Increased Tumor Cell Death Research Articles

Sensitivity of Papilloma Virus-Associated Cell Lines to Photodynamic Therapy with Curcumin-Loaded Liposomes.

Simple SummaryGlobally, the burden of papilloma virus-associated cancers is high. About 5% of all cancers worldwide are caused by the human papillomavirus (HPV). Photodynamic therapy (PDT) is considered as a useful therapeutic option to treat cancers, particularly those near the tissue surface, since it is typically well tolerated and less invasive with a lower risk of severe complications as compared to conventional treatment strategies. PDT requires the combination of a photosensitizer, light of a specific wavelength, and tissue oxygen. In the present study, we examined the effectiveness of PDT together with a curcumin (liposome)-based photosensitizer in three papilloma virus-associated cell lines. PDT with curcumin liposomes could inhibit proliferation, cell migration, and colony formation of the tested tumor cells. The results suggest that curcumin-encapsulated liposomes in conjunction with PDT could be a useful tool for the treatment of papilloma virus-associated tumors.Photodynamic therapy (PDT) is a minimally invasive therapeutic approach used in the treatment of various medical conditions and cancerous diseases, involving light, a photosensitizing substance, and oxygen. Curcumin, a naturally occurring compound, carries antitumor activities and potentially could be exploited as a photosensitizer in PDT. Only little is known about liposomal-encapsulated curcumin that could help in increasing the efficacy, stability, and bioavailability of this compound. This study investigates the in vitro effects of curcumin-loaded liposomes in combination with PDT. Three papilloma virus-associated cell lines were treated with curcumin-loaded liposomes corresponding to a curcumin concentration of 0–100 µmol/L for 4 h followed by illumination at 457 nm (blue) for 45, 136, and 227 s at a fluence of 220.2 W/m2 (100 mA) corresponding to 1, 3 and 5 J·cm−2. After 24 h, the biological outcome of the treatment was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), SYTO9/PI (propidium iodide), Annexin V-FITC (fluorescein isothiocyanate)/PI, clonogenic survival, and scratch (wound closure) assays. Photoactivation of curcumin-loaded liposomes led to a significant reduction in colony formation and migratory abilities, as well as to an increase in tumor cell death. The results point to the combination of curcumin-loaded liposomes with PDT as a potentially useful tool for the treatment of papillomavirus-associated malignancies.

The Combination of Tafasitamab and Rituximab Increases Cytotoxicity Against Lymphoma Cells In Vitro

Introduction Tafasitamab (MOR208) is a CD19-targeting, Fc effector function-enhanced antibody that has shown promising clinical activity in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). For patients newly-diagnosed with DLBCL, R-CHOP regimens combining the anti-CD20 antibody, rituximab (RTX) with chemotherapy is the standard of care; however, 30-40% of patients experience relapse with a high mortality rate. CD20 is not as broadly expressed as CD19 on B-cell lymphomas, and CD19 expression is preserved on CD20-negative tumor sub-populations and following CD20-targeting therapy (Horna, et al. EHA 2020). These findings support the evaluation of immunotherapies combining anti-CD19 and anti-CD20 antibodies to target all malignant lymphoma cells. To further explore the rationale for adding tafasitamab to a RTX-containing regimen, we analyzed the effect of this combination on antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and tumor B-cell viability in vitro. Additionally, protein expression of the oncogenic transcription factor c-Myc was investigated to gain deeper insight into the molecular effects mediated by RTX and tafasitamab. Methods A comprehensive panel of 11 aggressive lymphoma cell lines was analyzed (9 DLBCL and 2 Burkitt's lymphoma). For the ADCC assays, tumor cells were incubated with tafasitamab and/or RTX at saturating concentrations in the presence of natural killer cells from healthy human donors at effector-to-target (E:T) ratios of 0.5:1 to 3:1 for 2 hours. Cytotoxicity was assessed by quantification of DAPI-positive, CFSE-labeled target cells. For the ADCP assays, lymphoma cells were incubated with tafasitamab and/or RTX at saturating concentrations in the presence of in vitro differentiated macrophages derived from healthy human donors at an E:T ratio of 2:1 for 3 hours. Phagocytosis was assessed by quantification of double-positive events (CFSE-labeled macrophages and CellTrace Violet-stained target cells). C-Myc protein levels were measured following the treatment of tumor cells with tafasitamab and/or RTX for 17-48 hours. CD19/CD20 surface expression, ADCC and ADCP rates, as well as intracellular c-Myc protein levels were analyzed by flow cytometry. Direct effects of antibody treatment on cell viability were assessed by the determination of ATP levels upon incubation of lymphoma cells for 24-96 hours. Results Increased ADCC activity for the combination of tafasitamab and RTX compared with the respective monotherapies was observed in 4/11 cell lines. For the remaining cell lines, the combination activity was similar to the most active monotherapy (tafasitamab: 5/11, RTX: 2/11). Furthermore, in 5/11 cell lines, the combination of tafasitamab and RTX resulted in enhanced ADCP activity compared with the monotherapies. Similar to ADCC, the combination activity in the other cell lines was comparable to the most active monotherapy (tafasitamab: 2/11, RTX: 4/11). Interestingly, a slight correlation between ADCC/ADCP activity and CD19/CD20 surface expression levels was observed. Treatment of tumor cells with the respective antibodies without effector cells showed a beneficial combinatorial effect of tafasitamab with RTX on the reduction of cell viability in 3/8 cell lines, whereas 2 cell lines were primarily sensitive to tafasitamab and 3 were sensitive to RTX. This observed difference in the activity profile of tafasitamab versus RTX suggests different mechanisms for induction of direct cytotoxicity (Figure 1). To further elucidate the molecular basis of the observed direct effects on cell proliferation, expression levels of c-Myc were assessed. A correlation between decreased cell viability and reduction of c-Myc expression was demonstrated. This finding is in line with the role of c-Myc in the regulation of cellular processes, such as proliferation and apoptosis. Conclusions The co-treatment of tafasitamab with RTX demonstrated superiority compared with the respective monotherapies for at least one and up to all three of the modes of action tested (ADCC, ADCP, or direct impact on cell viability) in 9/11 lymphoma cell lines. These data demonstrate that the combination of tafasitamab and RTX mediates increased tumor cell death in vitro via different mechanisms of action and support the clinical evaluation of this antibody combination in patients with DLBqCL. Disclosures Patra: MorphoSys AG: Current Employment. Augsberger:MorphoSys AG: Current Employment. Ginzel:MorphoSys AG: Current Employment. Polzer:MorphoSys AG: Current Employment. Landgraf:MorphoSys AG: Current Employment. Bartel:MorphoSys AG: Current Employment. Ness:MorphoSys AG: Current Employment. Steidl:MorphoSys AG: Current Employment. Heitmüller:MorphoSys AG: Current Employment. Schanzer:MorphoSys AG: Current Employment. Endell:Morphosys: Current Employment.

