bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 617: Oncogenic CXCL10 triggers CD8+ T cell exhaustion in ovarian cancer
Authors: Long Runying , Ding Tao , Siu Michelle K.L , Chan David W , He Jiangnan , Cheung Annie NY , Man Kwan , NGAN Hextan YS , Chan Karen KL
Published: April 2023

Ovarian cancer is one of the most severe gynecologic malignancies with high mortality. Although programmed cell death 1 (PD-1) or PD-ligand 1 (PD-L1) immune checkpoint blockade (ICBs) has been used therapeutically in gynecologic cancers and has a clinically significant response in endometrial cancer, the response rate remains poor and has no different from the conventional therapies in ovarian cancer. The highly immunosuppressive microenvironment in ovarian cancer, consisting of inhibitory ligands, suppressive mediators, and cell subsets, leads to CD8+ T cell dysfunction and exhaustion and contributes to the limited efficacy of immunotherapeutic approaches. Correlative human studies have highlighted the potential importance of chemokines on the status of CD8+ T cell infiltration into tumors and on patient survival. Here, we show that CXCL10, an IFN-γ induced chemokine, derived from ovarian cancer cells, contributes to impairing CD8+ T cell antitumor immunity by regulating CD8+ T cell exhaustion in the tumor microenvironment. Clinically, we found that CXCL10 and its receptor CXCR3 had a strong positive correlation with exhausted T cell signature genes in ovarian cancer patients in TCGA and in-house cohort. Functionally, we showed that CD8+ T cell exhaustion was induced by CXCL10 activation by CRISPRa from ovarian cancer cells, whereas inhibition by CRISPRi restored CD8+ T cell antitumor immunity ex vivo and in vivo. Mechanically, ovarian cancer cells-derived CXCL10 induced the expression of CD8+ T cells exhaustion transcription factors in a CXCR3-dependent manner, driving PD-1 and TIM-3 expression on CD8+ T cells. Therapeutically, suppressing CXCL10 secretion from ovarian cancer cells enhanced the response to anti-PD-1 immunotherapy in the orthotopic mouse model. The results suggest that selectively inhibiting the oncogenic CXCL10 production may work effectively with ICBs in ovarian cancer immunotherapy. Our findings advance the understanding of the low response rate of immunotherapy in ovarian cancer and explore the potential mechanism underlying the induction of CD8+ T cell exhaustion in the tumor microenvironment.

Citation Format: Runying Long, Tao Ding, Michelle K.L Siu, David W Chan, Jiangnan He, Annie NY Cheung, Kwan Man, Hextan YS NGAN, Karen KL Chan. Oncogenic CXCL10 triggers CD8+ T cell exhaustion in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 617.
Abstract 6176: Enhancing treatment efficacy of glioblastoma cell lines by adding tumor treating fields (TTFields) to temozolomide and lomustine
Authors: Fishman Hila , Monin Roni , Dor-On Eyal , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram
Published: April 2023

Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. Concurrent maintenance temozolomide (TMZ) with TTFields was approved by the FDA in 2014 for treatment of patients with newly diagnosed glioblastoma (ndGBM). Recently, the addition of lomustine (CCNU) to TMZ demonstrated clinical benefit in ndGBM patients. The aim of the current study was to examine in GBM cells the effect of TTFields concurrent with TMZ and CCNU.

Methods: Human GBM cell lines (U-87 MG, LN229, U118, and LN18) with various MGMT expression levels were treated with TTFields (intensity of 0.83 V/cm RMS; frequency of 200 kHz) using the inovitro system. Effectiveness of TTFields concomitant with TMZ and/or CCNU was tested by measuring cell count and colony formation. Levels of DNA repair proteins in control and TTFields-treated cells were measured using Western blot.

Results: The cell lines displayed different sensitivity to TMZ, in accordance with their MGMT expression level, but showed a comparable response to TTFields. TTFields concomitant with TMZ displayed an additive effect on cell numbers, irrespective of MGMT expression levels. The effect of TTFields concomitant with CCNU was additive in cell lines expressing MGMT, whereas it trended toward synergy in cell lines not expressing MGMT. Application of TTFields to the cells downregulated expression of proteins from the Fanconi Anemia-BRCA pathway for DNA repair. TTFields concurrent with both TMZ and CCNU provided higher efficacy relative to TMZ plus CCNU or TTFields apart.

Conclusions: Efficacy of TMZ and CCNU may be enhanced by TTFields, with synergism seen for the latter case in cells with limited MGMT expression. The BRCAness state induced by TTFields rationalizes these outcomes, as DNA damage induced by CCNU requires the BRCA pathway for repair, especially in the absence of MGMT. The results suggest a potential benefit for concomitant TTFields with the TMZ/CCNU combination in ndGBM.

