bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2819: Suppression of NKG2A mediated inhibition in ex vivo expanded natural killer cells increases their cytotoxicity
Authors: Croom-Perez Tayler J. , Robles-Carrillo Liza D. , Dieffenthaller Thomas A. , Copik Alicja J.
Published: June 2022

In this study we propose to circumvent HLA-E/NKG2A mediated Natural Killer (NK) cell inhibition by suppression of NKG2A, to enhance cytotoxicity of ex vivo expanded NK cells. One mechanism employed by tumor cells to evade immunosurveillance is through induction of the surface expression of unconventional HLA ligands that agonize inhibitory receptors on immune cells. Specifically on some tumors, HLA-E is either expressed or its expression is known to be induced by IFNγ and is indicative of resistance to immunotherapy. HLA-E agonizes the CD94/NKG2A inhibitory complex on NK cells and some T cells to lessen their cytotoxicity, potentially decreasing the efficacy of cellular therapy with these immune cells. The CD94/NKG2A inhibitory complex is in balance with CD94/NKG2C stimulatory complex. HLA-E binds both of these complexes and the cytolytic activity of NK cells is influenced by the relative ratios of CD94 complexed to NKG2A or NKG2C. Thus, altering the ratio to increase NKG2C will increase NK cell cytotoxicity. In this study we show that NK cells ex vivo expanded with PM21-particle technology are highly cytotoxic and have elevated expression of NKG2A. These expanded NK cells also secrete high levels of IFNγ, inducing expression of HLA-E in tumor cells. To increase the cytotoxic potential of these ex vivo expanded NK cells, the potential to form CD94/NKG2A inhibitory complex was suppressed by either antibody blockade of NKG2A or deletion of the NKG2A gene by CRISPR/Cas9 editing. Cytotoxicity of NK cells against a lung cancer cell line stably expressing HLA-E were determined by kinetic live-cell imaging. Multiple NK cell:target (NK:T) ratios were used to show an increase in cytotoxicity over control NK cell conditions (alone or with isotype control) in the presence of NKG2A blocking antibodies with 39% increase observed at 1:10 NK:T ratio at 96h with anti-NKG2A and 56% increase with NKG2A knockout NK cells. These data suggest that blockade of CD94/NKG2A complex can improve cytotoxic activity of ex vivo expanded NK cells and could provide a promising effector population with the potential for enhanced therapeutic efficacy.

Citation Format: Tayler J. Croom-Perez, Liza D. Robles-Carrillo, Thomas A. Dieffenthaller, Alicja J. Copik. Suppression of NKG2A mediated inhibition in ex vivo expanded natural killer cells increases their 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 2819.
Abstract 282: Breast cancer microphysiological systems based on human breast tissue
Authors: Lau Frank H. , Byrne C. Ethan , Brown Loren , Fontenot Jake , Tiongco Rafael P. , Gurrala Rakesh R. , Cuccia Jonathan , DiNardo Andrew , Burow Matthew , Martin Elizabeth C.
Published: June 2022

Breast cancer is a heterogeneous disease that accumulates signaling cues from cell intrinsic and extrinsic factors. Many of these cues are derived from the tumor microenvironment (TME). The TME modulates cellular proliferation, survival, and resistance to therapy in breast cancer. The TME is a bio-mechanical and biochemical reservoir for extracellular matrix and signaling factors (cytokines, growth factors, lipids, and hormones). In addition, the TME houses a diverse array of cell populations including adipocytes, vasculature, stem cells, immune cells, and cancer cells. Currently models do not reprpoduce the TME in vitro. The goal of this study was to develop a complex and dynamic micro-physiological 3D tumor model for pre-clinical studies using the native human breast environment. We developed a technique to maintain healthy human breast tissue (HBT) alive ex vivo for up to 8 weeks in the presence of breast cancer (BC) cells creating a breast cancer micro-physiological system (BC-MPS). In this study, we demonstrate alterations to lipid accumulation, matrix fiber remodeling, and cytokine profile in our BC-MPS system. We used RNA sequencing to detect breast cancer cell line signatures retained in the BC-MPS system. Results demonstrate retention of the breast cancer cell line transcriptome for a minimum of 14 days in vitro. In addition we identify alterations to genes associated with the TME, specifically the extracellular matrix and metabolism. Our novel model will allow for 1) real-time examination of BC-HBT interactions and 2) the isolation of BC-specific factors vs. patient-specific factors on the development and progression of BC.

