bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1932: Pollock: Fishing for cell states
Authors: Storrs Erik , Zhou Daniel Cui , Wendl Michael C. , Wyczalkowski Matthew A. , Karpova Alla , Wang Liang-Bo , Li Yize , Southard-Smith Austin , Jayasinghe Reyka G. , Yao Lijun , Liu Ruiyang , Wu Yige , Terekhanova Nadezhda V. , Zhu Houxiang , Herndon John M. , Chen Feng , Gillanders William E. , Fields Ryan C. , Ding Li
Published: June 2022

The use of single-cell methods is expanding at an ever-increasing rate. While multiple algorithms address the task of cell classification, they are limited in terms of cross platform compatibility, reliance on the availability of a reference dataset, and classification interpretability. Here, we introduce Pollock, a suite of algorithms for cell type identification that is compatible with popular single cell methods and analysis platforms, provides a series of pretrained human cancer reference models, and reports interpretability scores that identify the genes that drive cell type classifications. Our model combines two important approaches, one each from machine learning and deep learning: a variational autoencoder (VAE) and random forest classifier, to make cell type predictions. Pollock is highly versatile, being available as a command line tool, Python library (with scanpy integration), or R library (with Seurat integration), and can be installed as a conda package, or in containerized form via Docker. To allow for easier pan-disease and pan-tissue analyses, Pollock also ships with a library of pretrained cancer type specific and agnostic modules that were trained on expertly-curated single cell data that are ready to “plug and play” with no additional annotation or training required. Conversely, Pollock also allows for the training of custom classification modules, if an annotated reference single cell dataset is available. These pretrained models were fitted on manually curated and annotated single cell data from eight different cancer types spanning three single cell technologies (scRNA-seq, snRNA-seq, and snATAC-seq). Pollock also provides feature importance scores that allow for cell type classifications to be traced back to the genes influencing a particular cell type classification, further promoting biological interpretability. These scores could allow for new, technology-specific biomarker discovery. We also demonstrate the utility of Pollock by applying it in a pan-cancer single cell immune analysis.

Citation Format: Erik Storrs, Daniel Cui Zhou, Michael C. Wendl, Matthew A. Wyczalkowski, Alla Karpova, Liang-Bo Wang, Yize Li, Austin Southard-Smith, Reyka G. Jayasinghe, Lijun Yao, Ruiyang Liu, Yige Wu, Nadezhda V. Terekhanova, Houxiang Zhu, John M. Herndon, Feng Chen, William E. Gillanders, Ryan C. Fields, Li Ding. Pollock: Fishing for cell states [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 1932.
Abstract 1943: Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning
Authors: Paidi Santosh K. , Troncoso Joel R. , Rajaram Narasimhan , Barman Ishan
Published: June 2022

Clinical benefit is observed in only a limited fraction of patients that undergo immunotherapy for a variety of cancer types. Therefore, the determination of robust biomarkers for accurate prediction of response to immunotherapeutic agents remains one of the grand challenges of 21st century. Our primary goal was to investigate if phenotypic differences due to treatment with ICI can be recognized with minimally perturbative molecular tools and guide personalization of immunotherapy. In this work, we provide first-in-class evidence that Raman spectroscopy (an optical method) and machine learning allows sensitive detection of early changes in biomolecular composition of tumors in response to immunotherapy with immune checkpoint inhibitors (ICI). By studying the widely investigated CT26 murine model of colorectal cancer treated with anti-CTLA-4 (n = 8) and anti-PD-L1 (n = 7) ICIs and controls (n = 10), we revealed new biological insights into the nature and degree of microenvironmental modifications induced by exposure to clinically relevant doses of ICI. Multivariate curve resolution-alternating least squares (MCR-ALS) decomposition of Raman spectral datasets revealed early changes in lipid, nucleic acid, and collagen content of the tumors following therapy. We trained supervised classification models - support vector machines and random forests on the spectral datasets and showed that the models provided excellent prediction accuracies for response to both ICIs and delineated spectral markers specific to each therapy, consistent with their differential mechanisms of action. On the basis of these findings, we sought to determine if the tumor microenvironment changes delineated by Raman spectroscopy correspond to proteomic alterations via quantitative mass spectrometry. Of the more than 6600 proteins identified, about 136 proteins were found to be significantly different in treated tumors relative to controls (p < 0.05 and log2 fold change of > 2). A subset of these differentially expressed proteins is known to regulate lipid metabolism and extracellular matrix composition, while others are known to control transcriptome dynamics or are associated with response to ICI therapy, thereby corroborating our Raman spectroscopic measurements. Our observation of biomolecular changes in the TME should catalyze detailed investigations for translating such markers and label-free Raman spectroscopy for clinical monitoring of immunotherapy response in cancer patients.

