bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5310: Chromatin accessibility landscape and transcriptome of castration resistant prostate cancers reveals novel subtypes and diverse master regulators
Authors: Tang Fanying , Wong Chen Khuan , Cohen Sandra , Lee Cindy , Liu Minwei , Bareja Rohan , Eng Kenneth , Beg Shaham , Puca Loredana , Sternberg Cora , Mosquera Juan Miguel , Beltran Himisha , Sboner Andrea , Chen Yu , Khurana Ekta
Published: September 2020

Castration-resistant prostate cancer (CRPC) is a heterogeneous disease with diverse drivers and mechanisms of resistance to androgen receptor (AR) therapy.

We generated ATAC-seq and RNA-seq data for twenty-four metastatic human prostate cancer organoids and cell lines. Integration of chromatin accessibility profiles and transcriptomes revealed four subtypes: androgen-receptor(AR)-dependent, neuroendocrine, Wnt-dependent and epithelial mesenchymal transition (EMT). The transcriptomic signatures obtained from these four subtypes enable the classification of 100 metastatic prostate cancer patient samples from Institute Precision Medicine (IPM) and 270 published samples from SU2C study, revealing potential therapeutic vulnerabilities. Furthermore, using novel computational algorithms we constructed regulatory networks and identified the master regulators of each subtype. Currently we're carrying out western blot and quantitative PCR to confirm the subtypes of all prostate cancer models we use, and using drug sensitivity test, CRISPR knockout and cell competition assay to validate the functions of candidates in each subtype.

Our study has characterized global chromatin accessibility landscape and transcriptome in the largest number of metastatic prostate cancer models, which revealed novel subtypes and corresponding tumor drivers. Collectively, these organoids, cell lines and matching sequence data provide a resource to the community to study various CRPC models. The molecular classification and corresponding master regulators reveal new drug targets and could potentially guide future therapeutic studies.

Citation Format: Fanying Tang, Chen Khuan Wong, Sandra Cohen, Cindy Lee, Minwei Liu, Rohan Bareja, Kenneth Eng, Shaham Beg, Loredana Puca, Cora Sternberg, Juan Miguel Mosquera, Himisha Beltran, Andrea Sboner, Yu Chen, Ekta Khurana. Chromatin accessibility landscape and transcriptome of castration resistant prostate cancers reveals novel subtypes and diverse master regulators [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 5310.
Abstract 5323: Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis
Authors: Fifield Amber , Ho Thai H. , Faigel Douglas O. , Svarovsky Sergei , Sergienko Eduard , Chung Thomas C. , Lake Douglas F.
Published: September 2020

Purpose: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme over-expressed by tumor cells. Our group and others have shown that QSOX1 is involved in invasion and metastasis. We previously reported that a small molecule, SBI-183, inhibited the enzymatic activity of QSOX1 and suppressed tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo. In our current work we tested SPX-009, an analog of SBI-183 with increased potency, to suppress invasion and metastasis in vitro and in vivo.

Methods: Commercially available analogs for SBI-183 were screened in a cell-free fluorescent screening assay for the ability to inhibit QSOX1. Analog compounds that inhibited QSOX1 in a concentration-dependent manner were then tested for the ability to inhibit growth and invasion of triple negative breast cancer (TNBC) cells (MDA-MB-231), sarcomatoid kidney cancer cells (RCJ-41T2) and pancreatic adenocarcinoma cells (MIAPaCa2). MTT assays were used to measure the effect of the compounds on tumor growth. 2D invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion. One potent analog (SPX-009) was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts.

Results: Three of 53 analogs inhibited the enzymatic activity of QSOX1 at levels similar to the parent compound. However, one of those compounds, SPX-009, suppressed invasion of breast, kidney and pancreas tumor cells in 2D and 3D invasion assays at >10-fold lower concentrations than the parent compound (SBI-183). In a non-metastatic xenograft of kidney cancer SBI-183 suppressed primary kidney tumor growth by 51%, but in a metastatic xenograft breast cancer model (MDA-MB-231-luc TNBC), SBI-183 reduced lung metastasis by 76% compared to control. Human-mouse xenograft studies are ongoing with the more potent analog (SPX-009) and will also be presented.

Conclusions: Chemical inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. This finding suggests that combining a QSOX1 inhibitor with a cytotoxic agent might provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents.

