bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3451: Microglial phagocytosis suppressed the development of metastatic tumors in the imaging model of brain metastasis
Authors: Tsuji Takahiro , Wake Hiroaki , Shindo Mariko , Kato Daisuke , Ozasa Hiroaki , Hirai Toyohiro
Published: June 2022

Brain metastasis of lung cancer is a crucial problem that causes poor clinical outcomes and alters patient quality of life. Previous evidence showed that lung cancer cells (LUCs) may escape from host immunity to form distant metastases. We previously used an original imaging model of brain metastasis (IBrM) to show a heterogeneous interaction between microglia, the tissue macrophages in the brain, and LUCs in the brain niche. The findings confirmed that microglia can phagocytose metastatic cancer cells. Here, we show that genetic deletion of the “don’t eat me” signals of cancer cells can promote phagocytic activity of microglia against LUCs. We injected an adenocarcinoma lung cancer cell line expressing mCherry (CMT167mC) via the internal carotid artery of the CX3CR1-EGFP mice, whose microglia had been specifically labeled with EGFP. The microglia and LUCs were visualized in vivo simultaneously by two-photon microscopy for 14 days. The tumor fate and microglial response against cancer cells were evaluated at single cell resolution in the IBrM. CD24, CD47, and/or PDL1 were deleted in CMT167mC cells using CRISPR/Cas9 to examine the effect of the “don’t eat me” signals. The dynamic behavior of LUCs and the microglial phagocytosis activity against LUCs were visualized in living mice. Microglial phagocytosis was significantly increased by single deletion of CD47 or CD24 compared with WT cells. Combined deletion of CD47 and CD24 also synergically increased microglial phagocytosis, resulting in reduced IBrM and prolonged mouse survival. By contrast, deletion of PD-L1 did not enhance microglial phagocytosis or prolong survival. The pharmacological or genetic conditional depletion of microglia canceled both the increased phagocytosis and the extended survival time, suggesting that the effect of “don't eat me” signals was microglia-dependent. These results indicate that suppression of both CD24 and CD47 in LUCs enhances microglial phagocytic activity and suppresses the metastatic formation of LUCs. Furthermore, the combined use of CRISPR/Cas9 in LUCs and in vivo imaging allows us to evaluate potential therapeutic targets associated with microglial phagocytosis. We are currently using single cell RNA-seq and cell-to-cell communication analysis of the brain tumor and surrounding niche to explore other signal pathways that regulate tumor phagocytosis by microglia and to identify potential therapeutic targets.

Citation Format: Takahiro Tsuji, Hiroaki Wake, Mariko Shindo, Daisuke Kato, Hiroaki Ozasa, Toyohiro Hirai. Microglial phagocytosis suppressed the development of metastatic tumors in the imaging model of brain metastasis [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 3451.
Abstract 348: Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma cells
Authors: Haribabu Yoga , Chien Raymond , Alobaidi Ryyan , Kuo Li-Wei , Islam Nusrat , Simbulan-Rosenthal Cynthia M. , Rosenthal Dean S.
Published: June 2022

Malignant melanoma is the most aggressive form of skin cancer. Although kinase inhibitors have proven to be efficient with promising clinical results, the development of drug resistance is one of the major issues in targeted therapy. CD133, also known asProminin-1, is a membrane glycoprotein studied extensively as a cell surface marker for cancer stem cells. In our previous studies, mebendazole, a repurposed broad spectrum anti-helminthic agent, inhibited the growth of NRAS mutant melanoma cells in combination with trametinib, a MEK inhibitor. However, a CD133-positive cancer stem cell population remain resistant to this targeted therapy. Use of RNA aptamers in diagnostic as well as therapeutics has been studied recently in different types of cancer cells. Drug delivery of aCD133 aptamer coupled with doxorubicin has been found to inhibit the growth of liver cancer cells. In this study, we targeted the cancer stem cells expressing CD133 with RNA aptamers, in combination with trametinib and mebendazole to sensitize the cells to the combination treatment. Patient derived melanoma cell lines, BAKP and POT, harboring BRAFWT/NRASQ61K and BRAFWT/NRASQ61R driver mutations, were co-transduced with aTet-on vector expressing CD133 and a Tet activator. The doxycycline-induced and uninduced cells were subjected to single agent treatment with 100 nM trametinib, 100 nM mebendazole or 100 nM CD133 RNA aptamer, as well as dual and triple agent combinations for 24 or 48 hours. XTT cell viability and apoptotic assays such as AnnexinV-PI flow cytometry analysis revealed that the triple combination with trametinib, mebendazole and the CD133 aptamer was the most effective in inducing apoptotic cell death, even in the doxycycline-induced CD133-overexpressing melanoma cells. Further studies will use preclinical in vivo mouse xenograft models to investigate the effects on tumor growth of CD133 RNA aptamers in combination treatments, potentially making it available for clinical trials aiming to improve patient response to therapeutics against drug-resistant melanoma cells.

