Significant Tumor Growth Delay Research Articles

Abstract A153: Targeting DNA damage response upregulates PD-L1 level and promotes antitumor immunity in small-cell lung cancer

Abstract Purpose of the Study: Despite recent advances in the use of immunotherapy, only a minority of small cell lung cancer (SCLC) patients respond to immune checkpoint blockade (ICB) with programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) antibodies as monotherapy or combination. Therefore, there is a critical need to develop strategies to enhance the efficacy of ICB in SCLC, an otherwise immunosuppressed disease with dismal 5-year survival rate of <5%, which has remained unchanged for >30 years. We have previously shown that prexasertib and olaparib treatment enhanced the protein (but not mRNA) and surface expression of PD-L1 in SCLC cell lines and tumor models. Here we elucidate the previously unexplored mechanism of how DNA damage response targeting enhances antitumor immunity in SCLC. Methods: SCLC cell lines and immune competent murine models were treated with inhibitors targeting DNA damage repair (DDR) proteins, checkpoint kinase 1 (CHK1) (by prexasertib), poly (ADP-ribose) polymerase (PARP) (by olaparib) either as single agents or in combination with anti-PD-L1. End point analyses were done by Western blot, real-time RT-PCR, multicolor flow cytometry and reverse phase protein array (RPPA). Result: We observed increased PD-L1 glycosylation post-prexasertib and olaparib treatment. Prexasertib and/or olaparib +/- anti-PD-L1 combination activates mTOR and inactivates GSK3β pathway thus providing mechanistic insight into DDR-targeting mediated PD-L1 glycosylation and stabilization. We treated tumor-bearing immune-competent B6129F1 mice with either IgG (control), prexasertib (10mg/kg, 2/7), anti-PD-L1 (300ug, 1/7) or combination (n=10/group). At Day 21, anti-PD-L1 did not cause tumor regression and prexasertib treatment delayed tumor growth [T/C=0.31(p<0.001)]. However, most notably, 60% mice treated with anti-PD-L1+ prexasertib had a complete tumor regression within 15 days of treatment and the other 40% had no further increase in tumor growth beyond baseline [T/C=0.05(p<0.001)]. Co-targeting CHK1+PD-L1 significantly increases the level of CD8+ cytotoxic-T-cell tumor infiltration and decreases exhausted T-cell population in SCLC models. CD8 depletion was able to reverse the antitumor effect of the combination demonstrating the role of cytotoxic T-cell infiltration in CHK1i+PD-L1 response. Next, we treated the tumor-bearing B6129F1 mice with either IgG (control), olaparib (100mg/kg, 4/7), anti-PD-L1 (300ug, 1/7) or combination (n=10/group). At Day 21, anti-PD-L1 or olaparib did not cause tumor regression. However, there was a significant delay in tumor growth when the mice were treated with olaparib+anti-PD-L1 combination [T/C=0.26(p<0.001)]. Immune profiling demonstrated increased CD8+ cytotoxic-T-cell tumor infiltration, decreases exhausted T-cell population in the olaparib+anti-PD-L1 combination group. Conclusions: Expression of PD-L1 is associated with tumors deficient in DNA damage repair and we, for the first time, provide rationale for investigating the role of immunotherapy in the context of DDR targeting in SCLC. This study further demonstrates the novel mechanism of DDR-mediated PD-L1 regulation in SCLC via GSK3β inactivation and stabilization. Moreover, our results demonstrating the remarkable efficacy of the combination of PD-L1 blockade with PARP and CHK1 inhibition provide a strong scientific rationale for combining these modalities in clinical trials for SCLC patients. Combining DDR inhibition with ICB increases CD8+ cytotoxic T-cell infiltration in SCLC models. The immune profiling and correlative biomarker data from our study further provide valuable mechanistic insight and indicate the subset of the patient population who are most likely to respond to these treatments. Because prexasertib, olaparib and other PARP inhibitors are already in clinical trials for SCLC, we expect that this hypothesis has the potential for rapid translation into the clinic. Citation Format: Triparna Sen, Bertha Leticia Rodriguez, Limo Chen, Naoto Morikawa, Junya Fujimoto, Lixia Diao, Youhong Fan, Jing Wang, Bonnie S. Glisson, Ignasio Wistuba, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. Targeting DNA damage response upregulates PD-L1 level and promotes antitumor immunity in small-cell lung cancer [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 A153.

TGFB1-driven mesenchymal stem cell-mediated NIS gene transfer.

