bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4050: Effect of benzofuran pharmacophores on CYP-activated duocarmycin bioprecursors
Authors: Jukes Zoe H.
Published: June 2022

Duocarmycins are natural DNA alkylators which have potent toxicity with no established mechanism of resistance. Prodrug versions need to be developed to ensure a sufficient therapeutic window for cancer treatment (1). A series of duocarmycin bioprecursors have been developed in-house which lack a key hydroxyl trigger group, required for spirocyclisation and subsequent activation. This hydroxyl group can be regioselectivity restored by specific Cytochrome P450 enzymes (CYPs), with a primary focus on cancer associated isoforms CYP1A1 and CYP2W1 (2). An exploratory library of analogues of our lead compound, ICT2700 (CYP1A1 and CYP2W1 activated), has been synthesized and elucidated aspects of the structure-activity relationship (SAR) between the compound and subsequent toxicity in CYP transfected and mock cell lines, using MTT assays. The data shows the importance of having an indole in the DNA recognition subunit (the non-alkylating subunit) for CYP1A1 and CYP2W1 activation. Further, a compound with thus far unexplained activity was identified. ICT11038 has a benzofuran instead of an indole in the recognition subunit and subsequently demonstrates inherent nanomolar toxicity consistently in all cell lines tested, including a range of cancerous and non-cancerous cells, which is not dependent on cytochrome expression. CYP1A1 has been shown to enhance the toxicity of ICT11038 further but is not required for nanomolar toxicity. Further SAR exploration and western blots confirm that the inherent toxicity is achieved through DNA damage despite lacking the key trigger group, suggesting a novel activation pathway. Additionally, our SAR studies show that replacing the indole of the DNA alkylation subunit with a benzofuran ablates CYP dependent activity, with one exception, ICT2724. ICT2724 is non-toxic in mock transfected cells but is toxic in CYP1A1 transfected cells, albeit it at higher concentration than ICT2700. ICT2724 is analogous to ICT2700, except for the direct indole to benzofuran substitution. Other compounds with benzofurans in the alkylating subunit compounds are not metabolized to a toxic compound by CYP1A1 or CYP2W1 despite their indole equivalents being good prodrug candidates. To conclude, the introduction of the benzofuran functional group into the CYP-activated duocarmycin bioprecursor pharmacophores produces an unexplained pattern of CYP activation and, in one example, an intrinsically toxic DNA damaging agent (ZYP-11038).

References (1) Z. Jukes et al, Drug Discov Today, 2020, 26(2), 577-584, https://doi.org/10.1016/j.drudis.2020.11.020 (2) H. Sheldrake et al, J. Med. Chem, 2013, 56(16); 6273-6277, dx.doi.org/10.1021/jm4000209

Citation Format: Zoe H. Jukes. Effect of benzofuran pharmacophores on CYP-activated duocarmycin bioprecursors [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 4050.
Abstract 4055: First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model
Authors: Guijarro Maria V. , Kellish Patrick C. , Dib Peter E. , Paciaroni Nicholas G. , Nawab Akbar , Andring Jacob , Kulemina Lidia , Borrero Nicholas V. , Modenutti Carlos , Bennett Richard L. , Shabashvili Daniil , Licht Jonathan D. , McKenna Robert , Roitberg Adrian , Huigens Robert W. , Kaye Frederic J. , Zajac-Kaye Maria
Published: June 2022

Thymidylate synthase (TS) inhibitors are an integral component of chemotherapy regimens for difficult to treat cancer subtypes. Despite initial therapeutic benefit, current inhibitors induce TS overexpression or alter folate transport metabolism feedback pathways that tumor cells exploit for drug resistance. Here we report a small molecule TS inhibitor that exhibits i) enhanced antitumor activity as compared to current fluoropyrimidines and antifolates without inducing TS overexpression, ii) is structurally distinct from classical antifolates, iii) extends survival in a pancreatic tumor mouse model, iv) and is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we confirm the compound is a multifunctional non classical antifolate and through a series of analogues identify structural features allowing direct TS inhibition while also maintaining the ability to inhibit dihydrofolate reductase (DHFR). Collectively, this work identifies new non classical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile highlighting potential for enhanced cancer therapy.

Citation Format: Maria V. Guijarro, Patrick C. Kellish, Peter E. Dib, Nicholas G. Paciaroni, Akbar Nawab, Jacob Andring, Lidia Kulemina, Nicholas V. Borrero, Carlos Modenutti, Richard L. Bennett, Daniil Shabashvili, Jonathan D. Licht, Robert McKenna, Adrian Roitberg, Robert W. Huigens, Frederic J. Kaye, Maria Zajac-Kaye. First in class multifunctional non classical antifolates inhibits thymidylate synthase and extends survival in pancreatic cancer model [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 4055.
Abstract 4072: Cytotoxic effects of LRRK2 inhibitors in combined treatment with chemotherapeutic agents on a large panel of cancer cell lines
Authors: Fimm-Todt Franziska , Lauterwasser Joachim , Egenter Eva-Maria , Weber Christian , Feger Daniel , Schaich Katharina , Ulrich Sarah , Siedentopf Oliver , Totzke Frank , Kubbutat Michael , Ehlert Jan Erik
Published: June 2022