Citation Format: Amber Fifield, Thai H. Ho, Douglas O. Faigel, Sergei Svarovsky, Eduard Sergienko, Thomas C. Chung, Douglas F. Lake. Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis [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 5323.
Abstract 5337: Analysis of drug combinations with the PKC inhibitor IDE196 support dual MEK and PKC inhibition as a rational combination in metastatic uveal melanoma
Authors: Frey Christian R. , Wagle Marie-Claire , Vaidya Kedar , Hambleton Julie , Lackner Mark , Mounir Zineb
Published: September 2020

Uveal melanoma (UM) is the most common primary intraocular tumor. The majority (>90%) of uveal melanoma harbor activating mutations in the GNAQ and GNA11 Ga subunits of G protein coupled receptors. These genetic driver mutations, which are also detected in other solid tumors, lead to continuous activation of protein kinase C (PKC) and downstream signaling pathways such as the mitogen-activated protein kinase (MAPK) signaling pathway. Inhibition of PKC with small molecule inhibitors is an attractive therapeutic approach in UM. IDE196 is a selective and potent protein kinase C inhibitor targeting the novel (δ, ϵ, η, θ) and classical (α, β) PKC isoforms and has shown anti-tumor activity as monotherapy in patients with metastatic uveal melanoma (MUM). Inhibiting downstream signaling pathways in addition to PKC inhibition with IDE196 may increase the anti-tumor activity in MUM patients. To address this question, we assessed pathway-focused drug combination partners for their potential to exert synergy with IDE196. In this study, we evaluated baseline-expression of different PKC isoforms in uveal melanoma or control cell lines and the response to PKC inhibition with IDE196 by western blotting (WB). We measured the sensitivity to several drug combinations utilizing a concentration matrix in a 3-day cell titer glow assay to measure cell viability and determined synergy of drug combinations. Modulation of PKC, MAPK pathway activity and other downstream markers were assessed by WB. The evaluated uveal melanoma cell lines expressed different phospho-PKC isoforms and were sensitive to increasing concentrations of IDE196 as demonstrated by decreased levels of PKC substrate p-MARCKS and reduced cell viability. Furthermore, we did not detect synergy of IDE196 with cyclin-dependent kinase (CDK) 4/6 inhibitors Palbociclib or Ribociclib in viability assay. In contrast, combination of IDE196 with MEK inhibitors Binimetinib or Trametinib showed synergy at clinically relevant doses for each compound. The observed synergy of IDE196 with MEK inhibitors is further substantiated by modulation of MARCKS and MAPK pathway activity as measured by WB. Additional work is underway in in vitro as well as in vivo models to clarify the potential benefit of combining IDE196 with MEK inhibitors in the clinic.

Citation Format: Christian R. Frey, Marie-Claire Wagle, Kedar Vaidya, Julie Hambleton, Mark Lackner, Zineb Mounir. Analysis of drug combinations with the PKC inhibitor IDE196 support dual MEK and PKC inhibition as a rational combination in metastatic uveal 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 5337.
Abstract 5373: Circulating tumor cell as a predictive marker for immunotherapy in advnaced non-small cell lung cancer
Authors: Park Cheol-Kyu , Yoon Joon-Young , Kim Min-Suk , Koh Bo-Gun , Cho Hyun-Ju , Oh In-Jae , Kim Young-Chul
Published: September 2020

Background: Circulating tumor cells (CTCs) have potential to provide minimally invasive way for the response monitoring of various cancer. This study aimed to investigate the feasibility of CTCs as a predictive marker for anti-PD-L1 immunotherapy in patients with advanced non-small cell lung cancer (NSCLC).

Methods: This study included 37 patients with advanced NSCLC receiving atezolizumab monotherapy as second-line or more from July to November 2019. Blood was collected in a K2-EDTA tube and CTCs were isolated and enriched by using CD-PRIMETM system which is antibody-independent size-based isolation method. By Bioview CCBS system, total CTCs (tCTCs) were identified by a sum of single EpCAM/CK-positive (sCTCs) and double EpCAM/CK-CD45-positive cells (dCTCs).

Results: Among 26 response-evaluable patients, objective response rate (ORR) and disease control rate (DCR) were 11.5% (3/26) and 57.7% (15/26), respectively. At cycle 1 (C1), patients with progression (PD) showed higher sCTC count (median, 8.6 vs. 4.3, p=0.065) and sCTC-to-tCTC ratio (median %, 35.9 vs. 9.8, p=0.024) than those without PD. During treatment, tCTC and sCTC count showed significant increase from C1 to C2 (p=0.019 and p=0.022). The subgroup with high sCTC increase from C1 to C2 (more than 2-folds) showed higher DCR than subgroup with low sCTC increase (less than 2-folds) (88.9% vs. 44.4%, p=0.066). Patients with high sCTC-to-tCTC ratio at C1 showed worse progression-free survival (median, 2.1 months) than those with low sCTC-to-tCTC ratio (not reached, hazard ratio 2.87, 95% confidence interval: 1.06-7.75, p=0.082).

Conclusion: The baseline sCTC count and sCTC increase (more than 2-folds) at the first 3-week after atezolizumab could be potential biomarkers to predict the disease progression and poor survival in advanced NSCLC.