Citation Format: Hila Fishman, Roni Monin, Eyal Dor-On, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Enhancing treatment efficacy of glioblastoma cell lines by adding tumor treating fields (TTFields) to temozolomide and lomustine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6176.
Abstract 618: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1 positive regulator for tumor immune evasion
Authors: Wang Shengnan , Lyer Radhika , Fang Deyu
Published: April 2023

Regulation of tumoral PD-L1 expression is critical to advance our knowledge in understating the tumor evasion of immunosurveillance and for improvement of antitumor immunotherapy. While several E3 ubiquitin ligases and ubiquitin-specific peptidases regulate PD-L1 protein stability, the deubiquitinases involved in PD-L1 transcription for cancer cell immune evasion have not been identified. Herein, we developed a CRISPR-based screening platform and identified ATXN3 as a top positive regulator for PD-L1 transcription. Analysis of TCGA database revealed a statistically significant positive correlation between ATXN3 and CD274 expression in more than 80% human cancers, indicating that ATXN3 is a positive regulator for PD-L1 transcription in broad spectra of human cancers. ATXN3 promotes PD-L1 transcription induced by multiple tumor microenvironmental (TME) factors including inflammatory cytokine IFN-γ and hypoxia through protecting their downstream transcription factors IRF1, STAT3 and HIF-2α. In addition, ATXN3 functions as a deubiquitinase of PD-L1 transcription factors YAP-1 and JunB, indicating ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells resulted in reduced PD-L1 expression and consequently enhanced antitumor immunity in mice, which are partially revised by reconstitution of PD-L1 expression. Further, tumoral ATXN3 suppression improves the preclinical efficacy of checkpoint blockade antitumor immunotherapy and partially dependent on CD8+ T cells. Importantly, ATXN3 expression is increased in human lung adenocarcinoma and melanoma and its levels are positively corelated with PD-L1 as well as its transcription factor IRF1 and HIF-2α. Collectively, our study identifies a previously unknown deubiquitinase ATXN3 as a positive regulator for PD-L1 transcription and provides a rationale for ATXN3 targeting in sensitizing checkpoint blockade antitumor immunotherapy.

Citation Format: Shengnan Wang, Radhika Lyer, Deyu Fang. CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1 positive regulator for tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 618.
Abstract 6184: Pre-clinical study of SYX3759, a novel PARG inhibitor for the treatment of homologous recombination deficient malignancies
Authors: Tang Xuzhen , Cui Yaqi , Zhang Lanqiao , Jin Huihui , Xu Zeqiong , He Hu , Ge Chongxun , Liu Song , Yu Xiaochun , Shi Song
Published: April 2023

Although PARP inhibitors (PARPi) are very successful on the treatment of BRCA-mutant cancer, the tumor spectrum targeted by PARPi is quite narrow. Interestingly, Poly(ADP-ribose) Glycohydrolase (aka PARG), the major dePARylation enzyme, has emerged as a promising therapeutic target for cancer treatment. Here, we have developed a cell membrane permeable PARG inhibitor with subnanomolar IC50. SYX3759 directly binds to the catalytic domain of PARG and induces significant accumulation of PARylation at DNA lesions, where it traps numerous DNA repair factors, such as XRCC1. Compared to PARPi, such as Olaparib, SYX3759 shows selective and potent cytotoxicity on a broader spectrum of homologous recombination deficient (HRD) tumors both in vitro and in vivo. Moreover, SYX3759 is well tolerated at the highest dose tested without obvious adverse effect on hematopoiesis. Collectively, SYX3759 is a potent PARG inhibitor both in vitro and in vivo warranting future clinical trials for the treatment of HRD cancers and beyond.

Citation Format: Xuzhen Tang, Yaqi Cui, Lanqiao Zhang, Huihui Jin, Zeqiong Xu, Hu He, Chongxun Ge, Song Liu, Xiaochun Yu, Song Shi. Pre-clinical study of SYX3759, a novel PARG inhibitor for the treatment of homologous recombination deficient malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6184.
Abstract 6191: Temozolomide-associated DNA damage processes related to glioblastoma-immune interactions
Authors: Choe Elizabeth Y. , White Forest M.
Published: April 2023

How do cancer cells convert an external perturbation into an interaction with the immune system? The processes associated with antigen presentation remain largely unknown, especially in cancer cells responding to treatment. These intracellular changes may be critical in cancers where immunotherapies need significant optimization, such as glioblastoma. Glioblastoma is also a particularly relevant cancer to characterize the cellular response to perturbations, since all patients currently receive temozolomide, an alkylating agent, regardless of their prognosis.

We used mass spectrometry methods to quantify temozolomide-associated changes to the repertoire of peptides presented on MHC class I molecules in vitro over time, as well as the associated changes that occur in DNA damage signaling and protein synthesis. We treated U87-MG cells with temozolomide and directly profiled the peptide-MHC repertoire, ATM/ATR kinase substrate phosphorylation, and newly-synthesized proteins via isotopically labeled and non-canonical amino acid-tagged proteins after 24, 48, and 72 hours.

These proteomics approaches identified several MHC-peptides, including phosphorylated peptides, whose presentation changes over time after treatment with temozolomide. We also found that the temporal dynamics of the DNA damage response mirrored that of immunopeptidome remodeling. Additionally, temozolomide alters translation at these time points, with newly-synthesized proteins reflecting internal cell stress processes.

These findings suggest potential treatment-associated antigens against which to design vaccines as well as particular signaling nodes that could be modulated with combination therapies to improve presentation of these peptides.