Modification of Mcl-1 alternative splicing induces apoptosis and suppresses tumor proliferation in gastric cancer.

Splicing dysregulation, which leads to apoptosis resistance, has been recognized as a major hallmark for tumorigenesis and cancer progression. Targeting alternative splicing by either increasing pro-apoptotic proteins or inhibiting anti-apoptotic proteins in tumor cells may be an effective approach for gastric cancer (GC) therapy. However, the role of modulation of alternative splicing in GC remains poorly understood. In this study, to the best of our knowledge, the unbalanced expression of the myeloid cell leukemia-1 (Mcl-1) splicing variants, Mcl-1L and Mcl-1S, was identified in GC patients for the first time. Increasing anti-apoptotic Mcl-1L and decreasing pro-apoptotic Mcl-1S expression levels were correlated with tumor proliferation and poor survival. In vitro data showed that a shift in splicing from Mcl-1L to Mcl-1S induced by treatment with Mcl-1-specific steric-blocking oligonucleotides (SBOs) efficiently decreased Mcl-1L expression, increased Mcl-1S expression, and accelerated tumor cell apoptosis in a dose-dependent manner. Additionally, mouse xenotransplant models confirmed that modification of Mcl-1 alternative splicing increased tumor cell death and suppressed tumor proliferation. This study demonstrated that the modification of Mcl-1 splicing might stimulate the pro-apoptotic factor and inhibit the anti-apoptotic protein to induce significant apoptosis. Thus, this finding provided a strategy for cancer therapy, according to which SBOs could be used to change the Mcl-1 splicing pattern, thereby inducing apoptosis.

Abstract 347: Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas

Abstract Introduction. Diffuse Intrinsic Pontine Glioma (DIPG) is a rare high-grade tumor of the brainstem that occurs almost exclusively in children. The significant mortality rates of DIPG stem largely from its capacity to invade adjacent normal brain. We have previously published studies identifying the axon guidance factor netrin-1 and its receptor neogenin as key drivers regulating brain tumor invasion in medulloblastoma and glioblastoma. Here we expand this work to investigate the role of netrin-1 and neogenin in DIPG and identify them as putative biomarkers and governors of tumor invasion. Methods. Urine of 49 DIPG patients and 42 age- and sex-matched controls was collected. Netrin-1 expression was quantified by ELISA. Results were subjected to univariate and multivariate statistical analyses and compared between clinical cohorts. In vitro experiments with established DIPG cell lines utilized RTqPCR, Western blot and flow cytometry to quantify expression of netrin-1 and neogenin. The effect of neogenin blockade on viability and invasion was assessed by flow cytometry (n=3) and transwell chamber assays (n=3) respectively. Results. Patient samples revealed a significantly low level of urinary netrin-1 compared to matched controls (p<0.005). This result was corroborated with cell lines in vitro. Interestingly, its receptor neogenin was upregulated in these cells when compared to control glial cells when quantified by mRNA, protein and FACS analysis. Blockade of neogenin in DIPG cell lines resulted in an increase of cell death and a significant decrease of cell invasion. Conclusion. Expanding on previous work, this data suggests that urinary levels of netrin-1 may have utility as a non-invasive biomarker for DIPG. Our data demonstrates the vital role of neogenin as a regulator of tumor invasion and survival in DIPG. The blockade of neogenin in DIPG cells also demonstrates the potential utility of neogenin as a cell-surface druggable target, capable of reducing tumor invasion and increasing tumor cell death. Citation Format: Julie Sesen, Jessica Driscoll, Edward Smith. Neogenin as an anti-invasive target for diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 347.