Citation Format: Frank H. Lau, C. Ethan Byrne, Loren Brown, Jake Fontenot, Rafael P. Tiongco, Rakesh R. Gurrala, Jonathan Cuccia, Andrew DiNardo, Matthew Burow, Elizabeth C. Martin. Breast cancer microphysiological systems based on human breast tissue [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 282.
Abstract 2830: Multiplexed shRNAs targeting FAS and PTPN2 enhance CAR T persistence and anti-tumor efficacy
Authors: Gagnon John , Litterman Adam J. , Hall Jason A. , Polyak Dina , Zhou Stanley , Joshi Sahil , Takacsi-Nagy Oliver , Pope Hans , silva luisa , Singh brenal K. , Granja jeffrey M. , DeTomaso david , Aristil-Lepe Edgar , Tan Michelle , Galvin Brendan , Zheng Grace X. , Santoro Stephen , Cooper Aaron , Bezman Natalie , Haining W Nicholas
Published: June 2022

Cell therapies have shown limited efficacy in solid tumors, in part due to limitations in CAR T cell survival, expansion, and resistance to immunosuppressive pathways operating in the tumor microenvironment (TME). To overcome these hurdles, we screened a library of 171 pair-wise gene knockouts using CRISPR-Cas9 to identify gene pairs that confer greater proliferation and persistence with repetitive stimulation. Combined KO of FAS and PTPN2 led to a significant improvement in T cell effector function and proliferative capacity. FAS, the receptor for FASLG, is highly expressed on patient-derived T cells and has been shown to induce T cell apoptosis and limit T cell persistence in the TME. PTPN2 is a phosphatase, in which deletion increases T cell proliferation and cytotoxicity. To develop this clinically, we generated CAR T cells with a shRNA module capable of multiplexed knockdown of both FAS and PTPN2. The FAS/PTPN2 shRNA module protected T cells from FAS-mediated apoptosis and resulted in a marked increase in T cell expansion over the course of a repetitive stimulation assay. Consistent with these observations, RNAseq analysis revealed enhanced transcriptome-wide signatures of cell cycle and T cell effector function. Finally, in a tumor xenograft model, CAR T cells containing FAS/PTPN2 shRNA module demonstrated significantly improved tumor control in addition to increased cell numbers in circulation compared with CAR T cells expressing an irrelevant shRNA module.

The use of a multiplexed shRNA module is a powerful approach to improve T cell intrinsic functionality in cellular therapies, enabling: 1) tailored levels of inhibition of multiple key regulators of T cell biology with constitutive or antigen-gated expression, and 2) a method for genetic perturbations in T cells with reduced risk of toxicity or transformation compared with multiple DNA edits. The FAS/PTPN2 shRNA module described here will be tested as a component of AB-1015, an integrated circuit T cell therapy for solid tumors expressing ALPG and mesothelin.

Citation Format: John Gagnon, Adam J. Litterman, Jason A. Hall, Dina Polyak, Stanley Zhou, Sahil Joshi, Oliver Takacsi-Nagy, Hans Pope, luisa silva, brenal K. Singh, jeffrey M. Granja, david DeTomaso, Edgar Aristil-Lepe, Michelle Tan, Brendan Galvin, Grace X. Zheng, Stephen Santoro, Aaron Cooper, Natalie Bezman, W Nicholas Haining. Multiplexed shRNAs targeting FAS and PTPN2 enhance CAR T persistence and anti-tumor efficacy [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 2830.
Abstract 2843: Development of a translatable CD19 CAR-T platform for drug discovery
Authors: Kelsey Lauren , Self-Fordham Jezrom , Oswald Eva , Aylott Ilona , Rocca Daniel , Nunan Robert , Harris David , Schueler Julia , Jenkinson Rhiannon
Published: June 2022

Chimeric Antigen Receptor (CAR)-T cell therapy has achieved great success with specificity, efficacy, safety and ingenuity. CAR cell technology is advancing rapidly, with ever increasing capabilities for cancer treatment both with regard to tumor types and the CAR cell used (NK, Macrophage, T). To date, the most successful application remains the treatment of B cell lymphoma using CD19-specific CAR-T cells. Here we describe how to efficiently generate and expand a CD19 CAR-T cell and validate their efficacy in in-vitro and in-vivo studies. HLA-A*02:01+ CD3+ T-cells were transduced to express a clinically tested CD19-specific CAR to >80% efficiency and reproducibly expanded to >68 fold over a 19-day period. After sufficient expansion, cells were validated in in-vitro studies to assess their efficacy, utilizing a HLA-A*02:01+ patient derived xenograft (PDX) CD19+ cell line, alongside human cancer cell lines RAJI (CD19+) and K562 (CD19-). Flow cytometry analysis revealed the potent ability of CD19-CAR-T cells to kill CD19+ targets in a dose-dependent fashion. Highly specific killing at all E:T ratios was observed, with negligible off target killing identified. Supportive supernatant analysis showed highly elevated pro-inflammatory cytokine concentrations, identifying enhanced activation of CAR-T cells in the presence of CD19+ target cells only. After successful in-vitro batch testing for on-target killing CAR-T cells were released for in-vivo survival studies. Mice were inoculated with the same strains of CD19+ cells used during in-vitro validation to produce a tumor burdened model. CAR-T cells or non-transduced cells from the same donor were transfused and survival rates, tumor progression and tolerability assessments were observed over several weeks. Ex-vivo tissue and sera analysis further characterized these highly efficacious human CAR-T cells. These assays represent the capability to produce, expand and study CAR-effector cells both in-vitro and in-vivo. Careful consideration of human target cells enables the efficient translation of in-vitro efficacy data to whole organism mouse models.