Citation Format: Yoga Haribabu, Raymond Chien, Ryyan Alobaidi, Li-Wei Kuo, Nusrat Islam, Cynthia M. Simbulan-Rosenthal, Dean S. Rosenthal. Efficacy of triple combination treatment with trametinib, mebendazole and CD133 RNA aptamer in recalcitrant NRAS-mutant melanoma 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 348.
Abstract 3587: Racial disparity in COVID-19 severity after adjusting for chronic illnesses and sociodemographic factors
Authors: Wu Xiao-Cheng , Yi Yong , Maniscalco Lauren S. , Hsieh Mei-Chin , Gruber DeAnn , Mendoza Lee , Sokol Theresa , Mederos Eileen T. , Ochoa Augusto
Published: June 2022

Background. Research has reported that African American (AA) cancer patients with COVID-19 had a higher hospitalization rate than their white counterparts. Because the severity of COVID-19 is partly related to existing chronic diseases, one of the speculations is that racial differences in COVID-19 severity are attributable to AA cancer patients having a higher prevalence of chronic illnesses. Our study aimed to assess the impact of existing chronic diseases on the racial differences in hospitalization and length of hospitalization in COVID-19 cancer patients in Louisiana.

Methods. We linked cancer cases diagnosed in 2015-2019 from the Louisiana Tumor Registry (LTR) with the statewide COVID-19 data to identify COVID-19 patients who had been previously diagnosed with cancer. We also identified chronic illnesses (i.e., heart disease, peripheral vascular and cerebrovascular diseases, pulmonary disease, renal disease, liver disease, diabetes, and others) from 2012-2020 hospital discharge data and LTR data. Age and census tract level poverty were at the time of COVID-19 diagnosis. Bivariate and multivariable logistic regressions were used to exam the association of race with hospitalization after adjusting for socio-demographic and chronic illnesses. The multivariable Poisson model was used to assess the racial disparity in length (in days) of hospitalization.

Results. Of 6,518 COVID-19 cancer patients, there were 30.8% AA, 68.4% whites, and 0.8% other races. AA, male, older, residing in high poverty, and patients with chronic illnesses were more likely (P<0.05) to be hospitalized. The odds of hospitalization was 87.2% higher among AA patients than white patients in bivariate analysis. After adjusting for age, gender, poverty, obesity, smoking status, and chronic illnesses, the odds of hospitalization was still higher for AA than white patients (OR=1.81;95% CI: 1.55-2.09). The length of hospital stay for AA was more (P<0.05) than whites After adjusting for the same covariates.

Conclusion. Sociodemographic factors and chronic illnesses are associated with the severity of COVID-19 among cancer patients. However, AA COVID-19 cancer patients have significantly higher odds of hospitalization and longer hospital stays even when controlling these factors. More research is warranted to determine underlying factors of the observed racial disparities.

Citation Format: Xiao-Cheng Wu, Yong Yi, Lauren S. Maniscalco, Mei-Chin Hsieh, DeAnn Gruber, Lee Mendoza, Theresa Sokol, Eileen T. Mederos, Augusto Ochoa. Racial disparity in COVID-19 severity after adjusting for chronic illnesses and sociodemographic factors [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 3587.
Abstract 366: Combinatorial effect of fisetin and paclitaxel encapsulated PBM nanoparticles in ovarian cancer therapy
Authors: McFadden Melayshia , Singh Santosh Kumar , Singh Rajesh
Published: June 2022

High-grade Serous Ovarian Cancer (HGSOC) accounts for 68% of Ovarian Cancers (OvCas). It is a clinically aggressive neoplasm that even after treatment, a substantial proportion of advanced OvCas will develop resistance within 18 months. Thus, it is imperative to formulate anti-cancer agents that target cancer cells without affecting normal cells. The proposed study evaluated a novel method for targeted delivery of fisetin alone or combined with paclitaxel (PTX) at desired rates. We have used a natural polysaccharide (starch; FDA approved) as a core for planetary ball milled (PBM) nanoparticles which binds high-affinity folate receptors on OvCa cells via folate conjugated poly (ε-caprolactone)/poly (ethylene glycol) copolymer coating of the PBM nanoparticles. Treatment of OvCa cells (CAOV-3 and OVCAR-3) with PBM-NPs has shown its cytotoxic effect. The cell viability (MTT) assay was performed to determine the optimal IC50 values of fisetin, PTX alone, and in combination. These particles are rapidly internalized and induce significant cancer cell death at lower doses than unformulated fisetin or PTX. Importantly, negative controls showed that PBM nanoparticles without fisetin or paclitaxel have no toxic effects on OvCa cells. The immunofluorescence study confirmed the effectiveness of the combined drug; dead nuclei were found more within the cells treated by the combination than fisetin or PTX alone. The combined drug treatment was most effective in inhibiting proliferation and inducing apoptosis; accordingly, it showed noticeable increases in pro-apoptotic (BAX, BID), apoptotic (PARP, Caspase-3), and decreases in anti-apoptotic genes (BCL-XL and MCL-1) gene in a western blot and RT-PCR analysis. Taken together, we observed that the combination of PTX and fisetin resulted in significant cytotoxicity and apoptosis as compared to alone. Our results suggest that fisetin can be a potential agent in enhancing the efficacy of PTX in OvCa treatment. These finding highlights the promising role of natural bioactive compounds and provide the rationale for further transitional research.

