bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3079: Characterization of ER-AF2 inhibitors in breast cancer
Authors: Foo Jane , Gentile Francesco , Lee Joseph , Morin Helene , Massah Shabnam , Guo Maria , Smith Jason , Ban Fuqiang , Cherkasov Artem , Lallous Nada
Published: April 2023

Up to 80% of breast cancers (BCa) rely on the estrogen receptor (ER) for their growth and progression. This dependence on ER has led to many hormonal therapies that target this receptor. However, almost 40% of these cancers will acquire resistance over the course of the treatment period. One potential cause of resistance is mutations in the estrogen binding site (EBS) of ER. As such, there is an increasing need for novel inhibitors that targets ER at a site separate from the EBS. Here, we propose targeting the activation-function (AF2) pocket of ER that is important for cofactor binding and transcription activation. Billions of compounds were screened through an in-silico deep docking method, and potential AF2 inhibitors were then validated in cell-based and biophysical assays. We tested the effect of potential AF2 inhibitors on ER transcriptional activity using luciferase reporter assay in ER-positive T47D-kbluc cells. We then evaluated the effect of molecules on cell viability of ER-positive T47D and ER-negative MDA-MB-231 cells using PrestoBlue assays in order to exclude off-target effects. From these cell-based assays, we identified several inhibitors that effectively reduced transcriptional activity and viability in ER-positive T47D cells at low micromolar concentrations. We conducted PGC-1α peptide displacement assay to confirm their AF2 binding and estradiol displacement assays to exclude any binding to the EBS. Proximity ligation assay (PLA) showed disruption of the interaction between ER and coactivator SRC-3 upon treatment with ER-AF2 inhibitors in T47D cells. Current work focuses on confirming the direct binding between the compounds and recombinant ER-ligand binding domain by various biophysical assays (MST, BLI, and ITC). Future work aims to solve the structure of ER-LBD in a complex with our lead compound by X-ray crystallography. We predict that the use of potent ER-AF2 inhibitors along with current treatments, will provide a novel tactic that can act as a complementary therapeutic to target treatment resistance in ER+ BCa.

Citation Format: Jane Foo, Francesco Gentile, Joseph Lee, Helene Morin, Shabnam Massah, Maria Guo, Jason Smith, Fuqiang Ban, Artem Cherkasov, Nada Lallous. Characterization of ER-AF2 inhibitors in breast 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 3079.
Abstract 3092: Optimization of deferiprone for the treatment of triple-negative breast cancer
Authors: Johnston Alexis , Oyelere Adegboyega K.
Published: April 2023

Triple-negative breast cancer (TNBC) is the leading cause of new cancer cases and the second leading cause of cancer deaths in American women. TNBC is one of the most difficult cancers to treat due to the lack of three receptors, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (HER2), found in regular forms of breast cancer (BCa). Currently, there is no targeted therapy for TNBC and treatment options to manage this lethal disease include surgery, adjuvant chemotherapy and radiotherapy. A better understanding of TNBC etiology has aided in unraveling the roles of two additional cellular targets - androgen receptor (AR) and histone lysine demethylase (KDM) - in the viability and survival of TNBC. Interestingly, AR overexpression is the most important driver of prostate cancer (PCa). A class of current medications used to treat PCa act as androgen receptor (AR) antagonists (or antiandrogens). It is therefore plausible to repurpose and optimize these antiandrogens for TNBC therapy. The proposed therapeutics are dual-acting antiandrogen-KDM inhibitors. The antiandrogen moiety of these agents will enable TNBC cell-targeting and translocation of the drug into the nucleus where inhibition of KDM will prevent transcription of KDMs that promote tumor. The three pharmacophores of these agents are (i) a surface recognition cap, (ii) a linker, and (iii) an Fe2+-binding group. Two classes of these compounds, alkyl-derivative and benzyl-derivative, vary in the design of the surface recognition cap. To optimize these agents, the linker length is varied from 2-7 methylene groups in each class and the optimal length will be determined through in vitro studies. The compounds are tested on five cancer cell lines: TNBC cell lines (MDA-MB-231, low AR+; and MDA-MB-453, high AR+), and ER+ BCa (MCF-7) while PCa cell lines (LNCaP PCa, AR+; and DU 145, PCa, AR-) serve as controls for AR dependency of the effects of test compounds. Longer linker length compounds exhibited low micromolar cytotoxicity and selectivity for MDA-MB-231 (TNBC) and LNCaP (PCa) cell lines due to the presence of the AR. Western blot analysis on lead compounds supported significant downregulation of AR in MDA-MB-231 cells confirming the predicted mechanism of action. Thus far, the designed dual-acting therapeutics have potential to revolutionize the design of anticancer drugs and improve treatment outcome for TNBC patients.

