involving DNA-damaging agents and BRAF inhibitors increase tumor cell death in vitro and in vivo, and impede MM regrowth after treatment cessation. We propose to reconsider the use of chemotherapy in combination with BRAF inhibitors for MM treatment.

Phase I study of functionalized hafnium oxide nanoparticles (NBTXR3) activated by radiotherapy in cisplatin-ineligible locally advanced HNSCC patients.

6051 Background: The non-surgical standard of care (SOC) for the treatment of locally advanced head and neck squamous cell carcinoma (LA HNSCC) patients is concurrent chemoradiation with high dose cisplatin or cetuximab in case of contra-indication to cisplatin. However elderly patients, and those with poor performance status, comorbidities, and/or intolerance, may not benefit from these SOC treatments and represent a high unmet need. New approaches are thus needed to improve clinical outcomes without adding toxicity. NBTXR3, a novel radioenhancer, composed of functionalized hafnium oxide nanoparticles, is injected once intratumorally and activated by radiotherapy (RT).NBTXR3 increases the RT energy deposit inside tumor cells and subsequently increases tumor cell death compared to RT alone, while sparing healthy tissues. We present here the results of the dose expansion part of the phase I study evaluating NBTXR3 plus intensity modulated radiation therapy (IMRT) in this population. Methods: Patients with stage III-IVA or T3/T4 (AJCC/UICC TNM staging system 8th ed.) HNSCC of the oropharynx or oral cavity, ineligible to cisplatin or cetuximab and amenable for RT, received a single intratumoral injection of NBTXR3 and IMRT (70 Gy in 35 fractions /7 weeks). A classical 3 + 3 dose escalation design has tested four doses of NBTXR3, equivalent to 5, 10, 15, and 22% of baseline theoretical tumor volume. The RP2D established as 22% of baseline tumor volume is further tested in the dose expansion part. The primary endpoints of the dose expansion part are objective response rate (ORR) and complete response rate (CRR) of the primary tumor, by imaging according to RECIST 1.1. Safety is also evaluated. Results: As of August 13, 2020, 43 patients have been treated in the phase I dose expansion part. The median age was 70.7 years old (range: 50.7- 89.9), 70% of patients had cardiac disorder risk, 44% had gastrointestinal disorder risk and 44% metabolic and nutrition disorder risk. The median tumor volume was 42.8 mL (range: 1.3 - 222.3). At a median time of 7.8 months after NBTXR3 injection, the ORR of the primary lesion was 83.9% and the CRR 67.7% in the evaluable population for efficacy (N = 31). Three patients (7%) experienced at least one serious adverse event (AE) related to the injection procedure and/or NBTXR3 which represented less than 1% of all reported AEs. RT-related toxicity was as expected with IMRT. Three deaths due to AEs related to RT and other causes were reported. The recruitment is ongoing and updated efficacy and safety results will be presented. Conclusions: NBTXR3 intratumoral administration followed by IMRT may represent an option in elderly patients or patients with multiple comorbidities with LA-HNSCC who have limited therapeutic options. NBTXR3 activated by RT showed promising anti-tumor efficacy, supporting further evaluation in a phase III randomized trial. Clinical trial information: NCT01946867.

Inhibition of DNA-PK with AZD7648 Sensitizes Tumor Cells to Radiotherapy and Induces Type I IFN-Dependent Durable Tumor Control.

Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.

NEUTROPHILS ALTER DSB REPAIR PATHWAY IN INFLAMED MUCOSA TO PROMOTE COLON CARCINOGENESIS

Abstract Due to exacerbated inflammation and recurring tissue injury patients with Inflammatory Bowel Disease (IBD) are at higher risk of developing colorectal cancer (CRC). Although, PMN infiltration of the intestinal mucosa is a hallmark of IBD, and is associated with tissue injury, the contribution of PMNs to CRC and more so to IBD associated CRC is not known. We recently showed that PMNs can acutely exacerbate tissue injury and promote genomic instability by promoting miR-23a and miR-155-dependent accumulation of double-strand breaks (DSBs) and inhibition of DSB-repair by homologous recombination (HR). We now demonstrate that in chronic gut inflammation and recurring epithelial injury, as seen in IBD, via similar miRNA-dependent mechanism, PMNs alter DNA damage responses (DDR) to promote CRC progression. In murine model of Colitis-associated CRC (Azoxymethane, AOM/Dextran sodium sulfate, DSS) and human CRC xenografts, intratumoral PMNs intriguingly, functional dualism, suppressing tumor onset, but promoting tumor cell survival and growth in progressive tumors. The observed PMN effects were mediated by persistent suppression of HR-mediated DSB repair. HR suppression by PMNs, initially resulted in elevated replication stress and increased tumor cell apoptosis, however, longer term, altered DDR transcriptional profile and facilitated the upregulation of DSB repair by non-homologous end-joining (NHEJ). NHEJ upregulation enhanced CRC progression and tumor cell survival. PMN depletion, CRSPER-mediated deletion of miR-155 responsive sequence in RAD51 (preserves HR activity) or inhibition of NHEJ by small molecule inhibitors increased tumor cell death and diminished tumor development. Collectively, our data define a novel link between PMN-mediated mucosal injury and colon carcinogenesis.