Citation Format: Melayshia McFadden, Santosh Kumar Singh, Rajesh Singh. Combinatorial effect of fisetin and paclitaxel encapsulated PBM nanoparticles in ovarian cancer therapy [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 366.
Abstract 369: Improving the nanomaterial delivery by using cell-penetrating peptides in the bystander manner
Authors: Li Yue-Xuan , Wei Yushuang , Pang Hong-Bo
Published: June 2022

While nanoparticles (NPs) can be useful to improve the diagnosis and treatment of various diseases including cancer, a major limiting factor for their applications is the poor efficiency of cell entry. A common solution to this problem is to covalently link NPs with cell-penetrating peptides (CPPs), such as the transactivator of transcription (TAT) peptide. A few cationic CPPs were previously shown to increase the cellular uptake of co-administered, but not covalently coupled, NP cargo through an endocytic pathway, macropinocytosis. This so-called bystander uptake process is of unique advantage for NP intracellular delivery as it bypasses the requirements of additional chemical modifications. Here, we set out to determine whether other classes of CPPs (e.g. hydrophobic and amphiphilic) exhibit similar bystander activities, and what physico-chemical properties of NPs affect the bystander uptake process. First, we discovered that transportan (TP) peptide, an amphiphilic CPP, can initiate similar bystander uptake process for a variety of NPs and solute tracers. TP-induced bystander uptake relies on macropinocytosis as well, and occurs in a variety of cell types and in physiological tissues. Second, using TAT-functionalized NPs to stimulate the bystander effect, we showed that the bystander activity depends on the size of bystander NPs, with an optimal range around 50 nm in diameter. Compared to rod-like and triangular NPs, spherical NPs showed higher uptake in the bystander manner. Additionally, we investigated how CPP-NPs, once inside cells, get out of one cell and enter another, and found again that NP sizes affect the efficiency of CPP-NP intercellular transport as well. Overall, these efforts lay the foundation to further understand the bystander activity of CPP-assisted cell entry and improve the efficacy of nanomedicine in cancer and other human diseases.

Citation Format: Yue-Xuan Li, Yushuang Wei, Hong-Bo Pang. Improving the nanomaterial delivery by using cell-penetrating peptides in the bystander manner [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 369.
Abstract 3822: Chromosomal instability shapes the tumor microenvironment through a chronic ER-stress response
Authors: Li Jun , Hubisz Melissa , Earlie Ethan , Duran Mercedes A. , Lettera Emanuele , Phyu Su M. , Amin Amit D. , Deyell Matthew , Kamiya Erina , Budre Karolina , Cavallo Julie-Ann , Garris Christopher , Wen Hannah , Izar Benjamin , Parkes Eileen , Laughney Ashley , Bakhoum Samuel
Published: June 2022

Chromosomal instability (CIN) is a cancer hallmark associated with cancer metastasis and immune evasion. Yet, it is unclear how CIN modulates the tumor-microenvironment (TME). Here we show that CIN results in a protumor TME with enrichment of immune-suppressive macrophages, a granulocytic infiltrate, and exhausted T cells. Using ContactTracing, a newly developed computational tool to infer conditionally dependent cell-cell interactions from single cell RNA sequence data, we identify tumor ligands induced by the ER stress response in cancer cells as central mediators of immune suppression. Mechanistically, CIN-dependent chronic activation of the cytosolic DNA sensing cGAS-STING pathway promotes ER-stress-dependent transcription. Suppression of CIN or depletion of cancer cell STING reduces ER-stress and restores CIN-induced changes on the TME. Correspondingly, chronic STING activation in human breast cancer patients is associated with reduced tumor infiltrating lymphocytes and increased metastasis. Remarkably, pharmacologic inhibition of chronically active STING or depletion of downstream ER stress signaling suppresses metastasis in syngeneic models of melanoma, breast, and colorectal cancers, thereby demonstrating a viable therapeutic strategy for chromosomally unstable cancers.