Citation Format: Cheol-Kyu Park, Joon-Young Yoon, Min-Suk Kim, Bo-Gun Koh, Hyun-Ju Cho, In-Jae Oh, Young-Chul Kim. Circulating tumor cell as a predictive marker for immunotherapy in advnaced non-small cell lung cancer [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 5373.
Abstract 5420: Multiomics approach identifies the SP8-FGF8 axis as an important promoter of metastatic features in hepatoblastoma
Authors: Wagner Alexandra , Häberle Beate , Schmid Irene , Vokuhl Christian , von Schweinitz Dietrich , Kappler Roland
Published: September 2020

Introduction

Hepatoblastoma (HB) is the most common pediatric liver tumor with a general good prognosis, but if associated with multifocal growth, vessel invasion or metastasis, outcome is still poor. The molecular mechanisms causing this high-risk profile are still unknown. The aim of our study was to gain insight into the neoplastic continuation towards more aggressive tumor phenotypes and to aid identification of biomarkers for high-risk patients.

Experimental procedures

Tumor specimens of seven non-metastasized and four metastasized HB patients were analyzed by RNA-sequencing and the Human Methylation 450 BeadChip microarray to assess global expression and methylation levels. Integration of both datasets identified new candidate genes, which were further verified in a cohort of 34 HB patients using real-time PCR. Gain and loss of function experiments of SP8 were conducted in five HB cell lines to evaluate its effects on proliferation, mobility, self renewal, migration and invasion. Chromatin-immunoprecipitation was used to assess binding of SP8 to the FGF8 promoter. Kaplan-Meier estimates of specific survival time in various groups were compared using the log-rank Mantel-Cox test.

Results

Global integration of expression and methylation levels identified the transcription factor SP8 and the fibroblast growth factor FGF8 as significantly upregulated genes accompanied with strong methylation changes in metastatic HB. In an independent cohort of 34 HB patients expression of both candidates was highly correlated (R² = 0.74, P < 0.0001) and could be positively associated with metastasis, the aggressive C2 subtype of the 16-gene signature and poor overall survival. Chromatin-immunoprecipitation identified binding of SP8 to the FGF8 promoter. Following SP8 overexpression in liver tumor cells, we found a significant increase in cell motility, self renewal and invasive growth together with the acquisition of a mesenchymal phenotype. In turn, CRISPRi-mediated knockdown of FGF8 rescued the phenotype.

Conclusions

We showed that elevated levels of SP8 induced cell motility, self renewal and mesenchymal characteristics by targeting FGF8, thereby implicating an important functional role of the SP8-FGF8 axis for the progression and metastasis of malignant childhood liver tumors. Moreover, SP8 might serve as a new biomarker in metastatic HB with poor prognosis.

Citation Format: Alexandra Wagner, Beate Häberle, Irene Schmid, Christian Vokuhl, Dietrich von Schweinitz, Roland Kappler. Multiomics approach identifies the SP8-FGF8 axis as an important promoter of metastatic features in hepatoblastoma [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 5420.
Abstract 5483: An image-based mechanistic computational model for early prediction of organ-confined untreated prostate cancer growth
Authors: Lorenzo Guillermo , Hughes Thomas J. , Reali Alessandro , Gomez Hector , Yankeelov Thomas E.
Published: September 2020

According to the American Cancer Society, prostate cancer (PCa) is the most common newly-diagnosed cancer and the second leading cancer-related cause of death among men in the US in 2019. The current clinical protocols of PCa are based on two key strategies: regular screening of men over fifty and patient triaging in risk groups. Prostatic tumors are usually detected at an early organ-confined stage, which poses a low to intermediate risk to the patient and may not produce any symptoms or require treatment for long time. However, most patients are prescribed a radical treatment immediately after diagnosis (e.g., surgery or radiation therapy), which may adversely impact their lives without necessarily prolonging their longevity or quality of life. Moreover, some patients who initially delay their treatment ultimately succumb to PCa due to an inaccurate initial diagnosis. Thus, while regular screening enables the detection of the majority of tumors at an early and mild stage, the limited individualization of patient monitoring and treatment has led to significant rates of both overtreatment and undertreatment.

To overcome these fundamental limitations in the clinical management of PCa, we propose the use of a mechanistic mathematical model for which we can perform computer simulations to forecast patient-specific tumor growth. This computational model is based on a set of partial differential equations that describe the main mechanisms involved in organ-confined PCa growth. The model is parameterized using longitudinal multiparametric magnetic resonance (MR) images and the available clinical data for each patient. Tumor growth is simulated over the patient's prostate extracted from T2-weighted MR images. We use isogeometric analysis to accurately and efficiently address the computational challenges arising in this application.

Our preliminary simulation results show that our computational technology can predict tumor growth and associated serum Prostate Specific Antigen (PSA, a key biomarker in clinical management of PCa) with reasonable accuracy. We also explore the potential of model parameters and variables to characterize tumor aggressivity. Thus, we believe that our imaging-based modeling approach could be a promising tool capable of being implemented in current PCa protocols to assist physicians in the clinical management of PCa.