Citation Format: Alexis Johnston, Adegboyega K. Oyelere. Optimization of deferiprone for the treatment of triple-negative breast 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 3092.
Abstract 3133: Loss of the Y chromosome as a candidate driver in solid tumors
Authors: Qi Meifang , Rheinbay Esther , Lane Andrew A. , Pang Jiali , Mitsiades Irene
Published: April 2023

It has long been recognized that certain cancer types afflict female and male patients disproportionately. The reasons for this disparity include differences in male and female physiology, effect of sex hormones, risk behavior and environmental exposures, and different copy number of the sex chromosomes X and Y. Loss of Y (LOY) is common in peripheral blood cells in aging men, and this phenomenon is associated with several diseases. However, the frequency and role of LOY in tumors is little understood. Here, we present a comprehensive catalog of LOY in about 5,000 primary tumors from male patients in the TCGA. We show that LOY rates vary by tumor type, and provide evidence for LOY being either a passenger or driver event depending on context. LOY in uveal melanoma specifically is associated with age, survival and is an independent predictor of poor outcome. LOY creates common dependencies on DDX3X and EIF1AX in male cell lines, suggesting that LOY generates unique vulnerabilities that could be therapeutically exploited.

Citation Format: Meifang Qi, Esther Rheinbay, Andrew A. Lane, Jiali Pang, Irene Mitsiades. Loss of the Y chromosome as a candidate driver in solid tumors [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 3133.
Abstract 3139: Pan-cancer splicing analysis reveals shared drivers of malignant transformation and survival
Authors: Bhattacharjee Anukana , Crowther Audrey , Li Guangyuan , Venkatasubramanian Meenakshi , Schnell Dan , Hay Stuart , Singh Preeti , Subramanian Krithika R. , Chetal Kashish , Chen Xiaoting , Kulkarni Aishwarya , Weirauch Matthew T. , Salomonis Nathan
Published: April 2023

Alternative splicing is one of the primary mechanisms used to achieve mRNA transcript and proteomic diversity in higher order eukaryotes. In cancer, altered mRNA splicing can lead to aberrant protein products that promote oncogenic transformation, metastasis and confer chemotherapy resistance, due to splicing factor mutation or mis-expression. We hypothesize that the role of splicing imbalance in cancer is grossly underestimated and is likely regulated by the same recurrent global disruptions observed across human cancers. To determine common and malignancy specific splicing subtypes across cancer we designed a novel integrated computational workflow that uses genome variant data from RNA-Seq in conjunction with fully unsupervised analyses (NMF and SVM) to identify novel patient splicing-defined subtypes (OncoSplice). Applied to 20 adult and pediatric cancers with large cohorts, we identified common-recurrent splicing subtypes associated with MYC-hyperactivation and TGF-beta signaling, lineage reprogramming, tumor infiltration and broad mutation impacts. Such recurrent and tumor specific subtypes were frequently associated with poor prognosis and novel dominantly regulated driving splicing events (e.g., transcription factors). Broad splicing subtypes were associated with circadian dysregulation, as determined from normal healthy tissues (GTEx) and in many cases were found to phenocopy well-described splicing mutation impacts or were associated with new RNA-binding proteins (RBPs) evidenced by orthogonal computational predictions. Splicing subtype associated RBPs were frequently undergo autoregulation, as further evidenced by CLIP-Seq and RBP knockdown. To enhance the RNA community’s ability to explore hundreds of known and novel splicing variation across cancers and healthy tissues, we provide an interactive online splicing explorer at oncosplice.org. Together, these data highlight previously unknown regulatory relationships and prognostic associations in cancer associated with broad and targeted splicing regulation.

Citation Format: Anukana Bhattacharjee, Audrey Crowther, Guangyuan Li, Meenakshi Venkatasubramanian, Dan Schnell, Stuart Hay, Preeti Singh, Krithika R. Subramanian, Kashish Chetal, Xiaoting Chen, Aishwarya Kulkarni, Matthew T. Weirauch, Nathan Salomonis. Pan-cancer splicing analysis reveals shared drivers of malignant transformation and survival [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 3139.
Abstract 3144: Mapping the functional interactions at the tumor-immune checkpoint interface
Authors: Bozorgui Behnaz , Kong Elisabeth K. , Luna Augustin , Korkut Anil
Published: April 2023

The interactions between tumor intrinsic processes and immune checkpoints can mediate the immune evasion by tumors and responses to immunotherapy. It is, however, challenging to identify functional interactions due to the prohibitively complex molecular landscape of the tumor-immune interfaces. We address this challenge with a statistical analysis framework, termed immuno-oncology gene interaction maps (ImogiMap). ImogiMap quantifies tumor-immune checkpoint interactions based on their co-associations with immune-associated phenotypes. The interactions are statistically validated for significance, robustness, and specificity. The outcome is a catalog of maps that quantify the interactions at the tumor-immune interface for diverse immune phenotypes and checkpoints. We demonstrate that applications of ImogiMap on immune checkpoints and T-cell dysfunction signatures genes recapitulates the previously reported interaction of SERPINB9 and immune checkpoints in the tumor microenvironment. Our analyses also suggest other immune checkpoint and T-cell dysfunction signatures interactions that are significantly associated with IFNγ expression in uterine corpus endometrial carcinoma and basal-like breast cancer. We also provide open-source software and a user-friendly web application to enable future applications and rapid adoption of ImogiMap. Such applications may guide the discovery of previously unknown tumor-immune interactions and help the identification of immunotherapy targets.