Results from the phase I dose-escalation study of the radiation enhancer NBTXR3 for the treatment of HCC and liver metastases.

319 Background: NBTXR3, a first-in-class radioenhancer composed of functionalized hafnium oxide nanoparticles, is administered by one time intratumoral injection (ITI) and activated by radiotherapy (RT), such as stereotactic body RT (SBRT). NBTXR3 increases RT energy deposit inside tumor cells and subsequently increases tumor cell death compared to RT alone, while sparing healthy tissues. SBRT is well-tolerated in hepatocellular carcinoma (HCC) or liver metastases (mets) and a valuable alternative for patients not eligible for surgery or radiofrequency ablation. However, energy dose deposit to tumor cells is limited due to potential toxicity to surrounding healthy tissues. Patients with unresectable liver cancers including patients with liver or renal dysfunction might benefit from NBTXR3. A Phase 1 clinical trial was conducted to evaluate the safety and Recommended Phase 2 Dose (RP2D) of NBTXR3 activated by SBRT [NCT02721056] in these patients, here we report the results. Methods: A 3+3 dose escalation scheme tested 5 NBTXR3 levels: 10, 15, 22, 33, and 42% of baseline tumor volume. NBTXR3 ITI was followed by SBRT (45 Gy / 3 fractions / 5-7 days or 50 Gy / 5 fractions / up to 15 days) in patients with HCC or liver mets. Primary endpoints included RP2D determination and early DLT incidence. Secondary endpoints include safety profile, liver disease scores evolution, and early efficacy by response rate (mRECIST for HCC/RECIST 1.1 for liver mets). Results: 23 patients were treated: 6 patients at 10% (2 SBRT doses tested due to organ constraints), 4 patients each at 15% and 22% (due to fiducial displacement and ITI shift), 3 patients at 33% and 6 patients at 42%. No early DLT was observed at any dose level. 1 SAE (grade 3 late onset bile duct stenosis) related to NBTXR3 and RT occurred at 22%. There were 11 AEs related to NBTXR3 and/or ITI, of which grade 3 AEs were: 2 abdominal pain (ITI related; 15%) and 1 late onset bile duct stenosis (22%), lesion close to the bile duct. No NBTXR3 or ITI related grade 4-5 AEs were observed. RT related AEs were as expected with SBRT. No clinically meaningful changes in Child-Pugh score or APRI were observed post-treatment. CT-scan showed NBTXR3 present within the tumor without leakage to surrounding healthy tissues. Best observed responses (MRI) in injected target lesions from HCC patients (n = 15) were 5 CRs (33.3%), 5 PRs(33.3%), 1 SD (6.7%), 2 NE (13.3%), 2 patients did not have post baseline MRI and for liver mets (n = 8) were 5 PRs (62.5%), 2 SD (25.0%) and 1 NE (12.5%). Conclusions: No early DLTs were observed and NBTXR3 demonstrated a good safety and tolerability profile. The RP2D was determined to be 42% of tumor volume. Initial efficacy is promising and highlights the potential for NBTXR3 to address an unmet medical need in patients with unresectable primary or metastatic liver cancers. Clinical trial information: NCT02721056.

Haloperidol Induced Cell Cycle Arrest and Apoptosis in Glioblastoma Cells.

Although several antipsychotic drugs have been shown to possess anticancer activities, haloperidol, a “first-generation” antipsychotic drug, has not been extensively evaluated for potential antineoplastic properties. The aim of this study was to investigate the antitumoral effects of haloperidol in glioblastoma (GBM) U87, U251 and T98 cell lines, and the effects of combined treatment with temozolomide (TMZ) and/or radiotherapy, using 4 Gy of irradiation. The viability and proliferation of the cells were evaluated with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis, using the annexin-propidium iodide (PI), and cell cycle, cluster of differentiation (CD) expression and caspase-8 activation were measured using flow cytometry. Treatment with haloperidol significantly reduced cell viability in U87, U251 and T98 GBM cell lines. Haloperidol induced apoptosis in a dose-dependent manner, inhibited cell migration and produced an alteration in the expression of CD24/CD44. The additional effect of haloperidol, combined with temozolomide and radiation therapy, increased tumor cell death. Haloperidol was observed to induce apoptosis and to increase caspase-8 activation. In conclusion, haloperidol may represent an innovative strategy for the treatment of GBM and further studies are warranted in glioma xenograft models and other malignancies.