Abstract 3449: A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors

Abstract Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. There is an urgent need of better therapeutics for improving survival and quality of life for these patients. Additionally, we need improved animal models to accelerate identification of novel therapeutics and improve through-put. In line with our long-term interests of validating therapies and developing model systems for pediatric brain tumors, we tested a repurposed drug quinacrine as a potential therapy using a new and faster model system in zebrafish. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We hypothesized that quinacrine will increase tumor cell death and decrease tumorigenicity of DIPG and ATRT. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). In all tumor cell lines, quinacrine caused a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P<0.01). Quinacrine had no effect on growth of normal hindbrain neural stem and progenitor cells. Using quinacrine fluorescence as a surrogate, we achieved micromolar concentration of quinacrine in the mouse and zebrafish brain without overt toxicity. Treatment of mice bearing ATRT flank tumors with quinacrine resulted in decreased tumor volume compared to vehicle-treated mice (P<0.05). To validate quinacrine orthotopically in DIPG, we developed a novel model in zebrafish wherein we injected fluorescent DIPG cells into the developing zebrafish blastula. Two days post injection, DIPG cells had homed to the zebrafish brain. Treatment of xenografted zebrafish with quinacrine for 72 hours decreased DIPG growth by 40% as measured by fluorescence, suggesting that minor groove binding drugs like quinacrine are a viable treatment strategy for these tumors. Using this new system, we were able to validate a potential therapeutic in 7 days versus 7 months in mice. Future studies are aimed at investigating the mechanism of quinacrine in these tumors and optimizing our zebrafish model for high-throughput screening of potential drugs. Citation Format: Harpreet Kaur, Huizi Guo, Kevin Emmerich, David White, Peter Green, Alara Michelle Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Allison Martin, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3449.

Abstract 1477: LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy

Abstract In cancer blood vessels are dysfunctional, poorly perfused and leaky. Malfunctioning vessels contribute to the pro-oncogenic environment and limit the efficacy of current systemically administered drugs. Normalizing the tumor vasculature to improve vessel permeability, reduce hypoxia and vascular leakage and enhance drug delivery, has become an experimental objective in cancer research. This study was carried out to investigate the effect of blocking the secreted glycoprotein leucine-rich alpha-2-glycoprotein 1 (LRG1) on tumor vascular function, and evaluate the impact it has on the efficacy of the common standard of care chemotherapeutic drug cisplatin. Under normal conditions LRG1 is mainly expressed in the liver but also in other tissues such as bone marrow and immune cells. LRG1 has been described in multiple reports to be a serum prognostic biomarker in several cancers, for example lung, prostate, colorectal and breast. LRG1 promotes dysfunctional vessel growth by disrupting TGFβ signaling. We demonstrate that in Lrg1-/- mice and following treatment with a LRG1 function-blocking antibody (15C4) tumor growth was inhibited. In addition, we show using RNAscope that following subcutaneous grafting of the B16F0 and LL2 tumor cell lines in mice, Lrg1 is induced in tumor endothelial cells. Despite having no effect on total vessel area, the density was decreased upon LRG1 blockade, with the persisting larger vessels exhibiting improved vessel structure as evidenced by increased pericyte and basement membrane endothelial cell coverage. Better mural cell association with tumor vascular endothelial cells and basement membrane coverage are also indicators of vessel stabilization and maturation. Using a systemically delivered fluorescent lectin tracer to mark perfused vessels, we observed a significant increase in tumor perfusion in mice treated with 15C4. Lastly, vessel normalization, through LRG1 antibody blockade, significantly enhanced the efficacy of cisplatin chemotherapy as shown by a slower tumor growth rate and increased tumor cell death compared to monotherapy. These data further corroborate the hypothesis that inhibition of LRG1 improves the delivery, and hence efficacy, of a cytotoxic drug. In conclusion, deletion or inhibition of LRG1 results in an improved vascular configuration and function, and the efficacy of chemotherapy. LRG1 blockade may therefore represent a novel strategy to enhance vessel health and improve the efficacy of cancer therapeutics. Citation Format: Camilla Pilotti, Marie N O'Connor, David Kallenberg, Laura Dowsett, Jestin George, Stephen E. Moss, John Greenwood. LRG1 blockade normalizes tumor vasculature and improves efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1477.

