bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4062: Activity of 3 Bromopyruvate in human tumor primary culture 3-dimensional explants using ex vivo analysis of programmed cell death
Authors: Nagourney Adam Jeremiah , D'Amora Paulo , Evans Steven Scott , Bernard Paula , Francisco Federico , Ko Young , Stockwell Thomas , Johnson Maxine , Nagourney Robert Alan
Published: June 2022

Altered metabolism is a hallmark of human malignancy. The “Warburg Effect “reflecting increased glycolytic activity in the presence of oxygen represents an opportunity to target specific features of cancer to induce selective toxicity to malignantly transformed cells. 3-bromopyruvate (3-BP) is a pyruvate analogue that inhibits hexokinase II at the mitochondrial level resulting in cancer cell death. Numerous studies have confirmed activity for 3-BP in human SW480 and HT 29 colon carcinoma, glioblastoma (U373MG), HTP-1 leukemic and other cell lines. Our interest in exploring the activity of 3-BP in human tumor primary culture explants led to the application of the ex vivo analysis of programmed cell death in 19 patient tumors. The intent was to develop concertation ranges for future studies and explore disease-specific activity.

Methods: Fresh Sterile tumor samples were mechanically and enzymatically disaggregated and 3-dimensional organoids of desired size isolated by density centrifugation. Blood samples were isolated by density centrifugation. Cell suspensions in modified RPMI media were exposed to serial dilutions of 3-BP for 72 hours and then assessed for cell death by morphology, staining characteristics and ATP content by luciferase. Viability was scored against saline-exposed controls, normalized to 100%.

Results: 3BP reveals activity in human tumor explants. Activity varied by cancer type. Hematologic tumors including NHL, CLL and AML were found sensitive as were several solid tumors including GI and lung cancers. Of interest lower degrees of activity were observed in breast and ovarian cancers that are generally considered chemo-sensitive . Activity in 2 fibrolamellar cancers could suggest a novel therapy for this drug refractory neoplasm. Studies are underway examining 3-BP correlations with other classes of drugs and metabolic inhibitors as well as 3-BP combinatorial potential. Supported by the Fibro fighters Foundation

Citation Format: Adam Jeremiah Nagourney, Paulo D'Amora, Steven Scott Evans, Paula Bernard, Federico Francisco, Young Ko, Thomas Stockwell, Maxine Johnson, Robert Alan Nagourney. Activity of 3 Bromopyruvate in human tumor primary culture 3-dimensional explants using ex vivo analysis of programmed cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4062.
Abstract 4065: Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel
Authors: Cruz-Collazo Ailed M. , Katsara Olga , Salvo Elizabeth , Ruiz Jean , Schneider Robert , Dharmawardhane Suranganie
Published: June 2022

Triple negative breast cancer (TNBC) represents a challenge because of its aggressiveness and resistance to standard chemotherapy. Since TNBC lacks targeted therapy options, the frontline therapy is Paclitaxel, which targets actively dividing cells inducing subsequent apoptosis. Since dormant cells or metastatic cancer cells are not targeted by Paclitaxel, more targeted treatment options are needed. MBQ-167, a small molecule developed by us to target the metastasis drivers Rac and Cdc42, is highly effective in reducing tumor growth and metastasis in mouse models of TNBC (Cruz-Collazo et al., 2021, Molecular Cancer Therapeutics). The purpose of this study is to test the hypothesis that MBQ-167 is a viable candidate for combined therapy with Paclitaxel in TNBC. Therefore, we tested the efficacy of MBQ-167 in combination with Paclitaxel in MDA-MB-231 human TNBC cells via MTT viability assays and caspase 3/7 apoptosis, using effective concentrations of MBQ-167 and/or Paclitaxel for 24, 48, 96 & 120 hrs. We found that individual MBQ-167 or the combination therapy was more effective at 48-120 hrs at reducing cell viability by ~80% compared to ~60% by Paclitaxel treatment alone and significatively increasing apoptosis. Next, we tested individual or combined MBQ-167 (5 mg/kg 5X a week) and Paclitaxel (10 mg/kg 1X a week) on SCID mice bearing MDA-MB-231 tumors, via intraperitoneal administration. We report a significant reduction in tumor growth and lung metastasis in response to combined MBQ-167 and Paclitaxel compared to either treatment alone. A more aggressive syngeneic model of 4T-1 mouse breast cancer cells in BALB/c immunocompetent model was used to determine the direct effect of MBQ-167 and Paclitaxel on metastasis. When 4T-1 tumors were surgically removed and MBQ-167, Paclitaxel, or the combination administered for 17 days, the combination treatment, but not individual compounds, significantly reduced lung metastases by ~80%. In conclusion, this study validates the clinical testing of MBQ-167 in combination with Paclitaxel as a potential therapeutic for TNBC.