Leucin-rich repeat kinase 2 (LRRK2) plays an important role in the onset of sporadic as well as familial Parkinson’s disease. Pathogenic gain-of-function mutations of LRRK2 are associated with aberrant LRRK2 hyperactivity which results in neurotoxicity and protein aggregation caused by dysfunctional autophagy and vesicle trafficking. Thus, the development of LRRK2 inhibitors represents a promising strategy for the treatment of Parkinson’s disease. Interestingly LRRK2 mutations have also been reported to increase the risk for the onset of different types of cancer (e.g. breast, thyroid, lung). Several studies suggest that LRRK2 is involved in the regulation of different cancer-related pathways (e.g. ATM-p53-p21-pathway, JNK pathway). Deregulation of LRRK2 activity caused by mutations has been shown to interfere with these pathways thereby increasing the risk to develop certain types of cancer. Since anti-cancer treatments mostly target the same pathways, we hypothesized that LRRK2 inhibitors may affect anti-cancer treatments in specific cancer cell types. In this study we report on the analyses of various inhibitors (e.g. MLi-2 and PF-06447475) on LRRK2 autophosphorylation at S935 in a cellular phosphorylation assay using the non-small cell lung cancer cell line A549 in comparison to biochemical LRRK2 activity assays. Furthermore, we compare the direct LRRK2 inhibitor effect on the proliferation of 140 cell lines, as well as their potential combinatorial impact on the potency of chemotherapeutic agents (e.g. Adriamycin). The observed effects can help to understand the implications of pharmaceutical LRRK2 inhibition in the treatment of both Parkinson’s disease and cancer.

Citation Format: Franziska Fimm-Todt, Joachim Lauterwasser, Eva-Maria Egenter, Christian Weber, Daniel Feger, Katharina Schaich, Sarah Ulrich, Oliver Siedentopf, Frank Totzke, Michael Kubbutat, Jan Erik Ehlert. Cytotoxic effects of LRRK2 inhibitors in combined treatment with chemotherapeutic agents on a large panel of cancer cell lines [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 4072.
Abstract 4098: Comparison of frontline therapeutic modalities in stages I/IE extranodal NK/T-cell lymphoma-nasal type (ENKTL-N): A network meta-analysis
Authors: Haddad Philip A. , Gallagher Kevin , Hammoud Dalia
Published: June 2022

Introduction: Despite many studies reporting on the optimal therapies for early stage ENKTL-N in the front-line setting, this area continues to be subject to significant controversy regarding single modalities versus combination or sequential approaches. All these comparative studies were small non-randomized studies that combined stages I & II. The results were often inconclusive and occasionally conflicting. In the absence of direct head-to-head randomized controlled trials in this clinical setting, a network meta-analysis was conducted to compare these therapeutic approaches and their respective impact on 5-year survival in stage I/IE ENKTL-N.

Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language, diagnosis of stage I/IE ENKTL-N, treatment with chemotherapy (CT), radiation (RT), sequential CT and RT (SEQ), and chemoradiotherapy (CRT), comparative studies that reported predominantly I/IE 5-year survival and disease-free survival rates. Studies that reported on mixed samples of early and advanced ENKTL were excluded. A frequentist network meta-analysis was conducted using the netmeta package and random-effects model.

Results: Seven studies comprising a total of 377 participants were included. Our network meta-analysis revealed that upfront RT tended to have better 5-year OS and DFS than CRT and SEQ, though it did not reach statistical significance. However, RT demonstrated significant 5-year OS and DFS when compare to CT alone in the frontline setting. Based on the pairwise and network meta-analyses, RT was ranked as the most effective first-line treatment approach followed by CRT, SEQ, and CT in decreasing order. Inconsistency analysis did not reveal any significant differences between direct and indirect estimates.

Conclusions: This is the first network meta-analysis to compare all commonly utilized upfront treatment modalities in stages I/IE ENKTL-N. It indicates that upfront RT alone may be sufficient as there was no signal that chemotherapy added any OS or DFS advantages. Adequately powered randomized trials are warranted.

Citation Format: Philip A. Haddad, Kevin Gallagher, Dalia Hammoud. Comparison of frontline therapeutic modalities in stages I/IE extranodal NK/T-cell lymphoma-nasal type (ENKTL-N): A network meta-analysis [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 4098.
Abstract 4173: Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR
Authors: Zhang Qi , Zhe Yang , Wu Xiaoqing , Lan Lan , Wei Lanjing , Xu Liang
Published: June 2022

Cancer immunotherapy, especially those targeting the immune checkpoint PD-1 and PD-L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)-mediated anti-tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA-binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs. In this study, we show the direct binding of HuR protein to PD-L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP-IP) and further validated by RNA pulldown assay. We also show that PD-L1 mRNA decay is greatly expedited in HuR-knockout cells, which underlines the stabilization function of HuR on PD-L1 transcripts. Immunoblotting results demonstrate that PD-L1 protein expression level is decreased in HuR-knockout cells while up-regulated in HuR-proficient cells in comparison with control. These data together clarify HuR is engaged in PD-L1 post-transcription regulation. Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti-PD1 antibodies to overcome the limitation by regulating the tumor immune microenvironment. KH-200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4-week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. A similar combination effect was further observed in Lewis lung syngeneic lung cancer model. In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows effectiveness in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy.