Citation Format: Behnaz Bozorgui, Elisabeth K. Kong, Augustin Luna, Anil Korkut. Mapping the functional interactions at the tumor-immune checkpoint interface [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 3144.
Abstract 3173: Integrative structural models of oncogenic mutants within the trans- and juxta-membrane region of human epidermal growth factor receptors (HERs)
Authors: Mercado Christina , Mamun Abdullah Al , Park Younghee , Lovly Christine M. , Meiler Jens , Ledwitch Kaitlyn
Published: April 2023

The trans- and juxtamembrane domains (TMD/JMD) of human epidermal growth factor receptors (HER/EGFR/ErbB) regulate the helix-helix dimerization interface to control receptor tyrosine kinase (RTK) activation. Patient-identified non-small cell lung cancer (NSCLC) mutations occur all along the EGFR (HER1) and HER2 TMD and JMD, but our structural understanding of how these patient-derived TMD/JMD mutations hijack wild-type RTK function to drive cancer is lacking. In this work, we integrate tools in experimental biophysics and computational modeling to evaluate the structural mechanism(s) driving these interactions in atomic detail. We use nuclear magnetic resonance (NMR) spectroscopy to assess how oncogenic mutations within the TMD/JMD perturb the architecture/interactions of the dimer interface. NMR-derived models are used to seed molecular dynamics (MD) simulations to probe how mutations affect TMD association and activation/stabilization via dimerization in a realistic membrane environment. We apply this strategy and focus our initial efforts on the EGFRA647T variant. We computed the free energy landscape from our simulations and show that the active state for the EGFRA647T-HER2WT heterodimer is stabilized by 10 kcal/mol. Conversely, the wild-type EGFR-HER2 heterodimer is stabilized by 5 kcal/mol for the inactive state suggesting the propensity of the EGFRA647T mutant favors the active state. These results are part of a multidisciplinary collaboration that share the common goal of constructing holistic models that detail how TMD/JMD variants are aberrantly activated in cancer.

Citation Format: Christina Mercado, Abdullah Al Mamun, Younghee Park, Christine M. Lovly, Jens Meiler, Kaitlyn Ledwitch. Integrative structural models of oncogenic mutants within the trans- and juxta-membrane region of human epidermal growth factor receptors (HERs) [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 3173.
Abstract 3216: Comparative characteristics of stage I non-small cell lung cancer (NSCLC) patients segregated by quality of surgery and survival in a population-based cohort
Authors: Shao Huibo , Lee Yu-Sheng , Fehnel Carrie , Akinbobola Olawale , Saulsberry Andrea , Dortch Kourtney , Matthews Anberitha T. , Faris Nicholas R. , Smeltzer Matthew , Ray Meredith A. , Osarogiagbon Raymond U.
Published: April 2023

Introduction. Poor quality NSCLC resection impairs survival, but some patients survive irrespective. We explored characteristics of patients in 4 cohorts defined by surgery quality and survival.

Methods. We analyzed stage I resections in a population-based cohort surviving >120 days post-op from 2009-2018. Using the International Association for the Study of Lung Cancer’s stringent definition of ‘complete resection’ and 3-year survival as ‘good’, we categorized patients into 4 dyads: 1) Good surgery/good survival; 2) Good surgery/poor survival; 3) Poor surgery/good survival (reference); 4) Poor surgery/poor survival. We used Chi-square and Kruskal-Wallis test for description, multivariable multinomial logistic regression to estimate Odds Ratio (OR) of surgery quality and survival.

Results. Of 1,515 patients, 31%, 7%, 51%, and 12% were in Groups 1-4, respectively. In the ‘mis-match dyads’ (Groups 2 and 3), Group 2 was more likely to be elderly (70 vs. 66, p<0.01), on Medicaid (p=0.01), have ever smoked (96% vs. 85%, p<0.01), have squamous cell carcinoma.

Older (OR=1.06), male (OR=1.78), Medicaid patients (OR=2.16) with smoking history (OR=3.25) had poorer survival despite good surgery rather than survival despite bad surgery. With poor surgery, male (OR=2.01) patients tumor 2-3 and >3 cm (OR=1.77 and 1.64) had poorer survival; commercially insured patients had 36% reduced risk of poor survival. Compared to open thoracotomy, patients with robotic-assisted surgery were >4-fold more likely to have good surgery rather than poor surgery but good survival.

Conclusion. Differentiating characteristics associated with good/bad survival after good/bad quality lung cancer surgery provide opportunity to compare biomarker profiles between dyads.