Citation Format: Santosh K. Paidi, Joel R. Troncoso, Narasimhan Rajaram, Ishan Barman. Elucidating early tumor microenvironmental changes due to immunotherapy with label-free Raman spectroscopy and machine learning [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 1943.
Abstract 1958: CTC surge
Authors: Xue Chengsen , Amukele Timothy K.
Published: June 2022

Circulating Tumor Cells (CTC) are a prognostic biomarker for some types of malignant epithelial cancers and have been correlated with poor clinical outcomes. However, the routine measurement of CTC in clinical practice is still very uncommon and some key features of CTC in clinical samples are only being discovered. One of these features that we have identified by supporting CTC clinical research for over 10 years, is the presence of two distinct patient populations: one with high levels of CTC >220/7.5mL blood and one with levels of CTC <160/7.5mL. The clinical significance of this observation is still unknown, but based on this report, we propose a new oncology phenomenon: CTC Surge. Methods: In the past ten years we worked on more than half dozen clinical study projects by using CellSearch Systems by Menarini Silicon Biosystems (formerly Veridex), the only CTC technology platform approved by FDA. The Cell Search system detects CTCs by staining with surface markers coated on nanoparticle beads. The detection of CTC is linear over the reportable range of 0 to 1238 tumor cells. The FDA-approved clinical significant cut-off lines are 5 CTCs for prostate and breast cancers, and 3 for colorectal cancer. Results: All CTC results are sorted by count from low to high. There are two distinct groups roughly above 220 and below 160 per 7.5 mL of peripheral blood. The significance of a CTC enumeration > 220 is that there are as many as 150,000 tumor cells in circulation at the moment of sample collection. This may be the reason why some patients’ condition suddenly worsens when there is an overwhelming presence of CTCs in the bloodstream. The CTC test is a great tool for clinicians and scientists to monitor cancer diseases progression. Unlike a low number of CTCs that may actively engage in intravasation through a set of genes, the mechanism of CTC Surge likely has a completely different set of driving forces, such as internal wound, tumor necrosis and open access to blood vessels inside or surround the tumor mass. Although we do not yet know the clinical significance of our observation, the CTC test offers a simple tool to monitor the rapidly changing tumor dynamics seen in CTC Surge.

Citation Format: Chengsen Xue, Timothy K. Amukele. CTC surge [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 1958.
Abstract 1975: Potential novel Immuno-oncology mechanism revealed during translational phase I Immuno-blood profiling of experimental ADC medicine OBT076 in a gastric cancer patient
Authors: Rohlff Christian , Sahebjam Solmaz , Mita Alain , Lee Rosen , Peddaboina Chander Sekhar , Bisht Arnima , Hudson Lindsey , Fridman Wolf , Fandi Abderrahim , Rixe Olivier
Published: June 2022

CD205 is a type I transmembrane glycoprotein, with unique characteristics that make it an ideal target for Antibody Drug Conjugate (ADC) therapy. Here we report on a potential novel immuno-oncology mechanism revealed during the Translational Phase I (NCT04064359) immuno-blood profiling of a chemo-refractory patient treated with OBT076, an experimental CD205-directed ADC.

A chemo-refractory advanced gastric cancer patient with 60% CD205 expression in the primary tumor via IHC and having previously undergone 2 lines of chemotherapy treatment (Docetaxel/cisplatin/5FU and Ramucirumab/Paclitaxel), received five 21-day cycles of OBT076 (one at 2.5mg/kg and four cycles at 2.0 mg/kg) followed by 1 cycle of Pembrolizumab (PZ; 200mg) ~4 weeks later. Clinical response was evaluated and immunological markers (CD45, CD205, CD4, CD8, and PD1) in peripheral blood cells were quantified using flow cytometry.

After 2 OBT076 cycles at 2.0 mg/kg, there was an ~40% shrinkage in the primary gastric tumor size and resolution of ascites and lymph node metastases were observed. Following 2 further cycles and PZ, complete response was achieved for the primary tumor. Flow cytometry showed (1) an initial decrease in the absolute numbers of dendritic cells by day 8, followed by a 2-fold increase in numbers by day 21 after treatment; (2) a near total decrease in the population of CD8+ CD205+ cells by day 8, no recovery in levels were observed; (3) a 3-fold increase in CD4+ and CD8+ T-cell numbers between days 8 and 21 and (4) an initial decrease in CD4+ PD1+ and CD8+ PD1+ T-cell numbers followed by an ~4-fold increase between days 8 and 21.

In summary our results show that increases in PD1+ T-cells, T-cell induction, and decreases in immuno-suppressive CD4+ CD205+ and CD8+ CD205+ cells occur simultaneously; coinciding with rapid resolution of the primary tumor, lymph node metastases and ascites. These findings suggest that OBT076 activates the patient’s immune response against the tumor through a potentially novel mechanism: drug-induced depletion of CD8+ CD205+ immuno-suppressive cells and subsequent T-cell activation. Additionally, our data support the use of immune checkpoint inhibitors in conjunction with OBT076 to achieve favorable clinical outcomes.

Citation Format: Christian Rohlff, Solmaz Sahebjam, Alain Mita, Rosen Lee, Chander Sekhar Peddaboina, Arnima Bisht, Lindsey Hudson, Wolf Fridman, Abderrahim Fandi, Olivier Rixe. Potential novel Immuno-oncology mechanism revealed during translational phase I Immuno-blood profiling of experimental ADC medicine OBT076 in a gastric cancer patient [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 1975.
Abstract 1992: Evaluating in vivo efficacy of peptide vaccines in humanized B-HLA-A2.1 mice
Authors: Yao Jiawei , Shang Chengzhang , Madaan Shivam , Nie Yanhui , Yang Yi
Published: June 2022