Based on their excellent tumor-homing capacity, genetically engineered mesenchymal stem cells (MSCs) are under investigation as tumor-selective gene delivery vehicles. Transgenic expression of the sodium iodide symporter (NIS) in genetically engineered MSCs allows noninvasive tracking of MSC homing by imaging of functional NIS expression as well as therapeutic application of 131I. The use of tumor stroma-activated promoters can improve tumor-specific MSC-mediated transgene delivery. The essential role of transforming growth factor B1 (TGFB1) and the SMAD downstream target in the signaling between tumor and the surrounding stroma makes the biology of this pathway a potential option to better control NIS expression within the tumor milieu. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by a synthetic SMAD-responsive promoter (SMAD-NIS-MSCs). Radioiodide uptake assays revealed a 4.9-fold increase in NIS-mediated perchlorate-sensitive iodide uptake in SMAD-NIS-MSCs after TGFB1 stimulation compared to unstimulated cells demonstrating the successful establishment of MSCs, which induce NIS expression in response to activation of TGFB1 signaling using a SMAD-responsive promoter. 123I-scintigraphy revealed significant tumor-specific radioiodide accumulation and thus NIS expression after systemic application of SMAD-NIS-MSCs into mice harboring subcutaneous tumors derived from the human hepatocellular carcinoma (HCC) cell line HuH7, which express TGFB1. 131I therapy in SMAD-NIS-MSCs-treated mice demonstrated a significant delay in tumor growth and prolonged survival. Making use of the tumoral TGFB1 signaling network in the context of MSC-mediated NIS gene delivery is a promising approach to foster tumor stroma-selectivity of NIS transgene expression and tailor NIS-based gene therapy to TGFB1-rich tumor environments.

A Novel Approach for Image-Guided 131I Therapy of Pancreatic Ductal Adenocarcinoma Using Mesenchymal Stem Cell-Mediated NIS Gene Delivery.

The sodium iodide symporter (SLC5A5/NIS) as theranostic gene would allow for non-invasive imaging of functional NIS expression and therapeutic radioiodine application. Genetically engineered mesenchymal stem cells (MSC), based on their tumor-homing abilities, show great promise as tumor-selective NIS gene delivery vehicles for non-thyroidal tumors. As a next step towards clinical application, tumor specificity and efficacy of MSCs were investigated in an advanced genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC). Syngeneic murine MSCs were stably transfected with a NIS-expressing plasmid driven by the CMV-promoter (NIS-MSC). In vivo 123I-scintigraphy and 124I-PET revealed significant perchlorate-sensitive NIS-mediated radioiodide accumulation in PDAC after systemic injection of NIS-MSCs. Active MSC recruitment into the tumor stroma was confirmed using NIS immunohistochemistry (IHC). A therapeutic strategy, consisting of three cycles of systemic MSC-mediated NIS delivery, followed by 131I application, resulted in a significant delay and reduction in tumor growth as compared to controls. Furthermore, IHC analysis of α-SMA and Ki67 revealed differences in the amount and behavior of activated fibroblasts in tumors of mice injected with NIS-MSCs as compared with saline-treated mice. Taken together, MSCs as NIS gene delivery vehicles in this advanced endogenous PDAC mouse model demonstrated high stromal targeting of NIS by selective recruitment of NIS-MSCs after systemic application resulting in an impressive 131I therapeutic effect. IMPLICATIONS: These data expand the prospect of MSC-mediated radioiodine imaging-guided therapy of pancreatic cancer using the sodium iodide symporter as a theranostic gene in a clinical setting.

Antitumor Activity of Auger Electron Emitter 111In Delivered by Modular Nanotransporter for Treatment of Bladder Cancer With EGFR Overexpression.

Gamma-ray emitting 111In, which is extensively used for imaging, is also a source of short-range Auger electrons (AE). While exhibiting negligible effect outside cells, these AE become highly toxic near DNA within the cell nucleus. Therefore, these radionuclides can be used as a therapeutic anticancer agent if delivered precisely into the nuclei of tumor target cells. Modular nanotransporters (MNTs) designed to provide receptor-targeted delivery of short-range therapeutic cargoes into the nuclei of target cells are perspective candidates for specific intracellular delivery of AE emitters. The objective of this study was to evaluate the in vitro and in vivo efficacy of 111In attached MNTs to kill human bladder cancer cells overexpressing epidermal growth factor receptor (EGFR). The cytotoxicity of 111In delivered by the EGFR-targeted MNT (111In-MNT) was greatly enhanced on EJ-, HT-1376-, and 5637-expressing EGFR bladder cancer cell lines compared with 111In non-targeted control. In vivo microSPECT/CT imaging and antitumor efficacy studies revealed prolonged intratumoral retention of 111In-MNT with t½ = 4.1 ± 0.5 days as well as significant dose-dependent tumor growth delay (up to 90% growth inhibition) after local infusion of 111In-MNT in EJ xenograft-bearing mice.

Resistance to Radiotherapy and PD-L1 Blockade Is Mediated by TIM-3 Upregulation and Regulatory T-Cell Infiltration.