Citation Format: Guillermo Lorenzo, Thomas J. Hughes, Alessandro Reali, Hector Gomez, Thomas E. Yankeelov. An image-based mechanistic computational model for early prediction of organ-confined untreated prostate cancer growth [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 5483.
Abstract 5488: Prediction of target receptor occupancy for ALX148, a CD47 blocker, using mechanistic PK/RO modeling
Authors: Vasileva Elena , Demin Oleg
Published: September 2020

Background: ALX148 is one of the anti-CD47 antibodies that are currently in clinical development. Understanding the target receptor occupancy (RO) in tumor can help with dose selection for ALX148 and other anti-CD47 antibodies. However, RO can be challenging to determine through direct experimental measurements. A mechanistic PK/RO model was developed to predict CD47 occupancy in tumor tissues and periphery for ALX148 based on its properties (PK, structure, and affinity).

Methods: Developed mechanistic PK/RO model describes PK of ALX148 in blood plasma, distribution to tumor tissues of Non-Hodgkin lymphoma (lymph nodes, spleen), non-linear clearance of ALX148 due to the binding with CD47 and further complex internalization. RO was described on red blood cells and tumor cells. The model takes into account CD47 expression (molecules per cell) and a number of cells expressing CD47. Parameters were fitted against clinical PK data. Clinical data on RO in the periphery was used for model validation.

Results: The model successfully described ALX148 clinical data on PK and RO in blood plasma. Concentrations in lymph nodes and spleen were predicted to be similar and in approximately 3 times lower than in blood plasma. Such high concentration in tumor tissues is caused by small size of ALX148 (molecular weight is 78 kDa). Dose and regimen chosen for combination treatment (10 mg/kg QW) result in 90-95% trough steady-state RO in tumor tissues. The model predicted that trough steady-state RO in lymph nodes and spleen was more than 95% after administration of maximal dose 30 mg/kg Q2W.

Conclusions: A mechanistic PK/RO model was built to capture ALX148 clinical PK data and then predicted more than 90% RO at the dose used in combination treatment cohort. This approach has the potential to be used to inform dose selection for other anti-CD47 antibodies taking into account their specific features as binding properties, size, etc.

Citation Format: Elena Vasileva, Oleg Demin Jr. Prediction of target receptor occupancy for ALX148, a CD47 blocker, using mechanistic PK/RO modeling [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 5488.
Abstract 5490: Scales and dynamics of intratumor heterogeneity
Authors: Ferrall-Fairbanks Meghan C. , Kimmel Gregory J. , Altrock Philipp M.
Published: September 2020

Current dogma stipulates that more heterogenous tumor cell populations results from compounding genetic and epigenetic changes and instability, ultimately driving unfavorable outcomes for these patients. Nonetheless, some cancers, including many pediatric cancers and some leukemias, have limited genomic diversity. As a result, we have a limited number of biomarkers available to stratify patients into high versus low-risk groups by their mutational cellular diversity or heterogeneity. For these cases, we have developed a method to quantify intratumor heterogeneity, for example at the transcriptomic level, using a generalized diversity index (GDI) from ecology. This index allows control of a scaling parameter called the order of diversity, q, to tailor the scale or resolution of the cellular diversity to a given context. However, the question remains, what role does the order of diversity play in the context of relative tumor age or disease stage? We show that the order of diversity parameter allows to either emphasize clonal richness (at low values of q), while high values shift the analysis toward the abundance of potential drivers of the tumor evolution. To gain deeper quantitative insight, we use evolutionary dynamics approaches that model the dynamics of heterogeneous cell populations over time, to understand how a patient's tumor diversity could emerge, persist, and change over time. Applying the replicator-mutator dynamics framework, we explore the effects and magnitude of frequency-dependent (positive or negative) selection on GDI. These analyses suggest that GDI often peaks at intermediate time points and, when run over a sufficiently long timescale, they reduce as the tumor population presumable becomes clonally dominant or otherwise stable before a major transition (e.g. metastasis). We explore GDI changes over time further in vitro in sequential single-cell RNA sequencing samples of fused breast cancer cells, where at earlier passages after fusion, the GDI peaks, and at later passages after fusion the GDI returns to levels similar to that of the parental breast cancer cell lines. Overall, GDI, as a means to quantify intratumor heterogeneity at select time points, can be a powerful tool to stratify high-risk patients. Our analyses show how computational analyses and mathematical modeling tools can be coupled with clinical and experimental data to understand eco-evolutionary dynamics of a patient's tumor.