HLA-A molecules are αβ heterodimers that belong to the human leukocyte antigen (HLA) class I major histocompatibility complex (MHC) in humans. Class I molecules play a central role in the immune system by presenting intracellular peptides derived from the endoplasmic reticulum lumen, enabling recognition by cytotoxic T cells. The heavy α chain structure of HLA-A, encoded by the HLA-A gene, contains the leader peptide, the α1 and α2 domains that both bind the peptide, and the α3 domain that binds CD8 molecules, the transmembrane region, and the cytoplasmic tail. Polymorphisms within α1 and α2 domains of the heavy chain are responsible for the peptide binding specificity of each class I molecule, while the light β2-microglobulin (B2M) chain encoded by the B2M gene, is not known to be polymorphic. Among more than 6,000 HLA-A alleles, HLA-A2.1 is the most commonly expressed, and its peptide-binding motif is well understood. We developed MHC I humanized mice (B-HLA-A2.1 mice) expressing human HLA molecules to establish an in vivo experimental model for evaluating human HLA restricted T cell-mediated vaccine efficacy in oncology applications. Our strategy was to replace the B2m gene of the mouse with the sequences encompassing the human B2M CDS and HLA-A2.1 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the mouse H-2Db gene encoding the α3, transmembrane and cytoplasmic domains. Using flow cytometry analysis, human B2M and HLA-A2.1 were detected in splenocytes of homozygous B-HLA-A2.1 mice but not in wild type mice. Mouse B2M was detectable in splenocytes of wild type mice but not in homozygous B-HLA-A2.1 mice. The percentage of T cells, B cells, CD4+ T cells, and CD8+ T cells within the spleens of homozygous B-HLA-A2.1 mice were similar to those in the wild type mice, demonstrating that introduction of human B2M and HLA-A2.1 does not change the overall development, differentiation, or distribution of these cell types. Next, in vivo efficacy evaluation of two peptide vaccines (V1 and V2) under development was performed with the humanized mice. Mice were inoculated with vaccines by intramuscular injections into the inner thigh (L and R) muscles and sacrificed three weeks after the last immunization. The splenocytes were extracted, stimulated with individual peptides, and measured for IFN-γ secretion. HLA-A2.1-restricted CTL responses of the vaccines were successfully detected in B-HLA-A2.1 mice, and no significant difference in body weight was observed among groups. Our results show that B-HLA-2.1 mice provide a powerful preclinical mouse model for in vivo evaluation of peptide vaccines.

Citation Format: Jiawei Yao, Chengzhang Shang, Shivam Madaan, Yanhui Nie, Yi Yang. Evaluating in vivo efficacy of peptide vaccines in humanized B-HLA-A2.1 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 1992.
Abstract 2003: Poor diet quality is associated with immune aging in survivors of pediatric acute lymphoblastic leukemia
Authors: Benmoussa Abderrahim , Kientega Tibila , Morel Sophia , Cardin Guillaume , Bérard Sophie , wajnberg Mickael , Valtchev Petko , Blondin-Masse Alexandre , Curnier Daniel , Krajinovic Maja , Laverdière Caroline , Sinnett Daniel , Levy Emile , Marcoux Sophie , Rodier Francis , Marcil Valérie
Published: June 2022

Rationale and objectives: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. Despite a 90% five-year survival rate, survivors of childhood ALL often suffer from late effects, including cardiometabolic disorders. Contributing factors such as inflammation and oxidative stress, combined with drug treatments, can induce premature aging and cellular senescence with a significant impact on cardiometabolic disorders. Premature aging can lead to decreased thymic T-cell production, resulting in decreased circulation of T-cell receptor excision circles (TRECs). Because diet has been associated with cardiometabolic disorders, we hypothesized that the quality of diet in children who had survived ALL was related to the immune aging biomarker TREC, in concert with inflammatory status.

Methods: Adolescent and young adult survivors of pediatric ALL of the PETALE cohort (n=241, 22.1 ± 6.3 years at diagnosis, 49.4% male) were examined in their profile for TREC levels (by qPCR) and for adherence to 6 diet quality indices.

Results: Adjusted linear regressions revealed that the Healthy Diet Indicator (HDI) was associated with TREC levels (β=50.0, p=0.005, adjusted p=0.03). After performing a conceptual relational analysis (CAR) for data mining of various biomarkers of inflammation, oxidative stress, endotoxemia, and endothelial or adipose dysfunction; interleukin-6 (IL-6) and C-reactive protein (CRP) were found to be negatively associated with TREC levels (β= -80 and -80.1, p=0.017 and 0.026, respectively) but not with HDI. Further analysis revealed that IL-6 and CRP levels were moderators, but not mediators, of the association between HDI and TREC.

Conclusion: This study supports the positive impact of a healthy diet on premature immune aging and the moderating role of inflammation in this association.