Purpose: Radiotherapy (RT) can transform the immune landscape and render poorly immunogenic tumors sensitive to PD-L1 inhibition. Here, we established that the response to combined RT and PD-L1 inhibition is transient and investigated mechanisms of resistance.Experimental Design: Mechanisms of resistance to RT and PD-L1 blockade were investigated in orthotopic murine head and neck squamous cell carcinoma (HNSCC) tumors using mass cytometry and whole-genome sequencing. Mice were treated with anti-PD-L1 or anti-TIM-3 alone and in combination with and without RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Depletion of regulatory T cells (Treg) was performed using anti-CD25 antibody.Results: We show that the immune checkpoint receptor, TIM-3, is upregulated on CD8 T cells and Tregs in tumors treated with RT and PD-L1 blockade. Treatment with anti-TIM-3 concurrently with anti-PD-L1 and RT led to significant tumor growth delay, enhanced T-cell cytotoxicity, decreased Tregs, and improved survival in orthotopic models of HNSCC. Despite this treatment combination, the response was not durable, and analysis of relapsed tumors revealed resurgence of Tregs. Targeted Treg depletion, however, restored antitumor immunity in mice treated with RT and dual immune checkpoint blockade and resulted in tumor rejection and induction of immunologic memory.Conclusions: These data reveal multiple layers of immune regulation that can promote tumorigenesis and the therapeutic potential of sequential targeting to overcome tumor resistance mechanisms. We propose that targeted Treg inhibitors may be critical for achieving durable tumor response with combined radiotherapy and immunotherapy. Clin Cancer Res; 24(21); 5368-80. ©2018 AACR.

Molecular Profiling of Head and Neck Cancer Patient Derived Xenografts Identifies FGFR As a Target for Radiosensitization

The fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3) are frequently altered or overexpressed in head and neck squamous cell carcinomas (HNSCC). Amplification of FGFR1 has been reported to be as high as 17% in this disease. FGFRs activate multiple pathways involved in the radiation response including RAS/RAF/MAPK, PI3K/AKT and STAT. AZD4547 is a potent and selective tyrosine kinase inhibitor of the FGFR family and is currently being investigated in clinical trials. We therefore examined the anti-tumor effects of AZD4547 in combination with radiation in HNSCC model systems. HNSCC patient derived xenografts were screened using FGFR1 and FGFR2 RNA in situ hybridization. A panel of HNSCC cell lines was screened for FGFR1/2 protein and RNA expression and response to AZD4547. Immunoblots were used to examine the effect of AZD4547 on downstream signaling proteins. Radiation clonogenic survival assays and xenograft growth delays experiments were performed to investigate radiosensitization. Out of the 22 HNSCC patient derived xenografts examined, three were found to have FGFR1/2 expression. This included one p16 negative tumor and two p16 positive tumors. Nine cell lines demonstrated varying degree of FGFR1/2 protein and RNA expression. The half-maximal inhibitory concentration (IC50) of AZD4547 in these cell lines ranged from 0.05 to 42.2 uM. Sensitivity to AZD4547 did not directly correlate with protein or RNA expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time dependent manner. In vitro clonogenic survival assays showed enhancement in radiosensitivity with AZD4547 in two HNSCC cell lines. AZD4547 induced significant growth inhibition in a FGFR1 expressing patient derived xenograft. Significant tumor growth delay was observed with the combination of radiation and AZD4547 compared to radiation or drug alone in a FGFR2 expressing cell line HNSCC xenograft model. These findings suggest that AZD4547 can augment the response of radiation HNSCC in vivo model systems. FGFR1 and FGFR2 expression may provide a potential target for radiosensitization in HNSCC. Additional studies are underway to understand the mechanism of radiosensitization.

Administration of Beta-Emitting Anti-CD37 Radioimmunoconjugate Lutetium (177Lu) Lilotomab Satetraxetan as Weekly Multiple Injections Increases Maximum Tolerated Activity in Nude Mice with Non- Hodgkin Lymphoma Xenografts

Lutetium (177Lu) lilotomab satetraxetan (177Lu-lilotomab) is a novel anti-CD37 radioimmunoconjugate (RIC) currently in Phase 2b clinical trial for treatment of non-Hodgkin lymphoma (NHL). The aim of the current study was to investigate tolerability and anti-tumor efficacy of multiple dosing of 177Lu-lilotomab in vivo. Nude mice with subcutaneous (s.c.) NHL (Ramos) xenografts were given 2, 3 or 4 weekly injections of 300 MBq/kg 177Lu-lilotomab or NaCl. Treatment tolerability was assessed by body weight, hematology and histopathological evaluation. A separate experiment in mice without xenografts was performed to collect long term toxicity data. Mice in this study were dosed more conservatively with the intention that all mice should survive until end of experiment and were administered 2 or 4 weekly injections of 200 MBq/kg 177Lu-lilotomab or NaCl. Total accumulated activity of 900 MBq/kg 177Lu-lilotomab given as three weekly injections was tolerated by nude mice with s.c. Ramos xenografts, which is 50 % higher than the maximum tolerated activity (MTA) of a single injection of 530-600 MBq/kg. In the long-term toxicity animals, the highest accumulated activity tested, 800 MBq/kg, was also well tolerated. Radioactivity-dependent transient reduction in white blood cell and platelet counts occurred in all treated groups, with nadir 1-3 weeks after last injection. This toxicity was radioactivity dependent and consistent with histopathological changes in hemolymphopoietic tissues. Significant tumor growth delay measured as time to reach 4 times intial tumor volume was observed in all groups given multiple injections of 300 MBq/kg 177Lu-lilotomab compared to NaCl control group (p<0.001). In conclusion, weekly injections of 177Lu-lilotomab increase the accumulated MTA from 530 to 900 MBq/kg in nude mice, allowing the total injected activity and hence the radiation dose to tumor to be increased without increasing the toxicity to normal tissues.