Citation Format: Jun Li, Melissa Hubisz, Ethan Earlie, Mercedes A. Duran, Emanuele Lettera, Su M. Phyu, Amit D. Amin, Matthew Deyell, Erina Kamiya, Karolina Budre, Julie-Ann Cavallo, Christopher Garris, Hannah Wen, Benjamin Izar, Eileen Parkes, Ashley Laughney, Samuel Bakhoum. Chromosomal instability shapes the tumor microenvironment through a chronic ER-stress response [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 3822.
Abstract 3829: Microenvironmental stiffness induces metabolic reprogramming in glioblastoma
Authors: Sohrabi Alireza , Lefebvre Austin , Condro Michael , Kornblum Harley , Digman Michelle , Seidlits Stephanie
Published: June 2022

Glioblastoma (GBM) is the most common and lethal type of brain cancer with 5-year survival of <15%. The unique microenvironment of GBM is a key factor in tumor aggression. Similar to other solid tumors, GBM cells experience different biomechanical forces than do cells in healthy brain tissue. Here, we use atomic force microscopy to perform micro-compression mechanical analysis of xenografted GBM tumors. We show that tumors have a relatively stiff core (5-6 kPa Young’s modulus) and a softer edge (1-2 kPa Young’s modulus), yet all tumor tissue was stiffer than adjacent brain tissue (0.5-0.8 kPa Young’s modulus). To understand the functional implications of this biomechanical landscape, GBM spheroids were encapsulated in 3D culture matrices tuned to mimic the measured stiffnesses of a tumor regions and neighboring, non-cancerous tissue. These 3D matrices are fabricated from hyaluronic acid to promote GBM/matrix interaction through CD44 and RHAMM. RNA-sequencing analysis revealed overexpression of mitochondrial-encoded genes in stiff conditions, specifically genes encoding proteins involved in oxidation phosphorylation (OXPHOS), when compared to soft hydrogels. Using fluorescence-life time imaging microscopy to assess metabolic states at a single-cell level, we found that metabolism in GBM cells cultured in stiff matrices was dominated by OXPHOS while those in softer matrices was dominated by glycolysis (GLY). Furthermore, we show that this stiffness-induced shift towards OXPHOS in stiff matrices was associated with increased proliferation while the shift towards GLY in soft matrices was associated with increased migratory activity. Finally, inhibition of ezrin, an adaptor molecule linking the HA receptor CD44 to the actin cytoskeleton, eliminated the shift towards GLY induced in soft matrices, indicating that HA-CD44 interactions are necessary for these matrix-induced metabolic shifts.

Citation Format: Alireza Sohrabi, Austin Lefebvre, Michael Condro, Harley Kornblum, Michelle Digman, Stephanie Seidlits. Microenvironmental stiffness induces metabolic reprogramming in glioblastoma [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 3829.
Abstract 3837: The physiological relevance of TIMP dimer formation
Authors: Lazaroff Carolyn , Peeney David , Gurung Sadeechya , Stetler-Stevenson William G.
Published: June 2022

Tissue inhibitors of metalloproteinases (TIMPs) are a family of four paralogous genes that modulate the activity of matrix metalloproteinases (MMPs) and are vital for tissue homeostasis. Additionally, TIMPs display activities that are independent of their MMP inhibitory activities. Dysregulation and perturbations within this system can lead to complex tissue-specific pathologies. TIMP2 is the most abundantly expressed member of the TIMP family and has been shown to display potential therapeutic uses in cancer and other diseases. We have noted the persistent presence of SDS-stable TIMP2 dimers/multimers in SDS-PAGE at a level of ~5% total protein. The goal of this study is to determine the stability and biological relevance of SDS-stable dimer formation of TIMP2 in in vitro and in vivo. We found that TIMP2 dimers display an enhanced ability to inhibit MMP2 activity in reverse zymography assays. Immunoblotting revealed gel extracted TIMP2 dimer entered an equilibrium with monomeric TIMP2, while the extracted monomer did not form new dimer. In addition, MMP2 activity assays do not corroborate the enhanced MMP2 inhibitory activity observed in reverse zymograms but are consistent with the observation that isolated TIMP2 dimers re-equilibrate with monomers. This implies that structural or sequence difference mediates the formation of dimers. Dimer/multimer formation is observed across the TIMP family. Mass spectrometry analysis of gel extracted TIMP monomers and dimers show that monomer and dimer forms of TIMPs display unique post-translation modification (PTM) profiles. We propose that understanding the interplay between TIMP multimers and PTMs will reveal new biological functions within this intriguing family of proteins. We are currently working to resolve the functional differences between monomer and dimer and reveal the underlying mechanisms at play.

Citation Format: Carolyn Lazaroff, David Peeney, Sadeechya Gurung, William G. Stetler-Stevenson. The physiological relevance of TIMP dimer formation [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 3837.
Abstract 3885: USP7 is an interaction partner of EZHIP and potential druggable target in PFA ependymomas
Authors: Jenseit Anne , Camgöz Aylin , Mauermann Monika , Pfister Stefan M. , Kool Marcel
Published: June 2022