Citation Format: Ailed M. Cruz-Collazo, Olga Katsara, Elizabeth Salvo, Jean Ruiz, Robert Schneider, Suranganie Dharmawardhane. Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4065.
Abstract 407: OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance
Authors: Yang Yuqi , Wu Zhuo-Xun , Wang Jing-Quan , Teng Qiu-Xu , Lei Zi-Ning , Lusvarghi Sabrina , Ambudkar Suresh V. , Ji Ning , Chen Zhe-Sheng
Published: June 2022

The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). OTS964 is a potent T-LAK cell-originated protein kinase (TOPK) inhibitor. Herein, we investigated the interaction of OTS964 and MDR-associated ATP-binding cassette sub-family G member 2 (ABCG2, breast cancer resistance protein/BCRP). The cell viability assay indicated that the effectiveness of OTS964 is limited in drug-resistant and gene-transfected cells overexpressing ABCG2. We found that the known ABCG2 inhibitor is able to sensitize ABCG2-overexpressing cells to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2, and in turn, affects the pharmacokinetic profile of other ABCG2 substrate-drugs. Furthermore, OTS964 upregulates ABCG2 protein and mRNA expression levels, resulting in enhanced resistance to ABCG2 substrate-drugs. The ATPase assay demonstrated that OTS964 stimulates ATPase activity of ABCG2 in a concentration-dependent manner, and that this stimulation can be antagonized by a verified ABCG2 ATPase inhibitor. Additionally, the computational molecular docking analysis combined with results from ATPase assay suggested that OTS964 interacts with drug-binding pocket of ABCG2 protein and has substrate-like behaviors. Thus, OTS964 is an MDR-susceptible agent due to its interactions with ABCG2, and overexpression of ABCG2 transporter may attenuate its therapeutic effect in cancer cells.

Citation Format: Yuqi Yang, Zhuo-Xun Wu, Jing-Quan Wang, Qiu-Xu Teng, Zi-Ning Lei, Sabrina Lusvarghi, Suresh V. Ambudkar, Ning Ji, Zhe-Sheng Chen. OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 407.
Abstract 4076: Single cell RNA analysis reveals heterogeneous sub populations in aggressive variants of prostate cancer
Authors: Urban Rosalia Quezada , Keerthikumar Shivakumar , Clark Ashlee K. , Porter Laura H. , Lawrence Mitchell G. , Taylor Renea A. , Risbridger Gail P. , Toivanen Roxanne , Goode David L.
Published: June 2022

Neuroendocrine prostate cancer (NEPC) represent an aggressive malignancy that results in poor patients outcomes. At a histological level, NEPC exhibits heterogeneous pathologies; nevertheless, the molecular features, drivers and therapeutic implications of this heterogeneity are poorly understood. Single cell technology enables the resolution needed to unveil the complex heterogeneity of NEPC that bulk RNA analysis could not previously detect. Therefore, we aim to characterize the molecular heterogeneity of NEPC at a single cell level to detect common phenotypes across patients and identify new therapeutic targets. We performed single cell RNA sequencing on a novel cohort of nine patient derived xenograft (PDX) models that recapitulate the pathological and clinical heterogeneity of NEPC. Downstream analysis of single cell data was first completed on individual samples to identify distinct subpopulations of cells within each tumor. Then, integration analysis was performed to identify common and unique neuroendocrine populations across the different tumors. We profiled the transcriptome of 19,361 cells captured from the eight NEPCs. On an individual sample analysis, we detected 3 to 8 different subpopulations in each tumor, where every subpopulation displayed distinct biological properties such as epithelial mesenchymal transition, quiescence and stemness. Data integration of all samples revealed 16 subpopulations of tumor cells across all patients, of which 10 populations were primarily unique to one patient. Enriched pathway analyses revealed heterogeneous expression of most cancer hallmarks and oncogenic pathways across subpopulations, although some pathways were common to several neuroendocrine subpopulations such as EMT, P53 and KRAS. The detection of heterogeneous subpopulations in NEPC provides novel insight into why finding effective treatments for these aggressive tumors is challenging. Future work should focus on further evaluation of common druggable pathways that are shared across NEPC subpopulations. Alternatively, efforts could look at combination therapies that could effectively target these heterogeneous tumors.

Citation Format: Rosalia Quezada Urban, Shivakumar Keerthikumar, Ashlee K. Clark, Laura H. Porter, Mitchell G. Lawrence, Renea A. Taylor, Gail P. Risbridger, Roxanne Toivanen, David L. Goode. Single cell RNA analysis reveals heterogeneous sub populations in aggressive variants of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4076.
Abstract 4107: Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor
Authors: Bhavar Prashant Kashinath , Surampudi Uday Kumar , Sarma Partha Pratim , Shidore Mahesh Mohan , Kshirsagar Anuj Ramesh
Published: June 2022