Oligosaccharyltransferase Inhibition Overcomes Therapeutic Resistance to EGFR Tyrosine Kinase Inhibitors.

Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling.Significance:EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094-106. ©2018 AACR.

Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating Mesenchymal-Epithelial Transition Factor (MET) Signaling.

Malignant gliomas are themost common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10-10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.

Abstract 2545: Antitumor efficacy of Type I polarized dendritic cells in combination with immune checkpoint blockade in a preclinical model of breast cancer

Abstract Breast cancer is the most commonly diagnosed cancer and a major cause of cancer death among women. Our group has shown progressive loss of the anti-HER2 Th1 immune response in HER2 positive breast cancer patients and administration of peptide-pulsed Type I polarized dendritic cell (DC1) vaccine induced strong anti-HER2 immune response with pathologic complete response rate (pCR) in DCIS (ductal carcinoma in situ) and early invasive breast cancer (IBC). Within the tumor microenvironment, presence of multiple factors, including expression of co-inhibitory immune checkpoint signals has been shown to inhibit anti-tumor immune responses and preventing complete tumor regression. Hence, there is a need to develop strategies that disrupt these negative regulators in the tumor microenvironment in order to achieve robust anti-tumor immune responses. In this study, we investigated whether blockade of immune checkpoints, PD-1 or PD-L1 in combination with DC1 vaccine enhance anti-tumor immune response in a preclinical model of HER2 positive TUBO breast cancer. Balb/c mice received TUBO cells subcutaneously on day 0. Two different treatment regimens were followed to examine anti-tumor efficacy. For regimen-1, TUBO- bearing mice received three doses of p66 (MHC class I) peptide-pulsed DC1 vaccine starting on day 7 at weekly intervals concurrently with intraperitoneal injection of anti-PD-1 or anti-PD-L1 monoclonal antibodies. For regimen-2, starting on day 7 mice received six doses of HER2 specific p66 peptide- pulsed DC1 vaccine twice a week. Monoclonal checkpoint antibodies were given after the last dose of DC1 vaccine. Mice that received DC1 vaccine followed by treatment with anti-PD-1 or anti-PD-L1 monoclonal checkpoint antibodies had significant delay in tumor growth compared to the mice that received DC1 or checkpoint antibodies alone. However, mice that received DC1 in combination with anti-PD-1 antibody had superior efficacy in delayed tumor growth and improved survival rate. TUBO- bearing mice that received regimen-1, with DC1 vaccine and checkpoint antibodies concurrently had no significant delay in the tumor growth compared to mice treated with DC1 alone. Induction of anti-tumor immune response using HER2 specific MHC class II peptide- pulsed DC vaccine in combination with immune checkpoint blockade is ongoing. Overall, these findings suggest that optimal scheduling of immune checkpoint blockade is critical in improving anti-tumor efficacy of DC1 vaccine. This could be a potential combinatorial approach for the treatment of HER2 positive breast cancer in clinical settings. Citation Format: Krithika N. Kodumudi, Doris Wiener, Amrita Basu, Brian Czerniecki. Antitumor efficacy of Type I polarized dendritic cells in combination with immune checkpoint blockade in a preclinical model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2545.

Abstract 1145: Humanized immune-PDX mouse models for 10 different tumor entities in translational immuno-oncology research

Abstract We established humanized mouse models by transplantation of human hematopoietic (stem) cells to immunodeficient mice. Engraftment results in the development of a functional human immune system. By co-transplantation of human patient-derived xenografts (PDX) we successfully generated a fully human tumor-immune-cell (TIC) model in mice. These humanized TIC-PDX models for melanoma and lung cancer were further characterized by treatment with immune therapy drugs like the CTLA4 inhibitor ipilimumab (Ipi) and the PD-1 inhibitor nivolumab (Nivo). In our current studies we monitored the effects of the human immune cells and evaluated concepts for combination therapies i.e. with chemotherapy or radiation. We further established new human TIC-PDX models from other tumor entities like lymphoma, pancreatic or breast cancer. Hematopoietic stem cell engraftment was monitored by regular FACS analysis for human immune cells in the blood. PD-L1 expression on tumors as a target for immunotherapy was determined by FACS and immunohistochemistry. PD-L1 positive and negative PDX from 10 different entities were transplanted on humanized mice and treated with Nivo or Ipi alone or in combination therapy with radiation. Blood and tumor samples were analysed by FACS and immunohistochemistry for immune cell infiltration and activation. The transplanted stem cells showed engraftment in immunodeficient mice with proliferation and differentiation and established a functional human immune system with T-cells, B-cells, NK-cells, monocytes, and dendritic cells. So far, we have transplanted more than 40 different PDX from 10 different tumor entities on humanized mice. Most of investigated PDX (>70%) successfully engrafted on humanized mice and showed no difference in tumor growth compared to growth on non-humanized mice. However, for a few PDX we observed a delayed tumor growth or a complete rejection. These results suggest an immune reaction of the engrafted human immune cells against the PDX. The tumor engraftment or rejection in the humanized mice seems to be independent from the tumor entity. To evaluate whether engraftment of the PDX in mice with a human immune system is in part enabled by checkpoint mediated immune-tolerance, we have treated over 30 PDX models with Ipi or Nivo alone or in combination. Significant tumor growth delay, observed in most models, accompanied by an increased number of T-cells in the blood and in the tumor, has functionally validated our immune-PDX model. Response to the checkpoint inhibitors seems to correlate with innate immune responsiveness and PD-L1 expression and can be further increased by combination with radiotherapy. Our humanized immune-PDX models enable appropriate preclinical translational research in tumor immune biology and the evaluation of new therapies and combinations, as well as the identification and validation of biomarkers for immune therapy. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Magdalena Paterka, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Humanized immune-PDX mouse models for 10 different tumor entities in translational immuno-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1145.