The role of innate immunity in the protection conferred by a bacterial infection against cancer: study of an invertebrate model

All multicellular organisms are exposed to a diversity of infectious agents and to the emergence and proliferation of malignant cells. The protection conferred by some infections against cancer has been recently linked to the production of acquired immunity effectors such as antibodies. However, the evolution of innate immunity as a mechanism to prevent cancer and how it is jeopardized by infections remain poorly investigated. Here, we explored this question by performing experimental infections in two genetically modified invertebrate models (Drosophila melanogaster) that develop invasive or non-invasive neoplastic brain tumors. After quantifying tumor size and antimicrobial peptide gene expression, we found that Drosophila larvae infected with a naturally occurring bacterium had smaller tumors compared to controls and to fungus-infected larvae. This was associated with the upregulation of genes encoding two antimicrobial peptides—diptericin and drosomycin—that are known to be important mediators of tumor cell death. We further confirmed that tumor regression upon infection was associated with an increase in tumor cell death. Thus, our study suggests that infection could have a protective role through the production of antimicrobial peptides that increase tumor cell death. Finally, our study highlights the need to understand the role of innate immune effectors in the complex interactions between infections and cancer cell communities in order to develop innovative cancer treatment strategies.

Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.

Macrophages genetically engineered to secrete IL-12 induce tumor killing and immune activation in human tumor slice cultures

Abstract Cell-based immunotherapies for cancer, such as chimeric antigen receptor (CAR) T-cells, are effective against certain hematologic malignancies, but less reliably overcome the immunosuppressive tumor microenvironment (TME) of solid tumors to effectively cause cancer remission. Preclinical studies testing interleukin-12 (IL-12), an immune stimulating cytokine that activates T-cells and NK cells, as an anti-tumor agent have been promising. However, clinical trials have demonstrated that IL-12 is toxic if delivered systemically. To achieve local, low-dose delivery of IL-12, we have made genetically engineered macrophages (GEMs) to produce IL-12. In vitro, IL-12 GEMs stably produce IL-12 for at least one month. To evaluate their ability to stimulate an immune response and induce tumor cell death in an intact immune environment, we co-cultured them with tumor slices from both human colorectal cancer liver metastases and pancreatic ductal adenocarcinoma. In both tumor subtypes, the addition of IL-12 GEMs increased tumor cell death and generated an inflammatory response within the tumor slice, as evidenced by an increase in interferon-gamma and C-X-C Motif Chemokine Ligand 9 and 10, when compared to control GEMs. We demonstrate our GEM platform as a promising way to locally and sustainably deliver IL-12 to solid tumors to overcome the immunosuppressive TME.

Treatment of hepatocellular carcinoma and liver metastases with hafnium oxide nanoparticles activated by SBRT: A phase I/II trial.

537 Background: Treatment of unresectable liver cancer or liver metastases (mets) by stereotactic body radiotherapy is well tolerated but limited by the need to preserve liver function. Increasing energy deposition in the tumor while at the same time maintaining the dose in healthy tissue remains a major challenge in radiation oncology that could be achieved by NBTXR3 (hafnium oxide nanoparticles) when activated by radiotherapy (RT). NBTXR3 augments energy dose deposit within tumor cells, increasing tumor cell death compared to RT alone, while sparing healthy tissues. Patients (pts) with hepatocellular carcinoma (HCC) or liver metastasis (mets) may benefit from the mode of action of NBTXR3. A phase I/II clinical trial has been conducted to evaluate NBTXR3 activated by SBRT in these pts [NCT02721056]. Methods: The Phase I used a 3+3 dose escalation scheme with 5 NBTXR3 dose levels: 10, 15, 22, 33, and 42% of baseline tumor volume. NBTXR3 was administered by intratumoral injection (ITI) followed by SBRT (45 Gy / 3 fractions / 5 to 7 days or 50 Gy / 5 fractions / 15 days). Primary endpoints were identification of the recommended Phase II Dose and early DLTs. Secondary endpoints included safety profile, liver function evaluated by Child-Pugh score (CPS), AST to Platelet Ratio Index (APRI), and early efficacy by response rate (mRECIST/RECIST 1.1). Results: The dose escalation levels of 10, 15, 22 and 33% are completed (n = 17): 6 pts at 10% (2 SBRT doses tested due to organ constraints), 4 pts each at 15% and 22% (due to fiducial displacement and ITI shift) and 3 pts at 33%. No early DLT was observed and only one SAE (bile duct stenosis) related to NBTXR3 and RT occurred. CPS and APRI did not show clinically meaningful changes post-treatment and CT-scan showed no leakage of NBTXR3 into surrounding tissues. Best response for HCC (n = 8) were 5CR, 3PR and for mets (n = 6) the results were: 3 PR, 3SD. Conclusions: ITI of NBTXR3 is feasible, demonstrated a very good safety and tolerability profile up to the 33% dose level. Recruitment needs to be finalized at the 42% dose level. Based on early efficacy results NBTXR3 has the potential to address an unmet medical need in pts with unresectable primary or metastatic liver cancer. Clinical trial information: NCT02721056.

