bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4903: Minimal mutational determinants of human squamous cell carcinoma
Authors: Planells-Palop Vicente , Koo Angie , Malave Heydi , Lee Katharine , Tward Aaron
Published: September 2020

Dozens of driver mutations and copy number changes have been implicated in human squamous cell carcinomas (SCCs), and each tumor harbors multiple putative driver mutations. However, the minimal set of mutations sufficient to transform a normal keratinocyte into squamous cell carcinoma is unknown. By analyzing TCGA data and previously published datasets, we have identified common combinations of driver mutations in SCCs. Among the most common driver events are mutations in TP53, CDKN2A, and NOTCH1. Using CRISPR editing at endogenous loci in primary human keratinocytes, we demonstrate that no single driver mutation is sufficient to transform keratinocytes, and in fact many single gene mutations appear toxic to keratinocytes. The combination of loss of function mutations in TP53 and CDKN2A are sufficient to immortalize both skin and oral primary human keratinocytes in 2D culture, and confer a selective advantage in competition assays. Simultaneous mutation of TP53, CDKN2A, and NOTCH1 demonstrates a very similar phenotype in 2D culture as mutation of TP53 and CDKN2A alone. However, using 3D organotypic squamous epithelial cultures consisting of genome-edited keratinocytes grown on decellularized dermis impregnated with fibroblasts and grown at an air-liquid interface demonstrates that keratinocytes with TP53 and CDKN2A mutation alone are sufficient to induce dysplasia, whereas keratinocytes harboring TP53, CDKN2A, and NOTCH1 mutation exhibit an SCC like phenotype including invasion through the basement membrane. RNAseq analysis demonstrates that the transition to SCC by NOTCH1 loss involves a partial-EMT like program. Targeted hybrid capture sequencing of edited keratinocytes revealed no other driver mutations or copy number changes other than the experimentally induced ones, consistent with the notion that TP53, CDKN2A, and NOTCH1 mutations alone are sufficient to reprogram normal keratinocyte into their malignant counterparts. Thus, despite a high mutation burden and frequent chromosomal instability, as few as three mutational events appear to be sufficient to induce SCC.

Citation Format: Vicente Planells-Palop, Angie Koo, Heydi Malave, Katharine Lee, Aaron Tward. Minimal mutational determinants of human squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4903.
Abstract 4973: Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients
Authors: Yaghoobi Vesal , Pelekanou Vasiliki , O'Meara Tess , Silber Andrea , Pusztai Lajos , Rimm David , Blenman Kim
Published: September 2020

Introduction: Although there are many possible causes, it has been observed that Triple Negative Breast Cancer (TNBC) has a worse overall outcome in African American (AA) than white patients. Recently, there is also evidence that the patients response to the tumor, or the biological response expressed as the tumor immune microenvironment, impacts outcome. The goal of this study was to compare the Tumor Microenvironment (TME) of TNBC in the two mentioned groups in order to seek molecular parameters that are different in TNBC in AA patients as compared to whites.

Methods: We selected N=50 AA and N=50 white TNBC samples from the Yale Pathology archives that were matched by diagnosis date. We measured CD45, CD3, CD8, CD20, CD14, CD68, PD-L1, THY1, aSMA and Fibroblast Activation Protein (FAP) expression in both the tumor and stromal compartments in whole slides from formalin fixed paraffin embedded (FFPE) tissues using multiplexed quantitative immunofluorescence (QIF). We also assessed the activation status of CD3-positive cells using Ki67 and Granzyme B markers. Median of each marker expression was calculated for each case by using quantitation from all Fields of View (FOV) that contained at least 10% tumor area.

Results: AA tumors contained a significantly higher CD45-positive cells in overall, within tumor and stromal compartments (p = 0.0102, 0.0007 and 0.0280 respectively). Being more specific about CD45 marker, AA also showed to have a significantly higher level of overall lymphocyte population, measured by quantitating CD45+ CD14- cells (p =0.0081). AA tumors also had a significantly higher level of CD8 expression (p <0.0001). Even though we could not find any differences in CD3 expressions between the two groups, AA tumors contained a significantly higher level of activated T-cells (p =0.0432), where activation is defined as CD3 cells with high Ki67 or Granzyme B. AA tumors contained a significantly higher level of CD68 positive cells (p < 0.0001) and PD-L1 expression in CD68+ cells (p =0.0101). All other markers showed no difference between the two groups.

Conclusion: Significant differences are seen in the TME of AA vs. white TNBCs. Further characterization of macrophages, fibroblasts, regulatory T-cells and Extracellular Matrix are underway to more specifically define TME and determine the relationship between TME features and outcome.