Citation Format: Elizabeth Y. Choe, Forest M. White. Temozolomide-associated DNA damage processes related to glioblastoma-immune interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6191.
Abstract 6192: MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC
Authors: Bychkov Igor , Deneka Alexander , Topchu Iuliia , Pangeni Rajendra P. , Lengner Christopher , Izumchenko Evgeny , Patel Jyoti , Karanicolas John , Makhov Petr , Boumber Yanis
Published: April 2023

Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide, accounting for >2.2 million of new cancer diagnoses and 1.8 million cancer-related deaths in 2020. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including adenocarcinomas and squamous cell carcinomas. Many lung adenocarcinomas have activating driver mutations in KRAS (~32% tumors), often co-occurring with mutations inactivating the TP53 tumor suppressor. Expression of the RNA-binding protein Musashi-2 (MSI2) MSI2 regulates target mRNA translation, and its expression is progressively elevated along with tumor stage in lung cancers. We previously showed that MSI2 actively promotes lung cancer growth, invasion, and metastasis, in part through promoting expression and activity of the TGFβR1/SMAD3 and EGFR signaling cascades. We now find that genetic MSI2 deficiency in a murine lung tumor model, Krasmut/Trp53KO/Msi2KO (KPM2) decreases both the total tumor number and tumor burden in comparison to control Krasmut/Trp53KO (KP) mice. Using a panel of KPM2 and KP cell lines we established from tumors from these mice, we found KPM2 cell lines demonstrate a significant decrease in proliferation compared to KP cell lines. Interestingly, reverse phase protein array (RPPA) comparison analysis of KPM2 and KP cell lines showed that MSI2 strongly and positively regulates expression of Ataxia-Telangiectasia Mutated (ATM) protein, with lower levels observed in KPM2 cells. The ATM serine/threonine protein kinase is a key transducer of DDR signaling in cases of double-strand breaks; ATM activation causes G1/S cell cycle arrest, contributes to efficiency of DNA repair, and triggers apoptosis in cases of repair failure. Notably, KPM2 cells showed significantly higher levels of γH2AX, suggesting inefficient DDR. Finally, untreated or cisplatin-treated KPM2 cells demonstrated G2/M arrest and had increased level of apoptosis as demonstrated by increase in caspase 3 cleavage, consistent with ATM loss and compensatory activation of Ataxia-telangiectasia and Rad3-related protein (ATR). Taken together, our data suggests that one of the consequences of MSI2 upregulation in NSCLC includes maintenance of an efficient DDR. Targeting MSI2 and its downstream signaling would be of therapeutic interest.

Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Rajendra P. Pangeni, Christopher Lengner, Evgeny Izumchenko, Jyoti Patel, John Karanicolas, Petr Makhov, Yanis Boumber. MSI2 regulates DNA damage response signaling and tumor progression in murine NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6192.
Abstract 6202: 6-Phosphofructo-2-kinase in regulating DNA damage and repair in EGFR-driven lung cancer
Authors: Lypova Nadiia , Lanceta Lilibeth , Dougherty Susan , Chesney Jason , Imbert-Fernandez Yoannis
Published: April 2023

Activating mutations of epidermal growth factor receptor (mutEGFR) are established drivers of lung tumor development, aggressiveness, and resistance to targeted therapies. While targeting mutEGFR with small-molecule tyrosine kinase inhibitors (EGFR-TKIs) dramatically improved progression-free survival in patients with non-small cell lung cancers (NSCLCs), intrinsic or acquired resistance to TKI therapy limits the duration of an effective response to TKIs. Alteration in DNA damage response (DDR) plays a vital role in genomic stability and cancer progression and helps cells to escape apoptosis, limiting the efficacy of targeted therapies.

Interestingly, lung cancer cells exposed to EGFR-TKIs demonstrate attenuated glycolytic flux - one of the major providers of precursors for de novo nucleotide synthesis. A key regulator of glycolysis is the enzyme 6-phosphofructo-2-kinase (PFKFB3). PFKFB3 synthesizes fructose 2,6-bisphosphate (F2,6BP), which is a potent allosteric activator of the glycolytic rate-limiting enzyme, 6-phosphofructo-1-kinase (PFK1). Accordingly, F2,6BP controls flux throughout the entire glycolytic pathway.

In preliminary studies, we provide evidence that PFKFB3 plays multiple roles in regulating the efficacy of DDR in lung cancer cells exposed to EGFR-TKIs. We show that PFKFB3 is required for EGFR-driven glucose metabolism, which provides a list of intermediates utilized in nucleotide synthesis. PFKFB3 regulates the expression of ribonucleotide reductase small subunit M2 (RRM2) of the ribonucleotide reductase (RNR), which is important for de novo onsite synthesis of deoxynucleotide triphosphate (dNTP) during DNA replication and repair. Moreover, a PFKFB3 inhibitor, PFK-158, increases oxidative stress in mutEGFR lung cancer cell lines and, as a result, promotes DNA damage. Importantly, we found that PFKFB3 plays an important role in the chromatin binding of scaffold proteins regulating the assembly of DNA repair complex.