Ependymomas (EPN) are tumors of the central nervous system (CNS) that can arise in the supratentorial brain (ST-EPN), hindbrain or posterior fossa (PF-EPN), or anywhere in the spinal cord (SP-EPN), both in children and adults. Molecular profiling has identified distinct subgroups and subtypes in each of the anatomic compartments. In the posterior fossa, three subgroups have been identified: PFA, PFB and PF-SE. Of them, PFA ependymomas are characterized by a young median age at diagnosis, an overall balanced genome and a bad clinical outcome (56% 10-year overall survival). Standard therapy consists of tumor resection and radiotherapy, but working chemo- or targeted therapies will be essential to improve patient outcomes. Recently, we and others identified enhancer of zeste inhibiting protein (EZHIP) as potential main driver of PFA tumorigenicity. By inhibiting EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), EZHIP prevents the distribution of the epigenetic repressor mark H3K27me3. However, since EZHIP does not possess any known enzymatic functions itself, it does not serve as the druggable target urgently needed for PFA tumors. Thus, interaction partners are one main focus of ongoing PFA research. In this project, we focused on the ubiquitin-specific protease 7 (USP7), a well-known regulator of cancer pathways with a variety of inhibitors available, which was observed to interact with EZHIP in non-PFA cell lines before. We were able to confirm this interaction of EZHIP and USP7 in PFA cells in vitro by co-immunoprecipitation and mass spectrometry, and could show that the EZHIP-USP7 interaction is independent of EZH2, a separate interactor of both. Functionally, we are testing the hypothesis that USP7 de-ubiquitinates EZHIP, thereby preventing its proteasomal degradation and thus stabilizing the protein. As EZHIP expression is essential to PFA cell survival, we aim to target EZHIP indirectly by interfering with its stability regulation via USP7. RNAi-based knockdown (KD) experiments have been used to test a susceptibility of PFA cells to a loss of USP7, focusing on effects on cell proliferation and apoptosis, but also investigating gene expression changes provoked by a change in USP7 levels. Moreover, multiple USP7 inhibitors already highly affect the survival of different PFA cell lines in vitro. Ongoing experiments with PFA patient-derived xenograft (PDX) models will hopefully confirm the strong effect of USP7 inhibitors in PFA, and help to improve targeted therapy for PFA patients.

Citation Format: Anne Jenseit, Aylin Camgöz, Monika Mauermann, Stefan M. Pfister, Marcel Kool. USP7 is an interaction partner of EZHIP and potential druggable target in PFA ependymomas [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 3885.
Abstract 3887: β3-adrenergic receptor sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma
Authors: Bruno Gennaro , Nastasi Nicoletta , Subbiani Angela , Boaretto Alessia , Tondo Annalisa , Favre Claudio , Calvani Maura
Published: June 2022

In this study we aimed to investigate the role of the β3-adrenergic receptor (β3-AR) in regulating the immune system response against neuroblastoma (NB) tumor. NB is a very heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via β-adrenergic receptors ligation, may affect different signaling pathways in the tumor microenvironment (TME). We previously demonstrated that the specific antagonism of the β3-AR on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB (A/J mice inoculated with the murine neuroblastoma cell line Neuro-2a), the β3-AR blockade ability to influence the number of immunoreactive and/or immunosuppressive cells in TME was investigated through flow cytometry analysis. Moreover, the involvement of the immune-checkpoint signaling axis PD-1/PD-L1 in mediating the anti-tumoral effects brought by a β3-AR antagonist administration was analyzed. Results demonstrated that β3-AR antagonism leads to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, β3-AR antagonism was able to increase the number of immune reactive CD8+, natural killer (NK) and dendritic cells (DCs), and to decrease the number of immune suppressive regulatory T cells (Treg) and Myeloid-derived suppressor cells (MDSC) in tumor mass. Moreover, β3-AR blockade on tumor infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates negatively with the patient’s clinical outcome compared to the low expression group, and that ADRB3 gene expression is also related to the expression of different immune-checkpoints. Overall, results indicate that β3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

Citation Format: Gennaro Bruno, Nicoletta Nastasi, Angela Subbiani, Alessia Boaretto, Annalisa Tondo, Claudio Favre, Maura Calvani. β3-adrenergic receptor sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma [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 3887.
Abstract 3917: A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins
Authors: Nitika  , Kline Jake , Fornelli Luca , Truman Andrew
Published: June 2022

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and inability of current technologies to distinguish direct vs bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the budding yeast Hsp70 protein interactome. In doing so, we have uncovered not only a new set of Hsp70 client proteins but show that these clients bind at multiple sites on Hsp70, including the N-terminal NBD. Notably, many of the Hsp70 interactions with client proteins are in close proximity to biologically important post translational modifications (PTMs). Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multi-point interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors which can be used to probe chaperone function in cells, we have also identified a suite of PTM-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins. All in all, our data suggest that our XL-MS approach to chaperone interactome characterization can also be used as a novel way to identify biologically important and previously unknown PTMs on client proteins.