Citation Format: Qi Zhang, Yang Zhe, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR [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 4173.
Abstract 4189: Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates
Authors: Nash Amanda , Aghlara-Fotovat Samira , Hernandez Andrea , Castillo Bertha , Lu Alexander , Pugazenthi Aarthi , Rios Peter , Ghani Sofia , Joshi Ira , Isa Douglas , Xu Chunyu , Sheth Rahul , Peng Weiyi , Oberholzer Jose , Jazaeri Amir , Jang Hee-Jin , Burt Bryan , Lee Hyun-Sung , Ghanta Ravi , Veiseh Omid
Published: June 2022

Pro-inflammatory cytokines have been approved as a cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years. However, widespread use of cytokine therapy in the clinic is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a clinically translatable cytokine delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These cells are able to produce a wide range of natural cytokines (IL2, IL7, IL10, or IL12) and allow for controlled and predictable dosing in vivo. In vivo administration of cytokine factories created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. Local, or tumor adjacent, administration of pro-inflammatory (IL-2-based) cytokine factories caused reduction of tumor burden by 70% after only 1 week of treatment when delivered as a monotherapy to mice with malignant pleural mesothelioma (MPM). Importantly, when administered in combination with local anti-PD1 injections, these cytokine factories lead to eradication of these highly aggressive tumors in 7/7 treated mice. To validate the translatability of this platform, we evaluated the safety profile in non-human primates. Significantly, this platform produced local and systemic T cell biomarker profiles that predict efficacy without renal, liver, or general toxicity in non-human primates. Our findings demonstrate the safety and efficacy of cytokine factories in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans.

Citation Format: Amanda Nash, Samira Aghlara-Fotovat, Andrea Hernandez, Bertha Castillo, Alexander Lu, Aarthi Pugazenthi, Peter Rios, Sofia Ghani, Ira Joshi, Douglas Isa, Chunyu Xu, Rahul Sheth, Weiyi Peng, Jose Oberholzer, Amir Jazaeri, Hee-Jin Jang, Bryan Burt, Hyun-Sung Lee, Ravi Ghanta, Omid Veiseh. Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates [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 4189.
Abstract 4208: In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade
Authors: Lim Raymond J. , Salehi-rad Ramin , Tran Linh M. , Dumitras Camelia , Abascal Jensen W. , Crosson William P. , Patel Tejas S. , Man Samantha , Li Rui , Krysan Kostyantyn , Liu Bin , Dubinett Steven M.
Published: June 2022

Lung cancer is the most common cause of cancer death worldwide with approximately 85% of patients having non-small cell lung cancer (NSCLC). Checkpoint blockade immunotherapy has evolved the current treatment landscape with robust and durable responses in approximately 20% of patients with progressive, locally advanced or metastatic NSCLC, as well as in treatment-naïve advanced disease. Inefficient tumor antigen presentation, diminished T cell infiltration into tumor and LKB1-inactivating mutations contribute to the mechanisms of resistance to programmed death-ligand 1 (PD-L1) or programmed cell death protein 1 (PD-1) blockade in NSCLC. LKB1 deficiency also facilitates an immunosuppressive environment that promotes tumor growth. One approach to overcome this immunosuppressive tumor microenvironment (TME) is to utilize in situ vaccination with gene-modified functional antigen presenting cells (APCs) to enhance tumor antigen presentation and promote tumor-specific T cell activation. It has been shown that the chemokines CXCL9 and CXCL10 (CXCL9/10) are important signaling molecules secreted by CD103+ dendritic cells (DCs) to orchestrate the anti-tumor immunity. Thus, we hypothesize that intratumoral (IT) injection of CXCL9/10-secreting DCs (CXCL9/10-DCs) can potentiate the response to anti-PD-1 monotherapy in LKB1-deficient NSCLC by promoting antigen presentation and inducing T cell infiltration. Our preliminary data shows that CXCL9/10-DC injection modulates systemic immunity and generates anti-tumor responses that are augmented by the combination treatment with anti-PD1. In an in vivo murine model of Lkb1-null lung cancer bearing high tumor mutational burden (TMB), CXCL9/10-DC facilitates recruitment of effector T cells into the TME and tumor-specific T cell activation. These studies will enhance our understanding of anti-tumor immunity mediated by in situ DC vaccination and facilitate the improvement of novel vaccine therapies.

