bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2826: Rewiring CAR T-cells for absolute solid tumor specificity and safety
Authors: Weist Michael R. , Sharbi-Yunger Adi , Yi Jason , Schnair Caitlin , Bassan David , Kim Tanya , Tabak Sarit , Lopesco Yael , Weinberger Leehee , Bujanover Nir , Chaim Neta , Vucci Kristina , Foord Orit , Calzone Frank J. , Kendall Rick , Adams Gregor B.
Published: June 2022

Translation of the unprecedented efficacy of chimeric antigen receptor (CAR) T-cell technology into solid tumors requires a solution to the problem of on-target damage of vital organs. We have developed an inhibitory CAR platform (iCAR) that imposes self-regulation in CAR T-cells to restrict inappropriate activation against normal cells without compromising tumor efficacy. The system consists of an iCAR that is co-expressed with a conventional activating CAR (aCAR) designed to activate T-cells. The iCAR and aCAR scFvs bind distinct cell-surface antigens that are ubiquitously expressed across all solid tumor histologies and normal tissues. The iCAR is allele-specific whereas the aCAR is pan-allelic. The iCAR technology was validated in functional assays and NSG models with cancer cell lines that mimic loss-of-heterozygosity (LOH) which is common in most human cancers due to increased chromosomal instability. In this context, loss of iCAR target expression due to LOH generates irreversible target specificity. Tumor-specific overexpression of aCAR and iCAR targets, a common barrier to the clinical translation of potent agents targeting the cell-surface proteins is not required. We have shown using in vitro and in vivo models that CAR T-cell activation and target killing are fully inhibited by dual iCAR + aCAR antigen engagement. This outcome validates the concept of iCAR-mediated protection of normal cells in vital organs. In contrast, tumors that mimic iCAR target LOH through CRISPR editing were fully eradicated with aCAR engagement alone. T-cell activation, proliferation, and cytotoxic activity after exposure to target cells lacking iCAR target antigen was found to be quantitatively indistinguishable from the results obtained with single input aCAR CAR T-cells. These results suggest that iCAR technology can enable treatment of cancer patients with solid tumors using CAR T-cells that maximize potency and efficacy by mitigating on-target CRS, neurotoxicity, and inevitable organ damage. The restriction of CAR T activation to the local tumor environment by iCAR-mediated self-regulation directly addresses a fundamental technology gap that cannot be solved by CAR T dosing or aCAR modification alone.

Citation Format: Michael R. Weist, Adi Sharbi-Yunger, Jason Yi, Caitlin Schnair, David Bassan, Tanya Kim, Sarit Tabak, Yael Lopesco, Leehee Weinberger, Nir Bujanover, Neta Chaim, Kristina Vucci, Orit Foord, Frank J. Calzone, Rick Kendall, Gregor B. Adams. Rewiring CAR T-cells for absolute solid tumor specificity and safety [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 2826.
Abstract 283: Reconstructed plate-based 3D screening assays that predict in vivo responses
Authors: Urs Sumithra , Steffey Michael , Qu Ying , Ryou Matthew , Kirshner Julia , Wise Scott
Published: June 2022

In preclinical drug development, there is an urgency to create models that are predictive and relevant at the same time. To support this effort, early drug development emphasizes establishing multiple strategies that address treatment response, resistance, and combination effects, all with rapid turnover. Given the ever-increasing relevance of 3D in vitro models, Labcorp employs 3D model systems co-developed with zPREDICTA. These organ-specific models can support tumor cell growth and mimic the composition and architecture of both solid tumors and hematological malignancies. While 2D in vitro assays are preferred for screening efforts of chemical libraries, mainly because of cost, they do not recapitulate the tumor architecture and the microenvironment that is more comparable to in vivo models. This can result in differences in pharmacological potencies of standard of care (SoC) agents when compared to the 3D and in vivo models. zPREDICTA 3D assays serve as a tool for in vitro screening of tumor cell lines, which in long term cultures develop similar characteristics to in vivo disease states. Specifically, this in vitro system correlates well with breast and lung carcinoma in vivo models when matching rank order of potencies of known SoC agents. zPREDICTA tumor-specific 3D systems as well as 2D cultures and in vivo models were used to compare and contrast the activity of a set of anticancer agents. Human MDA-MB-231 cells were sensitive to paclitaxel both in 2D and in the 3D r-Breast (IC50 of 7.9nM and 6.1nM, respectively) as well as in vivo, with complete growth inhibition at the dose of 15mg/kg. Paclitaxel was cytotoxic in 2D cultures of murine 4T1 breast tumor cells (IC50=59nM), but not in the 3D reconstructed mouse breast model (r-mBreast) or in vivo, where the syngeneic 4T1 model was refractory to paclitaxel up to 10mg/kg. Similarly, NCI-H460 cells were sensitive to paclitaxel in 2D (IC50=2.3nM), while minimal response was seen in the 3D r-Lung model, and in vivo there was minimal response seen at a 20mg/kg treatment. These same cells were refractory to erlotinib in 2D, 3D, and in vivo. Additional SoC agents were profiled in these models and will also be discussed. Taken together these data demonstrate that zPREDICTA’s organ-specific 3D models mimic in vivo response with a higher fidelity than the standard 2D cultures. Additional in vitro reconstructed 3D histotype models are being developed and validated with the goal of providing faster and more predictive in vivo results.