Citation Format: Lauren Kelsey, Jezrom Self-Fordham, Eva Oswald, Ilona Aylott, Daniel Rocca, Robert Nunan, David Harris, Julia Schueler, Rhiannon Jenkinson. Development of a translatable CD19 CAR-T platform for drug discovery [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 2843.
Abstract 2854: Logic gates controlled by priming receptors increase specificity and potency of CAR T cells
Authors: Williams Jasper , Martinez Kevin , Boroughs Angela , Lim Laura , Sandoval Marian , Sue Cate , Drever Matthew , Yao Anzhi , Choe Joseph , Sidorov Maxim , Phillips Sophia , Polyak Dina , Mohanty Suchismita , Santoro Stephen , Cooper Aaron , Haining W. Nicholas
Published: June 2022

CAR T cell therapies for solid tumors are limited by a paucity of tumor-specific single target antigens and insufficient potency due to limitations of CAR T biology. Logic gated (LG) CARs increase control of cell therapy activation and target cell killing by requiring recognition of two or more antigens through AND or AND-NOT Boolean logic. They represent an attractive strategy for cell therapy to improve the therapeutic index by increasing specificity for tumor antigens.

We created an AND logic gate cassette deliverable by CRISPR-mediated, non-viral, site-specific integration into human T cells. This logic gate activates expression of a MSLN-targeting CAR upon ALPP/G binding of a priming receptor (PrimeR) within high-grade serous carcinoma ovarian tumors. The PrimeR triggers proteolytic release of a chimeric, fully human transcription factor, which then translocates to the nucleus to induce expression of a minigene encoding a CAR. The PrimeR is built only from human protein sequences, reducing theoretical risk of cell therapy immunogenicity and rejection within a patient.

In order to test the specificity of our LG CARs, we integrated LG cassettes into a defined site in the T cell genome, and then co-cultured T cells with K562 cells engineered with only one target antigen (K562-ALPG or K562-MSLN), both target antigens (K562-ALPG/MSLN), or neither target antigen (K562). LG CAR T cells only killed K562-ALPG/MSLN cells. The specificity of this LG was also demonstrated in vivo in a dual-flank K562 model, in which only the ALPG/MSLN tumor was inhibited by LG CAR T cells. CAR expression was not observed on LG CAR T cells recovered outside the ALPG/MSLN tumor, and no growth inhibition was observed in K562-MSLN tumors, unlike a constitutive CAR T control. To model priming antigen heterogeneity expected in tumors, we mixed K562-MSLN cells with varying proportions of K562-ALPG/MSLN cells. We found that the presence of only 5-15% ALPG/MSLN cells was sufficient to effect killing of all target cells by the induced MSLN CAR. The ALPG/MSLN LG CAR will be tested as a component of AB-1015, a novel therapy for indications including ovarian, fallopian tube, or primary peritoneal cancer.

Citation Format: Jasper Williams, Kevin Martinez, Angela Boroughs, Laura Lim, Marian Sandoval, Cate Sue, Matthew Drever, Anzhi Yao, Joseph Choe, Maxim Sidorov, Sophia Phillips, Dina Polyak, Suchismita Mohanty, Stephen Santoro, Aaron Cooper, W. Nicholas Haining. Logic gates controlled by priming receptors increase specificity and potency of CAR 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 2854.
Abstract 286: Patient derived explant culture (PDEC) as a model for treatment resistant ERα positive breast cancers
Authors: Munne Pauliina , Räty Iiris , Peura Aino , Patrikainen Linda , Belitskin Denis , Klefstrom Juha
Published: June 2022

Breast cancer is the most frequent cancer and leading causes of women’s death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα+) phenotype that is initially responsive to antihormonal therapies, but often drug resistance emerges. Three-dimensional ex vivo cultures from patient-derived tumor tissue offer new opportunities for drug development and personalized treatment of cancer. However, one of the main challenges in establishing a tumor type faithful ex vivo tissue culture system is in optimization of biochemical and physical microenvironment to preserve desired phenotypic features. The ERα+ phenotype is not stable in cultured cells for reasons not fully understood. Together with Helsinki University Hospital we examined 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. In our current study we have utilized the PDEC model to identify novel biomarkers that could predict the emergence of tamoxifen resistance in primary breast tumors.

Citation Format: Pauliina Munne, Iiris Räty, Aino Peura, Linda Patrikainen, Denis Belitskin, Juha Klefstrom. Patient derived explant culture (PDEC) as a model for treatment resistant ERα positive breast 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 286.
Abstract 2888: Tumor-targeted CD28 bispecific POWERbody࣪ for safe and synergistic T cell-mediated immunotherapy
Authors: Liu Guizhong , Dai Zhengxi , Qu Jiagui , Shi Jianfeng , Zhao Qi , Cai Bin , Nguyen Aaron N. , Zheng Songmao , Xu Jiangchun , Li Yan , Du Felix , Luo Peter
Published: June 2022