Citation Format: Huibo Shao, Yu-Sheng Lee, Carrie Fehnel, Olawale Akinbobola, Andrea Saulsberry, Kourtney Dortch, Anberitha T. Matthews, Nicholas R. Faris, Matthew Smeltzer, Meredith A. Ray, Raymond U. Osarogiagbon. Comparative characteristics of stage I non-small cell lung cancer (NSCLC) patients segregated by quality of surgery and survival in a population-based cohort [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 3216.
Abstract 3217: Risk of thromboembolism in patients with non-small lung cancer (NSCLC) treated with immune check point inhibitors (ICIs) versus chemoimmunotherapy (ChemoIO)
Authors: Attia Doaa , Zhao Ran , Khorana Alok A. , Patil Prandya
Published: April 2023

Background: Immune checkpoint inhibitors with or without chemotherapy form the backbone of treatment for patients with advanced NSCLC without an actionable driver mutation. Emerging literature shows that ICIs increase the risk of venous thromboembolism (VTE), which is a major cause of death in patients with NSCLC. The mechanism is yet to be elucidated but is likely secondary to inflammation. Since chemotherapy is immunosuppressive, we sought to understand if the addition of chemotherapy to ICIs impacted the incidence of VTEs.

Methods: We performed a retrospective cohort study to evaluate thromboembolic outcomes in patients diagnosed with advanced NSCLC between 2012 and 2018 and treated with either ICIs or chemo immunotherapy at Cleveland Clinic Foundation. Diagnosis of thrombotic events (TE) (arterial/deep vein thrombosis and/or pulmonary embolism) was confirmed by imaging. Overall survival (OS) was estimated by Kaplan-Meier and compared using log rank test. Odds of developing TE was estimated and compared between ICI and ChemoIO groups, using Fisher's Exact Test.

Results: A total of 367 NSCLC patients (242 pts on ICI only and 125 pts on ChemoIO regimen), were identified with a median age 65 years ( range 58-72), 52% were men, 82% white, 85.3% smokers, 91.8% had ECOG ≤2, 73 % had adenocarcinoma and 18% had squamous cell carcinoma, 34% with brain metastasis and the distribution of PDL1expression was as follows- <1 (26.4%), 1-49% (48%) and ≥50% (25.6%). Adjusted response rates to treatment were (N, %): Complete Response (CR) (6/367, 1.9%), Partial Response (PR) (96/367, 30%), Stable Disease (SD) (76/367, 24%) and PD (143/367, 45%). TE was detected in 19% of the cohort (n=70) (8.4% DVT, 4.9% PE, 5.2 % both, and 0.5 % arterial TE. Rate of TE in NSCLC pts on ICI alone was 18% (44/242) Vs. 21% (26/125) for those on ChemoIO (OR 1.5, 95% CI 0.66-2.1, P 0.6). Development of TEs was not associated with survival differences with either regimen (P >0.05).

Conclusion: Rates of TE were high in patients with advanced NSCLC who received ICI therapy, but no different if treated with ICIs alone or ChemoIO. Further studies to better understand the pathophysiology of TE in patients treated with ICIs might allow us to identify subgroups of patients that are at the highest risk for TEs.

Citation Format: Doaa Attia, Ran Zhao, Alok A. Khorana, Prandya Patil. Risk of thromboembolism in patients with non-small lung cancer (NSCLC) treated with immune check point inhibitors (ICIs) versus chemoimmunotherapy (ChemoIO) [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 3217.
Abstract 3233: A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies
Authors: Wang Linlin , Liu Xiao , Jin James , Yu Yuxiang
Published: April 2023

Regulatory T cells (Tregs) play an immunosuppressive role in the tumor microenvironment (TME), such that altering or depleting Treg function has emerged as an important anti-tumor strategy. Targeting CCR8, which is highly expressed in tumor-infiltrating Tregs, has shown its potency in reducing tumor burden and increasing responsiveness to immune-checkpoint blockade therapy. Collectively, this suggests a need for preclinical models that support Treg-targeted therapies. To address this, Biocytogen developed a triple humanized (B-hPD-1/hPD-L1/hCCR8) mouse model to evaluate in vivo efficacy of combination therapies. In this model, the full coding sequence of murine Ccr8 was replaced by the full coding sequence of human CCR8, while murine Pd-1 and Pd-l1 IgV domains were replaced by their human counterpart gene sequences. Human PD-1 and PD-L1 protein expression was confirmed in B-hPD-1/hPD-L1/hCCR8 mice by flow cytometry, and human CCR8 protein expression was observed in MC38 tumors harvested from B-hPD-1/hPD-L1/hCCR8 mice. Finally, combination therapy using anti-human PD-1 and anti-human CCR8 antibodies were effective in controlling B-hPD-L1 MC38 tumor growth in B-hPD-1/hPD-L1/hCCR8 mice. Altogether, this line provides a powerful preclinical model for in vivo evaluation of therapeutic antibodies.