Citation Format: Sumithra Urs, Michael Steffey, Ying Qu, Matthew Ryou, Julia Kirshner, Scott Wise. Reconstructed plate-based 3D screening assays that predict in vivo responses [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 283.
Abstract 2838: Antigen-independent expansion enhances efficacy of CAR-T cells against solid tumor
Authors: Cao Zhiyuan , Pu Chengfei , Jiang Xianyang , Han Guiting , Peng Yuzhe , Wang Wensheng , Ding Wei , Shen Xiaogang , chen Dongqi , Jia Beibei , Xu Xiaoqiang , Huang Zhipeng , Huang Xi , Liu Wenbi , Zhu Ruihong , Tian Lee , Ballas Christopher , Lu Victor.X , Wu Zhao , Xiao Lei
Published: June 2022

One key hurdle for the CAR-T cell treatment of solid tumors is the limited accessibility of solid tumor antigens outside the tumor microenvironment, which prohibits the expansion of solid tumor-targeting CAR-T cells in patients. Here, we report the characterization of prostatic acid phosphatase (PAP) as a feasible CAR-T target for prostate cancer and a novel approach, named CoupledCAR, to expand solid tumor-targeting CAR-T cells lacking solid tumor antigens based on the observation of non-transduced T cells proliferating together with CD19 CAR-T cells during the treatment of acute lymphocyte leukemia. We demonstrated that CoupledCAR can significantly enhance the expansion and antitumor efficacy of PAP CAR-T cells both in vitro and in vivo. Furthermore, we showed that the expansion of solid tumor-targeting CAR-T cells does not depend on CAR/CD3ζ stimulation through direct antigen binding with CAR but enhances the memory status of CAR-T cells and causes little exhaustion. Since the CoupledCAR system does not rely on solid tumor antigens, we propose that it can be utilized in all CAR-T and T cell therapies for the treatment of solid tumors.

Citation Format: Zhiyuan Cao, Chengfei Pu, Xianyang Jiang, Guiting Han, Yuzhe Peng, Wensheng Wang, Wei Ding, Xiaogang Shen, Dongqi chen, Beibei Jia, Xiaoqiang Xu, Zhipeng Huang, Xi Huang, Wenbi Liu, Ruihong Zhu, Lee Tian, Christopher Ballas, Victor.X Lu, Zhao Wu, Lei Xiao. Antigen-independent expansion enhances efficacy of CAR-T cells against solid tumor [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 2838.
Abstract 2858: Development of a first-in-class humanized antibody targeting CXCL1 in bladder cancer
Authors: Murakami Kaoru , Sasaki Yuka , Furuya Hideki , Rosser Charles
Published: June 2022

Introduction and Objectives: Chemokine (C-X-C motif) ligand 1 (CXCL1) plays a crucial role in tumor initiation, promotion, and progression in bladder cancer (BCa) development. Previously, we reported on a monoclonal murine antibody (HL2401) towards CXCL1 which was capable of inhibiting growth in both in vitro and in vivo models. Herein, we have developed a first-in-class humanized monoclonal antibody neutralizing CXCL1 (NTC-001). This study is final step in pre-clinical stage. In this study, we aimed to evaluate efficacy and safety of the antibody to move into phase 1 clinical trials in near future.

Methods: We humanized mouse anti-human CXCL1 antibody resulting in NTC-001, and then tested for the following: molecular weight and pH, specificity, binding affinity to ligand and to albumin, ability to inhibit in vitro angiogenesis, ability to inhibit cellular proliferation alone or with chemotherapy, and pharmacokinetics and pharmacodynamics.

Results: NTC-001 had a molecular weight of 150kD, and was an acidic antibody (pH5.1). NTC-001 specifically bound human CXCL1 in both WB and ELISA. NTC-001 was bound to albumin and had minimal hepatic metabolism. NTC-001 significantly inhibited tube formation in HUVEC, and more efficiently with the formulation. NTC-001 significantly inhibited T24 cell proliferation (p<0.001). Inhibitory effect of NTC-001 on cell proliferation was correlated with the levels of CXCL1 expression in BCa cell lines. Compared to CXCL1 expression-low group (RT112 and UMUC-14), CXCL1 expression-high group (T24, 5637, 253J, 253J-BV) tended to show low IC50. Synergistic combination efficacy was observed in NTC-001 in combination with gemcitabine (combination index<1) though not with cisplatin. PK study demonstrated a half-life of 324 hours and 18 hours in CD-1 and NSG-SGM3, respectively.

Conclusions: NTC-001 is a first in class humanized neutralizing monoclonal antibody towards CXCL1, and could be a promising therapeutic agent in BCa. We are going to proceed to complete our investigational new drug enabling studies in both small and large animals.

Citation Format: Kaoru Murakami, Yuka Sasaki, Hideki Furuya, Charles Rosser. Development of a first-in-class humanized antibody targeting CXCL1 in bladder 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 2858.
Abstract 2896: DLL3-targeted T cell engager therapy (HPN328) for neuroendocrine prostate cancer
Authors: Ku Sheng-Yu , Yamada Yasutaka , Ng Patrick , Sun Laura , Beltran Himisha
Published: June 2022

Background: Neuroendocrine Prostate Cancer (NEPC) is an aggressive variant of prostate cancer that can arise de novo or emerge during prostate cancer progression as a mechanism of therapy resistance. The development of effective therapies for patients with NEPC is a clinical unmet need. Delta-Like Ligand 3 (DLL3) is a negative regulator of Notch signaling and aberrantly expressed on the cell surface in the majority of NEPC and other small cell carcinomas. HPN328 is a DLL3-targeted Tri-specific T cell Activating Construct (TriTAC) that binds to DLL3 on tumor cells, CD3 on T cells, and human serum albumin. Here, we evaluated the specificity and anti-tumor activity of HPN328 in NEPC preclinical models.