<p>SSBP1 Upregulation In Colorectal Cancer Regulates Mitochondrial Mass</p>

BackgroundColorectal cancers (CRC) are one of the most common forms of cancer seen worldwide, and also remain difficult to treat despite recent advances in chemotherapy. Although significant progress has been made in recent years towards precision medicine and mutation-guided therapy, common mechanisms that underlie tumor growth and progression remain incompletely understood.MethodsTumor tissue and nearby unaffected tissue were collected from >15 patients at each stage of CRC, from which we generated representative proteomics profiles of three stages. Bioinformatics analysis was performed to discover common differences that may be shared between the representative profiles and across larger cohorts. Flow cytometry was then used to identify functional consequences of SSBP1 depletion in cell lines, since its expression level was consistently increased in tumor cells across all of the datasets analyzed.ResultsDirect comparison of CRC tumor and unaffected tissue at each stage demonstrated that a number of proteins involved in mitochondrial function displayed significantly altered expression patterns. Depletion of SSBP1 in colon cancer cell lines was able to trigger loss of mitochondrial mass and an increase in tumor cell death, and this effect that was further accentuated in the presence of the common chemotherapy drug cisplatin.ConclusionMitochondrial biogenesis and maintenance may play an important part in tumor cell survival during CRC progression, and may be a useful target for directed inhibition or adjuvant targeting in the cases of cisplatin resistance.

756P - Phase I/II trial of NBTXR3 activated by SBRT in patients with hepatocellular carcinoma or liver metastasis

BackgroundTreatment of hepatocellular carcinoma (HCC) and liver metastasis (mets) is challenging due to presence of underlying disease, e.g. cirrhosis. Stereotactic body radiation therapy (SBRT) is a well-tolerated alternative for inoperable patients (pts), yet maximal dose to the tumor is limited by potential toxicity to surrounding healthy tissues. Otherwise inert, NBTXR3 (hafnium oxide nanoparticles) when activated by ionizing radiation (RT) augments dose deposit within tumor cells, increasing tumor cell death compared to RT alone. A phase I/II clinical trial is underway to evaluate NBTXR3 activated by SBRT in pts with HCC or liver mets [NCT02721056]. MethodsA 3+3 dose escalation was utilized in the phase I. Pts received a single intralesional injection (ILI) of NBTXR3 followed by SBRT (45Gy/3 fractions/5-7 days), with tested NBTXR3 dose levels of 10, 15, 22 and 33% of baseline tumor volume. Primary endpoints included recommended phase II dose(s) identification and DLT. Secondary endpoints included global safety profile assessment, liver function by Child-Pugh score (CPS), AST to Platelet Ratio Index (APRI), and response rate (mRECIST/RECIST v1.1). ResultsFour dose escalation levels are finalized (n=17): 6 pts at 10% (2 SBRT doses tested due to organ constraints), 4 pts at 15 and 22% (due to fiducial displacement and ILI site shift) and 3 pts at 33%. No NBTXR3 related DLTs were observed. Related AEs observed: one malaise (G2, 10%); 2 abdominal pain, (G3, 15%); one bilateral pleural effusion (G1, 22%), one bile duct stenosis (G3, 22%) with associated disease recurrence and SBRT; one fatigue (G1, 33%). There were no clinically meaningful changes to CPS or APRI and CT-scan demonstrated absence of NBTXR3 in surrounding healthy tissues. In 7 evaluable HCC pts, best mRECIST target lesion responses were: 3 CR, 4 PR. In 5 evaluable mets pts, best target lesion responses were: 2 PR, 1 SD, 2 PD. ConclusionsNBTXR3 was well tolerated and showed preliminary anti-tumor activity, supporting a protocol amendment to evaluate an additional NBTXR3 dose level (42%). This innovative approach has the potential to address an unmet medical need in pts with unresectable primary or metastatic liver lesions. Clinical trial identificationNCT02721056. Legal entity responsible for the studyNanobiotix SA. FundingNanobiotix SA. DisclosureM. Pracht: Honoraria (self): Nanobiotix. E. Chajon: Honoraria (self): Nanobiotix. Y. Rolland: Honoraria (self): Nanobiotix. T. de Baere: Honoraria (self): Nanobiotix. F. Nguyen: Honoraria (self): Nanobiotix. J. Bronowicki: Honoraria (self): Nanobiotix. V. Vendrely: Honoraria (self): Nanobiotix. A. Sa Cunha: Honoraria (self): Nanobiotix. A. Baumann: Honoraria (self): Nanobiotix. V. Croisé-Laurent: Honoraria (self): Nanobiotix. E. Rio: Honoraria (self): Nanobiotix. P. Said: Full / Part-time employment: Nanobiotix. S. Le Sourd: Honoraria (self): Nanobiotix. P. Gustin: Honoraria (self): Nanobiotix. C. Perret: Honoraria (self): Nanobiotix. D. Peiffert: Honoraria (self): Nanobiotix. E. Deutsch: Honoraria (self): Nanobiotix.