Citation Format: Abderrahim Benmoussa, Tibila Kientega, Sophia Morel, Guillaume Cardin, Sophie Bérard, Mickael wajnberg, Petko Valtchev, Alexandre Blondin-Masse, Daniel Curnier, Maja Krajinovic, Caroline Laverdière, Daniel Sinnett, Emile Levy, Sophie Marcoux, Francis Rodier, Valérie Marcil. Poor diet quality is associated with immune aging in survivors of pediatric acute lymphoblastic 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 2003.
Abstract 2007: Use of an anti-IL-10 antibody to improve Mifamurtide efficacy on aggressive osteosarcoma cells
Authors: Nastasi Nicoletta , Subbiani Angela , Taddei Maria Letizia , Scala Lucia , Tamburini Angela , Favre Claudio , Calvani Maura
Published: June 2022

Osteosarcoma is a primary malignant bone tumor highly aggressive and metastasizing, composed of mesenchymal cells producing osteoid and immature matrix. Osteosarcoma is the most frequent bone tumor and it mainly affects children and adolescents. The main treatment option is a combination of surgery and chemotherapy. Research into new therapeutic strategies to improve the quality of life of patients is currently underway. The development of the anti-tumor immunostimulant drug Mifamurtide is part of this research. There are very conflicting opinions on the therapeutic validity of this drug. Therefore, we tested experimentally the effectiveness of Mifamurtide in three cell lines of osteosarcoma (MG-63, HOS, 143B) with different grades of malignancy, grown in co-culture with macrophage to allow the correct internalization of the drug. Through in vitro experiments, we analyzed the effects of Mifamurtide on tumor cell viability, on cellular signaling pathway and on macrophage polarization. Results show that Mifamurtide loses its effectiveness as the tumor malignity increases. The most aggressive tumor cells are not affected by the therapy, probably because they implement methods of resistance to apoptosis. Furthermore, they would be able to affect the tumor microenvironment to their own advantage. To better understand Mifamurtide impact on tumor microenvironment, we also performed a cytokine assay on the culture medium in which the cells grew. The most significant results were identified in the interleukine-10 (IL-10): IL-10 is expressed in higher quantities in the highest-grade osteosarcoma cells. This anti-inflammatory cytokine can inhibit the synthesis of pro-inflammatory cytokine and suppresses the antigen presentation capacity of APC cells. This allows cancer cells to evade immune surveillance mechanisms. In vivo and in vitro results show that the synergic use of an anti-IL-10 antibody in combination with Mifamurtide can improve its efficacy. Here, we provide experimental evidence that the addition of an anti-IL-10 antibody to Mifamurtide causes a significant death rate in more aggressive osteosarcoma cells and lower metastasis, compared to Mifamurtide only. In conclusion, considering our data, it could be proposed a new treatment protocol for metastatic osteosarcoma that includes the use of an anti-IL10 antibody integrated with the administration of Mifamurtide to increase its effectiveness in this clinical context.

Citation Format: Nicoletta Nastasi, Angela Subbiani, Maria Letizia Taddei, Lucia Scala, Angela Tamburini, Claudio Favre, Maura Calvani. Use of an anti-IL-10 antibody to improve Mifamurtide efficacy on aggressive osteosarcoma 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 2007.
Abstract 2025: Spatial transcriptomic profiling of progression markers in clear cell renal cell carcinoma
Authors: Mo Chia-Kuei , Wu Yige , Nadezhda Terekhanova , Wagma Caravan , Lal Preet , Chen Siqi , Deen Nataly Naser AL , Liu Ruiyang , Zhao Yanyan , Sato Kazuhito , Yao Lijun , Serasanambati Mamatha , Shinkle Andrew , Jayasinghe Reyka G. , Ding Li , Chen Feng
Published: June 2022

Next-generation sequencing enabled molecular landscaping and the discovery of novel tumor markers in ccRCC. However, the spatial relationships and histological features of the tumor microenvironment were lost during this process. Combining snRNA-seq, snATAC-seq, Spatial Transcriptomics (ST) technology, and immunofluorescence labeling, we discovered novel ccRCC progression tumor markers, and uncovered their spatial dependent expression pattern in both human ccRCC tumors and patient-derived xenograft (PDX). Using snRNA-seq and snATAC-seq, novel tumor progression markers such as NDRG1, MGST1, ABCC3 and PCSK6 were discovered, and their expressions were validated in ST. We found that one of the key novel tumor markers, CP, exhibited specific spatial expression patterns on tissue slides. The elevation of CP expression region is associated with a higher degree of hyalinization in ccRCC human tumor samples. Similarly, using immunofluorescence labeling, we validated the co-expression of CP and PCSK6 canonical ccRCC marker CA9. Overall, this study revealed novel ccRCC progression markers and linked their spatial expression pattern with histological features.

Citation Format: Chia-Kuei Mo, Yige Wu, Terekhanova Nadezhda, Caravan Wagma, Preet Lal, Siqi Chen, Nataly Naser AL Deen, Ruiyang Liu, Yanyan Zhao, Kazuhito Sato, Lijun Yao, Mamatha Serasanambati, Andrew Shinkle, Reyka G. Jayasinghe, Li Ding, Feng Chen. Spatial transcriptomic profiling of progression markers in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2025.
Abstract 2036: Multi-omic dissection of immunotherapy response groups in non-small cell lung cancer (NSCLC)
Authors: Kulasinghe Arutha , Monkman James , Kim Honesty , Mayer Aaron , Mehdi Ahmed , Matigian Nicholas , Cumberbatch Marie , Bhagat Milan , Ladwa Rahul , Mueller Scott , Adams Mark , O'Byrne Ken
Published: June 2022