Methods: Immunohistochemistry (IHC) was used to evaluate DLL3 expression in prostate cancer preclinical models. We co-cultured DLL3-positive and DLL3-negative tumor cells in vitro with either human PBMCs or T cells and used CellTiter-Glo and live-cell imaging to examine tumor cell viability after the exposure to HPN328. We conducted flow cytometry to examine T cell activation. Patient-derived organoid xenografts were used to determine the anti-tumor activity of HPN328 in vivo and we applied digital spatial profiling (Nanostring) to examine effects on immune cell infiltration.

Results: HPN328 suppressed cell growth and induced cell death in DLL3-positive prostate cancer organoid cell line models at a range of 0.7nM - 10nM with the presence of human T cells. Flow cytometry analysis illustrated that T cell activation markers, such as CD25, were upregulated in a dose- and time-dependent manner. In vivo studies demonstrated robust anti-tumor activity of HPN328 in causing tumor regression and stable disease after stopping the treatment in DLL3-positive models, but not in DLL3-negative models. Sustained anti-tumor effects were observed without adverse events in mice.

Conclusions: The DLL3-targeted TriTAC HPN328 demonstrates robust and specific anti-tumor activity in DLL3-positive NEPC preclinical models in vitro and in vivo. T cell engagers targeting DLL3 represent a promising therapeutic strategy for NEPC.

Acknowledgements: This research is funded by a Helen Trailblazers Award from the Helen Gurley Brown Foundation/DFCI Presidential Initiative (H.B.).

Citation Format: Sheng-Yu Ku, Yasutaka Yamada, Patrick Ng, Laura Sun, Himisha Beltran. DLL3-targeted T cell engager therapy (HPN328) for neuroendocrine 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 2896.
Abstract 2999: Activation of NFKB is responsible for defective autophagy in cadmium-induced transformation of prostate cells
Authors: Shukla Vaibhav , Tyagi Ashish , Kolluru Venkatesh , Saran Uttara , Chandrasekaran Balaji , Ankem Murali K. , Damodaran Chendil
Published: June 2022

Background: Evidence from the epidemiological, laboratory and clinical studies suggest that chronic exposure to cadmium (Cd) leads to the development and progression of prostate cancer (CaP). Previously, we reported defective autophagy due to upregulation of Placenta Specific 8 (Plac8) (a phagosome and lysosome fusion regulator) is responsible for Cd-induced transformation of normal prostate epithelial cells. This study mechanistically dissects the upstream signaling that regulates Plac8 function, which drives defective autophagy during Cd-induced transformation.

Methods: To examine the molecular interaction between NFκB and Plac8, we developed NFκB/Plac8 overexpression and knockdown in normal prostate epithelial cells (RWPE-1) and cadmium transformed prostate epithelial cells lines (CTPE). In addition, we performed phenotypic, and molecular analysis including promoter-based studies, western blot, and in vivo analysis to determine the interaction between Plac8 and p65 in Cd-exposed prostate epithelial cells.

Results: Our results confirmed that overexpression of Plac8 in normal prostate epithelial cells mimics Cd-transformed prostate cells (CTPE) and induces defective autophagy by regulating autophagy-related genes (ATGs) and an enhanced autophagic flux. Conversely, either NFκB or Plac8 knockdown in CTPE cells abrogates autophagy signaling. Further, Cd-exposure failed to induce defective autophagy signaling in transformed cells. Silencing p65 either by genetically or pharmacological inhibitors downregulated Plac8 expression and its survival function in CTPE cells. Hence, we investigated the interaction between NFκB and Plac8 by promoter-based analysis. We found NFκB binding site in the Plac8 promoter, and mutating NFκB binding sites in the Plac8 promoter showed a decline in luciferase activity confirming our hypothesis that NFκB activation is necessary for Plac8 activation and autophagy signaling in CTPE cells. Finally, we confirmed that silencing Plac8 significantly inhibited the tumor growth of CTPE cells compared to vehicle control. Our ongoing studies on xenotransplanted stably knockdown of p65 in CTPE cells may confirm that NFκB activation is responsible for Plac8 function and tumor growth in mice models.

Conclusion: In conclusion, our results suggest that NFκB transcriptionally regulates Plac8 function, which drives pro-survival autophagy signaling in Cd-transformed prostate epithelial cells.