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

Citation Format: Raymond J. Lim, Ramin Salehi-rad, Linh M. Tran, Camelia Dumitras, Jensen W. Abascal, William P. Crosson, Tejas S. Patel, Samantha Man, Rui Li, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination of non-small cell lung cancer with CXCL9/10-engineered dendritic cells combined with PD-1 blockade [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 4208.
Abstract 4212: In situvaccination with Flt3l gene-modified CD103+type 1 conventional dendritic cells (cDC1) in murine models of non-small cell lung cancer (NSCLC)
Authors: Abascal Jensen W. , Lim Raymond J. , Salehi-Rad Ramin , Jing Zhe , Crosson William P. , Kahangi Bitta P. , Reyes Edgar Perez , Reyimjan Diana , Zhu Jessie , Tran Linh M. , Paul Manash , Krysan Kostyantyn , Liu Bin , Dubinett Steven M.
Published: June 2022

A major hurdle in treatment of Non-Small Cell Lung Cancer (NSCLC) with anti-PD-1 immune checkpoint blockade (ICB) therapy is a lack of response (primary resistance) and relapse after an initial response (acquired resistance). Recent studies reveal that responses to PD-1/PD-L1 blockade are associated with high tumor mutational burden (TMB), increased CD8+ T cell infiltration and high baseline PD-L1 expression within the tumor microenvironment (TME), while impaired tumor antigen presentation and the immunosuppressive TME have been associated with resistance to ICB. One approach to overcome anti-PD-1 resistance is to intratumorally vaccinate NSCLC tumors with gene modified conventional dendritic cells (cDC), specifically the type I conventional DC (cDC1) lineage. Recent studies have established that generation of an anti-tumor immune response driven by CD8+ T cells requires the cross presentation of tumor associated antigens and that cDC1s are the primary cross presenting APC subtype in vivo, which can license CD8+ T cells to initiate an adaptive anti-tumor immune response. In addition, previous studies have shown that intratumoral administration of the FMS-like tyrosine kinase 3 ligand (FLT3L) protein can expand endogenous CD103+ cDC1 cells in the TME and augment anti-tumor immune responses to ICB therapy. Here, we engineered murine CD103+ cDC1 cells to constitutively secrete soluble FLT3L (FLT3L_cDC1) and performed in situ vaccination studies on anti-PD1 resistant murine models of NSCLC with LKB1-deficiency and elevated TMB that better represents human disease. The FLT3L_cDC1 cells showed enhanced anti-tumor efficacy compared to non-modified cDC1 cells and synergized with anti-PD-1 ICB to inhibit tumor growth. Our data suggests intratumoral injection of FLT3L_cDC1 cells may represent a promising strategy to potentiate the efficacy of ICB and improve outcomes for patients with primary resistance PD-1/PD-L1 monotherapy.

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

Citation Format: Jensen W. Abascal, Raymond J. Lim, Ramin Salehi-Rad, Zhe Jing, William P. Crosson, Bitta P. Kahangi, Edgar Perez Reyes, Diana Reyimjan, Jessie Zhu, Linh M. Tran, Manash Paul, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situvaccination with Flt3l gene-modified CD103+type 1 conventional dendritic cells (cDC1) in murine models of 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 4212.
Abstract 5004: Integrative quality control of cancer somatic mutations with CaMutQC
Authors: Wang Xin , Ren Jian , Zhao Qi
Published: June 2022

The quality control of cancer somatic mutations is an essential step for eliminating false positive mutations from technical bias, and selecting key mutation candidates plays a crucial role in both downstream translation research and personal medical decisions. Existing tools, with complicated parameters and changeable filtering standards, may not be suitable specifically for cancer somatic mutations. In addition, previous genomic studies usually adopted different filtering criteria for processing raw sequencing datasets, which greatly restricts the efficiency of integrative exploration for cancer genomics.

In this study, we presented CaMutQC, a heuristic cancer somatic mutations (CAMs) quality control and filtration package for cancer genomic studies. CaMutQC provides two schemes, including filtration of false positive mutations generated by technical issues, and the screening of candidate cancer somatic mutations from single or multiple VEP-annotated files in VCF format. Specifically, for quality control, instead of directly discarding CAMs that failed to meet the thresholds of parameters collected from classic studies, CaMutQC labels them with customized tags to represent the filtering type, enabling portable subsequent analyses. And a vivid and well-structured filter report is generated after filtration or selection. In addition, we proved by applying an updated mutation QC strategy, which takes the union of CaMutQC-filtered cancer variants from multiple variant callers (eg. MuTect, MuSE and VarScan2) on published datasets that, CaMutQC can not only reduce the false positive CAMS, but also greatly rescue the false negative CAMS from each single caller or the overlap of multiple tools.

In summary, in this study, for the first time, we systematically implemented the parameters and criteria of CAMs quality control from published studies into the CaMutQC package, which we believe it would be a valuable tool for cancer genomic research. CaMutQC is implemented in R and is available at https://github.com/likelet/CaMutQC under the GPL-v3 license.