Citation Format: Linlin Wang, Xiao Liu, James Jin, Yuxiang Yu. A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies [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 3233.
Abstract 3242: Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with immunotherapy
Authors: Zhu Fang , Sun Hongyan , Lu Qian , Fang Yuan , Xu Jianming , Ju Cunxiang , Wang Hongyu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang
Published: April 2023

Antibody-drug conjugates (ADCs) are effective anti-tumor therapies with high specificity, with little toxicity and side effects. Xenograft tumor models based on immunodeficient mice have been used for evaluating the human anti-tumor efficacy of ADCs. However, the antibody-mediated immune response of ADCs is not well evaluated in immunodeficient mice. To this end, we have developed BALB/c-hPD1 humanized mouse model to evaluate the efficacy of ADCs in combination with immunotherapy.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is a HER2-targeting ADC. In vitro efficacy studies showed that T-DXd effectively bound to the HER2-positive cell line SK-BR-3, resulting in strong endocytosis and induction of apoptosis. It has also been shown to kill HER2-negative MDA-MB-468 cells through a bystander effect both in vitro and in vivo. In vivo efficacy studies have demonstrated that ADCs can effectively inhibit human tumor cell growth in immunodeficient mice. In addition to human tumor cell lines, the efficacy of ADCs was also studied in BALB/c-hPD1mice engrafted with murine colon cancer cells model, and the results showed that T-DXd and Keytruda could significantly inhibit CT26-hHER2 tumor growth, with the tumor growth inhibition rates being 57% and 66%, respectively, Tumor growth inhibition rate of the combined treatment group was about 91%, which was superior compared to the efficacy of single drug treatment. The evaluation of Fc segment-mediated effect (such as ADCC, CDC and ADCP) of ADCs antibodies was carried out in humanized immune system mouse models, such as NCG-hIL15 and NCG-hSGM3. In summary, we have established a drug evaluation platform using various humanized mouse models to study the synergistic anti-tumor effects of ADCs combined with immune checkpoint blockade antibodies.

Citation Format: Fang Zhu, Hongyan Sun, Qian Lu, Yuan Fang, Jianming Xu, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Efficacy evaluation by novel humanized mouse models for preclinical study of ADCs combined with 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 3242.
Abstract 3254: Combination of palbociclib, a potent CDK4/6 inhibitor, with anti-PD1 drug pembrolizumab treatment to promote T-cell mediated glioblastoma tumor cell death under hypoxia
Authors: Xia Yutong , Zhou Lanlan , Zhao Shuai , El-Deiry Wafik S.
Published: April 2023

Glioblastoma (GBM) is the most common malignant brain and other central nervous system (CNS) tumor, which comprises 47.7% of all cases. The standard treatment options are surgery, radiation therapy, and chemotherapy using temozolomide. GBM is fast-growing, aggressive, and results in poor overall survival, approximately 40% in the first year and 17% in the second year. Hypoxia, the lack of sufficient oxygen in tissues, is the hallmark of GBM and can induce drug resistance and inhibit anti-tumor immune responses. Previous studies show that CDK inhibitors can destabilize HIF1, a key regulator of hypoxia induced apoptosis and p53 stabilization. Palbociclib, a potent oral inhibitor of CD4/6, is undergoing clinical trials for GBM treatment. However, it was not an effective treatment for recurrent GBM when used as alone in a Phase II study. Thus, we are exploring the cytotoxicity of palbociclib in combination with an anti-PD1 drug pembrolizumab, which is proven to be highly effective in other cancer types. Preliminary studies use CellTitlerGLO viability assay to determine the IC50s for Palbociclib at 24, 48, and 72 hours in both normoxia and hypoxia conditions. Immune cell co-culture, comprising of GBM cell lines plus TALL104 human leukemia T cells, was used to determine the amount of tumor cell death with palbociclib, pembrolizumab and a combination at different time points in both normoxia and hypoxia conditions. Our results are providing insights into improving immune checkpoint therapy for GBM patients.

Citation Format: Yutong Xia, Lanlan Zhou, Shuai Zhao, Wafik S. El-Deiry. Combination of palbociclib, a potent CDK4/6 inhibitor, with anti-PD1 drug pembrolizumab treatment to promote T-cell mediated glioblastoma tumor cell death under hypoxia [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 3254.
Abstract 3428: Discovery of a highly potent and selective dual PROTAC degrader of CDK12 and CDK13
Authors: Chang Yu , Yang Jianzhang , Tien Jean Ching-Yi , Wang Zhen , Zhou Yang , Zhang Pujuan , Huang Weixue , Vo Josh , Apel Ingrid J. , Wang Cynthia , Zeng Victoria Zhixuan , Cheng Yunhui , Li Shuqin , Wang George Xiaoju , Ding Ke , Chinnaiyan Arul M.
Published: April 2023

Selective degradation of the cyclin-dependent kinases 12 and 13 (CDK12/13) presents a novel therapeutic opportunity for triple-negative breast cancer (TNBC), but there is still a lack of dual CDK12/13 degraders. Here, we report the discovery of the first series of highly potent and selective dual CDK12/13 degraders by employing the proteolysis-targeting chimera (PROTAC) technology. The optimal compound 7f effectively degraded CDK12 and CDK13 with DC50 values of 2.2 nM and 2.1 nM, respectively, in MDA-MB-231 breast cancer cells. Global proteomic profiling demonstrated the target selectivity of 7f. In vitro, 7f suppressed expression of core DNA damage response (DDR) genes in a time- and dose-dependent manner. Further, 7f markedly inhibited proliferation of multiple TNBC cell lines including MFM223, with an IC50 value of 47 nM. Importantly, 7f displayed a significantly improved anti-proliferative activity compared to the structurally similar inhibitor 4.The 2nd generation CDK12/13 degrader 9069 was further developed, which showed more potent degradation on both CDK12 and CDK13. Interestingly, multiple prostate cancer cell lines were sensitive to 9069, such as VCaP, 22RV1 and LAPC4 with IC50 values of 22.9, 90.3 and 152.1 nM, respectively. In vivo studies showed a potent tumor growth inhibition in both VCaP CRPC xenograft and PC310 PDX prostate cancer models. These results suggest the potential application of a CDK12/13 degrader for the treatment of TNBC and prostate cancer.