Targeting CD28 for systemic T cell activation caused severe cytokine storm and multiorgan failure in an anti-CD28 antibody TGN1412 phase 1 trial. However, tumor specific CD28 targeting in a tumor associated antigen (TAA)×CD28 bispecific format, similar to the clinically validated tumor specific CD3 bispecific T-cell engagers (TCEs), offers an attractive solution to mitigate the serious safety concerns associated with systemic CD28 activation while delivering potent T-cell costimulatory signal. Our goal is to combine tumor specific bispecific TCEs targeting both CD3 and CD28, the two essential pathways in T cell activation for safe and long-lasting T cell-mediated synergistic immunotherapies. Following our success in utilizing the CD3 POWERbody™ TCE platform for tumor targeting with tight safety control, we further applied our suite of antibody technologies to develop tumor specific bispecific CD28 POWERbody TCE programs. Our NEObody™ and SAFEbody® technologies enabled us to discover and engineer species cross-reactive anti-CD28 antibodies targeting a unique and conserved epitope of CD28 as well as precision masking of the antigen binding sites for conditional activation in tumor microenvironment. The engineered tailor-made anti-CD28 arm and the tumor specific antigen targeting arm were then integrated into our proprietary bispecific platform that has robust CMC properties. Toward this end we developed multiple TAA×CD28 bispecific TCEs, including B7-H3×CD28, HER2×CD28, and TROP-2×CD28, etc., which possess fine-tuned specificity and safety profiles to minimize their on-target off-tumor toxicities and to prevent the systemic cytokine release syndrome (CRS). These TAA×CD28 bispecific POWERbodies have no superagonist activities on their own and can be activated for tumor antigen binding by the TAA arm and T cell engagement by the CD28 arm. When combined with TAA×CD3 TCEs and/or checkpoint inhibitors, they show strong synergistic T cell-mediated antitumor responses with robust safety profiles. Taken together, our tumor specific CD28 bispecific POWERbody TCEs exhibit enormous potential to fulfill the promises of safe and durable T cell-mediated immunotherapies when combined with CD3 bispecific POWERbody TCEs and/or checkpoint inhibitors.

Citation Format: Guizhong Liu, Zhengxi Dai, Jiagui Qu, Jianfeng Shi, Qi Zhao, Bin Cai, Aaron N. Nguyen, Songmao Zheng, Jiangchun Xu, Yan Li, Felix Du, Peter Luo. Tumor-targeted CD28 bispecific POWERbody࣪ for safe and synergistic T cell-mediated immunotherapy [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 2888.
Abstract 2894: QL401, a dual PD-L1/CD47 blocker effective against both solid and hematological malignancies with improved blood safety
Authors: Tang Irene , Schwimmer Lauren , Gu Shenda , Prior Wei Wei , Tran Hieu V. , Chan Allan , McClain Anna , Chen Shihao , Cao Chuanzeng , Sun Chunyan , Si Meimei , Wang Guijiang
Published: June 2022

Upregulation of CD47, a “Do Not Eat Me” signal, is observed in nearly all solid and hematological malignancies. Engagement of CD47 on tumor cells with SIRPα on macrophages inhibits phagocytosis of tumor cells. Anti-CD47 antibodies block the CD47 - SIRPα engagement and reactivate phagocytosis of tumor cells by macrophages. Yet the ubiquitous expression of CD47 on normal cells, including red blood cells, presents a therapeutic challenge. Systemic targeting of CD47, by either anti-CD47 monoclonal antibodies or SIRPα-Fc fusion proteins, yielded only moderate clinical benefit due to severe adverse side effects, mainly anemia. QL401 is PD-L1 x CD47 bispecific antibody engineered to reduce binding to red blood cells while retaining potent phagocytic activation of macrophages in vitro and delayed tumor growth in vivo. QL401 comprises three functional components: a PD-L1 binding Fab arm, a CD47 binding scFv arm, and a human IgG4 backbone. The PD-L1 binding arm provides both tumor targeting and blocking of PD-1 for reactivating T cells. The CD47 arm blocks the binding of SIRPα, the “Do Not Eat Me” signal, while the IgG4 Fc retains Fc gamma receptor binding to provide a phagocytic “Eat Me” signal. Therefore, QL401 is multi-functional antibody that re-actives both the innate and adaptive immune response. In preclinical efficacy studies, QL401 potently blocked SIRPα to promote phagocytosis of tumor cells with sub-nanomolar potency. In vivo efficacy studies in mouse models of solid and hematological tumors showed QL401 to be comparable or superior to PD-L1 or CD47 monoclonal antibodies alone and in combination. In vitro safety evaluation of Q401 showed significantly reduced binding and phagocytosis of red blood cells, in contrast to CD47 monoclonal antibodies. In addition, Q401 did not induce hemagglutination. In non-human primates, QL401 was well tolerated up to 100 mg/kg without reduction of red blood cells below normal range. A phase 1 dose escalation and expansion study is expected to start Q1 2022 to evaluate the safety, tolerability, and early efficacy of QL401.

Citation Format: Irene Tang, Lauren Schwimmer, Shenda Gu, Wei Wei Prior, Hieu V. Tran, Allan Chan, Anna McClain, Shihao Chen, Chuanzeng Cao, Chunyan Sun, Meimei Si, Guijiang Wang. QL401, a dual PD-L1/CD47 blocker effective against both solid and hematological malignancies with improved blood 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 2894.
Abstract 2899: Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors
Authors: Kumar Roshan M. , Prigent Julie , Poullain Hombline , Kok Ayrin , George Carine , Ellouze Sami , Lu Yun-Yueh , Silver Rebecca , Monchecourt Clarisse , Fulton Ross B. , Zhang Qian , Beltraminelli Nicola , Moser Bernhard , Adrian Francisco , Schweizer Liang
Published: June 2022

Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform.

Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid 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 2899.
Abstract 2921: Probing PRMT5 inhibitors with distinct binding modes using surface plasmon resonance
Authors: Eells Rebecca , Baker Lynn K. , Bray Christopher , Ma Haiching
Published: June 2022

Epigenetic modifications are dynamic and reversible processes that regulate gene expression without altering the underlying sequence of the DNA. Gene expression is instead controlled by modification to the chromatin including DNA methylation, histone modifications, and chromatin remodeling. The proteins that perform these epigenetic modifications can be divided into three classes: writers, which perform the modifications; readers, which recognize the modifications; and erasers, which remove the modifications. While essential for normal cellular function, abnormal expression or alteration of the proteins involved in epigenetic regulation can lead to disease, such as cancer. As a result, epigenetic modifiers present attractive targets for the discovery and development of novel therapeutics. At Reaction Biology we offer a suite of services to enable epigenetic drug including the largest panel for epigenetic screening and profiling in the industry and biochemical and biophysical technologies to evaluate inhibition, potency, target occupancy, and even binding modes. Here we highlight our biophysical platform surface plasmon resonance (SPR) using the epigenetic target PRMT5 as an example. SPR was used to measure the binding kinetics of several small molecule inhibitors of PRMT5 and explore their distinct binding modes.

Citation Format: Rebecca Eells, Lynn K. Baker, Christopher Bray, Haiching Ma. Probing PRMT5 inhibitors with distinct binding modes using surface plasmon resonance [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 2921.
Abstract 2936: Multi-omic characterization of transitional cell populations in breast cancer
Authors: Iglesia Michael D. , Jayasinghe Reyka G. , Zhou Daniel Cui , Terekhanova Nadezhda V. , Herndon John , Karpova Alla , Chen Siqi , Deen Nataly Naser Al , Sato Kazuhito , Chen Feng , Veis Deborah J. , Fields Ryan C. , Gillanders William E. , Ding Li
Published: June 2022

Breast cancer is a heterogeneous collection of diseases grouped by hormone receptor status or by expression of key subtype-determining genes. Breast cancer subtypes, in particular basal-like breast cancer and the luminal breast cancer subtypes, differ by hormonal receptor status, proliferation, genomic instability and mutational signatures, treatment response, and prognosis. The highly distinct signatures of basal-like and luminal breast cancer suggest that they may have different cells of origin within the breast duct. As part of the Washington University Human Tumor Atlas Network (WU-HTAN) program, we generated multi-omic data for 53 samples from 37 breast cancer tumors and 4 normal adjacent tissues. Genomic subtyping was applied to both bulk and single-nucleus RNA sequencing. Analysis of single-nucleus gene expression and chromatin accessibility in epithelial cells underscores similarities between basal-like breast cancer and luminal progenitor cells within the breast duct, and between luminal breast cancer and mature luminal ductal cells. This study links distinct breast cancer subtypes to normal breast cell populations and suggests distinct cells of origin for these cancer types.

Citation Format: Michael D. Iglesia, Reyka G. Jayasinghe, Daniel Cui Zhou, Nadezhda V. Terekhanova, John Herndon, Alla Karpova, Siqi Chen, Nataly Naser Al Deen, Kazuhito Sato, Feng Chen, Deborah J. Veis, Ryan C. Fields, William E. Gillanders, Li Ding. Multi-omic characterization of transitional cell populations in 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 2936.
Abstract 2942: Robust sample recovery post-digital PCR for downstream genomic applications
Authors: Gutierrez Kimberley , Foret Benjamin , Jovelet Cécile , Mallory Allison C.
Published: June 2022

Droplet microfluidics technologies, and particularly, digital PCR have provided highly precise and sensitive detection of genetic targets. There is a strong interest for researchers to reuse sample due to sample scarcity, as well as, to further characterize or validate their experimental findings. Historically, recovery of PCR products for downstream genomic testing applications has been challenging due to the stability of the sample post-reaction. Stilla Technologies has developed Crystal Digital PCR™ and the naica® system, a flexible and highplex droplet and imaging-based digital PCR platform that allows for multi-color detection of genetic targets with the ability to also recover samples after the reaction is performed.

This study shows how the 6-color naica digital PCR system workflow can be combined with a simple protocol for sample recovery. DNA contained in the recovered emulsion is extracted using a standard chloroform protocol that takes approximately 1 hour. DNA recovery rate assessment showed high percentage recovery of droplet sample and on average, 70% of the total DNA present from the initial sample recovered. Recovered DNA from the droplet crystal was sequenced and the results were confirmed by qPCR.

The droplet and sample recovery protocol post-digital PCR has potential applications to support validation of Crystal Digital PCR results, concordance alongside a multitude of genomic platforms, and further characterization of biomarkers and mutations with profiling platforms such as NGS.