Citation Format: Xin Wang, Jian Ren, Qi Zhao. Integrative quality control of cancer somatic mutations with CaMutQC [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 5004.
Abstract 5040: Genetic predisposition of rectal neuroendocrine tumors
Authors: Saha Subrata , Grunn Adina , Califano Andrea
Published: June 2022

Rectal neuroendocrine tumors (RNETs) represent a clinically and molecularly distinct, yet poorly characterized subtype of gastroenteropancreatic neuroendocrine tumors (GEP-NET), with a virtually uncharted genetic landscape. Given their rare nature, compared with their small intestine (SI-NET) or pancreatic (PNET) counterpart, there have been no systematic efforts to elucidate the genetic landscape of RNETs and understand whether these tumors may present a common genetic etiology and predisposition. We have assembled a discover and a validation cohort comprising blood and tumor samples from 31 and 21 metastatic RNET patients, respectively, which was very challenging given the ultra-rare nature of these tumors. Whole-exome profiles were used to identify both rare and common germline variants that may be predictive of metastatic RNET predisposition, which is suspected to have a strong genetic component, given their frequent multifocal presentation. Among nonsynonymous rare germline variants (MAF < 0.05 in the general population), 314 were significantly over-represented in the discovery cohort (FDR ≤ 0.05), based on ethnicity-matched controls from the 1,000 Genome Project. Of these, 144 were also identified in samples from the validation cohort. Of the 40 that were statistically significant in both cohorts, 11 were deemed pathogenic by CADD Phred-scores analysis (≥ 20.0), including 8 in regulatory regions. This includes pathogenic variants harbored by ALKBH8, LRRC71, PSPH, MUC16, SCRT2, YARS, MICAL3, KDM6B, SYCP2L, and TTN. We validated six statistically significant variants in both cohorts by Sanger sequencing. Critically, a classifier trained on the 314 statistically significant variants in the discovery cohort produced perfect classification (area under the receiver operator curve AUROC = 1) in predicting the tumor bearing vs. healthy individuals in the validation cohort and in an equivalent number of ethnicity matched control samples, shuffled 1,000 times. Moreover, a weighted, graph-based biological pathway analysis method identified co-segregation of genes harboring tumor-predictive variants in cancer related pathways, including genome integrity, DNA replication fidelity, extracellular matrix organization, glycosylation, and the inflammasomes, among others. Taken together, these results support a common, uniquely distinctive genetic etiology for RNETs, suggesting a strong germline predisposition. More critically, the accuracy and sensitivity of the predictor suggests that these rare variants may be effectively used as a biomarker to identify individuals at risk of developing RNETs.

Citation Format: Subrata Saha, Adina Grunn, Andrea Califano. Genetic predisposition of rectal neuroendocrine 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 5040.
Abstract 5070: Prostate-specific membrane antigen (PSMA) targeted multiplexed siRNA-mediated gene silencing of CD46 and PD-L1 in prostate cancer
Authors: Chen Zhihang , Krishnamachary Balaji , Mironchik Yelena , Banerjee Sangeeta R. , Pomper Martin G. , Bhujwalla Zaver M.
Published: June 2022

Small interfering RNA are suitable for gene silencing through a sequence-specific RNA interference process. The redundancy and complexity of molecular pathways in cancer create a need for multiplexed targeting that can be achieved with multiplexed siRNA delivery. Here we delivered multiplexed siRNA with a PSMA-targeted biocompatible dextran nanoparticle (NP) to downregulate CD46 and PD-L1 siRNA in PSMA expressing prostate cancer cells. The multiplexed gene targets selected, PD-L1 and CD46, play important roles in the escape of cancer cells from immune surveillance. PSMA is abundantly expressed by prostate cancer cells allowing prostate cancer specific delivery of the NPs. The NP was decorated with acid cleavable amine functional groups through acetal bonds that undergo degradation under acidic conditions providing rapid release of siRNA. Cell imaging and flow cytometry studies confirmed PSMA-specific delivery of CD46 and PD-L1 siRNA to high PSMA expressing PC-3 PIP cells. Immunoblot, qRT-PCR and flow cytometry confirmed downregulation of CD46 and PD-L1 following treatment with multiplexed siRNA at a concentration of 50 nM. These studies pave the way for delivering multiplexed siRNA to tumors in vivo to unmask tumors to the immune system using PSMA targeted biocompatible NPs.

Citation Format: Zhihang Chen, Balaji Krishnamachary, Yelena Mironchik, Sangeeta R. Banerjee, Martin G. Pomper, Zaver M. Bhujwalla. Prostate-specific membrane antigen (PSMA) targeted multiplexed siRNA-mediated gene silencing of CD46 and PD-L1 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5070.
Abstract 5074: Investigating the cellular uptake of liposomal delivery system using cellular models of lymphoma
Authors: Bom Joyce Edlyne Chima , Van Le Tien , Togba-Bass Tracy , Kim Erica , Campbell Robert
Published: June 2022

Background: Lymphoma accounts for more than 4% of cancer diagnoses in the United States. Conventional treatment strategies for lymphoma are effective, but face a multitude of challenges limiting efficacy. Nanomedicine presents a viable option to optimize the efficacy of current anti-lymphoma therapies. To achieve most selective drug targeting, optimization of liposome preparations is key. To improve the selective targeting properties of conventional liposomes, cell membrane lipid-extracted nanoliposomes (CLENs) were prepared using lipids extracted from lymphoma target cells. Reported here is results of initial design and development, including physicochemical characterization and in vitro analyses.