KRASG12C is an important oncogenic driver mutation and occurs in about 13% of NSCLC and 1-3% of colorectal and other solid tumors. Lumakras࣪ (sotorasib), a KRASG12C inhibitor was recently approved for treatment of NSCLC, and others, such as, Adagrasib have shown relatively modest activity for KRASG12C.This modest activity may be partly due to incomplete target occupancy or due to unavailability of the drug at the target site, to elicit maximum clinical response. Clearly, this gives path for development of inhibitors which are potent, but also have desired drug like properties to achieve maximal clinical benefit in patients harboring KRASG12C mutation. Here, we report the identification of VRTX126, a novel, highly selective and potent inhibitor of the KRASG12C, discovered using fragment-based drug design. VRTX126 exhibited an IC50 of 10 nM in H358. VRTX126 also inhibited phospho-ERK with an IC50 of <20 nM and <10 nM in H358 and Mia-Paca-2 cell lines respectively, while demonstrating multi fold selectivity over KRAS wild-type or nonG12C mutant cells lines. In xenograft model harboring KRASG12C mutation, VRTX126 exhibited tumor growth inhibition at therapeutically relevant doses. Combination strategies with other targeted therapies such as, our proprietary SOS1 inhibitor, and other marketed or available MEK1/2, SHP2 inhibitors targeting Ras/Raf/MEK pathway is being explored. VRTX126 showed favourable oral properties with dose proportional pharmacokinetics. VRTX126 was found to have no adverse effect on hERG (10 uM). Additionally, early evaluation in acute (>1 g/kg (p.o.) and >0.2 g/kg (i.v.) was well tolerated, with no elevation in liver enzymes and repeat does studies indicated good safety margins, thus supporting further evaluation of VRTX126 in IND enabling studies. A first-in-human Phase 1 clinical study in KRASG12C mutated patients of NSCLC and mCRC will be planned upon completion of through safety evaluations.

Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4107.
Abstract 4121: Durable stable disease with nivolumab, PD-1 inhibitor, and subsequent bevacizumab, Anti-VEGF monoclonal antibody, in long standing recurrent respiratory papillomatosis with lung squamous cell carcinoma
Authors: Choi Horyun , Kim Jinah , Lee Yeun Ho , Kim Leeseul , Chae Young Kwang
Published: June 2022

Background: Recurrent respiratory papillomatosis (RRP) is a human papilloma virus (HPV) associated benign disease, which, in rare cases, can develop into lung squamous cell carcinoma. Treatment options are limited with no approved systemic adjuvant therapy and the disease requires frequent surgical resection to prevent airway obstruction. Therefore, expanding available treatment options remains a highly important, but unmet need in RRP. Herein, we present a case of RRP with squamous cell carcinoma of lung that demonstrated durable stable disease with nivolumab and subsequent bevacizumab.

Case presentation: A 48-year-old woman with long standing RRP since teenager, who underwent multiple microsuspension laryngoscopies, debridements including laryngeal lesions, and ultimately, 5 years ago, video assisted thoracic surgery (VATS) resection of right upper lobe mass, confirming squamous cell carcinoma, has been managed with debulking surgery on tracheal lesions by ENT on regular basis. Nivolumab, a programmed death-1 blockade, 3 mg/kg every 2 weeks was started given squamous cell carcinoma of lung. Stable disease (SD) in lung lesions was achieved on nivolumab for one year but she required more frequent debulking of tracheal lesions. After multidisciplinary discussion with Otorhinolaryngology, she was started on bevacizumab, vascular endothelial growth factor inhibitor, 10 mg/kg every 6 weeks. Follow-up CT scans in 10 weeks showed a decrease in size of a left lower lobe mass to 27 mm from 32 mm in the previous image. After one year, administration of bevacizumab was spaced out to every 12 weeks. She requires less debulking surgery and the last debulking surgery was approximately 3 years ago. She maintains SD for over 4 years since starting bevacizumab.

Discussion: Our patient with RRP and squamous cell carcinoma of lung demonstrated SD on nivolumab before she was switched to bevacizumab. In addition, her SD on bevacizumab of more than 4 years with 12 weeks of maintenance interval is encouraging as there is no current standard of care treatment for RRP. Despite difficulty in running clinical trials in RRP due to its rareness, additional studies evaluating the efficacy of PD-1 inhibitor or anti-VEGF monoclonal antibody on RRP are warranted.

Citation Format: Horyun Choi, Jinah Kim, Yeun Ho Lee, Leeseul Kim, Young Kwang Chae. Durable stable disease with nivolumab, PD-1 inhibitor, and subsequent bevacizumab, Anti-VEGF monoclonal antibody, in long standing recurrent respiratory papillomatosis with lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4121.
Abstract 4131: Antitumor effects of ensartinib in non-small cell lung cancer harboring MET exon 14-skipping mutations
Authors: Xia Yang , Lan Fen , Zhao Jing , Shen Hua-Hao , Selvaggi Giovanni , Li Wen
Published: June 2022

Background: Met proto-oncogene (MET) exon 14 skipping mutations are tumor drivers in approximately 3% of lung cancer patients. The antitumor efficacy of ensartinib, a novel multi-kinase inhibitor, against non-small cell lung cancer (NSCLC) harboring MET exon 14 skipping mutations was investigated in this study.

Methods: The MET-mutant binding affinity and antitumor activity of ensartinib were assessed in vitro and in vivo. Preliminary therapeutic activity of ensartinib in patient-derived organoids was observed. To explore clinical activity, 17 NSCLC patients with MET exon 14 skipping mutations were treated with ensartinib at 225 mg qd.