Our studies suggest that PFKFB3 plays an important role in DDR and provide a clear rationale to propose the use of PFKFB3 inhibitors in combination with EGFR inhibitors to increase the efficiency and/or overcome resistance to EGFR-TKIs.

Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan Dougherty, Jason Chesney, Yoannis Imbert-Fernandez. 6-Phosphofructo-2-kinase in regulating DNA damage and repair in EGFR-driven lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6202.
Abstract 6209: O6-methylguanine as the junction of MGMT and PARP1-mediated repair pathways
Authors: Cropper Jodie D. , Alimbetov Dauren S. , Brown Kevin T. , Robles Andrew , Likhotvorik Rostislav I. , Guerra James T. , Chen Yidong , Kwon Youngho , Kurmasheva Raushan T.
Published: April 2023

Temozolomide is the first-choice DNA alkylating agent and has been commonly used in oncology over the last several decades. The cytotoxicity that temozolomide elicits through the methylation of guanine and adenine residues often becomes the limiting factor in effective treatment. Despite growing evidence of dysregulated alkylating DNA damage repair as a driving force of genome instability leading to cancer, neurological diseases, and premature aging, little is currently known about the coordinated role of PARP1 and MGMT (O6-methylguanine methyltransferase) enzymes in the repair of temozolomide-induced methylated DNA lesions. A major PARP1 function in DNA damage is facilitation of repair of the methylated DNA lesions, N6-methyladenine and N7-methylguanine, via base excision repair (BER); while MGMT restores guanine in O6-methylguanine (O6meG), the most cytotoxic adduct, by a one-step catalysis. It is generally thought that BER and MGMT represent two distinct mechanisms for removing DNA damage induced by alkylating agents; however, using a number of advanced cell-free and cell-based approaches, we provided evidence for direct (DNA-independent) and indirect (through PARylation) interaction between PARP1 and MGMT and demonstrated a functional crosstalk between these repair pathways. Particularly, O6meG repair activity is increased once PARP1 PARylates MGMT. Further, longer (more clinically relevant) exposure to temozolomide induced stronger MGMT PARylation in cells, indicating that the PARP1-MGMT interaction is important for enhanced O6meG repair and cell survival. As PARP1-MGMT complex forms in a variety of cancer cell types, our findings have strong implications for the development of an effective cancer therapy for cells dependent on PARP1 and MGMT mediated DNA repair.

Citation Format: Jodie D. Cropper, Dauren S. Alimbetov, Kevin T. Brown, Andrew Robles, Rostislav I. Likhotvorik, James T. Guerra, Yidong Chen, Youngho Kwon, Raushan T. Kurmasheva. O6-methylguanine as the junction of MGMT and PARP1-mediated repair pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6209.
Abstract 6214: FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation
Authors: Li Fang
Published: April 2023

R loops arise from hybridization of RNA transcripts with template DNA during transcription, which are not only cause DNA damage in certain contexts as cancer hallmarks but also be important regulators of cellular processes. Recent findings revealed that DNA double-strand breaks (DSBs) elicit RNA-DNA hybrid formation. The RNA moiety of the R-loop displays an extraordinary potential to regulate multiple steps of homologous recombination (HR) by substituting for the double-stranded DNA (dsDNA) substrate on which DSB repair processes naturally occur. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCA with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate FANCA participates in forming R-loops in the initial DSBs repair stage and assists in removing R-loops later. Besides, we find that highly purified human FANCA anneals synthetic single-stranded RNA (ssRNA) and ssDNA species to R-loops and binds R-loop substrates with high affinity, preferring guanine-rich sequences in vitro. Importantly, we illustrate that FANCA promotes HR efficiency via catalyzation of DNA: RNA hybrids at DSBs sites. Finally, a series of RNA and R-loop substrates are found to stimulate ID2 monoubiquitination, with activity corresponding to remove transient R- loops in later HR stage. In summary, our results support a mechanism whereby FANCA promotes the formation of temporary R-loops by annealing ssRNA and ssDNA species at DSBs in transcribed regions, thereby stimulating HR.

Citation Format: Fang Li. FANCA promotes transcription-coupled homologous recombination by catalyzing R-loops formation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6214.
Abstract 6222: Clinical impact of p53 and AXL immunostaining on the efficacy of immune checkpoint inhibitor-based therapy after osimertinib treatment in patients with epidermal growth factor mutant non-small cell lung cancer
Authors: Morimoto Kenji , Yamada Tadaaki , Tokuda Shinsaku , Takayama Koichi
Published: April 2023

Background: Current evidence indicates that immune checkpoint inhibitors (ICIs) have a limited efficacy in patients with lung cancer harboring epidermal growth factor receptor (EGFR) mutations. However, there is a lack of data on the efficacy of ICIs after osimertinib treatment, and the predictors of ICI efficacy are unclear.

Methods: We retrospectively assessed consecutive patients with EGFR-mutant NSCLC who received ICI-based therapy after osimertinib treatment at 10 institutions in Japan, between March 2016 and March 2021. Immunohistochemical staining was used to evaluate the expression of p53 and AXL. The deletions of exon 19 and the exon 21 L858R point mutation in EGFR were defined as common mutations; other mutations were defined as uncommon mutations.