Citation Format: Yu Chang, Jianzhang Yang, Jean Ching-Yi Tien, Zhen Wang, Yang Zhou, Pujuan Zhang, Weixue Huang, Josh Vo, Ingrid J. Apel, Cynthia Wang, Victoria Zhixuan Zeng, Yunhui Cheng, Shuqin Li, George Xiaoju Wang, Ke Ding, Arul M. Chinnaiyan. Discovery of a highly potent and selective dual PROTAC degrader of CDK12 and CDK13 [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 3428.
Abstract 343: Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men
Authors: Chen Ching-Yi , Wu Pei-Ying , Van Scoyk Michelle , Simko Stephanie A. , Chou Chu-Fang , Winn Robert A.
Published: April 2023

Non-Hispanic Black/African American (Black/AA) men in the United States have disproportionally higher incidence and mortality rates of lung cancer compared to non-Hispanic White (NHW) men. Molecular determinants including biological and genetic factors are believed to play critical roles in driving disparities. Yet, several recent large-scale genomic studies fail to identify significant somatic differences in lung cancer driver genes contributing to observed disparities between Black/AA and NHW groups. These findings suggest that epigenetic mechanisms may contribute to lung cancer disparities in Black/AA men. Arginine methylation is a post-translational modification catalyzed by protein arginine methyltransferases (PRMTs) that has gained considerable interest as many cancer types display elevated expression of PRMTs correlating with poor prognosis. Here, we observed a significant difference in the expression levels of PRMT6 between Black/AA men vs. NHW men with non-small cell lung cancer (NSCLC) that may contribute to cancer health disparities. To uncover the molecular mechanism by which PRMT6 drives lung cancer, we performed co-immunoprecipitation, protein arginine methylation, and cell proliferation assays using several NSCLC cell lines. We demonstrated that PRMT6 formed a heteromer complex with PRMT1 and disruption of the complex with a competitive peptide inhibitor significantly reduced cancer cell proliferation. We further showed that PRMT1/PRMT6 heteromer complex recruited and catalyzed arginine methylation of interleukin enhancer-binding factor 2 (ILF2). Disrupting heteromer complex formation leaded to a significant reduction of ILF2 methylation and expression. Finally, downregulation of ILF2 expression reduced the positive effects of PRMT1/PRMT6 complex on cell proliferation. Our findings demonstrate the significant functionality of PRMT1/PRMT6 heteromer complex to positively regulate ILF2 expression that is essential for cancer cell proliferation. Furthermore, we uncover a category of substrates for this complex providing the new insights for more potential therapeutic targets that may set the stage for developing new alternative approach to PRMT inhibition. This approach would potentially allow the ability to overcome the current limitations of PRMT1 or PRMT6 inhibitors, thereby opening up new horizons for efficacious PRMT1/PRMT6-targeted agents that are expected to eliminate lung cancer health disparities in Black/AA men.

Citation Format: Ching-Yi Chen, Pei-Ying Wu, Michelle Van Scoyk, Stephanie A. Simko, Chu-Fang Chou, Robert A. Winn. Cooperation between PRMT1 and PRMT6 drives lung cancer health disparities among Black/African American men [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 343.
Abstract 3525: VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis
Authors: Watson Sarah , LaVigne Collette A. , Xu Lin , Surdez Didier , Cyrta Joanna , Calderon Delia , Cannon Matthew V. , Kent Matthew R. , Silvius Katherine M. , Kucinski Jack P. , Harrison Emma N. , Murchison Whitney , Rakheja Dinesh , Tirode Franck , Delattre Olivier , Amatruda James F. , Kendall Genevieve C.
Published: April 2023

Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that have not been functionally validated for their transformation capacity and biological activity. An example is the new fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implemented a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We found that in our transgenic zebrafish and mouse allograft models, VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle. Zebrafish and mouse VGLL2-NCOA2 tumors are consistent with the human disease histologically and transcriptionally, and express diagnostic rhabdomyosarcoma markers. We found a shared molecular feature of these tumors was a suppression of myogenic differentiation pathways and reactivation of developmental programs. To identify impacted developmental programs, we transcriptionally clustered zebrafish VGLL2-NCOA2 tumors with the trajectory of early zebrafish development. A subset of VGLL2-NCOA2 zebrafish tumors cluster with embryonic somitogenesis and pinpoint VGLL2-NCOA2 developmental targets, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse allograft, and patient tumors, ARF6 is highly expressed and is absent from mature skeletal muscle. ARF6 knockout in our C2C12-VGLL2-NCOA2 cell culture model suppresses VGLL2-NCOA2 soft agar colony formation capacity, suggesting a genetic cooperating event in the disease. Our data indicate that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that the reactivation or persistence of ARF6 could represent a therapeutic opportunity.