Methods: The CRL-1593.2 cell line was the chosen cellular model of lymphoma. CRL-1593.2 cells were cultured and expanded In vitro for cellular extraction purposes. The CRL-1593.2 cell lipid extract (LE) was included to prepare CRL-1593.2 CLENs. Conventional lipid ingredients used in preparation of CRL-1593.2-CLENs included DOPC, Cholesterol, and DPPE-Rhodamine (used as a fluorescence indicator) for fluorescence studies using a fluorescence microplate reader. Phase I studies included physicochemical characterization and cellular uptake studies of preparations of CLENs, including 0 to 40 mol% of CRL-1593.2 LE content. In Phase II, the optimal LE content as determined by Phase I was fixed, while cholesterol content varied from 0 to 40 mol%.. For in vitro studies, the control cell lines employed represented other hematological diseases, RPMI 8226 (multiple myeloma), CCRF-CEM (acute lymphoblastic leukemia), and K562-GFP (chronic myeloid leukemia).

Results: The optimized composition of CLEN preparation type consisted of DOPC: Cholesterol, lymphoma lipid extract (derived from CRL-1593.2 cells) in the ratio of 93:5:2. The mean particle size was 116 ± 10 nm (n=4) and the mean zeta potential was - 4.5 ± 5 mV (n= 4). For in vitro studies, the inclusion of 2 mol% LE in CRL-1593.2-CLENS demonstrated significant uptake when compared to 0% LE control preparations (P = 0.016). Additionally, the lymphoma cells also had a significantly higher uptake of the CRL-1593.2-CLENs when compared to the other hematological cell lines [(CCL-155; P = 0.0081), (K562-GFP; P = 0.0037) and, (CCRF-CEM; P = 0.0079)].

Conclusions: Early findings collectively suggest that the CRL-1593.2-CLENs is a highly selective drug delivery platform for the targeting of lymphoma cells.

Citation Format: Joyce Edlyne Chima Bom, Tien Van Le, Tracy Togba-Bass, Erica Kim, Robert Campbell. Investigating the cellular uptake of liposomal delivery system using cellular models of lymphoma [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 5074.
Abstract 5093: A predictive biomarker classififier in NSCLC treated with immunotherapy
Authors: Zhang Shixin , Wang Xintong
Published: June 2022

Background: Our Study have been focusing on investigating the immune components to increase immunotherapy efficacy and to assist patient stratification for immunotherapy NSCLC patients, we explored predict property of the mutations characteristics with immunotherapy survival benefit and Tumor Immune Microenvironment(TME)characteristics.

Methods: A training cohort of 344 immunotherapy NSCLC were obtained from MSK database with Clinical and biological data. intersecting Candidate genes related to survival(p_value < 0.05)with immunity-related genes (IRGs), following an independent cohort of 75 patients with immunotherapy NSCLC was assessed. Applying the same patients RNA-Seq data calculated TME by CIBERSORT from TCGA database.

Results: The result divided the patients into two groups: 54 samples in the MT group ( two or more of the 18 candidate genes mutated) and 290 samples in the WT group (less two mutations in the 18 genes).The TMB status frequency in WT_H/L and MT_H/L cohort were 11.9% vs 3.8% and 21.5% vs 62.8%(MT_H vs WT_H, P < 0.01 ; MT_L vs WT_L, P = 0.046 ). Discovery training cohort showed a high Overall Survial for patients with MT group which was 15.7% and 84.3% for WT group( P_value < 0.0001). The genes mutation signature degree of most immune cells (such as CD4\8+ T cells and M1 macrophages \natural killer ) were higher in the MT group.

Conclusions: Our study offers genes mutation biomarker for OS prediction and potential addition development of tumor exemption, to be served as a convenient tool for identifying high relapse individuals with immunotherapy NSCLC patients.

Citation Format: Shixin Zhang, Xintong Wang. A predictive biomarker classififier in NSCLC treated with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5093.
Abstract 5094: A pan-cancer analysis of GRIN2A as a potential biomarker for immune checkpoint therapy
Authors: Jiang Mei , Zhong Xuefeng , Huang Mengli
Published: June 2022

Background: GRIN2A is the encoding gene of GluN2A, which is a subunit of the N-methyl-D-aspartate receptor, and participates in the regulation of excitatory neurotransmission in the brain. GRIN2A is one of the key genes in epilepsy researches, however, only a few researches have been done on GRIN2A in cancer field. The existing studies showed that GRIN2A mutation was frequently found in melanoma and caused abnormal tumor suppressor function. Besides, GRIN2A mutation was associated with higher tumor mutation burden (TMB) in non-small-cell lung cancer, suggesting that GRIN2A-altered tumors might be more sensitive to immune checkpoint inhibitors (ICIs), but the correlation of GRIN2A mutation status with immune response in clinical remains unknown.