Abstract 3434: Selective inhibition of mTORC2 by disruption of mLST8 scaffolding function

Abstract Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that acts in two distinct complexes, mTORC1 and mTORC2, and is dysregulated in many diseases including cancer. mLST8 is a shared component of both mTORC1 and mTORC2, yet little is known regarding how mLST8 contributes to assembly and activity of the mTOR complexes. We assessed mLST8 loss in a panel of normal and cancer cells, finding little to no impact on mTORC1 activity, nor on assembly of mTOR with the mTORC1 co-factor Raptor. However, mLST8 loss blocked mTOR association with the mTORC2 co-factors Rictor and Sin1, and impaired mTORC2 kinase activity, including its phosphorylation of AKT at S473. We identified two sites within mLST8 governing its interaction with mTOR, finding that simultaneous mutation of these sites together, but not individually, disrupted not only mTOR-mLST8 interactions, but also blocked mTOR assembly with Rictor and Sin1, thus disrupting mTORC2 activity. Similary, a single mutation on mLST8 with a corresponding mutation on mTOR interfered with mTORC2 assembly, without affecting mTORC1 assembly or activity. We further discovered a direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor Sin1. Using PTEN-null prostate cancer xenografts, we found that mLST8 mutations disrupting the mTOR interaction motif inhibited AKT S473 phosphorylation, decreased tumor cell proliferation, and increased tumor cell death in vivo. Together, these data suggest that the scaffolding function of mLST8 is critical for assembly and activity of mTORC2, but not mTORC1, an observation which could enable therapeutic mTORC2-selective inhibition, while sparing the activity of mTORC1. Citation Format: Laura C. Kim, Yoonha Hwang, Wenqiang Song, Rebecca S. Cook, Jin Chen. Selective inhibition of mTORC2 by disruption of mLST8 scaffolding function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3434.

Mobilization of CD8+ T Cells via CXCR4 Blockade Facilitates PD-1 Checkpoint Therapy in Human Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA.See related commentary by Medina and Miller, p. 3747.

Abstract 2395: A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy

Abstract Background: Immunotherapies involving cytotoxic T lymphocytes (CTL’s) have been central to cancer immunotherapy. Bi-specific T cell engager antibody (BiTE) therapy has emerged as an effective immunotherapy by redirecting cytotoxic T cells against cell surface protein on tumor cells. Our BiTE antibody targets human tumor associated glycoprotein 72 (TAG-72). TAG-72 is a pan-carcinoma marker expressed on the surfaces of many cancer tissues including breast, prostate, ovary, endometrium, stomach, esophagus, and pancreas. It is not expressed in normal tissues making it an ideal target therapy for cancer. Our BiTE antibody can bind CD3 to activate the T-cell’s while simultaneously binding TAG-72 on the surface of the cancer cell leading to perforin induced cancer cell death. The in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells are presented here. Methods: We studied in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells; breast (DOX resistant MCF-7, SKBR3, MDA-MB-175 VII), ovarian (OVCAR-3), endometrium (KLE) and colon (Ls174T). In vitro cytotoxicity assay was performed by flow cytometry and immunofluorescence staining using live/ dead cell labelling fluorescent dyes. The cells were co-cultured with naïve human CD8 T cells with/ without BiTE antibody (1 µg, 5 µg and 10 µg) for different time intervals and assayed. In vivo study was carried out in NSG mice implanted with MCF-7 breast cancer cells + naïve human CD8 T cells and treated with a total of 4 doses of BiTE antibody (200µg/ dose). Results: Different tumor cells showed varying (%) of TAG-72 expression determined by flow cytometry, viz; DOX (55%), SKBR3 (16%), MDA 175 VII (8%), OVCAR-3 (27%), KLE (27%), Ls174T (30%). The BiTE antibody also showed dose dependent binding to these cells. Flow cytometry analysis showed that addition of the BiTE antibody to naïve CD8 T-cells and incubation for 4 days significantly increases tumor cell death as indicated by the Annexin V/ PI+ve signal from DOX (52.4% vs. 41.4% CD8 alone), SKBR-3 (47.9% vs. 30% CD8 alone), MDA 175 VII (23% vs 11% CD8 alone), OVCAR-3 (31.1% vs. 23.8% CD8 alone), KLE (27% vs 13% CD8 alone) and Ls174T (54% vs 29% CD8 alone) respectively (p<0.05). Further, a significant level of IFN-γ secreted by activated T-cells was only detected in the bispecific T-cell engager Ab + CD8 conditions. In vivo data showed good tumor regression at day 45 with tumors measuring (8 mm2) in group treated with BiTE antibody as compared to untreated group (31 mm2) (p<0.01). Conclusion: Our in vitro and in vivo data indicates that the TAG-72/ CD3 BiTE antibody can effectively redirect cytotoxic T cells against different cancer cells expressing TAG-72 glycoprotein and may be effective in the treatment of breast, ovarian, endometrium and colon cancers. Citation Format: Fatema Khambati, Hatem Soliman. A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2395.

Hafnium oxide nanoparticles NBTXR3 activated by radiotherapy as a new therapeutic option for elderly/frail HNSCC patients.