Citation Format: Sarah Watson, Collette A. LaVigne, Lin Xu, Didier Surdez, Joanna Cyrta, Delia Calderon, Matthew V. Cannon, Matthew R. Kent, Katherine M. Silvius, Jack P. Kucinski, Emma N. Harrison, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James F. Amatruda, Genevieve C. Kendall. VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. [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 3525.
Abstract 3530: Mechanisms underlying Ewing sarcoma-induced osteolysis
Authors: Williams James J. , Kitlinska Joanna B.
Published: April 2023

Ewing Sarcoma (ES) is an aggressive pediatric tumor, which often arises in bones and forms osseous metastases, both of which present as osteolytic lesions. Our previous studies indicated that neuropeptide Y (NPY), which is secreted from ES tumors, stimulates tumor-induced bone degradation via its effect on macrophage recruitment and their subsequent osteoclastic differentiation. This effect was dependent on the autocrine activation of the NPY Y5 receptor (Y5R) expressed by ES cells. However, the exact mechanisms of the osteolytic activity induced by the NPY/Y5R axis remained unknown. To identify the osteolytic factors released from ES cells due to the NPY/Y5R autocrine loop activity, we used shRNA to silence NPY or Y5R in SK-ES-1 ES cells and compared concentrations of known factors involved in the regulation of bone homeostasis in their conditioned media by an antibody array. Four secreted factors were found to be significantly decreased after silencing of NPY signaling: interleukin 8 (IL-8; p<0.005), matrix metalloproteinase 9 (MMP-9; p<0.05), intercellular adhesion molecule 1 (ICAM-1; p<0.005), and monocyte chemoattractant protein 1 (MCP-1; p<0.05). Among these proteins, IL-8 and MMP-9 were selected as the main candidates for mediators of NPY-induced osteolysis. To test their role in ES-mediated stimulation of osteolysis, we co-cultured mouse RAW 264.7 cells, that represent monocyte/macrophage lineage, with SK-ES-1 cells in the presence or absence of the MMP-9 inhibitor (JNJ-0966; 10-7M-10-5M) or anti-IL-8 neutralizing antibody (0.1-0.4µg/ml) and tested their effect on osteoclast differentiation detected by TRAP staining. Inhibiting MMP-9 activity significantly decreased osteoclastogenesis of RAW264.7 cells in dose-dependent manner, while blocking IL-8 had no significant effect on the number of formed osteoclasts. These findings implicate MMP-9 as the mediator of NPY osteolytic activity. Further studies are required to understand the exact mechanisms underlying these effects, as well as potential role of NPY-induced release of MCP-1 in macrophage recruitment to ES tumors.

Citation Format: James J. Williams, Joanna B. Kitlinska. Mechanisms underlying Ewing sarcoma-induced osteolysis. [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 3530.
Abstract 362: Development of PEP3-IR750 probe to identify adipose tissue and cachexia in murine models
Authors: Chakrabarty Sophia , Han Zheng , McNally Lacey R.
Published: April 2023

Background Cachexia is a multifactorial adipose and muscle wasting disease that is frequently identified in patients with cancer. Cachexia results from increased lipolysis, proteolysis, inflammation, and tumor/host secreted cytokines. Unfortunatly, cachexia is often identified in the clinic at late stages after substantial adipose and muscle loss has occured. Currently, the identification of cachexia in its adipose-wasting stages is difficult due to the lack of a diagnostic imaging tool that easily, objectively, and reproducibly monitors the loss of adipose tissue. Objective identification of fat stores could be used for the early identification and monitoring of cachexia.

Methods We used Fmoc chemistry to synthesize a peptide that binds to fat, i.e. PEP3, CPATAEPC, and scrambled control peptide, AQYLNPS. Following microwave peptide synthesis, we lyophilized, purified, and conjugated the peptides to the Hilyte Fluor750 succinimidyl ester using NHS reaction. Excess dye was removed using dialysis in water. IR750-PEP3 probe was assessed using HPLC and spectroscopy to determine the probe concentrations. Sixty nanomoles of probe reconstituted in 200 µL PBS (pH 7.4). Ex vivo interscapular, perirenal, and omental fat and control muscle tissues were incubated with 20 µM IR750-PEP3 and scrambled probes for 1 h in a 6-well plate, tissues washed with PBS to remove unbound probe, and imaged using near infrared fluorescence. In vivo testing assessed, 60 nmol intravenous injection into healthy female mice. Mice were imaged using MSOT 4 h post injection, images were reconstructed and spectrally unmixed based upon the spectrums of brown fat and IR750 dye. Parametric maps were generated via MATLAB to identify distributions of fat and injected probes. Secondary confirmation of probe biodistribution was conducted using NIR fluorescence of excised tissues.