Citation Format: Nitika, Jake Kline, Luca Fornelli, Andrew Truman. A novel multifunctional role for Hsp70 in binding post-translational modifications on client proteins [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 3917.
Abstract 3970: Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation
Authors: Martínez-Sabadell Alex , Arenas Enrique J. , Ruiz Irene Rius , Alonso Macarena Román , Escorihuela Marta , Luque Antonio , Fajardo Carlos Alberto , Gros Alena , Klein Christian , Arribas Joaquin
Published: June 2022

Elimination of cancer cells by effector immune cells represents the culmination of a complex cascade of events, and disruption of any of those events may result in resistance. T cell-engaging therapies, such as T cell bispecific antibodies (TCBs) or chimeric antigen receptors (CARs), are raising extraordinary expectations as future treatments for virtually all cancers. Encouraging these expectations, TCBs and CARs have been recently approved to treat some hematologic malignancies. In contrast, TCBs and CARs against solid tumors tested to date, have failed to show clinical efficacy. This failure prompted intense research and the subsequent identification of mechanisms of primary and acquired resistance. Different strategies are being implemented to overcome these mechanisms of resistance. All these mechanisms impinge on the ability of T cells to reach cancer cells and/or on the inhibition of T cells. However, little is known about putative intrinsic mechanisms of resistance of cancer cells. That is, mechanisms deployed by tumor cells to resist killing by fully active and correctly engaged T cells. Using in vitro and in vivo acquired resistant models to TCBs and CARs directed against HER2, we demonstrate that disruption in interferon-gamma (IFN-g) signaling in cancer cells is a mechanism of intrinsic resistance to killing by fully active, correctly engaged, T lymphocytes. Importantly, we have identified that the kinase JAK2, which transduces the signal initiated by IFN-g, is the component preferably disrupted to acquired resistance across all models used. These results unveil a novel mechanism of resistance to T-cell based therapies, and imply the potential use of JAK2 and IFN-g response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient interferon-gamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies.

Citation Format: Alex Martínez-Sabadell, Enrique J. Arenas, Irene Rius Ruiz, Macarena Román Alonso, Marta Escorihuela, Antonio Luque, Carlos Alberto Fajardo, Alena Gros, Christian Klein, Joaquin Arribas. Acquired cancer cell resistance to T cell bispecific antibodies and CAR T targeting HER2 through JAK2 down-modulation [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 3970.
Abstract 3993: ERO1α as a potential target in EGFR driven non-small cell lung cancer
Authors: Johnson Brennan D. , Chen Wei-Chih , Geldenhuys Werner J. , Hazlehurst Lori A.
Published: June 2022

Endoplasmic Reticulum Oxidoreductase-1 alpha (ERO1α) is an endoplasmic reticulum (ER) resident protein responsible for oxidation of Protein Disulfide Isomerase (PDI). The molecular interactions between ERO1α and PDI allow for proper protein folding to occur. Recent reports indicate that ERO1α expression is a poor prognostic indicator of survival in multiple cancer types including multiple myeloma, lung cancer, esophageal carcinoma, hepatocellular carcinoma, and diffuse large B-cell lymphoma. In this study we utilized genetic strategies to determine the role of ERO1α in EGFR driven Non-Small Cell Lung Cancer (NSCLC). Our data indicate that CRISPR knockout of ERO1α leads to decreased clonogenicity in both PC-9 and HCC4006 cell lines. The phenotype can be partially rescued upon addition of conditioned media derived from the control cell lines. The data suggest that ERO1α expression is required for correct protein folding of a secreted factor that contributes to clonogenicity and growth. PC-9 ERO1α knockout cells injected into SCID-Beige animals led to increased overall survival (OS) compared to animals injected with PC-9 control cells. (PC-9 Control OS = 100 days, PC-9 ERO1α Knockout OS =154 days p=0.0045 **). Interestingly, ERO1α knockout cell lines still maintained sensitivity to standard of care agent Osimertinib. We identified T151742 as an ERO1α inhibitor that was approximately 2-fold more potent compared to the previously published ERO1α inhibitor EN460. A cellular thermal shift assay was performed to determine specificity of T151742 toward ERO1α compared to other FAD containing enzymes. We show that T151742 had a higher specificity towards ERO1α in comparison to other flavoenzymes such as ERO1β and LSD-1. Finally, our data indicate that PC-9 ERO1α knockout cell lines were ~2-fold resistant to T151742 by MTT but were completely resistant when using a 3D cell culture model suggesting T151742 is reliant on ERO1α in a 3D system. Finally, treatment of PC9 and HCC4006 cells with T151742 in combination with Osimertinib was synergistic in vitro. Taken together, this suggests ERO1α may be a viable target for EGFR driven NSCLC and that future studies will elucidate the use of T157142 in vivo as a single agent and in combination with EGFR inhibitors using in vivo studies.