Results: A total of 36 patients with advanced or recurrent EGFR-mutant NSCLC were analyzed. In multivariate analysis, p53 expression in tumors was an independent predictor of PFS after ICI-based therapy (p = 0.002). In patients with common EGFR mutations, high AXL expression was a predictor of shorter PFS and overall survival after ICI-based therapy (log-rank test; p = 0.04 and p = 0.02, respectively).

Conclusion: The levels of p53 in pretreatment tumors may be a predictor of ICI-based therapy outcomes in patients with EGFR-mutant NSCLC after osimertinib treatment. High levels of AXL in tumors may also be a predictor of ICI-based therapy outcomes, specifically for patients with common EGFR mutations. Further prospective large-scale investigations on the predictors of ICI efficacy following osimertinib treatment are warranted.

Citation Format: Kenji Morimoto, Tadaaki Yamada, Shinsaku Tokuda, Koichi Takayama. Clinical impact of p53 and AXL immunostaining on the efficacy of immune checkpoint inhibitor-based therapy after osimertinib treatment in patients with epidermal growth factor mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6222.
Abstract 6242: Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma
Authors: Dorneburg Carmen , Galliger Celimene , Rasche Voker , Lechel Andre , Barth Thomas F. , Debatin Klaus-Michael , Beltinger Christian
Published: April 2023

Overexpression of survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB). Transcriptional inhibitors of survivin have been tested in clinical trials. However, results have been disappointing. We thus investigated the small molecules LQZ-7I and S12 that inhibit dimerization of survivin protein and compared their growth-inhibiting efficacy with that of markedly decreased survivin expression in a novel transgenic NB mouse. Mice hemizygous for BIRC5 were crossed with TH-MYCN mice to generate BIRC5+/−/MYCNtg/+ mice. NB incidence, growth and retransplantation capacity were assessed. In vitro, LQZ-7I and S12 were used to inhibit survivin protein interactions and the effects on proliferation and cell cycle of NB cells were investigated. BIRC5 hemizygous mice developed NB although mRNA and protein expression of survivin was markedly decreased. Surprisingly, incidence and latency of the NB that developed in the BIRC5+/−/MYCNtg/+ mice did not differ from the NB in the TH-MYCN mice, and tumor growth and survival of the mice was not affected. In stark contrast, the survivin interaction inhibitors LQZ-7I and S12 abolished anchorage-independent growth and proliferation of NB cell lines. Reduced growth of NB cells resulted from defects in mitosis induced by LQZ-7I or S12 or. LQZ-7I was the more efficient inhibitor of NB cells while S12 was not able to reduce tumor growth in a xenograft model. Taken together, these results support the notion that inhibiting survivin interaction is more efficacious in NB than inhibiting survivin transcription.

Citation Format: Carmen Dorneburg, Celimene Galliger, Voker Rasche, Andre Lechel, Thomas F. Barth, Klaus-Michael Debatin, Christian Beltinger. Inhibition of BIRC5/survivin by LQZ-7I inhibits neuroblastoma growth while hemizygosity of BIRC5 does not: implications for therapy of neuroblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6242.
Abstract 6271: Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor
Authors: Wang Xinghao , Ma Shichao , Xie Min (Bella) , Wang Lele , Wang Yajun , Zhang Song , Chen Sheng , Zhang Lynette , Zhou Yongqi , Zhao Mengxi , Li Dandan , Li Guobin , Fan Xiao , Zhang Hailong
Published: April 2023

Polycomb repressive complex 2 (PRC2) catalyze the writing of H3K27me3 epigenetic marker and suppresses the expression of genes including tumor suppressors. Activation mutation of EZH2, the catalytic subunit of the PRC2 complex, drives the progression of tumors such as DLBCL. The embryonic ectoderm development (EED) protein acts as the scaffolding and H3K27me3-binding subunit of PRC2 and is essential for its activity. We have developed potent, selective, and orally bioavailable EED inhibitor BR1733. BR1733 blocks the interaction between EED and H3K27me3, and inhibits catalytic activity of PRC2. BR1733 showed superior PK profile across species and exhibited tumor growth inhibition against several B cell lymphoma CDX models. Indication expansion studies demonstrated that BR1733 could inhibit solid tumors with SWI/SNF loss-of-function mutation only when both CDKN2A and TET1 are expressed. BR1733 also inhibited tumor angiogenesis and showed tumor growth inhibition in several CDX models of highly vascularized solid tumors such as HCC and RCC. BR1733 is currently in Phase I clinical trials.