Citation Format: Vaibhav Shukla, Ashish Tyagi, Venkatesh Kolluru, Uttara Saran, Balaji Chandrasekaran, Murali K. Ankem, Chendil Damodaran. Activation of NFKB is responsible for defective autophagy in cadmium-induced transformation of prostate 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 2999.
Abstract 301: Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant prostate cancer
Authors: Singh Santosh Kumar , Mishra Manoj Kumar , Singh Rajesh
Published: June 2022

Despite advanced treatment strategies and therapeutics for prostate cancer (PCa), the metastatic castration-resistant prostate cancer (mCRPC) mechanism remains obscure. Although docetaxel (DTX) and other anti-mitotic chemotherapeutic drug are considered the first line drugs for PCa patients; the resulting side effects and drug resistance increase the demand for an alternative such as natural compounds for effective treatment. However, the natural compounds that achieved remarkable height to overcome drug resistance, their bioavailability and targeted delivery during treatment has become a new challenge. As prostate-specific membrane antigen (PSMA) receptors are expressed on the surface of most PCa cells, we anticipated that these receptors may provide the potential target for effective therapy. Considering these, we employed the natural compound, thymoquinone (TQ)-encapsulated planetary ball-milled nanoparticles (PBM-NPs) formulated with starch (FDA approved, natural polysaccharides) inner core and biocompatible and biodegradable co-polymers poly (ε-caprolactone)/poly (ethylene glycol), functionalized with a PSMA aptamer (A10), which can selectively recognize and bind to PSMA membrane. The functionalities of this strategy were analyzed by targeting A10-TQ-PBM-NP in DTX-resistant cells (C4-2B-R and LNCaP-R), which overexpresses PSMA protein and multidrug resistance (MDR) gene ABCB1, and validated through flow cytometry, immunofluorescence, qRT-PCR, and western blot techniques. The results showed that the aptamer-based nano delivery of a TQ can modulate the cellular and molecular pathways responsible for DTX resistance in PCa cells through the EGFR and ZEB1 dependent mechanism. The A10-TQ-PBM-NPs counteract ATP-binding cassette (ABC)-transporter-mediated chemoresistance in PCa cells. In both cell lines, these NPs rapidly internalized and induced significant cell death at five times lower concentrations than non-conjugated PBM-TQ-NP. Further, A10-TQ-PBM-NPs have high selectivity to the PSMA receptor and downregulated the EGFR dependent pathway, Akt-1, ERK1/2, NF-kB, STAT3, and epithelial-mesenchymal transition (EMT) markers in both C4-2B-R and LNCaP-R cells, thus constraining cell survival, proliferation, and migration. The A10-TQ-PBM-NP bioconjugates also reveal remarkable efficacy and reduced toxicity as measured by body weight loss in the LNCaP-R cells xenograft mice. Altogether, our findings demonstrate that targeting PCa cells with PBM-NPs may reverse the MDR and could be used as potential therapeutic options for patients.

Citation Format: Santosh Kumar Singh, Manoj Kumar Mishra, Rajesh Singh. Repurposing engineered thymoquinone nanoparticles to inhibit drug-resistant 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 301.
Abstract 3015: Serine starvation silences estrogen receptor signaling through histone hypoacetylation
Authors: Li Albert M. , Li Yang , He Bo , Jiang Haowen , Lu Chao , Gruber Joshua J. , Rankin Erinn B. , Ye Jiangbin
Published: June 2022

Estrogen receptor (ER) plays important roles in regulating normal development and female reproductive system function. Loss of ER pathway activity is a hallmark of breast cancer progression, associated with accelerated tumor proliferation and resistance to endocrine therapy. How ER loss occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in solid tumors, downregulates estrogen receptor alpha (ERα) expression, represses transcriptional targets such as progesterone receptor (PR), and reduces sensitivity to antiestrogens, suggesting a transition of ER-positive (ER+) breast cancer cells to an ER/PR-negative (ER-/PR-) state. ER downregulation under serine starvation is accompanied by a global loss of histone acetylation. These chromatin changes are driven by metabolic reprogramming triggered by serine starvation, particularly lower glucose flux through glycolysis and the TCA cycle, leading to reduced acetyl-CoA levels and histone hypoacetylation. Supplementation with acetate or glycerol triacetate (GTA), precursors of acetyl-CoA, restores H3K27 acetylation and ERα expression under serine starvation. Therefore, a major consequence of serine starvation in breast cancer could be global chromatin changes that influence lineage-specific gene expression.

Citation Format: Albert M. Li, Yang Li, Bo He, Haowen Jiang, Chao Lu, Joshua J. Gruber, Erinn B. Rankin, Jiangbin Ye. Serine starvation silences estrogen receptor signaling through histone hypoacetylation [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 3015.
Abstract 3019: The epigenetic factor KDM2B alters the serine-glycine synthesis pathway and the one-carbon metabolism (SGOC) in triple-negative breast cancer
Authors: Chavdoula Evangelia , Anastas Vollter , La Ferlita Alessandro , Aldana Julian , Sircar Anuvrat , Freitas Michael A. , Sehgal Lalit , Tsichlis Philip N.
Published: June 2022