Abstract LB-054: FKBPL as a novel therapeutic target and prognostic biomarker in high grade serous ovarian cancer

Abstract FKBPL is a secreted protein with well-established anti-angiogenic and anti-cancer stem cell activity. A novel therapeutic peptide, ALM201, derived from this protein has successfully completed a phase I clinical trial and was granted orphan drug status, by the FDA, for the treatment of ovarian cancer. Ovarian cancer is the most lethal gynecological cancer and there are no prognostic biomarkers currently used in the clinic. It is associated with a high incidence of recurrent chemo-resistant disease and this has been attributed to a treatment-resistant subpopulation of CD44+ cancer stem cells (CSCs). Previously, we presented evidence that CD44 is a potential target of ALM201 and here, for the first time, we investigate its ability to target both angiogenesis and CSCs in HGSOC. Tumorsphere and flow cytometry assays demonstrated that ALM201 is effective at reducing CSCs in a range of HGSOC cell lines and primary patient samples in vitro. Clonogenic assays demonstrated that ALM201 is not cytotoxic, but rather mediates ovarian CSC differentiation. In vitro, ALM201 displayed potent anti-CSC activity in the HGSOC cell line, OVCAR3. However, OVCAR3 tumor xenografts are not dependent on angiogenesis for their growth and therefore ALM201 did not elicit a strong response in this model. Furthermore, OVCAR3 xenografts dramatically upregulated the inflammatory cytokines IL-6 and IL-8, which is consistent with the lack of anti-CSC activity of ALM201 in vivo compared to in vitro. In contrast, treatment of highly angiogenic Kuramochi tumor xenografts, another HGSOC cell line, resulted in a statistically significant delay in both tumor growth and tumor initiation. This was associated with disruption of the CD31+ vascular network and a 10 fold decrease in the CSC population. Finally, using HGSOC tissue microarrays we demonstrated that high endogenous expression of FKBPL correlated with increased progression free interval; indicating the potential for FKBPL to be used as a prognostic biomarker in HGSOC. HGSOC cell lines display marked differences in tumor vascularisation in vivo. ALM201, a peptide fragment of FKBPL, has potent anti-CSC and anti-angiogenic activity in an HGSOC xenograft which is dependent on angiogenesis for growth; an important finding for the clinical development of ALM201. Our data also strongly suggest that FKBPL is a potential novel prognostic biomarker in HGSOC, supporting its endogenous anti-tumor activity. Citation Format: Stephanie Annett, Gillian Moore, Amy Short, Neermeen Moustafa, Sudipto Das, Darran O'Connor, Cian McCrudden, Adrien Kissenpfenning, Laura Nelson, Ian Harley, Ken Arthur, Anita Yakkundi, Glen McCluggage, Andrea Marshall, Fiona Furlong, Helen O. McCarthy, Graham Cotton, Timothy Harrison, Lana McClements, Tracy Robson. FKBPL as a novel therapeutic target and prognostic biomarker in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-054.

Abstract 4169: Inhibition of EphB4-ephrin-B2 signaling results in enhanced sensitivity to cetuximab-radiation therapy in head and neck cancers