Citation Format: Vesal Yaghoobi, Vasiliki Pelekanou, Tess O'Meara, Andrea Silber, Lajos Pusztai, David Rimm, Kim Blenman. Comparison of tumor immune microenvironment in triple negative breast cancer (TNBC) of African American versus that of white patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4973.
Abstract 4984: Where do the T lymphocytes go in prostate cancer: Correlation with CD103, CD39 expression and clinical pathological parameters
Authors: Walter Beatriz , Gasmi Billel , Naizen Xu , Valera Vladimir , Pinto Peter , Merino Maria J.
Published: September 2020

Introduction: Many solid tumors such as prostate cancer have shown limited response to immunotherapy treatment. Whether this is due to absence of tumor specific antigens or poor immune infiltrate, the exact mechanism is not well understood. Our study evaluates the presence and spatial distribution in benign versus malignant tissue of CD4+ and CD8+ cells in whole mounted radical prostatectomies samples and characterized the subset of intraepithelial CD103+ and CD39+ cells, which have been previously shown to harbor more cancer specific T-cells.

Materials and Methods: Sixteen cases of whole mounted radical prostatectomy samples were used to asses CD4, CD8, CD103 (all abcam) and CD39 (Sigma) expression by IHC. Location of positive cells within epithelium and stroma in tumor and benign prostatic tissue was evaluated. The results were confirmed using QuPath Software, for positive detection of immune cells by color deconvolution and threshold adjustments.

Results: The CD4 and CD8 staining demonstrated that most of the immune cells were present in the stroma but not within the malignant glands. The whole prostate sections (mean size 51.4 × 44mm) generated around 1650 ROI's per/case. All the ROI's were included in the analysis. CD4+ and CD8+ were evaluated in whole gland vs tumor area. No significant difference = of CD4+ and CD8+ cell density was seen between whole prostate area and tumor areas (CD4+ 2.9 vs 1.9%) and (CD8+ 1.3 vs 1.2%). The density of CD4+ and CD8+ cells in tumors vs benign gland was approximately the same (means 0.93 for both). For the CD103+ analysis, the positive cells were rarely found in the tumor epithelium in comparison with the benign glands (mean 1.09 vs 5.41; representing 2 vs 9.3% of the total lymphocytes). The CD39 stain showed positive cells mostly at the benign epithelium. No correlation was found between our results and Gleason grade, stage or prognosis.

Conclusion: Our study suggests the possibility that in solid cancers such as prostate cancer, immune cells may not be able to reach appropriately the malignant epithelium with only a small subset of T cells reaching their target. This may have important clinical implication for the success of the immune therapy in this group of patients. Further studies are being performed to deeply characterized the tumor infiltrating cell population.

Citation Format: Beatriz Walter, Billel Gasmi, Xu Naizen, Vladimir Valera, Peter Pinto, Maria J. Merino. Where do the T lymphocytes go in prostate cancer: Correlation with CD103, CD39 expression and clinical pathological parameters [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4984.
Abstract 4993: Label-free metabolic imaging of T cell response to the tumor microenvironment
Authors: VanDommelen Ava , Gillette Amani , Patankar Manish S. , Skala Melissa
Published: September 2020

Numerous factors including low pH, low glucose, and high lactic acid levels in tumors create an immunosuppressive tumor microenvironment. This immunosuppression is partially due to the metabolic requirements of T cell activation, which are difficult to meet in the tumor microenvironment. The goal of this study is to use label-free optical metabolic imaging (OMI) of the metabolic co-factors NAD(P)H and FAD in CD3-conjugated (activated) and control T cells to monitor metabolic changes of T cells within tumor micro environmental conditions. Single-cell measurements include the optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD), which measures the oxidation-reduction state of the cell, and the mean fluorescence lifetimes (τm) of NAD(P)H and FAD, which measure enzyme-binding activity in the cell. Jurkat T cells were conjugated with CD3 antibody over a two-day incubation period to achieve activation. Control and CD3-conjugated cells were exposed to environments with increasing concentrations of lactic acid (0-20 mM), hydrochloric acid (HCl; pH 6.4- 7.4), and glucose deprivation to mimic the tumor microenvironment. OMI monitored metabolic changes on a single-cell level without confounding factors associated with cell surface marker labeling. Treatment with lactic acid resulted in a significant dose-dependent decrease in the FAD τm and optical redox ratio (p<0.005). Along with changes in metabolism, OMI also resolved impaired mobility of the T cells with increasing lactic acid concentrations. Lactic acid affects both pH and metabolism, so cells were separately exposed to different concentrations of HCl (pH 6.4 - pH 7.4) to monitor pH effects alone. In control cells, the optical redox ratio decreased with decreasing pH (p<0.005). Conversely, in the presence of CD3, the optical redox ratio increased with decreasing pH (p<0.005). CD3 conjugation in glucose deprived environments also resulted in increases in both optical redox ratio and NAD(P)H τm. Cellular viability assays indicate apoptosis only in the lowest pH environment (pH 6.4, p<0.005). These studies indicate that OMI is a promising label-free approach to investigate the effects of the tumor microenvironment on T cell metabolism, activation, and drug response.