Citation Format: Meghan C. Ferrall-Fairbanks, Gregory J. Kimmel, Philipp M. Altrock. Scales and dynamics of intratumor heterogeneity [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 5490.
Abstract 5548: Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line
Authors: Cerqueira Otto Luiz , Salomon Maria Alejandra , Cardoso Elaine Cristina , Tortelli Tharcisio Citrangulo , Strauss Bryan Eric
Published: September 2020

Immunoevasion adopted by tumor cells is an important cancer hallmark and understanding of its mechanisms has generated promising therapeutic approaches. In this context, triggering immunogenic cell death (ICD) is a promising idea since it alarms the immune system by attracting a myriad of cell populations that promote innate and adaptive immune responses. Since melanomas preserve wild-type p53 in over 80% of cases, our group developed a gene therapy strategy that utilizes an adenoviral vector with a p53-responsive promoter (PG). Here we present a critical advance for our approach, examining the immune activating potential of combined p14ARF and IFN-beta gene transfer in human melanoma cells. Our results indicate that IFN-beta alone or in combination with p14ARF was able to induce massive cell death accompanied by caspase 3/7 activation in the human melanoma cell line SKMEL147. In our animal model of in situ gene therapy, s.c. SKMEL147 tumors were established in Nod-scid mice before intratumoral virus injection, revealing tumor inhibition and improved survival in response to IFN-beta alone or in combination with p14ARF. However, this model cannot reveal immune activation and so far the benefit of p14ARF has not been revealed. Strikingly, three markers of ICD (exposition of calrecticulin, secretion of ATP and IFN-beta) were induced only when cells were treated with combined p14ARF and IFN-beta gene transfer. We then used PBMCs derived from normal donors as a source of DCs and lymphocytes. Coculture of DCs with previously-transduced SKMEL147 exhibit a typical phenotype of activation. Finally, coculture of these exposed DCs with lymphocytes results in high-level secretion of proinflammatory cytokines, followed by the increase of IL-10 in the samples treated with combination of p14ARF and IFN-beta. Collectively, our results indicate that the advantage of p14ARF and INF-beta delivered by our adenoviral system is the induction of cell death in human melanoma cells by an immune-activating mechanism. Future studies will include examination of this phenomenon in tumors from melanoma patients and using their own PBMCS. In parallel, the use of PDX model for in situ treatment will also be explored.

Citation Format: Otto Luiz Cerqueira, Maria Alejandra Salomon, Elaine Cristina Cardoso, Tharcisio Citrangulo Tortelli, Bryan Eric Strauss. Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line [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 5548.
Abstract 5607: Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade
Authors: Yokoyama Yumi , Wild Robert
Published: September 2020

We previously reported that DRP-104, a novel broad acting glutamine antagonist, has significant therapeutic potential in cancer via directly targeting tumor metabolism and inducing a potent antitumor immune response. Here we sought to further elucidate the immunomodulatory effect of DRP-104 on tumor growth inhibition as a single agent and in combination with PD-1/PD-L1 checkpoint inhibitors in the MC38 and CT26 colon cancer syngeneic models. We utilized multiple technologies to assess mechanistic effects on immune cells in vivoincluding flowcytometry, multiplex immunoassay, gene expression profiling and GeoMx™ digital spatial profiling. DRP-104 mediated tumor growth inhibition was associated with increased tumor-infiltrating leukocytes (TIL) including T, NKT, and NK cells, M1-polarized tumor associated macrophages, and decreased immune-suppressive cells such as MDSCs. Nanostring® IO360 analysis revealed broad immunological modulation such as increase in cytotoxicity/antigen presentation score, increase in tumor metabolic stress/apoptotic score and decrease in cell proliferation score. GeoMx™ profiling also showed increased tumoral T cells and granzyme B expression. Luminex analysis of tumor lysates revealed DRP-104 treatment decreased pro-tumorigenic proteins, such as VEGF and IL-8. DRP-104 showed significant dose dependent tumor growth inhibition and regressions as a single agent in mouse syngeneic tumor models, including those resistant to immune checkpoint inhibitors. Lastly, combination of DRP-104 with anti-PD-1 Ab or anti-PD-L1 Ab further reduced tumor growth resulting in significant improvement in survival with long-term durable cures. In summary, DRP-104 treatment resulted in broad remodeling of the tumor microenvironment including enhanced infiltration and function of multiple immune cells distinct from activities obtained by a checkpoint inhibitor treatment. Combination therapy of DRP-104 with anti-PD-1/PD-L1 achieved significantly enhanced anti-tumor efficacy including long-term durable cures even in checkpoint inhibitor resistant models. This unique and non-overlapping mechanism of action supports clinical development of DRP-104 alone and in combination with PD-1/PD-L-1 checkpoint inhibitors.