Immunotherapies have led to long term benefits in a subset of non-small cell lung cancer (NSCLC) patients. Developing predictive biomarkers of response to therapy is currently an unmet clinical need. The composition of the cellular content of the tumour microenvironment (TME) is an important characteristic driving treatment resistance. In this study, we utilised spatial transcriptomics methods, including Nanostring digital spatial profiling (DSP) and multiplex IHC, to define the tumour/stroma (compartment) specific proteome and transcriptome from a cohort of 2nd line immunotherapy treated NSCLC patients. We identified by mIHC that CD68+ macrophages with PD1+, FoxP3+ cells is significantly enriched in immunotherapy resistant tumours (p=0.012). Moreover, IL2 mRNA (p=0.001) in the stromal compartment was significantly increased in patients that were sensitive to ICI therapy, whilst CTLA-4 and IDO-1 were suppressed in responsive patients. Tumour compartment localised CD44 was depleted in the responder group and corresponded inversely with higher stromal expression of one of its ligands, SPP1 (p=0.008). Orthogonal validation of CD44 by multiplex immunofluorescence confirmed both its association with response and localisation to tumour cells rather than immune cell infiltrate. Cox survival analysis showed that tumour compartment CD44 expression was associated with a poorer prognosis (HR=1.61, p=0.01) whilst stromal localisation of CTLA-4 (HR=1.78, p=0.003) and ARG1 (HR=2.37, p=0.01) were associated with poorer outcome. Through a multi-omics approach, we demonstrate the utility of spatial mapping of NSCLC tumours and provide evidence for the role of a number of compartment-specific biomarkers which may aid in identifying those likely to respond to immunotherapy.

Citation Format: Arutha Kulasinghe, James Monkman, Honesty Kim, Aaron Mayer, Ahmed Mehdi, Nicholas Matigian, Marie Cumberbatch, Milan Bhagat, Rahul Ladwa, Scott Mueller, Mark Adams, Ken O'Byrne. Multi-omic dissection of immunotherapy response groups in non-small cell lung cancer (NSCLC) [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 2036.
Abstract 2043: The role of CCL20 in response to radiation in head and neck squamous cell carcinomas
Authors: Rutihinda Cleopatra , Oweida Ayman J. , Delage Patrick , Moreau Léanie , Chelighem Safia , Saidi Nour Elhouda
Published: June 2022

Background: Radioresistance is responsible for most cases of tumor progression and recurrence in head and neck squamous cell carcinomas (HNSCC). Combined radiotherapy (RT) and immunotherapy (IT) treatment has improved survival for a minority of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To enhance the response of radioresistant tumors to radiation, an understanding of the factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs and their depletion improves tumor response to RT. In this study, we investigated how radiation modulates Treg infiltration through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity and that blocking CCL20 could lead to improved response to RT.

Methods: Human (Cal27, SCC9) and mouse (MOC1, MOC2) cell lines with different immune phenotypes were irradiated at doses of 2Gy or 10Gy. Conditioned media (CM), RNA and protein were collected for the assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cell were were implanted into the right buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for the analysis of serum levels of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs.

Results: Cal27 and MOC2 tumors had a gene signature associated with immunosuppression and Treg infiltration whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed microenvironment. In vitro, high-dose radiation significantly induced CCL20 in Cal27 and MOC2 cells relative to control. Multiplex analysis of chemokines from conditioned media samples showed that induction of CCL20 was associated with increased secretion of GM-CSF by MOC2 and Cal27 cells. In vivo, inhibition of CCL20 alone resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. The combination of RT and anti-CCL20 showed a significant decrease in tumor volume compared to RT alone.

Conclusion: Our data suggests that high-dose radiation promotes the induction of CCL20 secretion by tumor cells which is responsible for attraction of Tregs. Inhibition of CCL20 secretion using anti-CCL20 antibodies can slow down tumor growth and enhance the response to radiation.

Citation Format: Cleopatra Rutihinda, Ayman J. Oweida, Patrick Delage, Léanie Moreau, Safia Chelighem, Nour Elhouda Saidi. The role of CCL20 in response to radiation in head and neck squamous cell carcinomas [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 2043.
Abstract 2067: NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity
Authors: Li Xuefeng , Huntoon Kristin , Wang Yifan , Walkey Carl , Kim Betty Y. , Jiang Wen
Published: June 2022

There is a growing interest in combining radiotherapy (RT), a standard of care treatment for many cancer types, with immunotherapeutic agents to improve efficacy. One such approach is RT in combination with high-dose recombinant interleukin 2 (IL-2), which elicits anti-tumor immune responses by stimulating T-cell and NK cell populations. Although effective for some patients, the overall clinical benefit of high-dose recombinant IL-2 is limited by toxicity and the expansion of regulatory T cells (Tregs) through signaling events mediated by IL2RΑ (CD25).

In this study, we examined RT in combination with NL-201, a highly potent and stable CD25-independent IL-2 and IL-15 receptor agonist with enhanced affinity for IL2RΒ and IL2RΓ. Single-agent NL-201 was well-tolerated in mice, yielded tumoricidal activity, expanded peripheral T cells, and enhanced the infiltration of effector T cells and dendritic cells (DCs) into murine breast cancers. In combination with RT, NL-201 enhanced activation of the cytosolic DNA sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, resulting in increased type I interferon (IFN) production in DCs and, consequently, greater tumor infiltration by T cells and more efficient priming of antigen-specific T cells by professional antigen-presenting cells (APCs). The immune stimulatory mechanisms triggered by the NL-201 and RT combination resulted in superior tumor growth inhibition and survival benefit in both localized and metastatic murine breast cancers, including those spread to the central nervous system. Genetic knockout of STING in host cells abrogated the activity of the NL-201 and RT combination, thus confirming the crucial role of innate immune sensors in the combination treatment’s antitumor responses. Additionally, when combined with PD-1 antibodies, NL-201 significantly improved growth inhibition of metastatic breast cancers that were previously resistant to checkpoint blockade.