Citation Format: Ava VanDommelen, Amani Gillette, Manish S. Patankar, Melissa Skala. Label-free metabolic imaging of T cell response to the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4993.
Abstract 500: Discovering epigenetic regulators of cells states in triple negative breast cancers
Authors: Burnett Joseph Patrick , Johnson Garrett , Truchan Nathan , Brooks Michael , Wicha Max S. , Sun Duxin
Published: September 2020

The implementation of targeted therapies have been instrumental in achieving clinical responses for ER, PR, and HER2 receptor expressing breast cancers. However, triple negative breast cancers in themselves do not respond to such therapies and are highly heterogeneous diseases with significantly worse progression and survival. Our preliminary data and literature have also shown that in TNBC different cancer cell subpopulations are present in any given tumor, each with its own unique chemo-response and resistance. To discover novel targets regulating cancer cell states in TNBCs, we have created a reporter cell line using CRISPR/Cas9 mediated knock-in of fluorescent reporter proteins after the endogenous loci of major cell type markers in SUM149. Using this reporter line allows for the direct visualization of ALDH1A3+/- and CD24+/- cells, facilities tracking of distinct cell state transitions in a given cell, and allows for lineage tracing following cell divisions. These reporter cells were subjected to HTS-FACS analysis after exposure to a siRNA library of known epigenetic regulators. Following secondary screening, three siRNA targets are able to uniquely inhibit distinct subpopulations of cells within the cell line DCAF1 (ALDH-/CD24- cells), MLL2 (ALDH-/CD24+ cells), and TRIM24 (ALDH+ cells). In combination, these siRNA's proportionally eliminated all cell types. Due to the inherent heterogeneity of TNBCs we then sought to determine if efficacy was consistent or unique across different cell lines with varying percentages of CD44, CD24, and Aldefluor+ cell populations. Most notably, in MDA-MB-468 which contains similar cell population percentages to SUM149 each siRNA was capable of reducing cell proliferation. Similarly, TRIM24 was also capable of reducing cellular proliferation in BT20, MLL2 in MDA-MB-231, and DCAF1 in HCC1937. Knockdown of TRIM24 consistently reduced Aldefluor+ cells in MDA-MB-468 and BT20 cells. Finally, coupled with sc-Omics technologies we utilized this efficacy data identify a subset of TNBC patients likely to respond to therapies which will target these genes in the future.

Citation Format: Joseph Patrick Burnett, Garrett Johnson, Nathan Truchan, Michael Brooks, Max S. Wicha, Duxin Sun. Discovering epigenetic regulators of cells states in triple negative breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 500.
Abstract 5031: Salmonella-mediated suppression of Akt/mTOR signaling inhibits metastasis via downregulation of Snail expression in melanoma
Authors: Pangilinan Christian R. , Lee Che-Hsin
Published: September 2020

The expression of EMT inducer Snail in many cancer types has been implicated in high-grade malignancies and correlated with poor prognosis. Tumor-intrinsic Snail not only has direct effects on metastasis but also impacts tumor microenvironment by altering secretomes. Our previous reports demonstrated that systemic administration of Salmonella significantly reduced tumor growth and metastatic nodules of tumor-bearing mice, in part, by coaxing changes in the tumor microenvironment. These led us to hypothesize that Salmonella targets metastasis inducers. Here, we use B16F10 and K1735 mouse melanoma cells to investigate the anti-metastatic potentials of Salmonella against melanoma. Immunoblotting revealed that the expression of Snail in both B16F10 and K1735 melanoma cell lines was significantly downregulated following Salmonella treatment in a dose-dependent manner. Likewise, the phosphorylation of Akt and mTOR was significantly reduced after Salmonella treatment. Our findings further revealed that in vitro metastatic activities of Salmonella-treated melanoma cells were significantly inhibited as demonstrated in the wound-healing and Transwell assays. When transfected with constitutively active AKT plasmid, B16F10 and K1735 markedly increased the phosphorylation of Akt and mTOR accompanied by an elevated Snail expression. Interestingly, Salmonella treatment reversed the Akt/mTOR and Snail upregulation of cells overexpressing Akt. Our data therefore suggest that Salmonella significantly reduces melanoma metastasis by downregulating Snail.