Abstract Introduction: The management of locally-advanced head and neck cancer patients relies on the combination treatment involving 7 weeks of radiation therapy (RT) with Cetuximab, an EGFR-inhibitor. Only a fraction of HNSCC patients, however, show response to cetuximab-radiation with an estimated 5-year overall survival of 46% compared to 36% with radiotherapy alone. This suggests potential involvement of redundant or compensatory tumor survival pathway that comes into action and blunts the therapeutic response. Based on our previous findings and published reports, elevated expression of EphB4, a member of Eph family of receptor tyrosine kinases and its membrane-bound ligand, ephrin-B2 might play a role in mediating this effect. In the current study, we hypothesized that EphB4-ephrin-B2 favors pro-tumorigenic survival signaling by altering the sensitivity to EGFR inhibitor and radiation. Therefore, combined inhibition of EphB4-ephrin-B2 signaling is critical to enhance sensitization of HNSCC towards anti-EGFR and radiation therapy. Materials and methods: To test the underlying hypothesis, we used a blocking protein targeting EphB4-ephrin-B2 axis in the absence and presence of EGFR inhibitor with radiation in HNSCC cell lines and patient-derived xenograft (PDX) model. Alteration in the protein levels in control and treatment groups was detected by Western blotting and immunohistochemistry. Circulating levels of cytokines was analyzed in vivo by Mesoscale U-plex assay. Tumor tissues were subjected to human PhosphoRTK array to assess changes in the activation status of downstream targets of EphB4-ephrin-B2 signaling mediating response in the triple combination groups. Results: Our data from locally advanced HNSCC patients treated with standard of care definitive Cetuximab-RT show high levels of both EphB4 and ephrin-B2 after failure of Cetuximab-RT. We observed significant tumor growth delay and increased response towards Cetuximab and radiation therapy following EphB4-ephrin-B2 inhibition resulting in better overall survival in PDX tumors and HNSCC cells. The tumor growth inhibition effects observed in vivo was accompanied by decrease in the levels of proliferation marker, angiogenic regulators, and increased apoptosis. Changes in levels of phopsho-receptor kinases and in circulating immune profile was also evident in tumors subjected to triple combination strategy. Conclusions: Overall, our data suggest that EphB4-ephrin-B2 and EGFR pathway cross-talk with each other to circumvent the therapeutic response resulting in uncontrolled tumor growth, apoptotic evasion, and immune escape. Therefore, development of combinatorial approaches targeting Eph-ephrin family of proteins might show promising outcome to enhance sensitivity towards anti-EGFR therapeutics and radiation in head and neck cancers. Citation Format: Shilpa Bhatia, Jaspreet Sharma, Sanjana Bukkapatnam, Ayman Oweida, Shelby Lennon, Parkash Gill, Antonio Jimeno, David Raben, Lynn Heasley, Sana D. Karam. Inhibition of EphB4-ephrin-B2 signaling results in enhanced sensitivity to cetuximab-radiation therapy in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4169.

Abstract 1725: A bi-functional mechanism of action: Activating the NLRP3 inflammasome and triggering apoptosis in cancer via a HK2-VDAC modulator

Abstract Introduction: Cancer cells undergo metabolic reprogramming to enable the efficient conversion of glucose needed for massive cell growth and proliferation, a well-documented phenomenon known as the Warburg effect. A key enzyme in this process is hexokinase 2 (HK2), which catalyzes the first step of glucose metabolism. Unlike HK1, which is ubiquitously expressed in normal cells, high levels of HK2 are found in cancer where it is required for cancer initiation and transformation. HK2 in cancer cells is attached to the outer mitochondrial membrane via the VDAC1 channel. VDAC1/HK2 association blocks pro-apoptotic signals as well as allows a continuous flux of mitochondrial ATP to HK, leading to apoptosis prevention and a high rate of glycolysis. Temporal high HK2 expression, and binding to VDAC, is also found in a variety of activated immune cells to support their changing metabolic needs. It has been recently published that detachment of HK2 from VDAC is one of the first events leading to the NLRP3-inflammasome activation, resulting in IL-1β and IL-18 secretion from macrophages (Cell 166:624 (2016)). Methods: A novel small molecule VDAC/HK2 modulator, VDA-1102, is being developed as a bi-functional drug for the treatment of solid tumors – triggering apoptosis in cancer cells while simultaneously activating the NLRP3-inflammasome in macrophages to induce an anti-tumoral immune response. Results: In vitro studies established that VDA-1102 selectively detaches HK2, but not HK1, from VDAC leading to cancer cell apoptosis, glycolysis inhibition, and prevention of cancer cell proliferation. VDA-1102 treatment of mouse primary bone marrow-derived macrophages and of human macrophage cell line, THP-1 cells, established a dose-dependent NLRP3-inflammasome activation and cytokine secretion. In vivo efficacy studies demonstrated significant tumor growth delay in syngeneic solid tumor models. Analysis of tumor-associated macrophages indicated a treatment-induced change in these macrophage phenotype from M2 to M1. This change was associated with a notable increase in spleen size, an increase ratio of naïve T cell in the spleen, and a significant increase in CD8+ and CD4+ tumor-infiltrating T cells. Conclusions: This data supports the notion that VDA-1102 is a bi-functional drug that targets both cancer and the innate immune system. In cancer cells it induces apoptosis, whereas in macrophages it activates the NLRP3-inflammasome machinery and stimulates an anti-tumor immune response. Our findings support further development of VDA-1102 to evaluate its potential as an anti-cancer therapy, either as a monotherapy or in combination with checkpoint inhibitors in high HK2-expressing solid tumors. Citation Format: Vered Behar, Reut Yosef Hamo, Eyal Dor-On, Oren M. Becker. A bi-functional mechanism of action: Activating the NLRP3 inflammasome and triggering apoptosis in cancer via a HK2-VDAC modulator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1725.