Citation Format: Kimberley Gutierrez, Benjamin Foret, Cécile Jovelet, Allison C. Mallory. Robust sample recovery post-digital PCR for downstream genomic applications [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 2942.
Abstract 2953: Subtype-specific and structure variation induced 3D genome alteration in acute myeloid leukemia
Authors: Xu Jie , Song Fan , Zhang Baozhen , Lyu Huijue , Kobayashi Mikoto , Zhao Ziyu , Hou Ye , Wang Xiaotao , Luan Yu , Jia Bei , Stasiak Lena , Wang Qixuan , Jin Qi , Jin Qiushi , Fu Yihao , Hardison Ross C. , Dovat Sinisa , Platanias Leonidas C. , Yang Yue , Yamada Tomoko , Viny Aaron D. , Levine Ross L. , Claxton David F. , Broach James R. , Zheng Hong , Yue Feng
Published: June 2022

Acute myeloid leukemia (AML) represents a set of heterogeneous myeloid malignancies hallmarked by mutations in epigenetic modifiers, transcription factors, and kinases that can cause epigenetic reshaping. It is unclear to what extent AML mutations drive chromatin 3D structure alteration and contribute to myeloid transformation. We first performed Hi-C and whole-genome sequencing in 25 AML patient samples and seven healthy donor samples, and identified recurrent alterations of A/B compartments, TADs, and chromatin loops that are unique to different subtypes. To investigate how altered chromatin organization contributes to transcriptional misregulation, we performed RNA-Seq, ATAC-Seq and CUT&ag for CTCF, H3K27ac, and H3K27me3 in the same AML samples. We identified extensive and recurrent AML-specific promoter-enhancer and promoter-repressor loops. We performed both CRISPR deletion and interference experiments and validated two repressor loops that downregulated cancer related genes IKZF2 and RTTN. Furthermore, by using our recently developed algorithm, we identified structural variation-induced enhancer-hijacking and repressor-hijacking events in AML samples. We further demonstrated the role of hijacked enhancers in AML cell growth by CRISPR screening, and the role of hijacked repressors by CRISPR de-repression. We performed whole-genome bisulfite sequencing in 20 AML and normal samples, and showed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Finally, by treating the AML cells with the DNA hypomethylating agent and performing triple knockdown of DNMT1/3A/3B, we demonstrated the impact of altered DNA methylation on gene expression and 3D genome organization. Overall this study provides an invaluable resource for leukemia studies and also highlighted the role of repressor-loops and hijacked cis-elements in gene regulation and human diseases.

Citation Format: Jie Xu, Fan Song, Baozhen Zhang, Huijue Lyu, Mikoto Kobayashi, Ziyu Zhao, Ye Hou, Xiaotao Wang, Yu Luan, Bei Jia, Lena Stasiak, Qixuan Wang, Qi Jin, Qiushi Jin, Yihao Fu, Ross C. Hardison, Sinisa Dovat, Leonidas C. Platanias, Yue Yang, Tomoko Yamada, Aaron D. Viny, Ross L. Levine, David F. Claxton, James R. Broach, Hong Zheng, Feng Yue. Subtype-specific and structure variation induced 3D genome alteration in 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 2953.
Abstract 2974: A complex of tumor-suppressor NF1 and SPRED2 promotes pancreatic cancer tumorigenesis
Authors: Kim Sunny , Kastan Jonathan P. , Ting Hsiu-Chi , Park Youngkyu , Tuveson Dave
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with poor prognosis due to late detection and lack of effective therapeutic options. >90% of PDAC tumors are driven by activating mutations in the KRAS oncogene, making it an appealing subject for further investigation into understanding PDAC biology and offering a potential genetic vulnerability to target in patients. Our laboratory has previously reported a mutant-KRAS-proximal complex at the plasma membrane that consists of tumor-suppressor Neurofibromatosis type 1 (NF1), Sprouty Related EVH1 Domain Containing 2 (SPRED2), and Ribosomal S6 Kinase 1 (RSK1). This complex mechanistically suppresses WT KRAS signaling via RSK1, a suppression which is then relieved upon mutant KRAS inhibition, ultimately promoting adaptive resistance for PDAC cells under these conditions. These data highlighted a potential paradoxical role for two canonical tumor-suppressors, NF1 and SPRED2, as promoters of PDAC tumorigenesis when mutant KRAS is inhibited. We find that knockout of NF1 in PDAC organoids induces a substantial growth defect in vivo, and our preliminary data indicates that SPRED2 knockout PDAC cells have reduced proliferation in vitro. Moreover, both SPRED2 and NF1 KO promotes a diminished migratory phenotype in vitro. Given NF1's well-established role as a tumor-suppressor, our observations of decreased tumorigenic properties upon NF1 deletion are unexpected. We hypothesize that NF1 and Spred2 promote tumorigenesis via recruitment of RSK1 to the plasma membrane, inducing the phosphorylation of putative substrates to stimulate a pro-migratory cellular behavior. Our work suggests the existence of a unique RSK1 program localized at the plasma membrane by mutant KRAS, providing a new facet of oncogenesis to investigate for fundamental understanding and potential therapeutic opportunities.