Citation Format: Christian R. Pangilinan, Che-Hsin Lee. Salmonella-mediated suppression of Akt/mTOR signaling inhibits metastasis via downregulation of Snail expression in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5031.
Abstract 5034: Effect of estrogen on heteronemin-induced anti-proliferation in breast cancer cells
Authors: Li Zi-Lin , Pan Yi-shin , Huang Tung-Yung , Shih Ya-Jung , Chen Yi-Ru , Tang Heng-Yuan , Whang-Peng Jacqueline , Lin Hung-Yun , Davis Paul J. , Wang Kuan
Published: September 2020

Estrogen, 17β-estradiol (E2) has multiple functions in breast cancers including stimulating cancer growth and interfering with chemotherapeutic efficacy. Heteronemin, a marine sesterterpenoid-type natural product, processes anti-proliferative effect in cancer cells. In the current study, we investigate the antitumor mechanism of heteronemin in breast cancer cells and further explore its molecular targets. Heteronemin exhibited potent cytotoxic effects against breast cancer cells. On the other hand, E2 stimulated cancer cell growth. Heteronemin and E2 showed different effects on gene expression of proliferation, angiogenesis or growth factor receptors in breast cancer cells. NanoString® analysis was further used to detect mRNAs, heteronemin repressed gene expression of transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (SMAD) and PCNA, whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. The results indicate that heteronemin was able to modulate cell adhesion, expression of extracellular matrix (ECM) receptors, TGF-β pathway, cell motility, membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in breast cancer cells. However, those effects were partially reversed by E2. Furthermore, Heteronemin and E2 altered several signaling transduction pathways. In summary, this study provides insight into the complex pathways by which anti-proliferation is induced by heteronemin in E2-depleted and -repleted environments.

Citation Format: Zi-Lin Li, Yi-shin Pan, Tung-Yung Huang, Ya-Jung Shih, Yi-Ru Chen, Heng-Yuan Tang, Jacqueline Whang-Peng, Hung-Yun Lin, Paul J. Davis, Kuan Wang. Effect of estrogen on heteronemin-induced anti-proliferation in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5034.
Abstract 5060: Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy
Authors: LIU Wai Nam , FONG Shin Yie , TAN Wilson Wei Sheng , TAN Sue Yee , LIU Min , IYER Narayanan Gopalakrishna , LIM Darren Wan-Teck , CHEN Qingfeng
Published: September 2020

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is common in Southern China and South-East Asia. Immune checkpoint blockade (ICB) is being studied in NPC and initial monotherapy ICB studies show early promise. Currently, lack of representative NPC models limit the preclinical studies to evaluate the efficacy and safety of novel ICB and combination regimens. Hence, there is a need to develop and characterize a mouse model with human immune system for testing immunotherapy. Here, we engrafted NPC-PDX in immunodeficient NSG mice, with a successful growth rate of 16.7% (6 out of 36), and humanized mouse NPC-PDX model was established subsequently. Of note, EBV latent membrane protein-1 was retained in the NPC in both NSG and humanized mice as revealed by IHC staining. There was an increase in tumor-infiltrating CD4+ T cells and a decrease in CD8+ cytotoxic T cells in humanized mice, when compared to the immune cells profile in circulation. Intriguingly, the expression levels of various exhaustion markers, namely PD-1, CTLA4 and TIM-3 were upregulated, particularly in the tumor-infiltrating T cells. Therefore, the use of ICB, either alone or in combination, may restore the function of anti-tumor immune effector cells. Taken together, we have established a humanized mouse NPC-PDX model, which plausibly provides a robust platform to test for the efficacy and safety of ICB and combination therapy regimens.

Citation Format: Wai Nam LIU, Shin Yie FONG, Wilson Wei Sheng TAN, Sue Yee TAN, Min LIU, Narayanan Gopalakrishna IYER, Darren Wan-Teck LIM, Qingfeng CHEN. Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5060.
Abstract 5062: Modeling NK cells activities in full human immune system mouse models
Authors: Yin Kiave Yune Ho Wang
Published: September 2020

The cytotoxic potential of NK-cells for the elimination of cancer cells represents the basis for many important ongoing clinical efforts. These efforts can now be supported by the humanized immune system mouse models (Hu-Mice), which offer an improved in vivo system for translating therapies to the clinic.

Using hydrodynamic injection of human IL-15, TransCure bioServices has developed a NK boosted hu-Mice (CD34+ engrafted NCG) providing sufficient and functional human NK cells. The transient and physiological expression of IL-15 allow the expansion of human NK cells in the context of a full and stable human immune system bringing numerous advantages compared to the NK infused mouse model in IL15 transgenic mouse models. One week after boost, three times more NK cells are present in peripheral blood of IL15-boosted hu-Mice and this level is stable up to five weeks, providing sufficient time to engraft tumor cells and test drug efficacy. A better infiltration of the tumor by human NK cells is also visible in IL-15 boosted mice. Furthermore, the efficacy of ADCC dependent drugs (administered prophylactically or therapeutically) have been highlighted in IL-15 boosted hu-Mice engrafted with melanoma and multiple myeloma cells.

In conclusion, the IL-15 boosted hu-Mice provide a reliable and validated in vivo model to better assess the efficacy and MOA of drug depending on NK cell activity.