Epigenetic and metabolic alterations in cancer cells are intertwined. The concentration of metabolites can influence the activity of chromatin modifiers, which in turn can act as metabolic sensors that translate changes in cellular metabolism to transcriptional reprogramming. In the present study, we investigated the role of histone demethylase KDM2B in the metabolic reprogramming of the triple-negative breast cancer (TNBC), in which KDM2B is selectively expressed at high levels. Knockdown of KDM2B in TNBC cell lines reduced their proliferation rate and tumor growth in vivo. Transcriptomic, proteomic, and metabolomic profiling demonstrated that the Serine-Glycine pathway and One Carbon metabolism (SGOC) and other amino acid biosynthetic and catabolic processes are downregulated by the knockdown of KDM2B. Additionally, we see reduction of metabolites produced via these pathways (purines, pyrimidines, formate, glutathione and NADPH). Importantly, the expression of the enzymes involved in the SGOC metabolic pathway (e.g. PHGDH, PSAT1, PSPH, SHMT2, MTHFD1L, MTHFD2 and DHFR) depends on c-MYC, NRF2, and ATF4 which our data show that they are under the positive regulatory control of KDM2B. The epistatic relationship between these factors, with the expression of the enzymes of the SGOC pathway and the effects of the KDM2B knockdown on chromatin occupancy and accessibility of the promoters of these factors is in progress and will be presented. Analysis of TCGA data showed positive and statistically significant correlations between KDM2B and the SGOC gene signature in TNBC patients. In addition, the metabolic pathway signature that distinguishes control and shKDM2B-transduced cells corresponds to the metabolic signature of a subset of TNBCs, which have been reported to carry poor prognosis. The present study highlights the role of the epigenetic factor KDM2B as an upstream regulator of the metabolic reprogramming of TNBC.

Citation Format: Evangelia Chavdoula, Vollter Anastas, Alessandro La Ferlita, Julian Aldana, Anuvrat Sircar, Michael A. Freitas, Lalit Sehgal, Philip N. Tsichlis. The epigenetic factor KDM2B alters the serine-glycine synthesis pathway and the one-carbon metabolism (SGOC) in triple-negative breast 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 3019.
Abstract 302: MEKi-FGFRi combination nanoparticles for use against KRASmt/FGFR-compensatory lung tumors
Authors: Izawa-Ishiguro Arianna R. , Sridharan Ramya , Wun Christopher , Sami Tabassum J. , Heller Daniel A.
Published: June 2022

Targeting oncogenic signaling in the MAPK pathway can be achieved with small molecules such as trametinib, a MEK inhibitor (MEKi). Trametinib-induced MEK inhibition in KRAS-mutant (KRASmt) lung cancer, however, results in compensatory MAPK pathway re-activation through fibroblast growth factor receptor 1 (FGFR1). Combining MEK inhibition with FGFR1 inhibition should thereby combat this compensation; nonetheless, systemic administration of combination therapy faces its own set of challenges. Such challenges include delivering appropriate kinase inhibitor concentrations to target tumor tissue while avoiding dose-limiting additive toxicities.

Although previous work has identified that combination therapy involving trametinib and FGFR inhibitor (FGFRi) ponatinib synergistically inhibits growth and proliferation of KRASmt cells, ponatinib interacts with a variety of targets and may mediate its synergistic effects via mechanisms not specific to FGFR1. To improve the efficacy at which we target KRASmt/FGFR-compensatory lung tumors, we encapsulated trametinib with a variety of FGFR1-specific inhibitors to form P-selectin-targeted nanoparticles. By formulating these nanoparticles, compensatory MAPK pathway activation through FGFR1 can be more efficaciously inhibited. Concurrently, greater concentrations of kinase inhibitor combinations can be locally delivered to lung tumors while mitigating toxic side effects that occur from systemic free drug. Here we show that these MEKi-FGFRi combination nanoparticles can be formulated to efficaciously inhibit growth and proliferation in KRASmt/FGFR-compensatory cancer cells.

Citation Format: Arianna R. Izawa-Ishiguro, Ramya Sridharan, Christopher Wun, Tabassum J. Sami, Daniel A. Heller. MEKi-FGFRi combination nanoparticles for use against KRASmt/FGFR-compensatory lung tumors [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 302.
Abstract 3021: Understanding the effects of a ketogenic diet against pancreatic cancer
Authors: Hajihassani Omid , Vaziri-Gohar Ali , Zarei Mehrdad , Hue Jonathan , Cheng Helen , Mudigonda Anusha , Katayama Erryk , Graor Hallie , Winter Jordan
Published: June 2022

With an overall survival of less than 3%, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related death in the U.S. at its advanced stages. With the most promising therapeutics, the overall survival after a stage IV PDAC diagnosis is only at 11.1 months. The marginal effects of current therapeutics emphasize the importance of developing new approaches to treat PDAC. In a wide range of cancer subtypes, a ketogenic diet has shown encouraging effects as combination therapy. However, the underlying anti-tumorigenic pathways involved in this diet alone are still not well understood. The ketogenic diet’s core elements and direct physiologic effects (ketone bodies, fatty acids, low glucose, low insulin levels) potentially alter PDAC biology that span from redox homeostasis to mitochondrial metabolism, and epigenetic modifications. We hypothesized that in carefully controlled in vitro and in vivo experiments, KD components delay growth of PDAC. Additionally, we hypothesize that understanding the underlying drivers of the anti-tumor effects of a ketogenic diet could be leveraged into the rational design of combination therapies that augment these anti-tumor effects. We show that in the subcutaneous mouse model of PDAC, tumor growth is markedly delayed under ketogenic diet restrictions Furthermore, in PDAC cell culture models, we observed anti-cancer effects when the ketogenic diet core elements are isolated. Specifically, fatty acids and ketone bodies inhibit PDAC cell growth, particularly under high glucose conditions. These effects are somewhat attenuated under low glucose, suggesting that these elements result in competing effects on PDAC cells (i.e., support some pro-survival pathways while inhibiting others). Future studies seek to better delineate the mechanistic impact of the principal ketogenic diet elements on metabolic pathways with a focus on mitochondrial metabolism, as well effects on epigenetic signaling.