Results: Molecular dynamic simulations revealed that ensartinib exhibited favorable binding to c-MET. Ensartinib was highly effective in inhibiting the kinase activity of the MET exon 14 deletion protein (IC50 = 7.9 nM). Furthermore, ensartinib potently suppressed the MET pathway and the growth of Hs746T cells that were subcutaneously implanted into mice. Most importantly, of 17 patients with 14 different types of MET exon 14 skipping mutations undergoing ensartinib treatment, 1 (6%) showed a complete response, 11 (65%) achieved a partial response, and 4 (24%) exhibited stable disease. Thus, the objective response rate (ORR) was 71% and the disease control rate (DCR) was 94%, and the ORR in MET-TKI naive patients was even higher (12/15, 80%). In 2 patients with brain metastasis without prior brain radiation, we observed one partial response in the brain, while in the other patient the brain lesions were stable for 6 months. The most frequently reported adverse events were rash, peripheral edema, and nausea; however, no fatal adverse events occurred.

Conclusions: These results provide the first evidence that ensartinib exhibits both preclinical and clinical antitumor activity against MET exon 14 skipping mutations with the potential of strong intracranial efficacy and warrant further evaluation in a planned phase II study thus providing more options to patients with MET exon 14 skipping mutations. Updated data including duration of response, PFS and and OS will be provided at the time of the presentation with additional few months of follow up.

Citation Format: Yang Xia, Fen Lan, Jing Zhao, Hua-Hao Shen, Giovanni Selvaggi, Wen Li. Antitumor effects of ensartinib in non-small cell lung cancer harboring MET exon 14-skipping mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4131.
Abstract 4183: Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity
Authors: Bhanu Krithikaa Rajkumar , Jayaprakash Priyamvada , Rice Meghan , Morrow Brittany , Marszalek Joseph R. , Gay Jason P. , Vellano Christopher P. , Cowen Benjamin R. , Welsh Dean J. , Curran Michael A.
Published: June 2022

We sought to determine whether various inhibitors of Oxidative Phosphorylation (OxPhos) could restore tumor infiltrating T cell functionality and longevity while compromising metabolic fitness of tumor cells. Using a combination of in vitro metabolic profiling with the Seahorse XFe96 Analyzer and in vivo tumor immunotherapy in the immunocompetent TRAMP-C2 prostate adenocarcinoma model, we determined the differential impact of multiple OxPhos inhibitors on both tumor immune metabolic fitness and functional capacity. Impact on the tumor immune microenvironment of TRAMP-C2 tumors was assessed through high parameter flow cytometry on a BD X-30. While OxPhos inhibition compromised initial T cell activation, we found that two of the inhibitors increased the glucose uptake, proliferation, and activation status of effector T cells. In contrast, the same two inhibitors decreased the mitochondrial fitness, proliferation and OxPhos metabolic capacity of TRAMP-C2 tumor cells themselves. To determine whether this selective inhibition of tumor versus T cell metabolic function could improve immunotherapy responses in vivo, we implanted TRAMP-C2 tumors and treated them with these OxPhos inhibitors with or without concomitant immune checkpoint blockade (ICB). The combination of ICB and OxPhos inhibition achieved highly significant and durable rates of tumor control and regression suggesting that these approaches are potentially synergistic. Analysis of the tumor microenvironment has identified the cellular mechanisms underlying these therapeutic effects which may provide useful biomarkers as this novel combination is translated to the clinic.

Citation Format: Krithikaa Rajkumar Bhanu, Priyamvada Jayaprakash, Meghan Rice, Brittany Morrow, Joseph R. Marszalek, Jason P. Gay, Christopher P. Vellano, Benjamin R. Cowen, Dean J. Welsh, Michael A. Curran. Differential modulation of tumor versus T cell oxidative phosphorylation potentiates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4183.
Abstract 4185: Targeting variant of CEACAM5 and CEACAM6 using NEO-201 and IL-15 in gynecologic cancers
Authors: Morelli Maria Pia , Fantini Massimo , Tsang Kwong Y. , Arlen Phillip , Fauceglia Paulette , Hernandez Lidia , Korrapati Soumya , Edmonson Elijah , Cole Christopher , Annunziata Christina M.
Published: June 2022

Background: Uterine and ovarian cancers express glycosylated forms of CEACAMs 5 and 6 that regulate interactions with the tumor microenvironment. NEO-201 is a novel monoclonal antibody that binds a glycosylated variant of these CEACAMs that is upregulated in cancer compared to normal uterine or ovarian epithelia. Previous work has shown that NEO-201 induced cancer cell death predominantly by antibody-dependent cellular cytotoxicity (ADCC) that can be enhanced by the addition of IL-15.

Methods: NEO-201 target expression was assessed on uterine and ovarian cancer cell lines, as well as a tissue microarray of primary uterine cancer samples. Cell lines were profiled with DNA and RNA sequencing. Chromium-release assays were performed to quantify ADCC without and with varying concentrations of IL-15 and varying target to effector ratios. In vivomodeling was performed using ovarian cancer cell line OV90, and mice were treated with NEO-201, with PBMC or NK cells, with or without IL-15.

Results: NEO-201 target antigen was highly expressed in uterine cancer cell line ACI158 and ovarian cancer cell line OV90. The antigen was expressed in primary uterine cancer samples as well. IL-15 consistently increased the in vitro killing of both cell lines and was further enhanced with the use of NK cells compared to PBMC. These findings were replicated in the in vivo mouse model, where mice survived longest with the combination of NEO-201, IL-15 and NK cells.