Citation Format: Kiave Yune Ho Wang Yin. Modeling NK cells activities in full human immune system mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5062.
Abstract 5079: Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy
Authors: Agarkova Irina , Buschmann Nicole , Strebel Silvan , Wolf Armin , Chiovaro Francesca
Published: September 2020

Background: Immunotherapy has revolutionized cancer treatment, but clinical success is still limited to a subset of patients, underlining the need for novel therapeutic targets. Available ex vivo animal platforms for drug screening show limitations due to only partial recapitulation of tumor-intrinsic features and of the interactions of tumors with stromal/immune cells. Here, we review our 3D InSightTM tumor models, a complex in-vitro platform allowing to study the efficacy of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) and to acquire insights on the modulatory capacity of immune checkpoint inhibitors.

Aim: In the present study we have developed a 3D tumor - immune cell coculture model and evaluated its application to study the effect of immune checkpoint inhibitors on tumor viability, cytokine secretion, and immune cell infiltration.

Material and Methods: Lung carcinoma GFP-A549 cells were cultured with dermal fibroblasts and PBMCs in Akura 96/384-well plates. Immune cells were pre-treated using different activation protocols to induce priming and pT-cell exhaustion. We assessed the effect of PD-1 inhibitors in combination with cytokines for potential synergistic anti-cancer effects. Killing activity and effector function of T cells were evaluated by measuring tumor microtissue size by automatic stage fluorescence microscopy and measuring release of IL-6, TNF, IFNγ and GM-CSF with a MAGPIXTM Luminex system over time. Immune tumor infiltration was quantified using fluorescent staining of CD3+ cells and the effect of the immune cell attack on the tumor cells was assessed by IHC staining.

Results: Combination treatment with cytokines and anti-PD-1 led to higher release of IL-6/TNF/IFNγ/GM-CSF and increased tumor microtissue infiltration by CD3+ lymphocytes, stronger expression of Granzyme B, resulting in enhanced tumor killing and apoptosis.

Conclusion: Our 3D InSightTM tumor models can be successfully employed for optimal high throughput screening of biologics to unveil the efficacy and anti-tumor efficacy of immune-targeting therapeutic agents.

Citation Format: Irina Agarkova, Nicole Buschmann, Silvan Strebel, Armin Wolf, Francesca Chiovaro. Immuno-competent 3d in vitro tumor models as novel platform to assess combinatorial biologics therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5079.
Abstract 510: Novel glutamine antagonist JHU-395 suppresses MYC-driven medulloblastoma growth and induces apoptosis
Authors: Pham Khoa , Maxwell Micah , Sweeney Heather , Alt Jesse , Rais Rana , Slusher Barbara S. , Eberhart Charles G. , Raabe Eric H.
Published: September 2020

Medulloblastoma is the most common malignant pediatric brain tumor. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. MYC upregulates glutamine metabolism, and medulloblastoma tumors with evidence of high glutamate have a poor prognosis. We modified the naturally occurring glutamine analog 6-diazo-5-oxo-l-norleucine (DON) to increase its lipophilicity and its stability in plasma, creating a prodrug termed JHU-395. We hypothesized that this prodrug would have enhanced cell penetration compared to DON and would kill MYC-expressing medulloblastoma. JHU-395 treatment caused decreased growth and increased apoptosis in human MYC-expressing medulloblastoma cell lines at lower concentrations than DON. In D283MED for example IC50 for DON is 20 uM while JHU 395 IC50 is 1 uM (p value < 0.05). Similar results were obtained with other high-MYC medulloblastoma cell lines, including D425MED and D341MED. JHU-395 induced apoptosis at low micromolar concentrations as measured by cleaved PARP Western blot and cleaved caspase 3 (CC3) immunofluorescence (60% CC3+ cells compared to 30% CC3+ in control p<0.05). Parenteral administration of JHU-395 in Nu/Nu mice led to the accumulation of micromolar concentrations of DON in brain as measured by mass spectrometry. These data suggest that JHU-395 may have activity against high MYC medulloblastoma in vivo, and testing this prodrug against MYC-driven medulloblastoma orthotopic xenografts is currently underway.