Citation Format: Brennan D. Johnson, Wei-Chih Chen, Werner J. Geldenhuys, Lori A. Hazlehurst. ERO1α as a potential target in EGFR driven 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 3993.
Abstract 4004: Interrogation of cancer gene dependencies reveals novel paralog interactions of autosome and sex chromosome encoded genes
Authors: Köferle Anna , Schlattl Andreas , Hörmann Alexandra , Spreitzer Fiona , Popa Alexandra , Thatikonda Venu , Puchner Teresa , Supper Verena , Ravichandran Madhwesh C. , Oberndorfer Sarah , Wieshofer Corinna , Corcokovic Maja , Reiser Christoph , Wöhrle Simon , Popow Johannes , Pearson Mark , Mair Barbara , Neumüller Ralph A.
Published: June 2022

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of these functional redundancies can create de novo vulnerabilities that are specific to cancer cells. In this regard, paralog genes are of particular interest, as the loss of one paralog gene can render tumor cells dependent on a remaining paralog. To systematically identify cancer-relevant paralog dependencies, we searched for candidate dependencies using CRISPR screens and publicly available loss-of-function datasets. Our analysis revealed >2,000 potential candidate dependencies, several of which were subsequently experimentally validated. We provide evidence that DNAJC15-DNAJC19, FAM50A-FAM50B and RPP25-RPP25L are novel cancer relevant paralog dependencies. Importantly, our analysis also revealed unexpected redundancies between sex chromosome genes. We show that chrX- and chrY- encoded paralogs, as exemplified by ZFX-ZFY, DDX3X-DDX3Y and EIF1AX-EIF1AY, are functionally linked so that tumor cell lines from male patients with Y-chromosome loss become exquisitely dependent on the chrX-encoded gene. We therefore propose genetic redundancies between chrX- and chrY- encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y-chromosome.

Citation Format: Anna Köferle, Andreas Schlattl, Alexandra Hörmann, Fiona Spreitzer, Alexandra Popa, Venu Thatikonda, Teresa Puchner, Verena Supper, Madhwesh C. Ravichandran, Sarah Oberndorfer, Corinna Wieshofer, Maja Corcokovic, Christoph Reiser, Simon Wöhrle, Johannes Popow, Mark Pearson, Barbara Mair, Ralph A. Neumüller. Interrogation of cancer gene dependencies reveals novel paralog interactions of autosome and sex chromosome encoded genes [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 4004.
Abstract 4011: Prognostic value of transcriptomic analysis in residual post neo-adjuvant triple negative breast cancer tumors
Authors: Aguilar-Mahecha Adriana , Majedi Yasamin , Elebute Oluwadara , Lafleur Josiane , Papadakis Andreas , Lan Cathy , Pelmus Manuela , Rashid Touhidur , Jenna Sarah , Basik Mark
Published: June 2022

Background: Triple negative breast cancer is the most aggressive type of breast cancer. Approximately 50% of TNBC patients respond to pre-operative neo-adjuvant chemotherapy (NAC), however those patients with residual disease (non-pathological complete response or non-pCR) have a very poor prognosis. Recently, the use of capecitabine in the adjuvant setting was shown to increase disease-free survival in non-pCR patients. However, there are no biomarkers to identify patients who may benefit from adjuvant capecitabine. Moreover, novel therapeutic targets are needed to treat patients with non-pCR.

Methods: RNA extracted from 32 Formalin Fixed Paraffin Embedded (FFPE) residual post-NAC tumor samples underwent NanoString nCounter gene expression analysis using the BC360 panel as well as RNAseq.

Results: Comparison of tumors from poor (RFS<2 yrs) and good prognosis (RFS>2 yrs) patients showed 65 genes differentially expressed (≥1.5 fold change and p<0.05) by nCounter. 40 of these genes had differential expression also validated by RNAseq (R2= 0.96) and were selected for further pathway analysis. Gene Set Enrichment Analysis (GSEA) showed an enrichment for biological pathways involved in cell cycle, with all genes in this set upregulated in the poor prognosis group. GSEA analysis of 310 upregulated genes identified by RNAseq showed an enrichment for biological pathways associated with cell population proliferation and skin development. Interestingly, one of the upregulated genes identified was TYMS, encoding thymidylate synthase, the target of capecitabine, which showed 2-fold increased expression in the poor prognosis group (p≤0.005).

Conclusion: Our results suggest that residual tumors from TNBC patients who relapse within two years of surgery show transcriptomic evidence of increased proliferation and that expression levels of TYMS may be a potential biomarker to select patients for capecitabine in the adjuvant setting.