We demonstrated that the CD25-independent IL-2 and IL-15 receptor agonist, NL-201, in combination with RT is well-tolerated and elicits robust anti-tumor activity through both innate and adaptive responses, including in checkpoint resistant tumors and brain metastases. Furthermore, we identify a unique mechanism in which NL-201 synergizes with RT, likely via the direct activation of STING in DCs. Taken together, the results provided herein support further preclinical and clinical investigation of this novel combination regimen in both localized and metastatic settings.

Citation Format: Xuefeng Li, Kristin Huntoon, Yifan Wang, Carl Walkey, Betty Y. Kim, Wen Jiang. NL-201, a de novo engineered IL2/IL15 mimic, synergizes with radiation to generate potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2067.
Abstract 2124: ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient non-small cell lung cancer
Authors: Crosson William P. , Li Rui , Salehi-Rad Ramin , Lim Raymond J. , Abascal Jensen W. , Kahangi Bitta P. , Reyes Edgar Perez , Dumitras Camelia , Jing Zhe , Krysan Kostyantyn , Tran Linh M. , Liu Bin , Dubinett Steven M.
Published: June 2022

Lung cancer remains the deadliest form of cancer, claiming the lives of 1.8 million worldwide in 2020. While treatment options have improved with the advent of immune checkpoint inhibitors (ICI), many patients do not respond to immunotherapy or develop resistance following initial response. A significant subset of patients harbor somatic mutations in both Kirsten rat sarcoma virus (KRAS) and Liver kinase 1 [LKB1, also known as serine/threonine kinase 11 (STK11)] genes. LKB1 loss has been identified as a driver of primary resistance to ICI therapy. Tumors from patients that possess these mutations are characterized by an increase in the population of myeloid derived suppressor cells (MDSCs). MDSCs are thought to play a critical role in promoting tumor progression by blocking the tumor killing function of cytotoxic T cells in the tumor microenvironment (TME). Our studies revealed all-trans retinoic acid (ATRA), a metabolite derived from vitamin A, sensitized murine models of non-small cell lung cancer (NSCLC) with LKB1 and KRAS co-mutations to PD-1 blockade. Combination therapy with anti-PD-1 and ATRA improved local and systemic T cell proliferation and generated systemic tumor-specific immunity. We observed that ATRA augmented anti-PD-1 efficacy in additional murine NSCLC models. We demonstrated that ATRA suppresses tumor growth through the inhibition of immunosuppressive MDSCs in the TME, and preliminary data indicates that ATRA increases intracellular reactive oxygen species (ROS) and IFN signaling in MDSC. Our findings implicate MDSCs as a potential mediator of immunosuppression in Lkb1-deficient NSCLC and provide a rationale for utilizing ATRA in combination with anti-PD-1 therapy in patients with Lkb1-deficient NSCLC refractory to ICIs.

* B. Liu and S. M. Dubinett contributed equally to this work

Citation Format: William P. Crosson, Rui Li, Ramin Salehi-Rad, Raymond J. Lim, Jensen W. Abascal, Bitta P. Kahangi, Edgar Perez Reyes, Camelia Dumitras, Zhe Jing, Kostyantyn Krysan, Linh M. Tran, Bin Liu, Steven M. Dubinett. ATRA mediated inhibition of myeloid-derived suppressor cells overcomes resistance to immune checkpoint inhibition in murine models of LKB1-deficient 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 2124.
Abstract 2135: Targeting N-Myc in neuroblastoma cells with selective Aurora kinase A degraders
Authors: Tang Jian , Moorthy Ramkumar , Demir Ozlem , Baker Zachary D. , Naumann Jordan A. , Jones Katherine F. , Grillo Michael J. , Haefner Ella , Shi Ke , Levy Michaella J. , Aihara Hideki , Harris Reuben S. , Amaro Rommie E. , Levinson Nicholas M. , Harki Daniel A.
Published: June 2022

Overexpression of the MYCN proto-oncogene is one of the strongest biomarkers for poor prognosis in neuroblastoma patients. N-Myc, encoded by MYCN, is a highly attractive yet “undruggable” target in neuroblastoma drug discovery. N-Myc is a short-lived protein in normal neuronal progenitor cells; however, in MYCN-amplified neuroblastoma cells, overexpressed N-Myc is stabilized by high levels of Aurora kinase A (Aurora-A). Several Aurora-A allosteric modulators have been developed to disassociate N-Myc binding and promote its degradation. These compounds function through a competitive mechanism against N-Myc, and clinical trial results suggest limited efficacy in high-risk neuroblastoma patients where N-Myc is highly expressed. To develop Aurora-A modulators with greater effects in destabilizing N-Myc, targeted degradation of Aurora-A was investigated in this study. First, we developed a highly selective Aurora-A-binding ligand by chemically modifying ribociclib, which targets CDK4/6. Next, a series of potent Aurora-A/N-Myc degraders were developed using proteolysis targeting chimaera (PROTAC) technology. Our lead compound, HLB-0532259, was found to substantially decrease N-Myc levels in neuroblastoma cells following the degradation of Aurora-A with nanomolar potency. Mechanistic studies suggest that HLB-0532259 binds to Aurora-A in an uncompetitive manner with respect to N-Myc, which contributes to its high potency for degrading the Aurora-A/N-Myc pool. Mass spectrometry analysis demonstrates that HLB-0532259 possesses remarkable proteome-wide selectivity for Aurora-A and N-Myc degradation. Further studies have revealed that HLB-0532259 exhibits nanomolar cytotoxicities against a panel of MYCN-amplified neuroblastoma cells, good pharmacokinetic properties, and favorable therapeutic outcomes in a xenograft neuroblastoma mouse model. Taken together, our data strongly support HLB-0532259 as a promising lead compound for neuroblastoma therapy.