Methods: Next generation sequencing (NGS) and clinical data of pan-cancer patients were obtained from two MSK-IMPACT Clinical Sequencing cohorts (MSK-2017, N=10336; MSK-2019, N=1661). The MSK-2019 cohort, which was treated with ICIs, were analyzed to explore the association between GRIN2A alteration and therapeutic efficacy of ICIs. Moreover, mRNA expression data of 10201 pan-cancer patients were obtained from the Cancer Genome Atlas (TCGA) database to explore the association between GRIN2A expression level and immune cell infiltration.

Results: In total, 3.74% (387/10336) of pan-cancer patients in MSK-2017, and 7.47% (124/1661) in MSK-2019, carried GRIN2A alteration. In MSK-2017, the top three GRIN2A alteration frequency cancer types were melanoma (68/365, 18.63%), skin cancer (non-melanoma, 22/148, 14.86%) and small cell lung cancer (11/82, 13.41%), respectively. And the commonly mutation types of GRIN2A were missense mutation. In MSK-2019 treated with ICIs, the TMB level of GRIN2A-altered group was significantly higher than wild group (median TMB, altered-type vs. wild-type = 25.09 vs. 5.90 Muts/Mb, P<0.0001) and the overall survival (OS) of GRIN2A-altered group were significantly longer than wild group (OS, median, 44.00 vs. 15.00 months; HR=0.5812 [95%CI: 0,4474-0.7550], P=0.0008). But in MSK-2017 without immunotherapy, there was no difference in OS between GRIN2A-altered group and wild group (OS, median, 26.00 vs. 26.13 months; HR=1.194 [95%CI: 0.9588-1.487], P=0.0849). Furthermore, the GRIN2A expression level was positively correlated with the degree of immune cell infiltration in some tumor types, such as prostate adenocarcinoma (PRAD), while some tumors did the opposite, such as sarcoma (SARC).

Conclusion: The results indicated that GRIN2A gene alteration was associated with a higher TMB level in pan-cancer patients, and patients carrying GRIN2A alteration might easily benefit from ICIs.

Citation Format: Mei Jiang, Xuefeng Zhong, Mengli Huang. A pan-cancer analysis of GRIN2A as a potential biomarker for immune checkpoint 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 5094.
Abstract 5098: Genetic and immunologic characteristics of biliary tract cancer patients with LRP1B mutation
Authors: Chen Weiwei , Zhang Hushan , Dong Hongmin , Wang Gang , Li Xiaokai , Chen Wanghua , Li Guodong , Bai Saixi , Chen Juan , Wang Wenling
Published: June 2022

Background: Although the initial clinical results of immune checkpoint inhibitors (ICI) have been obtained in biliary tract cancer (BTC), and previous researches explored potential biomarkers for predicting response to ICI in BTC, and gene LRP1B was found as one of predictive biomarkers. Here we evaluated the mutation information of LRP1B in Chinese patients with BTC, and analyzed the potential explanation for more benefit from ICI treatment in patients with LRP1B mutation.

Methods: The formalin-fixed paraffin-embedded specimens of 1324 cancer patients who have underwent next-generation sequencing (NGS) from 2016 to 2021, were included in this study. NGS was performed to detect gene mutation, and tumor mutational burden (TMB) measured by a 733 gene panel. PD-L1 expression detected by using Dako PD-L1 IHC 22C3 pharmDx.

Result: The NGS results revealed that 12.76% (169/1324) patients with BTC have LRP1B mutation. Among these 1324 patients, 931 (patients with or without LRP1B mutations were 129 and 802, respectively) patients were available for analysis of TMB level, 820 ((patients with or without LRP1B mutations were 114 and 706, respectively)) patients for analysis of PD-L1 expression, and 1163 (patients with or without LRP1B mutations were 172 and 991, respectively) patients for analysis of MSI-H. TMB level and PD-L1 expression level were compared between groups with and without LRP1B mutation, and results showed that both these two biomarkers were higher in group with LRP1B mutation than without LRP1B mutation, especially TMB level was significantly higher (p<0.001).Further analysis revealed that the median TMB level, proportion of strong positive PD-L1 expression, and proportion of MSI-H were all higher in group of LRP1B mutation than wild type.

Conclusion:These results suggest that LRP1B can be considered as one of effective biomarkers for ICI treatment in BTC, the underly mechanism may be related to the high TMB level.

Citation Format: Weiwei Chen, Hushan Zhang, Hongmin Dong, Gang Wang, Xiaokai Li, Wanghua Chen, Guodong Li, Saixi Bai, Juan Chen, Wenling Wang. Genetic and immunologic characteristics of biliary tract cancer patients with LRP1B mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5098.
Abstract 5101: Urine based dynamically monitoring reflect pathologic response in MIBC patients with neoadjuvant immunotherapy
Authors: Yang Xiao , Cai Lingkai , Cao Qiang , Shi Min , Liang Yiming , Qu Yurong , Lu Qiang
Published: June 2022

Background: The recommended standard of care treatment for nonmetastatic muscle-invasive bladder cancer (MIBC) is radical cystectomy (RC). Even with urinary diversion, radical cystectomy has a major impact on patients' quality of life. Although high rates of overall and disease-specific survival were observed in patients managed with bladder preservation, who achieved a clinically complete response to neoadjuvant therapy. Frequent evaluation of tumor response based cystoscopy implementing personalized bladder-sparing treatment paradigms.