Conclusions: The ADCC activity of NEO-201 monoclonal antibody was enhanced with the addition of IL-15, especially in the presence of purified NK cells. NEO-201 with IL-15 shows promising results as a potential treatment for biomarker-selected gynecologic cancers.

Citation Format: Maria Pia Morelli, Massimo Fantini, Kwong Y. Tsang, Phillip Arlen, Paulette Fauceglia, Lidia Hernandez, Soumya Korrapati, Elijah Edmonson, Christopher Cole, Christina M. Annunziata. Targeting variant of CEACAM5 and CEACAM6 using NEO-201 and IL-15 in gynecologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4185.
Abstract 419: The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia
Authors: Glynn Daniel Joseph , Crampton Rosie , Pesnot Thomas , Scott Andrew , Nassoy Anne-Chloe , Kirk Ralph , Narducci Daniele , Nelson Gary , Ongiri Lynette , Begum Habiba , Rao Vincent , Bingham Matilda , McPherson Rhoanne , Turner Darryl
Published: June 2022

Acetylation of histones and additional nuclear proteins is a key mechanism in the regulation of gene expression. Aberrant acetylation has been linked to a wide range of diseases including cancer, inflammation, and neurodevelopmental disorders. Histone acetylation is introduced by histone acetyltransferases complexes (HATs), where substrate specificity is dramatically enhanced by scaffolding proteins that activate and target them to specific chromatin sites. A protein of interest with both epigenetic acetyl reader and scaffolding function is the protein Bromodomain and PHD finger-containing protein 1 (BRPF1). The Protein contains domains of two plant homeodomain (PHD) fingers separated by a zinc knuckle (PZP domain), a bromodomain, and a proline-tryptophan-tryptophan-proline (PWWP) Tudor domain. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as the catalytic MYST-family acetyltransferase subunit and the stable complex with Moz-Tif2 is known to lead to the development of human acute myeloid leukemia (AML). Given this, we chose to pursue the development of BRPF1 degraders to probe cancer disease biology especially in AML, where new therapies are required to overcome several unmet needs such as less-toxic treatments and relapsed/refractory disease paradigms. Here we present the design and synthesis of a range of BRPF1 degraders in highly desirable physicochemical space utilizing in-silico modeling. The prepared degraders which utilize multiple E3 ligases were then screened against cell lines harboring Mixed-lineage leukemia (MLL) translocations specifically the THP-1 cell line. In addition to this, we investigated the ability of the compounds to effectively degrade the target and suitability of our degraders for potential in-vivo exposure through a panel of routine ADMET assays.

Citation Format: Daniel Joseph Glynn, Rosie Crampton, Thomas Pesnot, Andrew Scott, Anne-Chloe Nassoy, Ralph Kirk, Daniele Narducci, Gary Nelson, Lynette Ongiri, Habiba Begum, Vincent Rao, Matilda Bingham, Rhoanne McPherson, Darryl Turner. The development of BRPF1 degraders as a potential treatment for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 419.
Abstract 4199: rhIL-7-hyFc (efineptakinalpha; NT-I7) enhances the anti-tumor response when combined with hIL-2/TCB2c complex
Authors: Baek Seungtae , Im Sun-Kyoung , Lee Minji , Ji Mankyu , Kim Miyoung , Kim Daeun , Lee Jun Young , Choi Donghoon
Published: June 2022

rhIL-7-hyFc (efineptakin-alfa; NT-I7) is a potent T cell amplifier, with a homodimeric interleukin-7 (IL-7) fused to the hybridizing IgD/IgG4 immunoglobulin domain. Previous work has shown that in mice, NT-I7 dramatically increases tumor-infiltrating CD8+ T cells while reducing the frequency of PD-1+ CD8+ T cells in the tumor. There is also significant expansion of Central Memory (CM)-phenotype CD8+ T cells (CD62L+CD44+) in the tumor and tumor draining lymph node. Here, we investigated the anti-tumor effect of NT-I7 in combination with a T cell activator, SLC-3010 (hIL-2/TCB2c complex), in MC38 tumor-bearing mice. Because TCB2 is an antibody specific for IL-2 that blocks interaction of IL-2 and IL-2Rα (CD25), SLC-3010 can selectively activate T cells while disfavoring Treg activation. Mice were administered a single dose of NT-I7 or SLC-3010 via intramuscular or intravenous injection, respectively. The combination of NT-I7 with SLC-3010 enhanced the anti-tumor response with increased number and frequency of CD8+ T cells as well as granzyme B expression in the tumor. The number of CD8+ T cells peaked at day 4 and day 7 by SLC-3010 and NT-I7, respectively. The number of Tregs in the tumor was slightly increased by NT-I7 and SLC-3010, but it was not statistically significant. Interestingly, NT-I7, but not SLC-3010, increased the frequency of PD-1+TCF-1+TOX- stem-like CD8+ T cells in the draining lymph node. Meanwhile, SLC-3010 significantly increased the number of PD-1+ CD8+ T cells in the tumor. Our data suggests that NT-I7 can be applied in combination with other immunotherapies such as IL-2 to enhance the anti-tumor response.