Citation Format: Omid Hajihassani, Ali Vaziri-Gohar, Mehrdad Zarei, Jonathan Hue, Helen Cheng, Anusha Mudigonda, Erryk Katayama, Hallie Graor, Jordan Winter. Understanding the effects of a ketogenic diet against pancreatic 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 3021.
Abstract 3241: IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro
Authors: Punyamurtula Ujwal , Zhang Shengliang , Brown Thomas , De La Cruz Arielle , Raufi Alexander , Strandberg Jillian , Carlsen Lindsey , Zhou Lanlan , El-Deiry Wafik
Published: June 2022

The 50% inhibitory concentration (IC50) is a widely used measure of how effective a given anti-cancer therapeutic is at reducing cancer cell viability in vitro. It is known that IC50 of a given therapeutic agent is dependent on the seeding density of the treated cells (in that more densely-seeded cells tend to be more resistant to therapeutic agents). We performed experiments using the CellTiter Glo cell viability assay to assess the cytotoxicity of various chemotherapeutic drugs towards various cancer cell lines. To elucidate the role the extracellular environment of densely-seeded cells plays in cancer cell chemoresistance, we tested conditioned media from cells cultured at high density on cells seeded at low densities. The resulting impact on the IC50 of the low-density cells was determined. The IC50 of the various therapeutic agents tested was cell density-dependent in each cancer cell line examined. Based on this analysis, we propose the IC50-seeding density slope (ISDS), which relates seeding density to the effectiveness of a drug and that we believe could be a standardized quantitative measurement of how a given cancer cell line responds to a given therapeutic treatment. Our results from the conditioned media experiments consistently indicated that the environment of densely-seeded tumor cells conferred chemoresistance to sparsely-seeded cells, suggesting that extracellular cytokines and growth factors may be involved in density-dependent chemoresistance mechanisms. We are currently exploring the effect of serum deprivation on in vitro chemoresistance, in order to further understand how the presence of extracellular nutrients impacts observed IC50 trends. In addition, we are examining the role that cell-cell contact, integrin and FGF signaling pathways play in giving rise to density-dependent chemoresistance. Moreover, to analyze the manner in which conditioned media gives rise to chemoresistance and how this effect relates to intracellular survival pathways, we are performing cytokine profiling experiments. Overall, we describe density-dependent IC50 variations as a hallmark of cancer cells and propose that ISDS may serve as a standardized method of assessing chemosensitivity.

Citation Format: Ujwal Punyamurtula, Shengliang Zhang, Thomas Brown, Arielle De La Cruz, Alexander Raufi, Jillian Strandberg, Lindsey Carlsen, Lanlan Zhou, Wafik El-Deiry. IC50-Seeding Density Slope (ISDS)-measurement as a standardized methodology for assessing anti-cancer therapeutic activity in vitro [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 3241.
Abstract 3304: Gene expression profiling of full-thickness skin after FLASH proton radiotherapy
Authors: Velalopoulou Anastasia , Karagounis Ilias V. , Skoufos Giorgos , Verginadis Ioannis I. , Kim Michele , Shoniyozov Khayrullo , Hatzigeorgiou Artemis G. , Diffenderfer Eric , Dong Lei , Metz James , Koumenis Constantinos , Cengel Keith A. , Maity Amit , Busch Theresa M.
Published: June 2022

Purpose: To investigate the transcriptomic changes induced by FLASH proton radiotherapy (F-PRT) that could be responsible for the protection of normal epithelial tissues by radiation-induced toxicities as have been previously shown by us and others.

Methods: C57BL/6J mice received 30 Gy of F-PRT or S-PRT to the hind leg at respective dose rates of 69-124 Gy/sec or 0.39-0.65 Gy/sec. RNA sequencing was performed using full-thickness leg skin at 5 days after radiation revealing major pathways regulated by F-PRT and S-PRT. In an endeavor to identify the full repertoire of cells and gene expression profiles that are involved in the sparing effects of FLASH PRT, we expanded our studies to include single-cell RNA sequencing (sc-RNA seq) and examined additional time points such as Day 2 and Day 10 after radiation. Single-cell transcriptome libraries were generated on a 10X Genomics Chromium system. Datasets were acquired from cell samples derived and sequenced from pooled skin samples of three mice per group. Skin from the sequenced mice was also embedded for spatial analysis of gene expression.

Results: RNA sequencing revealed that F-PRT uniquely upregulates almost four times more genes compared to S-PRT (F-PRT-uniquely upregulated 489 genes vs S-PRT-uniquely upregulated 129 genes). Also, F-PRT uniquely downregulated 178 genes, compared to the 125 genes uniquely downregulated by S-PRT. GO analysis demonstrates that the keratinization and apoptosis pathways are uniquely upregulated by S-PRT, whereas F-PRT uniquely upregulates genes involved in vascular development pathway. During submission of the abstract, analysis of sc-RNA seq samples was pending.

Conclusion: Our comprehensive studies inform on the transcriptomic profiling of skin cell populations that are affected by F-PRT vs S-PRT; this insight will further spur discoveries on the biology of FLASH radiotherapy effects.