PO-424 modulating hexokinase 2 (HK2) as a novel approach to target metabolic immuno-oncology

IntroductionMetabolic reprogramming is one of the hallmarks of cancer, to support their needs for massive growth and proliferation. One major metabolic reprogramming is from oxidative phosphorylation to aerobic glycolysis, a well-documented phenomenon known as the Warburg effect. A key enzyme in this process is hexokinase 2 (HK2), which catalyses the first step of glucose metabolism and is overexpressed in many cancer types. Unlike HK1, which is ubiquitously expressed in normal cells, HK2 is required for cancer initiation and transformation even though their catalytic activity is highly similar. HK2 in cancer cells is attached to the outer mitochondrial membrane via the VDAC1 channel. VDAC1/HK2 association blocks pro-apoptotic signals, is less sensitive to feedback inhibition by the HK product, glucose-6-phosphate, as well as allows a continuous flux of mitochondrial ATP to HK, leading to apoptosis prevention and a high rate of glycolysis.Temporal high HK2 expression, and binding to VDAC, is also found in a variety of activated immune cells to support their changing metabolic needs. Detachment of HK2 from VDAC1 in activated immune cells leads to a range of responses ranging from glycolysis inhibition, NLRP3-mediated inflammasome activation, and metabolic reprogramming to activate immune pathways.Material and methodsA novel small molecule VDAC/HK2 modulator, VDA-1102, is being developed as a bi-functional drug for the treatment of solid tumours – triggering apoptosis in cancer cells while simultaneously enhancing an immune-mediated anti-tumour response by regulating immune cell metabolism.Results and discussionsIn vitro studies established that VDA-1102 selectively detaches HK2, but not HK1, from VDAC1 leading to cancer cell apoptosis, glycolysis inhibition, and prevention of cancer cell proliferation. In vivo efficacy studies demonstrated significant tumour growth delay and prolongation of survival in syngeneic solid tumour models. Analysis of tumor-associated macrophages in vivo indicated a treatment-induced change in these macrophage phenotype from M2 to M1.ConclusionThis data supports the notion that VDA-1102 is a bi-functional drug that targets both cancer and the innate immune system. In cancer cells it induces apoptosis, whereas in macrophages it stimulates an anti-tumour immune response. Our findings support further development of VDA-1102 to evaluate its potential as an anti-cancer therapy, either as a monotherapy or in combination with checkpoint inhibitors in high HK2-expressing solid tumours.

Abstract 4173: Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Survival rates are dismal, at only 8% five year survival. Radiation is one treatment option for PDAC, although historically its efficacy has been controversial. Our research goal is to improve the efficacy of radiation for PDAC patients. An ancillary study of the recent LAP07 trial showed that patients with neutrophilia were more resistant to chemoradiation. Additionally, when patients with systemic inflammation were excluded, one year local control was 80% in the chemoradiation arm compared to 54% in the chemotherapy arm. The EphB4 receptor and ephrinB2 ligand have been shown to play a role in neutrophil mobilization from the bone marrow and infiltration into melanoma tumors. We hypothesize that inhibition of EphB4-ephrinB2 signaling will enhance the efficacy of radiation in PDAC by reducing inflammation by neutrophils. Materials/Methods: To study tumor growth delay, Athymic nude mice were implanted in the flank with a patient derived xenograft (PDX) model of PDAC. To inhibit EphB4-ephrinB2 signaling, mice were administered with 20mg/kg of B11, a human single chain Fv antibody fragment against ephrinB2. Tumors were irradiated with 12Gy and volume was monitored by caliper. For mechanistic studies, C57/BL6 mice were injected in the flank with 10^6 FC1242 cells, which are derived from the pancreata of KPC mice. To inhibit EphB4-ephrinB2 signaling, mice received a hydrodynamic tail vein injection of 20ug/2mL TNYL-RAW-Fc plasmid. TNYL-RAW is a peptide antagonist of EphB4, and it is fused to human Fc. Tumors were irradiated with 12Gy and harvested four days later for flow cytometry analysis. Blood was also collected at various time points and analyzed by blood film white blood cell differential and flow for neutrophils. Plasmid transduction efficiency was confirmed by analyzing plasma samples collected at different time-points on an Fc protein-based ELISA assay. Results: We observed a significant tumor growth delay in PDX implanted mice who received B11 and radiation. An ELISA for human Fc confirmed transduction of the TNYL-RAW-Fc plasmid in FC1242 tumor bearing mice. Flow analysis on FC1242 tumors showed a significant decrease in percentage of infiltrating neutrophils when treated with TNYL-RAW and radiation, and blood film white blood cell differential and flow analysis on blood showed a significant decrease in circulating neutrophils in the same mice. Conclusions: Our findings support the hypothesis that EphB4-ephrinB2 signaling contributes to inflammation by neutrophils in PDAC, and inhibiting this signaling will increase the efficacy of radiation. This data can contribute toward a clinical trial design for combination therapy of EphB4-ephrinB2 inhibition and radiation in patients with pancreatic ductal adenocarcinoma. Citation Format: Shelby Lennon, Shilpa Bhatia, Ayman Oweida, Jaspreet Sharma, Andy Phan, Sanjana Bukkapatnam, Nomin Uyanga, Todd Pitts, Stephen Leong, Karyn Goodman, David Raben, Elena Pasquale, Jorge Martinez-Torrecuadrada, Sana Karam. Inhibition of EphB4-ephrinB2 signaling increases efficacy of radiation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4173.

Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors

Methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation [methionine-deficient diet (MDD)] at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In this study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and probably most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Hyperthermia-mediated drug delivery induces biological effects at the tumor and molecular levels that improve cisplatin efficacy in triple negative breast cancer

Triple negative breast cancer is an aggressive disease that accounts for at least 15% of breast cancer diagnoses, and a disproportionately high percentage of breast cancer related morbidity. Intensive research efforts are focused on the development of more efficacious treatments for this disease, for which therapeutic options remain limited. The high incidence of mutations in key DNA repair pathways in triple negative breast cancer results in increased sensitivity to DNA damaging agents, such as platinum-based chemotherapies. Hyperthermia has been successfully used in breast cancer treatment to sensitize tumors to radiation therapy and chemotherapy. It has also been used as a mechanism to trigger drug release from thermosensitive liposomes. In this study, mild hyperthermia is used to trigger release of cisplatin from thermosensitive liposomes in the vasculature of human triple negative breast cancer tumors implanted orthotopically in mice. This heat-triggered liposomal formulation of cisplatin resulted in significantly delayed tumor growth and improved overall survival compared to treatment with either non-thermosensitive liposomes containing cisplatin or free cisplatin, as was observed in two independent tumor models (i.e. MDA-MB-231 and MDA-MB-436). The in vitro sensitivity of the cell lines to cisplatin and hyperthermia alone and in combination was characterized extensively using enzymatic assays, clonogenic assays, and spheroid growth assays. Evaluation of correlations between the in vitro and in vivo results served to identify the in vitro approach that is most predictive of the effects of hyperthermia in vivo. Relative expression of several heat shock proteins and the DNA damage repair protein BRCA1 were assayed at baseline and in response to hyperthermia both in vitro and in vivo. Interestingly, delivery of cisplatin in thermosensitive liposomes in combination with hyperthermia resulted in the most significant tumor growth delay, relative to free cisplatin, in the less cisplatin-sensitive cell line (i.e. MDA-MB-231). This work demonstrates that thermosensitive cisplatin liposomes used in combination with hyperthermia offer a novel method for effective treatment of triple negative breast cancer.

Gemcitabine loaded microbubbles for targeted chemo-sonodynamic therapy of pancreatic cancer

Pancreatic cancer remains one of the most lethal forms of cancer with a 10-year survival of <1%. With little improvement in survival rates observed in the past 40 years, there is a significant need for new treatments or more effective strategies to deliver existing treatments. The antimetabolite gemcitabine (Gem) is the most widely used form of chemotherapy for pancreatic cancer treatment, but is known to produce significant side effects when administered systemically. We have previously demonstrated the benefit of combined chemo-sonodynamic therapy (SDT), delivered using oxygen carrying microbubbles (O2MB), as a targeted treatment for pancreatic cancer in a murine model of the disease. In this manuscript, we report the preparation of a biotin functionalised Gem ligand for attachment to O2MBs (O2MB-Gem). We demonstrate the effectiveness of chemo-sonodynamic therapy following ultrasound-targeted-microbubble-destruction (UTMD) of the O2MB-Gem and a Rose Bengal loaded O2MB (O2MB-RB) as a targeted treatment for pancreatic cancer. Specifically, UTMD using the O2MB-Gem and O2MB-RB conjugates reduced the viability of MIA PaCa-2, PANC-1, BxPC3 and T110299 pancreatic cancer cells by >60% (p < 0.001) and provided significant tumour growth delay (>80%, p < 0.001) compared to untreated animals when human xenograft MIA PaCa-2 tumours were treated in SCID mice. The toxicity of the O2MB-Gem conjugate was also determined in healthy non-tumour bearing MF1 mice and revealed no evidence of renal or hepatic damage. Therefore, the results presented in this manuscript suggest that chemo-sonodynamic therapy using the O2MB-Gem and O2MB-RB conjugates, is potentially an effective targeted and safe treatment modality for pancreatic cancer.

Antitumor effect of antibiotic resistance gene-free plasmids encoding interleukin-12 in canine melanoma model

The electrotransfer of interleukin-12 (IL-12) has been demonstrated as an efficient and safe treatment for tumors in veterinary oncology. However, the plasmids used encode human or feline IL-12 and harbor the gene for antibiotic resistance. Therefore, our aim was to construct plasmids encoding canine IL-12 without the antibiotic resistance genes driven by two different promoters: constitutive and fibroblast-specific. The results obtained in vitro in different cell lines showed that following gene electrotransfer, the newly constructed plasmids had cytotoxicity and expression profiles comparable to plasmids with antibiotic resistance genes. Additionally, in vivo studies showed a statistically significant prolonged tumor growth delay of CMeC-1 tumors compared to control vehicle-treated mice after intratumoral gene electrotransfer. Besides the higher gene expression obtained by plasmids with constitutive promoters, the main difference between both plasmids was in the distribution of the transgene expression. Namely, after gene electrotransfer, plasmids with constitutive promoters showed an increase of serum IL-12, whereas the gene expression of IL-12, encoded by plasmids with fibroblast-specific promoters, was restricted to the tumor. Furthermore, after the gene electrotransfer of plasmids with constitutive promoters, granzyme B-positive cells were detected in the tumor and spleen, indicating a systemic effect of the therapy. Therefore, plasmids with different promoters present valuable tools for focused therapy with local or systemic effects. The results of the present study demonstrated that plasmids encoding canine IL-12 under constitutive and fibroblast-specific promoters without the gene for antibiotic resistance provide feasible tools for controlled gene delivery that could be used for the treatment of client-owned dogs.