Citation Format: Jian Tang, Ramkumar Moorthy, Ozlem Demir, Zachary D. Baker, Jordan A. Naumann, Katherine F. Jones, Michael J. Grillo, Ella Haefner, Ke Shi, Michaella J. Levy, Hideki Aihara, Reuben S. Harris, Rommie E. Amaro, Nicholas M. Levinson, Daniel A. Harki. Targeting N-Myc in neuroblastoma cells with selective Aurora kinase A degraders [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 2135.
Abstract 2147: Synthetic lethality of VRK1 in VRK2-methylated cancers
Authors: So Jonathan , Mabe Nathaniel , Englinger Bernhard , Kwon Jason , Shim Brian , Filbin Mariella , Stegmaier Kimberly , Hahn William
Published: June 2022

Paralogs are a source of synthetic lethal interactions which lend themselves to novel bio-marker linked targeted therapeutics. From genome-wide essentiality measurements in over 900 cancer cell lines, we find VRK1 dependency in VRK2-methylated adult and pediatric gliomas and neuroblastomas. VRK2 methylation was mainly seen in IDH-mutant gliomas and H3.3-G34R-mutant DIPG. Knockout of VRK2 was able to sensitize cells to VRK1 knockout. Overexpression of kinase-active but not kinase inactive VRK2 rescued VRK1 knockout. Global phosphoproteomics in VRK1 and VRK2 knockout cells demonstrate increased phosphorylation of targets involved with DNA damage and decreased phosphorylation of nuclear membrane targets. VRK1 and VRK2 were found to phosphorylate BANF1, which is involved in nuclear membrane re-formation during mitosis. Taken together, we show that VRK1 is a viable target in VRK2-methylated adult and pediatric gliomas, and neuroblastomas.

Citation Format: Jonathan So, Nathaniel Mabe, Bernhard Englinger, Jason Kwon, Brian Shim, Mariella Filbin, Kimberly Stegmaier, William Hahn. Synthetic lethality of VRK1 in VRK2-methylated 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 2147.
Abstract 2148: Pre-clinical models recapitulating the spectrum of response of BRCA associated pancreatic cancer
Authors: Golan Talia , Stossel Chani , Raitses-Gurevitch Maria , Atias Dikla , Beller Tamar , Gallinger Steven , Berger Raanan
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease, with the majority of patients diagnosed at an advanced stage. Comprehensive genomic analysis identified the homologous recombination deficiency (HRD) subgroup which is predominantly enriched in patients harboring germline BRCA1/2 mutations (glBRCA) and presents ~7% and up to 15% in selected high-risk populations. Tumors with HRD are susceptible to DNA-damaging agents and PARP inhibition. However, not all patients demonstrate a similar response, and a spectrum is observed. We analyzed the clinical outcome of ninety-one glBRCA PDAC patients treated at Sheba Medical Center. We identify three main subgroups of response spanning from ~25% patients refractory to first line platinum agents and ~9% patients with durable long-term response with no evidence of disease for over three years. The majority of the patients display a prolonged response to platinum and PARPi maintenance (>24 months) followed by acquired resistance. We generated patient derived xenograft (PDX) models and an ex-vivo culture system (EVOC) from naïve to treatment tissue and at clinical resistance. In vivo efficacy to platinum agents and PARPi demonstrates that these models recapitulate the specific clinical spectrum of response. We demonstrate the utility of both pre-clinical models in their ability to predict response to platinum-based agents and PARPi, with EVOC having a potential to assist in medical decisions while treating the patient. Whole genome sequencing of the tumors revealed that BRCA monoallelic tumors are associated with innate resistance while biallelic tumors reflect sensitivity to platinum agents and PARPi. In the acquired resistance models, the main identified mechanism of resistance was due to reversion mutations in 4/6 of samples. Whole genome analysis demonstrates high mutational and neo-antigen load in majority of the glBRCA tumors. In a preliminary study, we show the efficacy of anti PD1 in a novel humanized glBRCA PDAC PDX model. This extensive preclinical and clinical collection of BRCA associated PDAC, enables further understanding and investigation of this unique subtype in aim to develop alternative treatments to overcome resistance.