Citation Format: Xinghao Wang, Shichao Ma, Min (Bella) Xie, Lele Wang, Yajun Wang, Song Zhang, Sheng Chen, Lynette Zhang, Yongqi Zhou, Mengxi Zhao, Dandan Li, Guobin Li, Xiao Fan, Hailong Zhang. Discovery of BR1733 for the treatment of SWI/SNF deficient or highly vascularized solid tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6271.
Abstract 6381: Quantitative multiplex analysis of soluble and cellular immune checkpoint proteins in response to interferon gamma across multiple murine tumor models
Authors: Lie Wen-Rong , Bucktrout Ryan , Chakraborty Sanjukta , Orlando Andrea , Serganova Inna , Kornmeier Christine , Hoberg James , Zappasodi Roberta
Published: April 2023

Immune checkpoint proteins are key regulators of the immune system and drug targets of cancer immunotherapy. Immune checkpoint molecules have been found on the cell surface of various tumor cells and immune cells as immune activating or inhibitory receptors and ligands. Recent studies have shown these immune checkpoint proteins can undergo alternative splicing or proteolytic cleavage leading to soluble isoforms. IFNγ is a pleiotropic and multifunctional cytokine commonly released by cytotoxic immune cells. IFNγ production can be enhanced in response to immunotherapy and further modulates the expression of immune checkpoints in the tumor microenvironment. To better understand the immune checkpoint signatures in tumors, we developed a 28-plex mouse checkpoint protein multiplex immunoassay for quantitative analysis of soluble (secreted) and cellular immune checkpoint proteins. Using this immunoassay, we tested 16 mouse cell lines of tumor (n=13) and normal tissues as control (n=3): 2 melanomas (B16F10, YUMM1.7), 3 mammary carcinoma (4T1, NT5, AT-3), 1 lung carcinoma (LLC), 1 prostate cancer (MycCaP), 1 fibrosarcoma (MCA205), 1 ovarian cancer (ID8), 1 bladder cancer (MB49), 1 glioma (CT-2A), 2 blood cancer (RAW, EL4), 1 dendritic cells (DC2.4), 1 myoblast cells (C2C12), and 1 endothelial cells (bEnd.3). We analyzed changes in immune checkpoint protein expression and secretion in these cell lines cultured in normal conditions or upon exposure to IFNγ to reflect an immune inflamed microenvironment. Cells were cultured in 96-well plate, 100 mm, or 150 mm Petri dishes, and treated with either 10 ng/ml or 100 ng/mL mouse IFNγ. Cell culture supernatants and cell lysates were harvested after 6h, 12h, or 24h incubation with IFNγ and processed for multiplex Luminex bead-based immunoassay. The soluble and cellular checkpoint proteins most substantially modulated by IFNγ treatment in these cell lines included: CD137, BTLA, HVEM, PD-L1. The tumor-specific signature of cellular and soluble checkpoint proteins and its response to IFNγ may have implications in the tumor microenvironment and in the outcome to immunotherapy.

Citation Format: Wen-Rong Lie, Ryan Bucktrout, Sanjukta Chakraborty, Andrea Orlando, Inna Serganova, Christine Kornmeier, James Hoberg, Roberta Zappasodi. Quantitative multiplex analysis of soluble and cellular immune checkpoint proteins in response to interferon gamma across multiple murine tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6381.
Abstract 6391: Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction
Authors: Susukida Takeshi , Sasaki So-ichiro , Shirayanagi Tomohiro , Aoki Shigeki , Ito Kousei , Hayakawa Yoshihiro
Published: April 2023

[Purpose] Tumors with low T cell infiltrates (poor immunogenicity) display lower probability of clinical response to immune checkpoint inhibitors. To achieve higher immunotherapy efficacy, it is imperative to establish a novel strategy for improving immunogenicity. Abacavir (ABC), an anti-HIV drug, binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activate CD8+ T cells by presenting altered abnormal peptides. In this study, we considered the immune interaction between ABC and HLA-B*57:01 improving immunogenicity in poorly immunogenic tumors leading to a potent anti-cancer effect.

[Methods] Mice were inoculated subcutaneously with tumor cells into the right flanks and interperitoneally treated with either ABC 5 mg or vehicle (water) for consecutive 9 days (B16F10) or 13 days (3LL) after tumor inoculation. Each tumor size was measured using vernier calipers on every 2 days. Activation, proliferation, and IFN-γ secretion of tumor infiltrating CD8+ T cells were analyzed by FACS.

[Results and Discussion] ABC treatment showed significant tumor growth inhibition associated with CD8+ T cell infiltration into tumor in mice inoculated with HLA-B*57:01 expressing B16F10 melanoma or Lewis lung carcinoma (3LL) cells, but neither in HLA-B*57:03 (negative allele)-expressing nor control tumors inoculated mice. Moreover, the tumor infiltrating CD8+ T cell were immune-activated and proliferated, with secreting IFN-γ. In contrast, ABC-induced anti-tumor effect was abrogated in CD8+ T cell-depleted mice, IFN-γ KO mice, and CXCR3-blocked mice. The combination therapy with anti-PD-1 antibody treatment induced better inhibition of tumor growth than ABC monotherapy in HLA-B*57:01 expressing B16F10 inoculated mice. These results suggested that ABC/HLA-B*57:01 interaction could improve tumor immunogenicity associated with CD8+ T cell-mediated anti-tumor effect in poorly immunogenic tumors. This finding lays a foundation to develop a novel drug-induced cancer immunotherapy targeting immune resistant tumors.