6069 Background: New therapeutic approaches are needed for elderly or frail head and neck squamous cell carcinoma (HNSCC) patients (pts) ineligible for standard of care treatment. NBTXR3, a crystalline solution of hafnium oxide nanoparticles may represent such an option. Injected intratumorally, NBTXR3 enters tumor cells and yields an increased cell-localized energy deposit upon exposure to radiotherapy (RT), leading to increased tumor cell death compared to the same dose of RT alone. Methods: Phase I study of NBTXR3 activated by RT in pts ≥70 years old or ≥65 years old and unable to receive cisplatin, eligible for exclusive RT with stage III or IV HNSCC of the oral cavity or oropharynx [NCT01946867]. A 3+3 dose escalation design was implemented with dose levels corresponding to 5%, 10%, 15% and 22% of baseline tumor volume, followed by an expansion phase. Pts received an intratumoral (IT) injection of NBTXR3 and intensity modulated RT (IMRT; 70 Gy/35 fractions/7 weeks). Determination of Recommended Phase 2 Dose (RP2D) and Dose Limiting Toxicities (DLT) were primary endpoints of phase I. Absence of NBTXR3 leakage and preliminary efficacy using RECIST 1.1 principles were also evaluated. Results: The dose-escalation is complete. Nineteen pts were enrolled: 3 at 5%, 3 at 10%; 5 at 15% and 8 at 22% with no observed DLT or SAE related to NBTXR3 or IT injection. One grade 1 NBTXR3-related AE (asthenia at 22%) and four IT injection-related AE (grade 2 oral pain; grade 1 tumor hemorrhage; grade 1 asthenia, and grade 1 injection site hemorrhage) were reported. RT-related toxicity was as expected with IMRT. RP2D has been determined to be 22%. CT-scan assessment between 24h and 7 weeks post-IT injection demonstrated absence of NBTXR3 leakage in the surrounding tissues. Among 13 evaluable pts treated at doses ≥10%, 9 achieved a complete response of the injected lesion. Conclusions: These results show that NBTXR3 activated by RT is safe and well tolerated at all doses with preliminary encouraging efficacy results. It thus represents a promising future treatment for frail and elderly pts with locally advanced HNSCC with limited therapeutic options. Expansion phase has started at the RP2D. Clinical trial information: NCT01946867.

Inhibition of Sphingosine Phosphate Receptor 1 Signaling Enhances the Efficacy of VEGF Receptor Inhibition.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Hepatocellular carcinoma and liver metastasis treated by hafnium oxide nanoparticles activated by SBRT: A phase I/II trial.

340 Background: Hafnium oxide nanoparticles, NBXTR3, were developed to increase the tumor-localized high energy deposit once activated by ionizing radiation such as stereotactic body radiotherapy (SBRT) and thus to increase tumor cell death compared to the same dose of radiation. NBTXR3 is characterized by a single intratumor/intralesional (IL) administration and fits into standard RT schedule with no change in patient’s flow, treatment protocol or equipment. Herein the preliminary results of a phase I/II clinical trial evaluating this combination in patients (pts) with hepatocellular carcinoma (HCC) or liver metastasis (mets). Methods: HCC and liver mets patients were treated with an IL injection of NBTXR3 followed by SBRT (15 Gy∗3 fractions). The phase I part of the trial follows a 3+3 dose escalation design at dose levels of NBTXR3 corresponding to 10%, 15%, 22%, 33% of the baseline tumor volume. This study aims primarily to identify the Recommended Dose and the incidence of early Dose Limiting Toxicities (DLTs) of NBTXR3 activated by SBRT. Secondary endpoints include assessment of global safety profile, liver function evaluated by Child-Pugh score (CPS), AST to Platelet Ratio Index (APRI), and response rate (mRECIST/RECIST 1.1). Results: Enrollment is at the last dose level, 33%, and completed at 10% (6 pts), 15% (4 pts) and 22% (4 pts). So far, no early DLTs nor severe adverse events related to NBTXR3 were observed. Both CPS and APRI did not reveal important variations in accordance to NBTXR3 low toxicity. The best observed target lesions responses, among 7 evaluable HCC pts for response (mRECIST), were: 3 complete responses, 3 partial responses (PR) and 1 stable disease (SD) and among 5 evaluable liver mets pts: 1 PR, 3 SD and 1 progressive disease (RECIST 1.1). Conclusions: NBTXR3 is well tolerated at the 22% dose level with an overall positive safety profile. This innovative approach might constitute a valuable solution for pts with liver tumors beyond standard treatment lines. NBTXR3 was successful in a phase II/III in soft tissue sarcoma [NCT02379845] and is currently evaluated in head and neck [NCT01946867; NCT02901483], prostate [NCT02805894] and rectum cancers. Clinical trial information: NCT02721056.