Citation Format: Yumi Yokoyama, Robert Wild. Broad acting glutamine antagonism remodels the tumor microenvironment, induces distinctive immune modulation, and synergizes with immune checkpoint blockade [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 5607.
Abstract 5613: Human interleukin 15 (IL15) humanized NCG mice support the human natural killer cells reconstitution and development
Authors: Ju Cunxiang , Zhang Mingkun , Wu Dan , Wang Dingyu , Sun Hongyan , Tang Jin , Li Shuai , Zhao Jing , Gao Xiang
Published: September 2020

The potential of NK cells in cancer therapy has aroused increasing attention. While highly immune deficient mice, NCG,with null mutation on Prkdc and IL2Rγ,is ideal for human immune reconstitution, the reconstitution of human NK cells is still challenging. This is mostly because species barrier on cytokines which are necessary for the proliferation and maturation of NK cells. To this end, we established a human IL-15 (hIL-15) knock-in NCG (NCG-hIL-15), which produces hIL-15 under physiological level and pattern.NCG-hIL-15 implanted with either purified human NK cells or hematopoietic stem cells (HSCs) could give rise to a longer-term maintenance of human NK cell when compared to NCG. These long-lived NK cells retained the ability of migration, as evidenced by their wide distribution in various organs (e.g. spleen, liver, and lung). We also tested the expression profiles of killer cell immunoglobulin-like receptor (KIR) molecules, as well as the secretion of granzyme and perforin, results suggested normal activity and function of these reconstituted NK cells. Moreover, after human NK cell reconstitution, efficient tumor growth inhibition of human NK-specific agonist was shown in tumor engrafted NCG-hIL15. Taken together, NCG-hIL-15 mice is a novel mouse model for studying human NK cell biology and human NK-mediated cancer immunotherapy in vivo.

Citation Format: Cunxiang Ju, Mingkun Zhang, Dan Wu, Dingyu Wang, Hongyan Sun, Jin Tang, Shuai Li, Jing Zhao, Xiang Gao. Human interleukin 15 (IL15) humanized NCG mice support the human natural killer cells reconstitution and development [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 5613.
Abstract 5621: Humanized rat model
Authors: Adedeji Bisoye Towobola , Noto Fallon K. , Jamling Tseten Yeshi , Argawal Yash , Beatty Cole Jamison , Ho Sara Grace , Das Antu , Salunke Rajeev Kishore , Bility Moses Turkle
Published: September 2020

The development of Novel cancer immunotherapies has relied on the use of immune humanized mice. The field of study can be enhanced using an immune humanized rat, which will provide several advantages over the currently available humanized mouse models.

The SRG OncoRat®; A Rag2/Il2rg double-knockout rat supports the growth of larger tumors for serial fine needle biopsies to assess immune infiltration and serial blood draws for assessing human immune development and tumor biomarkers in real-time throughout an efficacy study. Using this rat, we developed a novel autologous human skin and immune cells-humanized rat model by co-engrafting full-thickness human-fetal skin and autologous fetal lymphoid organoids under the kidney capsule along with intravenous injection of autologous fetal-liver derived hematopoietic stem cells, thus termed, human skin-immune system humanized rat model (hSIS-humanized rat). We demonstrated the development of adult-like, full-thickness human skin and human lymphoid organoids along with human immune cells. Methicillin-resistant Staphylococcus aureus inoculation in the human skin results in infection and skin pathology, thus recapitulating clinical outcomes. This model will enable in vivo mechanistic studies for development and evaluation of novel therapeutics for skin infectious disease and may also provide a model for establishing skin grafts of patient-derived melanoma tumors to investigate melanoma metastasis and response to therapies. In addition, engrafting the rat with human lymphoid organs and human immune cells may provide a similar platform to the BLT mouse for immunotherapy studies.

Furthermore, we demonstrated the presence of Human CD45+, CD3+, and CD20+ cells in peripheral blood, spleen, and bone marrow of the SRG OncoRat® engrafted with human PBMCs, hence humanizing the rat's immune system. These immune humanized rat models may be beneficial for evaluating immunotherapies in human cancer models, including assessment of immune cell infiltration through fine needle biopsies.

Citation Format: Bisoye Towobola Adedeji, Fallon K. Noto, Tseten Yeshi Jamling, Yash Argawal, Cole Jamison Beatty, Sara Grace Ho, Antu Das, Rajeev Kishore Salunke, Moses Turkle Bility. Humanized rat 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 5621.
Abstract 5689: CH7233163, a mutant-selective EGFR inhibitor, overcomes osimertinib resistant EGFR-T790M/C797S
Authors: Kashima Kenji
Published: September 2020

EGFR tyrosine kinase inhibitors (TKI) show dramatic improvement of treatment for EGFR-mutated lung cancer, but these clinical efficacy is limited by acquired drug resistance. Osimertinib is a clinically approved covalent EGFR-TKI and has been demonstrated to overcome the EGFR-T790M, the most relevant acquired resistance to first-generation EGFR-TKIs. However, the C797S mutation, which impairs the covalent binding between EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutics to inhibit EGFR triple-mutation (C797S/T790M/activating-mutation). In this study, we identified CH7233163, a novel EGFR TKI, capable of overcoming the EGFR triple-mutation. CH7233163 inhibited preferentially enzymatic activity of EGFR triple-mutation, and showed potent antitumor activity against the tumor with EGFR triple-mutation in vitro and in vivo. Crystal structure of EGFR triple mutant in complex with CH7233163 shows that this compound interacted with multiple amino acids located in the ATP pocket of EGFR. This binding mode suggests that CH7233163 non-covalently but potently competes ATP without steric hindrance with T790M. Consistent with this insight, CH7233163 also showed potent antitumor activity against the tumors harboring EGFR single activating-mutation (deletion 19 or L858R) and double-mutation (T790M/activating-mutation) in vitro and in vivo. These result suggested that CH723316 may become a potential therapeutics to overcome osimertinib resistance in EGFR-mutated NSCLC.