Citation Format: Sunny Kim, Jonathan P. Kastan, Hsiu-Chi Ting, Youngkyu Park, Dave Tuveson. A complex of tumor-suppressor NF1 and SPRED2 promotes pancreatic cancer tumorigenesis [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 2974.
Abstract 2979: ElevatedNRASexpression as a potential driver of DCIS progression to basal-like invasive breast cancer
Authors: Zheng Ze-yi , Elsarraj Hanan , Hong Yang , Lei Jonathan T. , Anurag Meenakshi , Shen Yichao , Lo Flora , Feng Long , Zhao Zifan , Zhang Xiang H. , Behbod Fariba , Chang Eric C.
Published: June 2022

Background: Ductal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives a subset of luminal DCIS to transition to basal invasive breast cancers is unclear. Basal-like invasive breast cancers are aggressive and associated with the shortest overall survival and time to distant metastasis. We have previously shown that N-Ras is highly expressed selectively in the basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers.

Methods: Wilcoxon rank-sum test was conducted to assess the relative expression of NRAS in DCIS compared to invasive breast cancers in a patient cohort (GSE59248). NRAS levels were also determined by fluorescence in situ hybridization (FISH) in a collection of 21 patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and NRAS’s association with DCIS and invasive breast cancer was assessed by Wilcoxon signed rank test. How NRAS levels correlated with basal biomarkers in the TMAs, as well as in patient datasets GSE59248 and GSE3369 was determined by Pearson correlation. Gene expression changes in cell line were assessed by RNA-seq.

Results: Invasive breast cancers showed higher NRAS mRNA levels, as compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signature, lower ER, higher p53 protein levels, and higher Ki67 levels. We have shown previously that N-Ras is a driver for tumor growth in SUM102 cells, which is a DCIS-like cell line model displaying basal-like features. Here we found that NRAS-silencing in these cells induced a shift to a luminal gene expression pattern as determined by PAM50.

Conclusions: These data suggest that the rise of N-RAS levels in DCIS can not only mark but also control the emergence of basal-like features leading to more aggressive tumor activities. Targeting N-Ras and/or its downstream pathway may prevent the emergence of basal invasive breast cancers.

Citation Format: Ze-yi Zheng, Hanan Elsarraj, Yang Hong, Jonathan T. Lei, Meenakshi Anurag, Yichao Shen, Flora Lo, Long Feng, Zifan Zhao, Xiang H. Zhang, Fariba Behbod, Eric C. Chang. ElevatedNRASexpression as a potential driver of DCIS progression to basal-like invasive 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 2979.
Abstract 2989: FAP facilitates ovary tumorigenesis through the NF-κB/survivin funcational axis
Authors: Li Bin , Huang Shuang
Published: June 2022

Ovarian cancer is the deadliest gynecologic cancer, mainly attributing to lack of early diagnosis of the diseases. Current mainstay treatment for ovarian cancer is surgical removal followed by chemotherapy. Although patients initially respond well, most of patients suffer from relapse and die from metastasis. Therefore, developing successful approaches combatting metastasis is most likely to prolong patients’ survival and improve their life quality. Toward this goal, we analyzed The Cancer Genome Atlas ovarian cancer patient dataset to reach for targetable genes preferentially elevated in advanced ovarian cancer. Among them, we found that knockdown of fibroblast activation protein alpha (FAP) led to dramatic apoptosis in mesenchymal-like ovarian cancer cells. To elucidate the underlying mechanism, we performed RNAseq and identified that TNF-alpha/NF-κB signaling pathway was impaired in FAP-knockdown cells. NF-κB signaling pathway is well characterized to promote cell survival by elevating the expression of pro-survival genes. In fact, the level of survivin was diminished upon FAP-knockdown. As forced expression of survivin almost completely abrogated apoptosis elicited by FAP-knockdown and survivin inhibitor also induced robust apoptosis in ovarian cancer cells, we reason that FAP participates in ovarian cancer development by promoting cell survival through activation of NF-κB/survivin axis. With the aid of EpCAM aptamer as delivering cargo, we demonstrated that FAP siRNA was able to suppress intraperitoneal ovarian cancer development. This study suggests that FAP is an ideal therapeutic target against mesenchymal-like ovarian cancer.

Citation Format: Bin Li, Shuang Huang. FAP facilitates ovary tumorigenesis through the NF-κB/survivin funcational axis [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 2989.
Abstract 3: Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice
Authors: Shen Yuelei , Yao Jiawei , Kokabee Mostafa , Guo Yanan , BaI Yang , Shang Chengzhang
Published: June 2022