Citation Format: Seungtae Baek, Sun-Kyoung Im, Minji Lee, Mankyu Ji, Miyoung Kim, Daeun Kim, Jun Young Lee, Donghoon Choi. rhIL-7-hyFc (efineptakinalpha; NT-I7) enhances the anti-tumor response when combined with hIL-2/TCB2c complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4199.
Abstract 4205: Staged anti-PD-1 therapy with intratumoral recombinant calreticulin improves anti-tumor immunity and survival in glioblastoma mouse models
Authors: Taori Suchet , Noffsinger Breanna , Miller Charlotte A. , Xiao Aizhen , Manigat Laryssa , Zhong Qing , Harris Tajie , Purow Benjamin
Published: June 2022

Background: Glioblastoma (GBM), the most common and lethal primary brain tumor, has a median survival of a mere 15 months and leads to approximately 12,000 deaths in the US annually. Targeted and combinatorial-based clinical trial therapies have shown poor efficacy in GBM treatment, partly due to the restrictive nature of the blood-brain barrier, an immunosuppressive tumor microenvironment, GBM’s heterogeneity and adaptability, and GBM’s ability to metastasize and invade critical regions of the brain. However, promising recent literature has indicated that neoadjuvant anti-PD-1 checkpoint-inhibition immunotherapy - i.e., starting it right before surgery for recurrence - improves survival outcomes in human GBM patients.

Results: Here, we demonstrate a proposed mechanism of action wherein localized intratumoral danger-associated molecular pattern (DAMP, a known immunogenic driver) injection of calreticulin - used to mimic natural DAMP release from necrotic cells during surgery - combined with neoadjuvant anti-PD-1 immunotherapy leads to better survival outcomes in both orthotopic mouse CT2A and CT2A-Luc GBM models. This survival benefit is also seen in a more aggressive (larger tumor inoculation size) orthotopic CT2A-Luc GBM model. Flow cytometry indicates increased microglia cell counts and activation marker expression, and increased myeloid activation marker expression in mice brains treated with our combination immunotherapy in a CT2A GBM model. Additionally, in vivo treatment with our combination immunotherapy led to increases in the local T and NK cell numbers, the CD8:CD4 ratio, and the proliferation of CD4 T cells in mice brains of a CT2A GBM model. In vitro results suggest that co-culture with CT2A cells increased PD-1 expression in macrophages and microglia and that our combination treatment of calreticulin and anti-PD-1 immunotherapy reduces the viability of mouse GBM cells when mixed with macrophages.

Significance: This project paves the path for a novel immunotherapeutic approach to tackle GBM and other cancers. Future studies could incorporate relevant DAMP’s into nanoparticles for sustained release after intratumoral injection and possibly viral delivery of DAMP’s that are constitutively secreted, thereby prolonging an anticipated immune response.

Citation Format: Suchet Taori, Breanna Noffsinger, Charlotte A. Miller, Aizhen Xiao, Laryssa Manigat, Qing Zhong, Tajie Harris, Benjamin Purow. Staged anti-PD-1 therapy with intratumoral recombinant calreticulin improves anti-tumor immunity and survival in glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4205.
Abstract 4217: MEKi in combination with anti-OX40 generates metabolically fit effector CD8+ T cells, enhances stem cell-like memory CD8+ T cells, and leads to strong anti-tumor activity
Authors: Gaur Pankaj , Verma Vivek , Elayyan Mays Alreem , Ramlaoui Zainab , Barry Simon T. , Valge-Archer Viia E. , Smith Paul D. , Gupta Seema , Khleif Samir N.
Published: June 2022

In the tumor microenvironment (TME), T cell exhaustion and short persistence of effector cells are crucial factors that limit the efficacy of endogenously generated or adoptively transferred effector cells. Regenerative stem-cell memory CD62LhiCD44lowSca1+ CD8+ CD8+ T cells (TSCM) persist longer and produce cells with more vital effector functions. OX40, a member of TNFRSF4, has been shown to promote the expansion and proliferation of activated effector T cells, leading to the generation of robust anti-tumor responses and immune memory. However, treatment with anti-OX40 as a single therapeutic modality has not generated desirable clinical outcomes. A combination of immune modulators has been found to enhance the therapeutic efficacy synergistically. Recently, we reported that inhibition of MEK1/2 using selumetinib (AZD6244) leads to a significant increase in the numbers of CD8+ TSCM cells with self-renewability, enhanced multipotency and proliferative capacity, leading to potent anti-tumor effects. Therefore, here we combined selumetinib with anti-OX40 to investigate if enhanced immune-mediated anti-tumor responses could be obtained. Using three mouse tumor models (TC1, B16, and CT26), we found that combination of MEKi with anti-OX40 enhances the anti-tumor activity by increasing the expansion of total and antigen-specific CD8+ T cells in the TME. These CD8+ T cells have higher functional activity as seen by increased Granzyme B and INF-γ production. We also found similar results after adoptive cell therapy (ACT) where infusion of MEKi-treated cells in B16 tumor-bearing mice synergized with anti-OX40 agonist treatment. Interestingly, anti-OX40 treatment of MEKi-treated CD8+ T cells enhanced the metabolic fitness as demonstrated by low mitochondrial potential and enhanced Oxygen Consumption Rates (OCR), Spare Respiratory Capacity (SRC), and Extra-cellular Acidification Rates (ECAR) compared to anti-OX40 treatment alone. Furthermore, OX40 agonist significantly enhanced the longevity of MEKi-induced CD8+ TSCM cells. In summary, our data provide a novel strategy to utilize MEKi to enhance the efficacy of ACT and combination immunotherapy using anti-OX40 agonist.