Citation Format: Khoa Pham, Micah Maxwell, Heather Sweeney, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Novel glutamine antagonist JHU-395 suppresses MYC-driven medulloblastoma growth and induces apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 510.
Abstract 5139: An atlas of perturbed functional proteomics profiles of cancer cell lines
Authors: Zhao Wei , Li Jun , Chen Mei-Ju , Akbani Rehan , Lu Yiling , Mills Gordon , Liang Han
Published: September 2020

In recent years, tremendous efforts have been made to systematically characterize the molecular profiles of tumor tissues from individuals with cancer, laying a critical foundation for elucidating the molecular basis of tumorigenesis and developing biomarker-based diagnostic, prognostic and therapeutic approaches. In particular, cancer genomic data at the DNA or RNA level are being accumulated at an unprecedented speed. However, it remains to be a big challenge in cancer research to systematically understand causality and mechanisms underlying the behaviors of cancer cells. To address it, perturbation experiments are a very powerful approach in which the cells are first modulated by perturbagens and the downstream consequences are then monitored. Recently, large-scale compendia of the phenotypic and cellular effects of perturbed cancer cell lines have been established. However, similar resources for the proteomic responses of perturbed cancer cell lines have yet to be established. Reverse-phase protein arrays (RPPAs) is a powerful targeted functional proteomics approach to studying cancer mechanisms, biomarkers and therapies. This quantitative antibody-based assay is able to assess a large number of protein markers in many samples in a cost-effective, sensitive manner. More recently, we have applied this technology to quantify the protein expression levels of large patient cohorts and cancer cell lines (>8,000 patient samples of 32 cancer types from The Cancer Genome Atlas, >650 cell lines across 19 lineages). Here, using RPPAs, we have generated and compiled the perturbed functional proteomic profiles of >12,000 cancer cell line samples in response to >150 drug compounds and other perturbagens using reverse-phase protein arrays. We show that integrating protein response signals substantially increases the predictive power for drug sensitivity and gains insights into the mechanisms of drug resistance. We build a comprehensive map of “protein-drug” connectivity and develop an open-access, user-friendly data portal for community use. Our study provides a valuable proteomic resource for a broad range of quantitative modeling and biomedical applications.

Citation Format: Wei Zhao, Jun Li, Mei-Ju Chen, Rehan Akbani, Yiling Lu, Gordon Mills, Han Liang. An atlas of perturbed functional proteomics profiles of cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5139.
Abstract 5161: Validation of drug candidates targeting dormant cancers in live cells as a key for successful translation into the clinic
Authors: Belenky Alexei , Kuznetsova Alexandra , Frid Michael , Chapovsky Felix , Duey Marc , Vilenchik Maria
Published: September 2020

Thermal shift assay was utilized to demonstrate target engagement of drug candidate compounds to their specific targets. We explored novel cancer dormancy targets: DYRK1B and AXL kinases and assessed interaction with corresponding small molecule inhibitors. We demonstrated the correlation of direct target binding with ASMS (affinity selected mass spectrometry) and mechanism of action with cell-based phenotypic assays (using flow cytometry) for previously identified and current clinical candidates.

We demonstrated that the above described approach can characterize and potentially predict clinical activity of novel molecules. Herein we investigated DYRK1B inhibitors from an azaindole-quinoline series. It was shown earlier that these molecules have picomolar activity in in vitro assays against DYRK1 kinases (DYRK1B and DYRK1A) and now we confirmed their DYRK1B specificity in cells as well as their activity against dormant cancers.

We suggest that described inhibitors are valid clinical candidates for targeting cancer dormancy based on direct target binding and engagement in live cancer cells. These compounds in combination with chemotherapeutic agents, have the potential to improve patient outcomes and prolong survival.

Citation Format: Alexei Belenky, Alexandra Kuznetsova, Michael Frid, Felix Chapovsky, Marc Duey, Maria Vilenchik. Validation of drug candidates targeting dormant cancers in live cells as a key for successful translation into the clinic [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5161.
Abstract 5165: Novel approach to colorectal cancer drug target discovery in human FFPE tissue using the Adaptive Dynamic Artificial Polyligand Targeting System
Authors: Williams Stephanie , Sima Chao , Hornung Tassilo , Rosenow Matthew , Tinder Teresa , Maher Varun , Kassner Michelle , Ortiz Gerri , Mok Perrin , Helle Nicholas , Logie Stephen , ONeill Heather , Miglarese Mark , Spetzler David
Published: September 2020

Identification of novel biomarkers and drug targets for colorectal cancer remains a critically unmet medical need. Target identification has historically been driven by preclinical models which poorly recapitulate the complex biology of human cancers. We describe a unique approach to drug target identification and comprehensive profiling directly in clinical specimens. Using the Adaptive Dynamic Artificial Polyligand Targeting (ADAPT) system, we aimed to identify such disease specific drug targets using a highly complex library of single-stranded oligodeoxynucleotides (ODNs). To generate the library, the ODNs were enriched using FFPE tissue specimens from pathologically determined colorectal cancer (CRC) and counter-selected against normal adjacent tissue (NAT) regions. Once the specificity of the library was verified via differential binding on independent CRC cases, it was used to affinity purify cognate biding partners from 393 CRC FFPE lysates with appropriate representation of cases with either KRAS, NRAS or BRAF mutations. Greater than 700 proteins were identified by mass spectrometry exclusively in the CRC FFPE tissue lysates and not in the NAT FFPE tissue lysates for all 393 CRC cases. Classification of these proteins into the druggable protein classes revealed G-protein coupled receptors, transporters, voltage-gated ion channels, proteases, and protein kinases. In addition, subsequent verification studies confirmed novel candidate cell surface protein targets appropriate for targeting by antibody-drug conjugates (ADC) or other biologic modalities. mRNA expression corresponding to the 700 potential targets was analyzed in over 1600 CRC FFPE tumor samples and >9000 other cancer types as well as in a broad panel of normal tissues by Whole Transcriptome Sequencing (WTS) and immunohistochemistry (IHC). We conclude that the unbiased ADAPT platform provides a robust and novel approach to drug target discovery with highly selective expression profiles directly from FFPE clinical specimens.