Citation Format: Kenji Kashima. CH7233163, a mutant-selective EGFR inhibitor, overcomes osimertinib resistant EGFR-T790M/C797S [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 5689.
Abstract 569: Rational combination approaches with CRM-1 inhibitor in ovarian cancer
Authors: Handley Katelyn F. , Rodriguez-Aguayo Cristian , Ma Shaolin , Kim Mark S. , Ram Prahlad T. , Sood Anil K.
Published: September 2020

Background: After the frontline treatment, the majority of patients diagnosed with ovarian cancer at an advanced stage will develop recurrence (80%); thus, there is an urgent need for novel therapeutic approaches.

Purpose: Selinexor is a CRM1 inhibitor, which is a selective inhibitor of nuclear export and has demonstrated antitumor effects in ovarian cancer. Here, we examined the efficacy of selinexor alone and in combination with PARP inhibitors in ovarian cancer. As Selinexor reduces the expression of DNA damage repair proteins, we hypothesized that this combination should reduce the number of DNA damage repair proteins, resulting in synergistic tumor cell death.

Methods: Western blot was performed to assess baseline CRM1 expression in eleven ovarian cancer cell lines. We tested the efficacy of selinexor alone and in combination with olaparib in ovarian cancer cells (A2780ip2, OVCAR5). Combination Indices (CI) were calculated with CompuSyn and were considered synergistic when CI less than 1 for classic MTT arrays. We used the annexin V-FITC/PI assay to assess apoptosis as a result of selinexor and olaparib alone or in combination.

Results: Baseline CRM1 expression was positive in eight of eleven ovarian cancer cell lines tested. The average IC50 in A2780ip2 for selinexor and olaparib was 0.09uM and 0.97uM, respectively. The median combination index was 0.81 in A2780ip2 and 0.92 in OVCAR5 cells. Flow cytometry demonstrated an additive effect of selinexor and olaparib in A2780ip2 cell apoptosis (7.34% and 7.93%, respectively, v. 17.86%).

Conclusions: Selinexor demonstrates synergistic activity with olaparib, and this combination warrants further investigation.

Citation Format: Katelyn F. Handley, Cristian Rodriguez-Aguayo, Shaolin Ma, Mark S. Kim, Prahlad T. Ram, Anil K. Sood. Rational combination approaches with CRM-1 inhibitor in ovarian cancer [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 569.
Abstract 5702: A spatially resolved single cell atlas of the human breast
Authors: Kumar Tapsi , Nguyen Quy , Nee Kevin , Bai Shanshan , Hu Min , Sei Emi , Wood Anita , Wiley Jie , Chen Hui , Contreras Alejandro , Teshome Mediget , Chen Ken , Lim Bora , Lawson Devon , Navin Nicholas , Kessenbrock Kai
Published: September 2020

The human breast consists of lobules connected to an intricate network of ducts that are surrounded by fatty tissues, designed to produce and transport milk to nourish offspring. Histopathology has identified 10 major cell types based on morphological features but have provided limited information on cell states - the transcriptional programs of cell types that reflect different biological functions. In this study we have generated an unbiased ‘cell atlas' of the normal human breast to define the cell types and cell states using single cell RNA sequencing and spatial transcriptomics methods. We performed 3' microdroplet based single cell and nuclei RNA sequencing of 248,687 stromal cells and 89,301 nuclei from 24 women with pathologically normal breast tissues that were collected from mastectomies and reduction mammoplasties. Unbiased expression analysis identified four major cell types: adipocytes, epithelial cells (luminal and basal), fibroblasts and endothelial cells and defined their transcriptional programs. Additionally, 8 minor cell types were identified in the breast, including macrophages, T-cells, natural killer cells, mast cells, pericytes, apocrine cells, neurons and smooth muscle cells. Our data revealed hundreds of novel markers of these cell types and defined their transcriptional programs and cell states. Most cell types had multiple transcriptional programs including luminal epithelial cells (hormone receptor positive and secretory), basal epithelial cells (myoepithelial or basal), endothelial cells (E1, E2, E3), myeloid cells, T-cells and fibroblasts (F1-F4) and provided insight into developmental lineages. We further delineated the spatial organization of these cell types and cell states within the tissue architecture using spatial transcriptomics assay. The breast cell atlas data provides an invaluable normal reference for the research community to understand how normal cell types are reprogrammed in diseases such as breast cancer.