Case Presentation: Here, we report on a case of urine-based dynamically monitoring during neoadjuvant immunotherapy in a MIBC patient treated with RC. A 57-year-old male presented macrohematuria and was diagnosed with bladder urothelial carcinoma by transurethral resection of bladder tumor (TURBT) with clinical stage IIIA (high-grade, multiple, cT2N0M0). He received 3 cycles of Tislelizumab monotherapy and sequential radical cystectomy. The RC specimen showed a complete pathological response (pCR). Four urine samples were collected prior to TURBT, on a day after TURBT, at beginning of neoadjuvant immunotherapy, and on the day of CR. Mutations status in 17 genes and methylation level of ONECUT2 were analyzed by a high-throughput sequencing-based urine test panel (genetron-uro-V1). In urine sediment DNA, which was collected before TURBT, the TERT promoter mutations C228T, TP53 D281H, and ERBB2 D277Y were detected in urine sediment DNA at variant allele frequencies (VAF) of 31.8%, 10.5%, and 0.5%, respectively. After removing in most parts of tumor, the VAF of TERT C228T decreased to 19.6% and mutation in TP53 and ERBB2 could not be detected. Without neoadjuvant immunotherapy, the VAF of TERT C228T still stayed above 12%. After 3 cycles of Tislelizumab monotherapy on the day of CR, all mutation was below the detection limit in urine sediment DNA, which was consistent with pCR in the pathological report of RC specimen.

Conclusions: This case suggested that urine-based dynamically monitoring reflected pathologic response, which could support personalized care and help select patients for bladder-sparing treatment.

Citation Format: Xiao Yang, Lingkai Cai, Qiang Cao, Min Shi, Yiming Liang, Yurong Qu, Qiang Lu. Urine based dynamically monitoring reflect pathologic response in MIBC patients with neoadjuvant immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5101.
Abstract 5125: Biological and clinical value of OCT4A/SPP1C axis in endometrial and lung adenocarcinoma
Authors: Irie Kyohei , Mizuno Nobuhiko , Miyamoto Tomoyuki , Ohtsuka Aiji , Yamamoto Hiromasa , Toyooka Shinichi , Masuyama Hisashi , Kosaka Mitsuko
Published: June 2022

Background: It is largely unclear whether octamer-binding transcription factor 4A (OCT4A, OCT3/4A) functions physiologically or pathologically in adult somatic tissues and related cancers. We have recently identified OCT4A translation in a human cancer cell subpopulation that might exacerbate cell migration and invasion. Here, we focus on biological and clinical features of OCT4A and its relationship with secreted phosphoprotein 1(SPP1), which is strongly related to the malignant progression of numerous tumors.

Methods: RT-PCR or qPCR analysis was used to identify full-length spliced forms of OCT4A and SPP1 transcripts in non-tumor and tumor human cells. An OCT4A-FLAG-tagged genomic transgene identified OCT4A-positive cancer cells. Cell-ablation of OCT4A-positive cells using promoter-driven diphtheria toxin A was used to examine the potential role of OCT4A in lung and endometrial adenocarcinoma cells. OCT4A and SPP1 transcripts in early-stage lung adenocarcinoma (stage I, fifty-eight cases) were analyzed and compared with pathohistological and prognostic features.

Results: We found that OCT4A and SPP1C are co-expressed in highly aggressive human lung and endometrial adenocarcinoma cell lines but not in mesothelioma or non-tumor cell lines. Ablation of OCT4A-positive cells significantly decreased cancer-cell migration and SPP1C mRNA levels. The OCT4A/SPP1C axis was found in primary, early-stage, lung adenocarcinomas, and all four cases of recurrence and/or metastasis out of 58 cases were included in the OCT4A and SPP1C positive group.

Conclusions: Co-expression of OCT4A and SPP1C may correlate with human cancer cell migration and the OCT4A/SPP1C axis may have a prognostic value in high-risk, early-stage lung adenocarcinomas. References: Stem Cells (2018), 36:1341-54. BMC Cancer (2020), 20:521.

Gene expression of OCT4A and SPP1 variants in 58 cases of early-stage lung adenocarcinoma OCT4A(-) OCT4A(+) OCT4A(++) SPP1A(+),SPP1C(+) 0 cases 9 cases 17cases(including 4 cases of recurrence or metastasis ) SPP1A(+),SPP1C(-) 3 cases 6 cases 7 cases SPP1A(-),SPP1C(-) 13cases 3 cases 0 cases

Citation Format: Kyohei Irie, Nobuhiko Mizuno, Tomoyuki Miyamoto, Aiji Ohtsuka, Hiromasa Yamamoto, Shinichi Toyooka, Hisashi Masuyama, Mitsuko Kosaka. Biological and clinical value of OCT4A/SPP1C axis in endometrial and lung adenocarcinoma [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 5125.