Citation Format: Takeshi Susukida, So-ichiro Sasaki, Tomohiro Shirayanagi, Shigeki Aoki, Kousei Ito, Yoshihiro Hayakawa. Development of a novel strategy for cancer immunotherapy based on tumor immunogenicity improvement by HLA-drug interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6391.
Abstract 6420: Harnessing immune responses in prostate MMRd tumors treated with immune checkpoint blockade
Authors: Badia Marcel Arias , Fan Zenghua , Lwin Yee May , Chen PeiXi , Srinath Aahir , Fong Lawrence
Published: April 2023

There is increasing evidence correlating tumor mutational burden with response to immunotherapy in many different types of cancer. Some tumors show high microsatellite instability (MSI-H) as a consequence of the loss of function of mismatch repair (MMR) genes, which would resolve DNA damage in normal conditions. MSH2 plays a critical role in MMR, and several studies have identified mutations in MSH2 as key drivers of MSI in tumors. In preclinical mouse models, MMR deficiency and high microsatellite instability have been previously shown to translate into higher sensitivity to anti-PD-1 treatment. Despite the advances led by the recent tumor-agnostic approval of checkpoint inhibitors for highly mutated tumors, response rates among MMR-deficient (MMRd) tumors in humans are variable, and the mechanisms behind such heterogeneity are still poorly understood. We successfully knocked out the MSH2 gene in the murine prostate adenocarcinoma TRAMP-C2 cell line by CRISPR gene editing. By sequential passaging, we generated a MSH2-KO subclonal population that presents MSI and MMR deficiency, validated by increased tumor mutational burden (TMB) observed by exome sequencing. In studies where we challenged immunodeficient and immunocompetent mice with MMR-proficient (MMRp) and MMRd tumors, the latter showed increase immunogenicity, which led us to interrogate and better characterize such immune responses in the context of immune checkpoint blockade by anti-PD-1.In our studies, MMRd tumor-infiltrating T cells presented significantly enhanced effector and cytotoxic profiles, which likely led to improved tumor control with or without anti-PD-1. We observed an intriguingly lower frequency of SPAS-1 (a well-known TRAMP-C2 immunodominant epitope) antigen-specific T cells in the tumor-reactive fraction of MMRd infiltrates compared to MMRp, which prompted us to interrogate tumor-infiltrate gene expression, TCR repertoire and the potential expansion of multiple antigen-reactive subsets against MMRd tumors in successful immune responses by single cell RNA sequencing and functional ex vivo studies. We also interrogated TCR repertoire data from patients harboring MMRd tumors to add translatability to our findings in mice.Our results constitute a relevant addition to the understanding of immune responses in MMRd tumors and the potential features that could be exploited to improve response rates in immunotherapeutic strategies targeting such malignancies.

Citation Format: Marcel Arias Badia, Zenghua Fan, Yee May Lwin, PeiXi Chen, Aahir Srinath, Lawrence Fong. Harnessing immune responses in prostate MMRd tumors treated with immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6420.
Abstract 6423: Targeting an autophagy regulator augments antitumor immunity and immunotherapy
Authors: Bao Yi , Qiao Yuanyuan , Chinnaiyan Arul
Published: April 2023

Immunotherapy has strikingly changed the landscape of cancer treatment, yet many patients fail to respond to the therapy exhibiting primary resistance. Accumulating evidence has supported that autophagy inhibition may sensitize tumor to immunotherapy. Here, we found that pharmaceutical or genetic inhibition of an autophagy regulator induces MHC class I surface expression in cancer cells. In a co-culture of OT1 CD8+ T cells and ovalbumin-expressing cancer cells, inhibition of the regulator enhances CD8+ T cell killing and increases functional and proliferative CD8+ T cells. Concordantly, genetic depletion or inhibition of the regulator elevates MHC class I in tumor cells, reduces tumor growth, and increases intratumoral functional CD8+ T cells in syngeneic mouse models. Furthermore, we found that inhibition of the regulator sensitizes syngeneic tumors to anti-PD1 therapy. Collectively, inhibition of this autophagy regulator augments antitumor immunity and immunotherapy via inducing MHC class I surface expression in cancer cells.

Citation Format: Yi Bao, Yuanyuan Qiao, Arul Chinnaiyan. Targeting an autophagy regulator augments antitumor immunity and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6423.
Abstract 6488: Correlation of availability of primary care physicians and colorectal cancer-related mortality in the United States: Do racial disparities exist
Authors: Grewal Udhayvir S. , Gaddam Shiva J. , Aggarwal Manik , Garikipati Subhash C. , Kumar Prabhat , Fei Naomi
Published: April 2023

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States (US). Primary care physicians (PCP) are critical towards a successful CRC screening program. However, the impact of availability of PCP on CRC-related mortality is not well studied.

Materials and Methods: We extracted data on crude incidence and mortality rates of CRC among all 50 states and District of Columbia (D.C.) from the Center for Disease Control (CDC) WONDER database for the year 2019. Data on number of actively practicing PCPs in all 50 states and D.C. for the year 2020 were obtained from Association of American Medical Colleges (AAMC) State Physician Workforce Data Report. The correlation between the ratio of number of PCPs per CRC case with age-adjusted mortality rate (AAMR) for CRC was studied by state and within White and African American (AA) populations from 2017-2019. Pearson’s co-efficient was used to study correlations and T-test was used for comparing independent samples. Statistical analysis was performed using VassarStats.