Glucagon-like peptide-1 (GLP1) is a pleiotropic hormone released from gut enteroendocrine cells following nutrient ingestion. In addition to promoting insulin secretion and inhibiting glucagon secretion, GLP1 acts via the glucagon-like peptide-1 receptor (GLP1R) to reduce gastric emptying and food intake. Therefore, development of GLP1R agonists appears to be a promising therapeutic option for type 2 diabetes. To evaluate the efficacy of GLP1 analogs in preclinical studies, Biocytogen has successfully generated a novel humanized GLP1R (B-hGLP1R) knock-in mouse line to support related drug development studies. In this mouse model, the full coding sequence of the human GLP1R gene was inserted into the mouse Glp1r gene locus. Human GLP1R mRNA expression was detected by RT-PCR in B-hGLP1R mice but not in wild-type mice. Protein expression of human GLP1R in B-hGLP1R mice was confirmed by western blot analysis and immunohistochemistry using pancreatic tissues. Furthermore, flow cytometry analysis of leukocyte subpopulations showed that B-hGLP1R mice do not exhibit a change in the overall development, differentiation, or distribution of immune cell types in the spleen, blood, and lymph nodes. Additionally, our in vivo efficacy study confirmed Dulaglutide, a GLP1 receptor agonist, reduced the non-fasting blood glucose, fasting blood glucose, glucagon, and food intake levels in B-hGLP1R obese mice and increased plasma insulin and GLP1 secretion. In B-hGLP1R mice, glucose tolerance (GTT) was also improved by Dulaglutide treatment, similar GTT results were observed after treatment with PF-06882961, a phase II agent specifically recognizing human GLP1R. Altogether, B-hGLP1R mice provide an efficacious preclinical animal model to evaluate novel GLP1 based therapeutic drugs. Keywords: Humanized mice, GLP1R, agonist

Citation Format: Yuelei Shen, Jiawei Yao, Mostafa Kokabee, Yanan Guo, Yang BaI, Chengzhang Shang. Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice [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 3.
Abstract 3005: Inflammasome-independent pyroptosis mediates DHA-led ovarian cancer cell death
Authors: Calbay Ozlem , Huang Shuang
Published: June 2022

Docosahexaenoic acid (DHA) is a natural compound, which exhibits anti-cancer capability in various cancer types in experimental models. Similar to previous reports in other cancer cell types, we demonstrated that DHA inhibited ovarian cancer cell growth by inducing cell death. In our model, distinct from the effect of DHA on other cancer cell types, we revealed that DHA-induced cell death was only slightly blocked by apoptosis inhibitor (caspase 3 inhibitor DEVD). We characterized the mechanism associated with DHA-led cell death and found that DHA-induced cell death was effectively blocked by caspase 1 inhibitor, indicating that DHA kills ovarian cancer cells by inducing pyroptosis. This mechanism was further supported by the observation that Disulfiram, a Gasdermin D inhibitor, prevented DHA-induced cell death and DHA treatment leads to the cleavage of Gasdermin D. Surprisingly, known inflammasome inhibitors did not affect DHA-induced cell death and Gasdermin D cleavage, ruling out the involvement of the inflammasome for caspase 1 activation. Instead, DHA remarkably elevated the amount of both caspase I and cleaved caspase 1 in ovarian cancer cells. This suggests that DHA activates caspase 1 by augmenting the abundance of caspase 1. Furthermore, we showed that DHA increased the level of intracellular reactive oxygen species (ROS). Use of ROS inhibitor (NAC) abrogated DHA-induced pyroptosis. This study uncovers pyroptosis as the mechanism for DHA-induced ovarian cancer cell death and reveals a previously unknown mechanism for caspase 1 activation.

Citation Format: Ozlem Calbay, Shuang Huang. Inflammasome-independent pyroptosis mediates DHA-led ovarian cancer 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 3005.
Abstract 3014: PHGDH- A therapeutic vulnerability in melanoma
Authors: Jasani Neel N. , Karreth Florian
Published: June 2022

Recurrently amplified oncogenes may represent melanoma vulnerabilities that can be exploited for therapy. The genomic locus containing the Phosphoglycerate Dehydrogenase (PHGDH) gene at chromosome 1p12 is frequently amplified in melanoma. PHGDH is the rate limiting enzyme that converts the glycolytic intermediate 3-Phospho Glycerate (3-PG) to serine through the serine synthesis pathway (SSP). Serine is utilized to make glycine, glutathione, one carbon metabolites, proteins and lipids. PHGDH inhibition suppresses proliferation of 1p12 amplified melanoma cells. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. We observed that 1p12 non-amplified melanoma cell lines have enhanced expression of the SSP enzymes as compared to melanocytes cultured in tetradecanoyl-phorbol acetate (TPA, a growth factor that stimulates MAPK pathway). Moreover, TPA starvation in melanocytes increased PHGDH expression. This led us to hypothesize that activation of oncogenic MAPK signaling leads to high expression of PHGDH which can serve as therapeutic vulnerability in melanomagenesis. Acute expression of oncogenic BRAFV600E in melanocytes resulted in upregulation of SSP enzymes, while treatment of melanoma cells with either BRAF or MEK inhibitor resulted in downregulation of SSP enzymes. We confirmed that PHGDH is regulated by BRAF-ERK-mTOR axis. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation and colony formation ability in vitro. We also observed that Phgdh knockdown in a BrafV600E/WT and Pten-/- (BPP) melanoma mouse model significantly increased survival. Furthermore, serine-glycine starvation (external source of Serine-glycine) in BPP chimeras did not affect the survival of these mice while on the other end, it suppressed the growth of the xenograft tumors injected in the NSG mice, which warrants further investigation. These results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. The exact mechanism of PHGDH regulation by BRAFV600E and the therapeutic effect of PHGDH inhibition in melanoma initiation and progression are the focus of our studies.

Citation Format: Neel N. Jasani, Florian Karreth. PHGDH- A therapeutic vulnerability in melanoma [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 3014.