Citation Format: Adriana Aguilar-Mahecha, Yasamin Majedi, Oluwadara Elebute, Josiane Lafleur, Andreas Papadakis, Cathy Lan, Manuela Pelmus, Touhidur Rashid, Sarah Jenna, Mark Basik. Prognostic value of transcriptomic analysis in residual post neo-adjuvant triple negative breast cancer 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 4011.
Abstract 4033: A functional genome-wide screen to identify the ER degradation machinery in ER+ breast cancer cells
Authors: Sandusky Zachary
Published: June 2022

Targeted protein degradation (TPD) is a new pharmaceutical strategy to hijack the ubiquitin-proteasome system to degrade target proteins. Many TPD drugs are discovered serendipitously, and we are only beginning to harness their potential. Understanding how TPD drugs co-opt the proteolysis pathway is important, as it could allow molecule re-engineering for TPD of new proteins (ie. neo-substrates). A rising example of TPD drugs are the selective estrogen receptor degraders (SERDs) - small molecule antagonists of estrogen receptor alpha (ER) that target ER protein for degradation. SERDs are the next generation of therapy for metastatic ER+ breast cancer. Currently, fulvestrant is the only FDA approved SERD, but fulvestrant is limited by poor pharmacokinetics and resistance conferred by ER mutations (ie. ER-Y537S). An intense pharmaceutical effort has developed five novel SERDs in phase three clinical trials. These novel SERDs have diverse side chains for inducing ER degradation but share advantages including non-steroidal structure, oral bioavailability, and activity against ER-Y537. Based on the diverse molecular properties of SERDs, I hypothesized they utilize different ubiquitin-proteasome pathway genes (such as E3 ligases) for ER degradation. To identify the machinery for SERD-mediated ER degradation, I designed a functional CRISPR/Cas9 screen, with highlights including a genome-wide gRNA library and measurement of endogenous ER protein in ER+ breast cancer cells. Initially focusing on genes required for fulvestrant-mediated ER degradation, we identified the proteolysis pathway as a top hit (q<5x10-6) including central genes in the ubiquitin-proteasome pathway (UBC, PSMA6, and NEDD8) and the ubiquitin-like SUMO pathway (UBC9 and RWDD3). Ubiquitin E3 ligases were also identified, including FBXO45 and VHL, which have been previously implicated as E3 ligases for ER. These hits were validated using inhibitors of ubiquitin-, NEDD8-, and SUMO-activating enzymes. These results demonstrate that multiple proteolysis mechanisms are sufficient for ER protein degradation. Currently, I am applying this screening approach to novel SERDs (such as elacestrant, amnecestrant, and giredestrant) to identify overlapping and unique mechanisms of degradation for wild-type ER and ER-Y537S. Importantly, understanding the mechanisms of these exciting new drugs will allow anticipation of resistance mechanisms in advanced ER+ breast cancer patients.

Citation Format: Zachary Sandusky. A functional genome-wide screen to identify the ER degradation machinery in ER+ breast cancer 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 4033.
Abstract 404: Identification of combination partners to combat acquired resistance to ulixertinib (ERK1/2 inhibitor) using transcriptomics
Authors: Reddy Anupama , Sorrell David , Knoerzer Deborah , Emery Caroline M.
Published: June 2022

Drug resistance is arguably the rate-limiting step in the successful clinical utility of MAPK inhibitors. Several mechanisms of resistance to BRAF and MEK inhibitors have been proposed, including feedback mechanisms and activation of complementary signaling pathways, which have led to the development of combination therapies. Ulixertinib (BVD-523), a first-in-class and ERK1/2 inhibitor, is clinically effective in patients with tumors harboring alterations in the MAPK pathway. In this project, we have developed resistance clones to ulixertinib and have identified mechanisms of acquired resistance using RNA-seq analysis, thus enabling us to identify potential combination partners to combat resistance.

In vitro experiments were deployed to develop resistance models to ulixertinib. Resistance clones were developed by culturing A375 cell line (melanoma; BRAF V600E) in escalating concentrations of ulixertinib.

RNA-sequencing was performed on the parental model and the resistance clones following varying treatment conditions with ulixertinib. RNAseq and principal component analysis revealed strong differences between parental and resistance clones, with further differentiation between acute high concentration and chronic treatment models compared to models treated with acute, lower concentrations of ulixertinib. These results highlight the presence of a dose-dependent resistance phenotype. Differential gene expression analysis revealed enrichment of several pathways, including MAPK, autophagy, focal adhesion, JAK/STAT, and VEGF. Interestingly, we observed rewiring of PI3K/AKT by shifting the signaling from EGFR to ERBB2 in the resistance models. The magnitude of changes in these genes and pathways also exhibited a dose-dependent effect, with higher concentration models showing a higher fold-change compared to the lower concentration models. We explored these hypotheses by performing combination experiments with ulixertinib in the generated resistance clones. Synergistic combination targets in the ulixertinib resistance clones included ERBB2, and FAK. We also validated combinations with autophagy inhibitors, one of which is now being tested in a Phase I clinical trial (NCT04145297).

In summary, we have identified some potential resistance mechanisms to ulixertinib and have validated key genes/pathways which may act as synergistic combination targets. This work not only informs future clinical development for ulixertinib but also improves our understanding of the complex interplay between the MAPK and other signaling pathways.

Citation Format: Anupama Reddy, David Sorrell, Deborah Knoerzer, Caroline M. Emery. Identification of combination partners to combat acquired resistance to ulixertinib (ERK1/2 inhibitor) using transcriptomics [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 404.