Citation Format: Anastasia Velalopoulou, Ilias V. Karagounis, Giorgos Skoufos, Ioannis I. Verginadis, Michele Kim, Khayrullo Shoniyozov, Artemis G. Hatzigeorgiou, Eric Diffenderfer, Lei Dong, James Metz, Constantinos Koumenis, Keith A. Cengel, Amit Maity, Theresa M. Busch. Gene expression profiling of full-thickness skin after FLASH proton radiotherapy [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 3304.
Abstract 3319: Purified poloxamer 188 mitigates radiation-induced lung toxicity
Authors: Dennis-Little Sarah G. , Ngwa Wilfred , Laiho Marikki , Warren Graham , Yost Michael J.
Published: June 2022

The dose-limiting factor in radiation therapy (RT) is damage to healthy tissues, which presents as radiation-induced lung toxicity (RILT). Acute RILT, pneumonitis, is an early inflammatory-driven toxicity that develops within weeks to months following RT. Purified poloxamer 188 (PP188) is a triblock copolymer that associates with and protects cell membranes from oxidative insult, minimizing secondary inflammatory damage. This study aimed to investigate the effects of PP188 to mitigate the onset of acute radiation pneumonitis in a rat model of RILT, and to test the effect of PP188 on the efficacy of RT on tumor growth of a syngeneic CT26.WT murine colon carcinoma in Balb/c mice. To evaluate the efficacy of PP188 to mitigate RILT in healthy tissues, Sprague Dawley rats were given 200mg/kg PP188 or vehicle via IV 1hr prior to a single dose of partial volume (6mm) 20Gy X-irradiation to the right lung, and then were followed for 6 weeks. To validate PP188 treatment does not interfere with RT efficacy to reduce tumor volume, Balb/c mice were implanted with CT26.WT murine colon adenocarcinoma in the high axilla space, given 5Gy SARRP irradiation, and tumor volume was monitored. Rats were evaluated for histopathological changes indicative of acute RILT, including leakage of proteins into alveolar space, thickening of alveolar septa, and alteration of bronchial epithelium and capillaries; for presence of inflammatory infiltrate (Hanker-Yates Peroxidase Leukocyte kit); and for immunohistochemical expression of alpha-smooth muscle actin (SMA). Without PP188, significant inflammatory infiltrate was observed in all lungs bilaterally (p<0.001) contributing to significant bronchiole epithelium thickening (p<0.001) and alveolar septa thickening (p<0.001). Within the thickened alveoli there was significant SMA expression (p<0.001) indicative of cells undergoing a myofibroblast transition that was not observed in the PP188+RT animals or controls. Morphological assessment demonstrated that PP188 significantly attenuated pathological changes in the functional subunits of the lung, alveoli, as well as the vasculature including bronchiole epithelium and small capillaries, with no significant differences from control animals. Finally, it was shown that PP188 does not abrogate radiation-induced tumor volume reduction. The unique mechanism of PP188 provides radioprotection to healthy tissue without interfering with tumor control, and has the potential to mitigate radiation-induced toxicities in the majority of cancers treated with RT.

Citation Format: Sarah G. Dennis-Little, Wilfred Ngwa, Marikki Laiho, Graham Warren, Michael J. Yost. Purified poloxamer 188 mitigates radiation-induced lung toxicity [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 3319.
Abstract 3323: Discovery of a small-molecule inhibitor that can target EGFR with C797S mutation
Authors: Kim Dongsu , Son Woo Seung , Jang Anna , Lee Yeri , Kim Donggeon , Choi Changyu , Min Kyung Hoon , Choi Sung Pil , Lim Sang Kyun
Published: June 2022

EGFR is a transmembrane protein that functions as a receptor tyrosine kinase (RTK). Upon ligand binding or by activating mutations (Exon19 deletion, L858R mutation, and others), EGFR turns on the downstream signals that include oncogenic RAS/MEK/ERK, PI3K/AKT/mTOR, and JAK/STAT pathways. EGFR gene mutations and amplifications are frequently found in various human cancers and, in non-small cell lung cancer (NSCLC), the EGFR gene is mutated with 10-15% frequency (about 50% in Asian patients). While the 1st and 2nd generation EGFR inhibitors are effective in targeting EGFR mutants with Exon19 deletion and L858R mutation, additional T790M mutation in the EGFR gene causes resistance. The 3rd generation EGFR inhibitor (Osimertinib) works against EGFR mutants with T790M mutation; however, another EGFR mutation occurs at the amino acid 797 position (C797S), which makes Osimertinib ineffective. This is one of the major resistance mechanisms in Osimertinib-treated patients, and the 4th generation EGFR inhibitor that can target the C797S mutant is needed. We developed a compound that effectively disables various EGFR mutations, including Del19/T790M/C797S, L858R/T790/C797S, Del19/C797S, and L858R/C797S. In vitro kinase assay and cellular assays showed high potency and selectivity. In vivo PK/PD and efficacy tests confirmed the great therapeutic potential of this inhibitor for patients with EGFR mutations.