Citation Format: Talia Golan, Chani Stossel, Maria Raitses-Gurevitch, Dikla Atias, Tamar Beller, Steven Gallinger, Raanan Berger. Pre-clinical models recapitulating the spectrum of response of BRCA associated pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2148.
Abstract 2157: SCR-6277, a potent and high selective arginine methyltransferase 5 (PRMT5) inhibitor with high tumor/plasma distribution ratio demonstrated robust anti-tumor activities and decreased hematological toxicities
Authors: Zhou Feng , Yang Guimei , Tang Feng , Xue Liting , Yang Wenqing , Chen Ping , Tang Renhong
Published: June 2022

Protein arginine methyltransferase 5 (PRMT5) is observed dysregulation in both solid and hematological tumors. Increasing evidences demonstrated that PRMT5 is a potential therapeutic target. Given the essential role in maintenance of adult hematopoietic cells, inhibition of PRMT5 leads to hematological toxicity. Anemia is the most common treatment related adverse event observed by PRMT5 inhibitor, such as GSK3326595, which limited the dose escalation. Therefore, developing a PRMT5 inhibitor with tumor-biased distribution could be an effective approach to avoid these target related toxicities. In this study, a novel and high selective PRMT5 inhibitor, SCR-6277 was identified. SCR-6277 inhibited the PRMT5 enzyme with an IC50 of 1.5 nM, and showed a high selectivity against other five methyltransferase isoforms (selectivity folds >1000). SCR-6277 potently inhibited the PRMT5-mediated SDMA production in Z-138 cells (IC50, 0.6 nM) and the cell proliferation (IC50, 3 nM). SCR-6277 also demonstrated robustly anti-proliferation activities on multiple PRMT5 proficient solid tumor cell lines (IC50 values ranging from 20 - 130 nM) and no inhibitory effects on PRMT5 deficient cell lines. SCR-6277 had good oral bioavailability and favorable pharmacokinetic properties. In Z-138 and 5637 xenograft models, SCR-6277 robustly inhibited tumor growth. In contrast to GSK3326595, SCR-6277 showed tumor biased distribution. Exposure of SCR-6277 in tumors were 50 folds higher than that in plasma. The extremely high T/P ratio made SCR-6277 achieve the better anti-tumor activities at lower blood exposure (Table 1). In conclusion, SCR-6277 is a novel, potent and high selective PRMT5 inhibitor. And higher tumor/plasma distribution ratio predicts SCR-6277 is a more tolerant and effective PRMT5 inhibitor in human.

Table 1. Distribution and anti-tumor activity of SCR-6277 Dose TGI Plasma Conc. (ng/mL) * Tumor Conc. (ng/g) * T/P ratio GSK3326595 50 mpk,BID 87% 45 106 2 SCR-6277 15 mpk,BID 117% 20 668 50

Citation Format: Feng Zhou, Guimei Yang, Feng Tang, Liting Xue, Wenqing Yang, Ping Chen, Renhong Tang. SCR-6277, a potent and high selective arginine methyltransferase 5 (PRMT5) inhibitor with high tumor/plasma distribution ratio demonstrated robust anti-tumor activities and decreased hematological toxicities [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 2157.
Abstract 2167: Machine learning-based tissue of origin classification for cancer of unknown primary diagnostics using genome-wide mutation features
Authors: Nguyen Luan , Schipper Luuk , Roepman Paul , Monkhorst Kim , van Hoeck Arne , Snaebjornsson Petur , Cuppen Edwin
Published: June 2022

Tumor tissue of origin (TOO) is an important factor for guiding treatment decisions. However, TOO cannot be determined for ~3% of metastatic cancer patients that present in the clinic and are categorized as cancers of unknown primary (CUP). As whole genome sequencing (WGS) of tumors is now transitioning from the research domain to diagnostic practice in order to address the increasing demand for biomarker detection, its use for detection of TOO in routine diagnostics also starts becoming within reach. While proof of concept for the use of genome-wide features has been demonstrated before, more complex WGS mutation features, including structural variant (SV) driver and passenger events, have never been integrated into TOO-classifiers even though they bear highly characteristic links with tumor TOO. Using a uniformly processed dataset containing 6820 whole-genome sequenced primary and metastatic tumors (ICGC/PCAWG and Hartwig cohors), we have developed Cancer of Unknown Primary Location Resolver (CUPLR), a random forest based TOO classifier that employs 502 genome-wide features based on simple and complex somatic driver and passenger mutations. Our model is able to distinguish 33 cancer (sub)types with an overall accuracy of 91% and 89% based on cross-validation (n=6139) and hold out set (n=681) predictions, respectively. We found that SV derived features increase the accuracy and utility of TOO classification for specific cancer types. To ensure that predictions are human-interpretable and suited for use in routine diagnostics, CUPLR reports the top contributing features and their values compared to cohort averages. The comprehensive output of CUPLR is complementary to existing histopathological procedures and may thus improve diagnostics for patients with CUP. CUP is the first reimbursed indication for WGS in The Netherlands. We will report on the prospective use and clinical utility of WGS for CUP patients in routine diagnostics but also highlight the added value of CUPLR for unsolved differential diagnosis cases and incidental cases where the independent WGS analysis was discrepant with the original tumor diagnosis.

Citation Format: Luan Nguyen, Luuk Schipper, Paul Roepman, Kim Monkhorst, Arne van Hoeck, Petur Snaebjornsson, Edwin Cuppen. Machine learning-based tissue of origin classification for cancer of unknown primary diagnostics using genome-wide mutation features [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 2167.