Citation Format: Stephanie Williams, Chao Sima, Tassilo Hornung, Matthew Rosenow, Teresa Tinder, Varun Maher, Michelle Kassner, Gerri Ortiz, Perrin Mok, Nicholas Helle, Stephen Logie, Heather ONeill, Mark Miglarese, David Spetzler. Novel approach to colorectal cancer drug target discovery in human FFPE tissue using the Adaptive Dynamic Artificial Polyligand Targeting System [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5165.
Abstract 5196: CSFR-1R a potential therapeutic target in 4T1 xenograft breast cancer model
Authors: Elia Jeanne , Zhang Hong , Wen Chiaokai , Yang Xifeng
Published: September 2020

The 4T1 cell line is a mouse mammary carcinoma which can mimic stage IV breast cancer in humans. When these cells are xenografted into a BALB/c mouse there is an infiltration of macrophages into the spleen, bone marrow and blood which was observed 11 days after engraftment. Increased cell proliferation was also detected in the spleen and blood. We found that 4T1 cells produced high levels of M-CSF in culture. In this study we attempt to neutralize the secreted cytokine from the tumor cells with anti-M-CSF (clone, A10603L) or to block the receptor with anti-M-CFR (CSF-1R) (clone, AFS98). Mice were xenografted with 4T1 cells then given one dose of 100 ug of neutralizing antibodies to either anti-M-CSF or anti-CSF-1R two days post tumor engraftment. Animals were sacrificed at multiple time points between 1-3 weeks. Treatment with one dose of anti-M-CSF showed minimal to no difference compared to the 4T1 xenograft group. Treatment with anti-CSF-1R showed reduced macrophage infiltration in the bone marrow, spleen and blood at day 11. BrdU incorporation levels dropped in the spleen and blood of these animals. Spleen size and total cell numbers were also reduced by 40% compared to the 4T1 xenograft group.

Citation Format: Jeanne Elia, Hong Zhang, Chiaokai Wen, Xifeng Yang. CSFR-1R a potential therapeutic target in 4T1 xenograft breast cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5196.
Abstract 521: CD45 targeted engineered toxin bodies deplete hematopoietic and malignant cells
Authors: Iberg Aimee , Robinson Garrett L. , LeMar Sara , Dekker Joseph D. , Zhao Jay , Ramos Hilario J. , Singh Melissa M. , Willert Erin K.
Published: September 2020

CD45 is highly expressed on the cell surface of all nucleated hematopoietic cells, including malignant cells of B, T and myeloid lineage. CD45, a receptor tyrosine phosphatase that has important roles in antigen receptor signaling, has multiple isoforms that are differentially expressed on immune cell subsets. Targeting of CD45 with antibody-based therapies, tested in conjunction with reduced dose chemotherapy and radiation regimens, has shown promise in preclinical models and clinical trials as a conditioning therapy for bone marrow transplant (BMT), although these approaches are associated with safety limitations. A single agent, targeted conditioning method could increase patient safety and eliminate genotoxic effects associated with the conditioning regimens. Conditioning therapy by depletion of CD45 positive cells specifically has potential applications in oncology as well as non-oncologic, hematopoietic disease settings where engraftment of donor or modified stem cells could provide benefit. Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Targeted ETBs are being developed to specifically target and destroy CD45 expressing cells. ETBs targeting CD45 specifically bind to, internalize into and then trigger cell death of the target cells in vitro. ETBs have been designed using antibody fragments that recognize all isoforms of CD45. The CD45 targeted ETB has shown high potency in immortalized blood cancer cell lines of T cell, B-cell and eosinophil origin, including leukemia and lymphoma cell lines. Additionally, the ability of the CD45 targeted ETBs to deplete primary cells from healthy donors has been demonstrated. The direct cell kill activity of the CD45 targeted ETBs have the potential to deplete immune cells in the periphery and the bone marrow, as well as to deplete malignant hematological cells expressing CD45. In vivo depletion of human immune cells in murine models by CD45 targeted ETBs will be evaluated.