Results: The median AAMR for CRC from 2017-2019 in the US was 13.2 (IQR 11,15.4) per 100,000 population. AAMR for CRC was significantly higher among AA versus White populations (t=5.79, p<0.001). The national median PCP per CRC case ratio was 2.14 (IQR 1.46, 2.82). Higher state-wide PCP per CRC case ratio correlated with lower state CRC-related mortality (r=-0.36, p=0.011). PCP per CRC case ratio was significantly lower among AA as compared to White populations (t=-15.95, p<0.0001). Higher PCP per CRC case ratio correlated with lower CRC-related mortality among both White (r=-0.64, p<0.0001) and AA (r=-0.57, p=0.0002) populations. Using Fisher r-to-z transformation, we found no significant difference between the correlation of PCP per CRC case ratio with CRC-related mortality in White versus AA populations (z=-0.51, p=0.61).

Conclusion: Results of this study suggest that greater availability of primary care physicians correlates with lower CRC-related mortality. AA have a significantly higher AAMR for CRC with a lower PCP per CRC case ratio. This may indicate a lower access to primary care among AA populations with CRC. Our results underscore the need to expand access to primary care among AA populations with the goal of minimizing disparities in CRC-related mortality.

Citation Format: Udhayvir S. Grewal, Shiva J. Gaddam, Manik Aggarwal, Subhash C. Garikipati, Prabhat Kumar, Naomi Fei. Correlation of availability of primary care physicians and colorectal cancer-related mortality in the United States: Do racial disparities exist. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6488.
Abstract 6515: Pancreatic ductal adenocarcinoma (PDAC) early detection
Authors: Hinestrosa Juan P. , Dhani Harmeet , Schroeder Gregor , Lewis Jean M. , Balcer Heath I. , Kurzrock Razelle , Keith Dove , Sears Rosalie , Billings Paul
Published: April 2023

Introduction Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and is one of the deadliest cancer in the U.S., with a 5-year survival rate of just 11%. It is expected that ~62,000 new cases will be diagnosed in the US in 2023 and >70% of those will be at the regional or distant state, when curative resection is no longer an option. It is imperative that tools enabling early disease detection (stages I and II) are developed to improve patient survival outcomes. Here, we report the results of a study on the utility of extracellular vesicles (EVs) for early PDAC detection.

Methods Our case-control training set was comprised of 75 pathologically confirmed PDAC cases (Stage I = 31, Stage II = 44) and 640 controls (including 11 subjects with pancreatitis) for EV biomarker and algorithm selection via machine learning. EVs from plasma were isolated using a proprietary technology, then EV-bound proteins of interest were analyzed via an immunoassay. A second independently collected cohort of 20 confirmed PDAC cases (Stage I = 10, Stage II = 10) and 27 controls (including 9 patients newly diagnosed with diabetes) was used for validation.

Results The machine learning analysis using EV-isolated proteins generated a signature that utilized 8 different biomarkers, with an AUC of 0.989 (95% CI: 0.980 - 0.998) in the training set and an AUC of 0.987 (CI: 0.964 - 1.000) in the validation set. For the validation set, the sensitivity was 95% (CI: 76.4% - 99.1%) and the specificity was 92.6% (CI: 76.6% - 97.9%), with 10 out of 10 stage I cases and 9 out of 10 stage II cases correctly classified.

Conclusions The ability to alter the future outcomes of PDAC patients will be predicated on development of a new generation of early-detection biomarkers and technologies. The use of EV protein signatures, a new class of cell-free biomarker, permits detection at high sensitivity and specificity, as demonstrated for the independently collected validation cohort. Studies aimed to assess this test for high-risk patients (family history of PDAC, known germline mutations, precursor lesions, hereditary pancreatitis, new onset diabetes will continue towards the establishment of real-world evidence.

Citation Format: Juan P. Hinestrosa, Harmeet Dhani, Gregor Schroeder, Jean M. Lewis, Heath I. Balcer, Razelle Kurzrock, Dove Keith, Rosalie Sears, Paul Billings. Pancreatic ductal adenocarcinoma (PDAC) early detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6515.
Abstract 6525: Colon cancer screening and treatment with engineered probiotics
Authors: Gurbatri Candice R. , Radford Georgette , Im Jongwon , Coker Courtney , Arpaia Nicholas , Woods Susan L. , Worthley Daniel L. , Danino Tal
Published: April 2023

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate selective colonization of CRC tumors with the orally-delivered probiotic E. coli Nissle 1917 (EcN) in genetically-engineered and orthotopic murine models of CRC, and human CRC patients. We subsequently engineer EcN to produce screening and therapeutic molecules, resulting in non-invasive detection of precursor CRC lesions and reduction of tumor burden through local induction of robust anti-tumor immunity.

Citation Format: Candice R. Gurbatri, Georgette Radford, Jongwon Im, Courtney Coker, Nicholas Arpaia, Susan L. Woods, Daniel L. Worthley, Tal Danino. Colon cancer screening and treatment with engineered probiotics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6525.