Results Following injecting 60 nmol of IR750-PEP3, MSOT images confirmed that the PEP3-IR750 dye localized to areas of fat in the interscapular, and perirenal areas in vivo compared to scrambled control (p=0.05, Mann-Whitney U Test). The higher capability of PEP3-IR750 to localize to fat tissue was further confirmed by ex vivo evaluation of PEP3-IR750 and scramble probes-IR750 (p=0.05, Mann-Whitney U Test). Notably, there was a complete lack of signal in the control muscle tissue.

Conclusion PEP3-IR750 can specifically identify fat in mice, and our data supports its potential use in the early identification of cachexia.

Citation Format: Sophia Chakrabarty, Zheng Han, Lacey R. McNally. Development of PEP3-IR750 probe to identify adipose tissue and cachexia in murine 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 362.
Abstract 3621: Identification of podocalyxin-positive tumor buds in pancreatic ductal adenocarcinoma: Implications for solid tumor collective invasion
Authors: Bell Erin M. , Kalloger Steve E. , Dean Pamela M. , Kopp Janel L. , McNagny Kelly M. , Roskelley Calvin D.
Published: April 2023

High expression of the single-pass transmembrane sialomucin, podocalyxin, has been shown to predict poor disease outcome in a number of solid tumor types, including pancreatic ductal adenocarcinoma (PDAC) (Taniuchi et al., 2016). In addition to the classical TMN staging measures of disease progression (tumor size, lymph node involvement, and distant metastasis) local invasion at the primary tumor site is a valuable indicator of solid tumor progression. Local invasion has been observed histologically as tumor buds, defined by the presence of small cohesive clusters of tumor cells in the invasive tumor-stromal interface. To date, tumor budding has been characterized as an indicator of poor prognostic outcome in colorectal carcinoma (CRC), pancreatic ductal adenocarcinoma (PDAC), invasive ductal breast carcinoma (IDC), lung adenocarcinoma (LUA), stomach and esophageal carcinomas, oral squamous cell carcinoma, periampullary adenocarcinoma, and head and neck squamous cell carcinoma (HNSCC). Our group has previously modelled tumor budding experimentally using the MCF7 human breast cancer cell line (Graves et al., 2016). We showed that forced over-expression of podocalyxin was a driver of local collective invasion and tumor budding in vitro and in vivo. Because podocalyxin is highly expressed in PDAC we sought to determine if its expression was involved in the process of tumor budding in this tumor context as well. We assessed large format histological sections from a cohort of patient PDAC tumors and identified high podocalyxin expression in tumor buds. Further, we generated podocalyxin-null MiaPaCa2 PDAC cell lines and demonstrated that podocalyxin-expression is required for collective invasion in 3D culture conditions. We are currently evaluating in vivo xenograft tumor models with these podocalyxin-expressing and podocalyxin-null MiaPaCa2 cells to determine whether changes in 3D culture invasion are representative of tumor budding in this model. Expression of podocalyxin by tumor buds provides rationale to support investigation into the impact of therapeutically targeting these invasive cells.

Citation Format: Erin M. Bell, Steve E. Kalloger, Pamela M. Dean, Janel L. Kopp, Kelly M. McNagny, Calvin D. Roskelley. Identification of podocalyxin-positive tumor buds in pancreatic ductal adenocarcinoma: Implications for solid tumor collective invasion. [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 3621.
Abstract 3659: RAS drivesmalignancy through stem cell crosstalk with the microenvironment
Authors: Yuan Shaopeng
Published: April 2023

Squamous cell carcinomas (SCCs) are triggered by marked elevation of RAS/MAPK signaling and progression from benign papilloma to invasive malignancy. At tumor-stromal interfaces, a subset of tumor-initiating progenitors, the cancer stem cells (CSCs), obtain increased resistance to chemo and immunotherapy along this path. The distribution and changes in CSCs during progression from a benign state to invasive SCC remain elusive. Here, we show that following HRASG12V activation, CSCs rewire their gene expression program and trigger a self-propelling, aberrant signaling crosstalk with their tissue microenvironment that drives their malignant progression. Surprisingly, the non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways often mutated in advanced metastatic SCCs with high mutational burden. Coupling our clonal skin HRASG12V model with single-cell transcriptomics, chromatin-landscaping, lentiviral reporters and lineage-tracing, we show that aberrant crosstalk between CSCs and their microenvironment triggers angiogenesis and TGFβ-signaling, creating conditions ripe for hijacking leptin and leptin receptor (LEPR)-signaling, which in turn launches downstream PI3K-AKT-mTOR signaling at the benign-malignant transition. By functionally interrogating each step in this pathway, we unravel how dynamic temporal crosstalk with the microenvironment and orchestrated by the stem cells (SCs) profoundly fuels this path to malignancy. These new insights suggest broad implications for cancer therapeutics.

Citation Format: Shaopeng Yuan. RAS drivesmalignancy through stem cell crosstalk with the microenvironment. [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 3659.