Citation Format: Aimee Iberg, Garrett L. Robinson, Sara LeMar, Joseph D. Dekker, Jay Zhao, Hilario J. Ramos, Melissa M. Singh, Erin K. Willert. CD45 targeted engineered toxin bodies deplete hematopoietic and malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 521.
Abstract 5212: The benefits of Didox in triple negative breast cancer treatment targeting mutant p53
Authors: Sultana Nahid , Wilson Elizabeth A. , Shah Khyati N. , Elford Howard L. , Faridi Jesika S.
Published: September 2020

Triple-negative breast cancer (TNBC) is an aggressive histological subtype of malignancy with limited treatment options. Frequently, doxorubicin (DOXO)- based chemotherapy is utilized in this patient population due to the lack of molecular targets available. While DOXO is an effective chemotherapeutic agent, its efficacy is limited due to acquired drug resistance and cardiotoxicity. Therefore the identification of other treatment options for TNBC is needed. TNBC is a heterogeneous malignancy, with 70% of cases classified as a basal subtype, which further complicates the search for an effective molecular target. However, it has been identified that 80% of TNBC samples carry TP53 (tumor protein p53) mutations, which leads to a mutant p53 protein no longer able to act as a tumor suppressor and thus failing to halt cell growth. Mutant p53, when present in malignant cells, is far less susceptible to degradation than its wild-type counterpart, and thus accumulates within the cell. As such, the TP53 is an attractive therapeutic target for TNBC. We have previously identified that ribonucleotide reductase M2 (RRM2) is upregulated in TNBC cells and is a key contributor to acquired drug resistance. The ribonucleotide reductase inhibitor Didox has been shown to be a powerful free radical scavenger and iron chelator. Through these mechanisms, Didox alleviates the non-selective cytotoxic effects of DOXO on non-cancerous tissue, including cardiotoxicity and potentiates DOXO's efficacy against malignant cells. We have previously reported that Didox in combination with DOXO effectively treats TNBC by enhancing tumor inhibition and minimizing DOXO-induced heart damage. Our current study shows that Didox alone or in combination with DOXO downregulates mutant p53 protein levels in the mutant p53 TNBC cell lines MDA-MB 468, MDA-MB 231 and BT-20. This effect is notably absent in breast cancer cell lines with wild-type p53, including MCF7, A3B5 and ZR751, where p53 successfully acts as a tumor suppressor allowing for cell cycle arrest or apoptosis after DNA damage. We hypothesize that mutant p53 is a valid therapeutic target in TNBC. As shown here, the addition of Didox downregulates mutant p53 levels and thus enhances tumor inhibition while alleviating DOXO-associated cardiotoxicity.

Citation Format: Nahid Sultana, Elizabeth A. Wilson, Khyati N. Shah, Howard L. Elford, Jesika S. Faridi. The benefits of Didox in triple negative breast cancer treatment targeting mutant p53 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5212.
Abstract 5230: Real-time monitoring of PROTAC and molecular glue targeted degradation in living cells
Authors: Cosby Neal C. , Riching Kristin M. , Mahan Sarah , Caine Elizabeth A. , Daniels Danette L. , Urh Marjeta
Published: September 2020

The emergence of targeted protein degradation as a broad, new therapeutic modality has greatly expanded the opportunities for treatments of many diseases. Currently, small molecule degrader compounds fall under two major categories; molecular glues and heterobifunctional PROTACs. The two types of compounds both facilitate and induce a ternary complex consisting of an E3 ligase-degrader-target protein, bringing into proximity the machinery proteins required to ubiquitinate and ultimately degrade the target protein. Significant challenges exist in characterization and rank-ordering of degradation compounds in live cells given the differences in dynamics of protein loss and recovery among compounds. Currently, the availability of technologies to interrogate real-time protein degradation is severely lacking. Here, we present a live-cell, luminescence-based technology platform with these capabilities. We employ CRISPR/Cas9 endogenous tagging of target proteins with the small peptide, HiBiT, which has high affinity for and can complement with the LgBiT protein to produce NanoBiT luminescence. This allows for sensitive detection of endogenous protein levels in living cells, and can also serve as a BRET energy donor to study protein:protein or protein:small molecule interactions. We demonstrate the power of this technology in continuous 24-hour monitoring of endogenous target protein levels, and the ability to quantify key degradation parameters for compound ranking including rate, Dmax, and DMax50. We further show the ability to measure the mechanistic steps important for the degradation including kinetics of PROTAC- or molecular glue-induced ternary complex formation and target ubiquitination. These studies facilitate discernment of individual parameters required for successful degradation, ultimately enabling chemical design strategies for optimization and rank ordering of therapeutic degradation compounds.

Citation Format: Neal C. Cosby, Kristin M. Riching, Sarah Mahan, Elizabeth A. Caine, Danette L. Daniels, Marjeta Urh. Real-time monitoring of PROTAC and molecular glue targeted degradation in living cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5230.