Citation Format: Pankaj Gaur, Vivek Verma, Mays Alreem Elayyan, Zainab Ramlaoui, Simon T. Barry, Viia E. Valge-Archer, Paul D. Smith, Seema Gupta, Samir N. Khleif. MEKi in combination with anti-OX40 generates metabolically fit effector CD8+ T cells, enhances stem cell-like memory CD8+ T cells, and leads to strong anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4217.
Abstract 4223: Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform
Authors: Carralot Jean-Philippe , Ruiz Matilde Arévalo , Tam Robert , Armentani Eric , Pattabiraman Vijaya R. , Kreft Bertolt
Published: June 2022

Immunocytokines (IC) provide the opportunity for targeted delivery of cytokines as payloads to tissues and cells to improve safety and efficacy of cytokine-based therapies. ICs have gained significant interest in recent years and several antibody-cytokine fusion proteins have entered clinical trials. Compared to recombinant fusion proteins, we have developed an entirely different approach to IC generation based on the site-specific, chemical conjugation of synthetic cytokines to antibodies. Bright Peak generates enhanced and conjugatable cytokines using a novel protein engineering platform based on solid-phase peptide synthesis and subsequent chemical ligation of protein segments. Our synthetic cytokines can then be readily chemically conjugated to specific lysine residues in the Fc region of an existing IgG1, IgG2 or IgG4 antibody without the need for prior antibody engineering. Chemical conjugation of cytokine payloads is rapid, enabling the flexible generation of ICs based on different antibodies and payloads within weeks. We applied our technology to more than 10 antibodies and found that neither antigen binding nor payload potency and selectivity are affected by chemical conjugation. Importantly, binding of the Fc domain of ICs to Fc gamma receptors or FcRn is not significantly affected. We are initially focusing on the development of PD-1-targeted ICs to achieve dual-targeting of PD-1+ effector T cells (cis-signaling). Using several anti-PD-1 antibodies and various synthetic IL-2 variants as payloads, we created ICs with different drug-antibody ratios and explored alternative conjugation sites within the Fc region. Resulting PD-1/IL-2 ICs are highly active showing significantly enhanced potency due to avidity resulting from binding of the cytokine to PD-1+ effector T cells in cis. Our PD-1/IL-2 ICs induce strong pharmacodynamic effects in vivo, and we are currently optimizing the pharmacological profiles of PD-1-targeted ICs for clinical application. In addition, we are actively exploring cis-signaling ICs targeting different surface receptors and immune cells.

Citation Format: Jean-Philippe Carralot, Matilde Arévalo Ruiz, Robert Tam, Eric Armentani, Vijaya R. Pattabiraman, Bertolt Kreft. Cis-activation of PD-1+ effector T cells with dual-targeting immunocytokines generated using a novel chemical conjugation platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4223.
Abstract 4230: Modeling the cDC1 ex vivo and in vitro: Development and comparison of a conventional dendritic cell culture system for industry
Authors: Goodfield Laura L. , Lahdenranta Johanna , Scott Heather , Ashworth Tamera , Luus Lia , Licht Anna F. , McDonnell Kevin , Brandish Phil , Keen Nicholas
Published: June 2022

Conventional dendritic cells (cDC) are innate immune cells specialized in antigen sampling and subsequent cross-presentation to immune cells and are critical for an effective anti-tumor immune response. While crucial to immunity against intracellular pathogens, viruses, and tumors, cDC type I (cDC1) are a relatively rare population and constitute about 0.05% of cells in the blood. Using these rare cells in screening assays can be a challenge, as very small proportions are found in peripheral human blood and since they are difficult to isolate with high viability and purity. Existing methods to generate acceptable numbers of cDC1s require a feeder cell line, which is not accessible to many laboratories.

Using a systematic approach, we developed a scalable differentiation system to generate a mixed culture containing cDC1 cells from CD34+ hematopoietic stem cells isolated from cord blood that does not require feeder cells. This culture of cells has been phenotypically and functionally characterized to express high levels of CD141 and Clec9a, as well as responsiveness to various activating stimuli including poly (I:C). Cells derived from this culture method express similar levels of CD141 and Clec9a compared to those observed in peripheral blood of healthy donors. Our effort to find a less laborious, more efficient system includes comparison to cDC1 responses from an in vitro differentiation system to cDC1s isolated from humanized mouse bone marrow, with evaluation of both surface marker phenotype and responsiveness to activating stimuli. We also compared the responses of these cells to monocyte derived dendritic cells that express CD141 and Clec9a. These data demonstrate that the HSC-derived cDC1 cultures provide a comparison of unique dendritic cells that may be utilized for screening assays in an industry setting.