Abstract B057: Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown

Abstract Triple-negative (TNBC) and HER2+ breast cancers (BCs) are especially aggressive tumors, which are prone to relapse and metastasize post chemo- or targeted therapy. Immunotherapy provides a promising, but unproven, alternative approach for treating poor-prognosis BCs. BCs have relatively low nonsynonymous mutation rates, suggesting that many BCs will respond poorly to immunotherapy. Novel strategies to increase BC cell immunogenicity and improve tumor-antigen specific T-cell responses are needed to enhance the efficacy of BC immune therapy. We aim to enhance the immunogenicity of breast tumor by taking advantage of the unique strength of EpCAM-aptamer conjugated small interfering RNAs (AsiCs), which can knock down gene expression selectively in EpCAM+ BC cells, to make aggressive BCs visible to T-cells and improve T-cell tumor recruitment and function. EpCAM is a tumor-specific antigen expressed at several logs higher levels on all epithelial cancers and cancer stem cells relative to normal epithelia. The EpCAM aptamer our lab developed binds with high affinity to both mouse and human EpCAM. Using this platform, we investigated the use of EpCAM-AsiCs for BC immune modulation by targeting genes controlling different functional processes: 1) knocking down the nonsense-mediated mRNA decay (NMD) gene UPF2, to elicit tumor neoantigen expression to improve BC tumor recognition by the immune system; 2) knocking down CD47 to promote phagocytosis of cancer cells and enhance cross-presentation of tumor antigens; 3) knocking down DNA repair enzyme PARP1 to produce more DNA damage and consequent genetic mutations, thereby introducing tumor-specific neoantigens to the immune system; 4) knocking down PLK1 which is essential for cell mitosis, and MCL1, which is a critical survival factor in TNBC, to directly kill tumor cells. The increased tumor cell death induced by PLK1 and MCL1 knockdown could potentially promote tumor antigen cross-presentation to CD8+ T-cells. Our data indicate that each of these AsiCs potently silences gene expression within mouse or human BC cell lines in vitro, and efficiently reduce target gene expression in EpCAM+ tumor cells in vivo. When treating mice bearing 4T1E (4T1 cell line with high EpCAM expression) tumors, EpCAM-AsiCs targeting UPF2, CD47, PARP1, PLK1 and MCL1 each significantly suppressed 4T1E tumor growth, increased CD8+ T-cell tumor infiltration, CD8+ T-cell/regulatory T-cells (Treg) ratio, the effector functions and degranulation capacity of CD8+ TILs compared to those of the control group. The capacity of these CD8+ TILs to directly kill tumor cells was also improved. Tumor-associated macrophages (TAMs) from CD47-AsiC treated tumors showed improved phagocytic capacity as determined by in vitro and in vivo phagocytosis assays. CD47-AsiC also increased the ratio of M1 (tumor suppressive) to M2 (tumor promoting) TAMs. Dendritic cells (DCs) from CD47-AsiC treated tumors show enhanced CD40 and CD86 expression, suggesting an improved immunostimulatory capacity. Using antibody depletion assay, we further show that CD4+ T-cells, CD8+ T-cells and macrophages all contribute to the tumor inhibition capacity of CD47-AsiC; and TAMs/DC play an important role in stimulating the functions of CD8+ TILs. In addition, we show that CD47-AsiC outperforms the anti-CD47 antibody in suppressing 4T1E tumor progression. Finally, we show that UPF2, CD47, PARP1, PLK1 and MCL1 AsiCs work in synergy to further delay tumor growth, and even lead to tumor regression, compared to single AsiC treatment. The combination therapy significantly increased the amount of CD8+ TILs, reduced the Tregs and myeloid-derived suppressor cells (MDSCs) in the tumor, and improved the functions of both CD4+ and CD8+ TILs. Our data suggest that these novel EpCAM-AsiCs, with their small size and high selectivity, have great potential to improve therapeutic efficacy and reduce toxicity for BC patients compared to current checkpoint inhibitors. Citation Format: Ying Zhang, Judy Lieberman. Enhancing immunotherapy for triple-negative and HER2+ breast cancer using EpCAM aptamer-siRNA mediated gene knockdown [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B057.

NEO412: A temozolomide analog with transdermal activity in melanoma in vitro and in vivo.

Despite new treatments introduced over the past several years, metastatic melanoma remains difficult to cure. Although melanoma in situ (MIS) has better prognosis, it relies heavily on thorough surgical excision, where ill-defined margins can pose a challenge to successful removal, potentially leading to invasive melanoma. As well, MIS in the head and neck area can create serious aesthetic concerns with regard to the surgical defect and substantial scar formation. Toward improved treatment of localized melanoma, including the targeting of unrecognized invasive components, we have been studying a novel agent, NEO412, designed for transdermal application. NEO412 is a tripartite agent that was created by covalent conjugation of three bioactive agents: temozolomide (TMZ, an alkylating agent), perillyl alcohol (POH, a naturally occurring monoterpene with anticancer properties), and linoleic acid (LA, an omega-6 essential fatty acid). We investigated the anti-melanoma potency of NEO412 in vitro and in mouse models in vivo. The in vitro results showed that NEO412 effectively killed melanoma cells, including TMZ-resistant and BRAF mutant ones, through DNA alkylation and subsequent apoptosis. in vivo, NEO412 inhibited tumor growth when applied topically to the skin of tumor-bearing animals, and this effect involved a combination of increased tumor cell death with decreased blood vessel development. At the same time, drug-treated mice continued to thrive, and there was no apparent damage to normal skin in response to daily drug applications. Combined, our results present NEO412 as a potentially promising new treatment for cutaneous melanoma, in particular MIS, deserving of further study.