Citation Format: Dongsu Kim, Woo Seung Son, Anna Jang, Yeri Lee, Donggeon Kim, Changyu Choi, Kyung Hoon Min, Sung Pil Choi, Sang Kyun Lim. Discovery of a small-molecule inhibitor that can target EGFR with C797S mutation [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 3323.
Abstract 3337: Preclinical activity of NVL-655 in ALK-driven cancer models beyond non-small cell lung cancer
Authors: Tangpeerachaikul Anupong , Bigot Ludovic , Friboulet Luc , Pelish Henry E.
Published: June 2022

The ALK receptor tyrosine kinase can be aberrantly activated by gene fusions, point mutations, or amplification to promote cancer. ALK fusions are detected in approximately 5% of advanced non-small cell lung cancers (NSCLC), 50% of inflammatory myofibroblastic tumors, 50% of adult and 90% of childhood anaplastic large cell lymphomas, and rare cases of cholangiocarcinomas. Over 90 fusion partners of ALK have been identified, each exerting a different influence on the biochemical properties of the fusion protein and its preclinical sensitivity to ALK inhibitors. Besides fusions, ALK point mutations and amplification account for up to 14% and 2% of sporadic neuroblastomas, respectively, with most activating mutations occurring at residues F1174, F1245, or R1275. Some activating mutations, such as those occurring at F1174 and I1171, also confer resistance to FDA-approved ALK inhibitors. Because ALK plays an oncogenic role in diverse cancer types, it is an important therapeutic target for other indications beyond NSCLC.

NVL-655 is a novel, brain-penetrant ALK-selective inhibitor that exhibits preclinical activity against ALK alterations, including resistance mutations, while also sparing inhibition of TRKB. TRKB inhibition in the central nervous system has been implicated in adverse events observed with FDA-approved ALK inhibitor lorlatinib. We previously reported the preclinical activity of NVL-655 in several NSCLC models bearing ALK-fusions, including human cell lines, a human cell-line derived xenograft, and a patient-derived xenograft. Here we report a broader characterization of NVL-655 in ALK-driven cancer models beyond NSCLC, alongside other ALK inhibitors that are either FDA-approved or in development.

In biochemical assays, NVL-655 showed potent inhibition (IC50 < 10 nM) of ALK with mutations at residues 1151, 1174, or 1275, all of which have been identified in neuroblastoma. In cell-based assays, NVL-655 was observed to inhibit proliferation of a human anaplastic large cell lymphoma cell line harboring NPM1-ALK fusion and human neuroblastoma cell lines harboring ALK activating mutations or amplification. In conclusion, the preclinical profile of NVL-655 supports its potential to address a medical need for patients with ALK-driven disease in both NSCLC and other cancers such as anaplastic large cell lymphoma and neuroblastoma.

Citation Format: Anupong Tangpeerachaikul, Ludovic Bigot, Luc Friboulet, Henry E. Pelish. Preclinical activity of NVL-655 in ALK-driven cancer models beyond non-small cell lung 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 3337.
Abstract 3379: Multi-cohort study shows HCC is associated with a relative regional reduction in TGFBR2 in liver biopsies from patients with cirrhosis
Authors: Bhowmick Krishanu , Amdur Richard , Xiang Xiyan , Yu Herbert , Wong Linda L. , Rao Shuyun , He Aiwu R. , Amin Karan , Zaheer Daewa , Narayan Raj K. , Satapathy Sanjaya K. , Guha Chandan , Latham Patricia S. , Shetty Kirti , Gough Nancy R. , Mishra Lopa
Published: June 2022

Background: Early detection of hepatocellular carcinoma (HCC) is challenging partly because most patients also have cirrhosis. Furthermore, patients with cirrhosis are at high risk of developing HCC. Loss of function of the transforming growth factor β (TGF-β) pathway is associated with HCC. The goal of this study was to determine if a reduction in TGF-β receptors (TGFBR1, TGFBR2) was able to reliably and reproducibly predict HCC in cirrhotic tissue.

Methods: We performed quantitative immunohistochemical (IHC) analysis of biopsy tissue from patients with cirrhosis with or without HCC using 3 separate sets of samples. We developed an automated image analysis pipeline to quantify IHC of TGFBR1 and TGFRB2 in liver biopsies. Using logistic regression modeling, we evaluated the predictive power of staining intensity of TGFBR1, TGFBR2, or the combination in differentiating HCC from cirrhosis.

Results: We found a consistently significant reduction in TGFBR2 in HCC (p < 0.02) among independent patient cohorts from 3 separate institutions. With the data, we developed an artificial intelligence-based image analysis pipeline that confirmed the significant reduction in TGFBR2 staining intensity in HCC compared to cirrhotic tissue (p < 0.002). Unfortunately, logistic regression modeling of the quantitative data showed that there is not a simple threshold of TGFBR2 staining intensity that is diagnostic of HCC. Instead, in 84% of samples with HCC, HCC was associated with a relative regional reduction in TGFBR2 compared to tumor-adjacent cirrhotic tissue.

Conclusions: The qualitative and quantitative analyses indicate that evaluating biopsy samples for a reduction in TGFBR2 abundance relative to the surrounding cirrhotic tissue could be an early indicator of the development of HCC in high-risk groups undergoing surveillance for HCC. Furthermore, an automated image analysis pipeline for quantitative IHC of TGFBR staining could improve the reliability of image analysis across institutions participating in clinical trials. 

Citation Format: Krishanu Bhowmick, Richard Amdur, Xiyan Xiang, Herbert Yu, Linda L. Wong, Shuyun Rao, Aiwu R. He, Karan Amin, Daewa Zaheer, Raj K. Narayan, Sanjaya K. Satapathy, Chandan Guha, Patricia S. Latham, Kirti Shetty, Nancy R. Gough, Lopa Mishra. Multi-cohort study shows HCC is associated with a relative regional reduction in TGFBR2 in liver biopsies from patients with cirrhosis [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 3379.