Citation Format: Laura L. Goodfield, Johanna Lahdenranta, Heather Scott, Tamera Ashworth, Lia Luus, Anna F. Licht, Kevin McDonnell, Phil Brandish, Nicholas Keen. Modeling the cDC1 ex vivo and in vitro: Development and comparison of a conventional dendritic cell culture system for industry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4230.
Abstract 4233: Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity
Authors: Dufort Fay J. , Leitheiser Christopher J. , Mudd Gemma , Kristensson Julia , Rezvaya Alexandra , Gaynor Katie , Uhlenbroich Sandra , Urbonas Liudvikas , Scott Heather , Chen Liuhong , Harrison Helen , Skynner Michael , McDonnell Kevin , Brandish Philip E. , Keen Nicholas
Published: June 2022

The tumor specific activation of natural killer (NK) cells is an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies). NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles® are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to identify Bicycles® that bind specifically to the key activating receptor, NKp46. When chemically coupled to tumor antigen binding Bicycles, this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules NK-TICAs and we will describe herein their discovery and evaluation.We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner.In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for the application of our Bicycle technology to drive NK cell-mediated tumor immunity.

Citation Format: Fay J. Dufort, Christopher J. Leitheiser, Gemma Mudd, Julia Kristensson, Alexandra Rezvaya, Katie Gaynor, Sandra Uhlenbroich, Liudvikas Urbonas, Heather Scott, Liuhong Chen, Helen Harrison, Michael Skynner, Kevin McDonnell, Philip E. Brandish, Nicholas Keen. Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4233.
Abstract 4238: A novel immunotherapy approach by inhibiting nanotube-mediated mitochondrial trafficking between cancer cells and T cells
Authors: Saha Tanmoy , Jaybalan Ruparoshni , Jang HaeLin , Sengupta Shiladitya
Published: June 2022

Trafficking of mitochondria from immune cells to cancer cells via physical nanotubes is a new mechanism by which cancer cells undergo immune evasion. This results in depletion of T cell, which can limit the efficacy of classical immune checkpoint inhibitors. Here we introduce a novel pharmacological inhibitor that inhibits nanotube-mediated mitochondria transfer from immune cell to cancer cell. Our preliminary study implicates the exocyst complex in the nanotube assembly. We performed molecular dynamic simulations to identify the potential inhibitors for the exocyst proteins. We have identified a drug candidate, TS-1, which successfully inhibits the mitochondria transfer from immune cell to cancer cell. Currently we are exploring the modification of the structural component to increase the inhibitory potential of the drug. TS-1-loaded liposome significantly inhibited tumor growth in a 4T1 metastatic breast cancer mouse model. We observed considerable increase in intratumoral T cell population with treatment. Furthermore, the use of TS-1 in combination with an anti-PD1 inhibitor resulted in effective tumor reduction in the 4T1 model, which typically responds poorly to anti-PD1 therapy alone.

Conclusion: Taken together, the inhibition of nanotube-mediated mitochondrial hijacking from immune cells emerges as an approach for the development of next generation of immunotherapy agents.

Citation Format: Tanmoy Saha, Ruparoshni Jaybalan, HaeLin Jang, Shiladitya Sengupta. A novel immunotherapy approach by inhibiting nanotube-mediated mitochondrial trafficking between cancer cells and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4238.
Abstract 4244: Using lysosomal immunogenic cell death to target cancer via xanthine oxidase
Authors: Taritsa Iulianna , Fossel Eric , Neote Kuldeep
Published: June 2022

Lysosome-induced immunogenic cell death (LIICD) is a powerful mechanism of targeting cancer cells that kills circulating malignant cells and primes the host’s immune cells against future remission. Current immunotherapies for cancer are limited in preventing recurrence - a gap which can be bridged by training the immune system to recognize cancer neoantigens. Lysosomal leakage can be induced therapeutically to traffic antigens from dying cells to dendritic cells which can later present those tumorigenic antigens to T cells. We demonstrate that the oxidative agent native bovine xanthine oxidase can induce early markers of lysosome-induced immunogenic cell death in an in vitro human cancer cell line (CCL2). Specifically, we measured overall cell death, as well as surface-expressed calreticulin, extracellular ATP release, and HMGB1 production. These markers are consensus indicators of ICD. Flow cytometry, luminescence assays, and ELISA were used respectively to quantify biomarker levels between treated versus untreated cells. We also included a positive control group of cells dosed with doxorubicin (a known inducer of LIICD). We looked at each marker at various time points after cancer cells were treated with xanthine oxidase, doxorubicin, and an untreated group. Upregulated biomarkers after treatment with the protein indicate an immunogenic response. Further, we used confocal imaging to show lysosomal membrane permeabilization after treatment with Native Bovine Xanthine Oxidase using Lucifer Yellow staining. We thus show the potential for this protein to induce an anticancer effect paired with an adaptive immune response against tumor cells. Our research in human cell lines here provides evidence for the success of the same therapeutic method in patients and serve as the gateway to developing a new treatment approach against breast cancer.

Citation Format: Iulianna Taritsa, Eric Fossel, Kuldeep Neote. Using lysosomal immunogenic cell death to target cancer via xanthine oxidase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4244.