Citation Format: Tapsi Kumar, Quy Nguyen, Kevin Nee, Shanshan Bai, Min Hu, Emi Sei, Anita Wood, Jie Wiley, Hui Chen, Alejandro Contreras, Mediget Teshome, Ken Chen, Bora Lim, Devon Lawson, Nicholas Navin, Kai Kessenbrock. A spatially resolved single cell atlas of the human breast [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 5702.
Abstract 5722: Acquired stemness by luminal cells
Authors: Karthaus Wouter , Hofree Matan , Choi Danielle , Linton Eliot L. , Turkekul Mesruh , Bejnood Alborz , Carver Brett , Gopalan Anuhandra , Laudone Vincent , Biton Moshe , Chaudhary Ojasvi , Masilionis Ignas , Mazutis Linas , Pe'er Dana , Regev Aviv , Sawyers Charles
Published: September 2020

Rare cell types in the prostate are reported to have stem cell properties based on organ regeneration potential following castration. Here, we use single cell RNA-seq (scRNA-Seq) to characterize these populations from the murine and human prostate in hormonally intact and androgen deprived conditions. Prostate cells from hormonally intact mice partitioned into one large subset of basal epithelial cells, another large subset of luminal epithelial cells, which we designate luminal 1 and two rare luminal populations: luminal 2 and luminal 3. Luminal cells that persist following castration display enhanced organoid regeneration potential, particularly within 1-2 days of androgen addback, and contribute equipotently to prostatic regeneration as revealed by lineage tracing. This regeneration is mediated, in part, through the orchestrated expression of Nrg2, Igf1, Fgf10 and Rspo3 by distinct populations of androgen-responsive mesenchymal and smooth muscle cells. Thus, luminal cells that persist post-castration undergo a cell state change that primes a proliferative response to microenvironment signals, analogous to other models of tissue injury such as liver damage.

Citation Format: Wouter Karthaus, Matan Hofree, Danielle Choi, Eliot L. Linton, Mesruh Turkekul, Alborz Bejnood, Brett Carver, Anuhandra Gopalan, Vincent Laudone, Moshe Biton, Ojasvi Chaudhary, Ignas Masilionis, Linas Mazutis, Dana Pe'er, Aviv Regev, Charles Sawyers. Acquired stemness by luminal 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 5722.
Abstract 5723: Loss of stemness and lineage commitment in the intestinal crypt
Authors: Habowski Amber , Flesher Jessica , Bates Jennifer , Ganesan Anand , Edwards Robert , Shi Yongsheng , Hertel Klemens , Waterman Marian
Published: September 2020

The stem cell niche in intestinal crypts maintains multi-potent stem cells that constantly replenish all cell types in the mucosal lining of normal tissue. As daughter cells exit this niche, a loss of high Wnt signaling triggers rapid differentiation and lineage commitment to secretory or absorptive trajectories. Although signaling pathways such as Wnt and Notch are known to play a role in this differentiation and commitment, the mechanism that allows for such rapid action is not well understood. To investigate this, we used a novel flow sorting protocol to sort mouse colon crypt cells and then performed deep bulk RNA-sequencing. During differentiation we uncovered a gradual loss of stem cell markers and a sharp increase in alternatively spliced and alternatively polyadenylated genes. This indicates mRNA processing may be one of the first loss-of-stemness signals in the intestine. We also were able to find lineage specific activity for the unfolded protein response and further resolution on other signaling pathways. This unique sorting procedure will aid further study of crypt dynamics and this rich dataset provides an unprecedented resolution of the intestinal crypt processes including differentiation and lineage commitment along with a baseline plasticity potential of crypt cells.

Citation Format: Amber Habowski, Jessica Flesher, Jennifer Bates, Anand Ganesan, Robert Edwards, Yongsheng Shi, Klemens Hertel, Marian Waterman. Loss of stemness and lineage commitment in the intestinal crypt [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 5723.
Abstract 574: In vitro TTFields enhance the response to temozolomide or radiation in GBM cells from a newly diagnosed patient
Authors: Michelhaugh Sharon K. , Mittal Sandeep
Published: September 2020

Background: Tumor treating fields (TTFields) are FDA-approved for treatment of newly diagnosed and recurrent glioblastoma and are typically initiated after patients have undergone surgical resection, temozolomide, (TMZ) and radiotherapy. In this study, GBM cells generated from a tumor resected from a newly diagnosed glioblastoma patient were treated in vitro with TTFields ± TMZ or treated with in vitro TTFields followed by irradiation to determine if TTFields alter the response to the standard-of-care treatment modalities.

Methods: Single-cell suspensions generated from tumor tissue obtained from a newly diagnosed patient were cultured in DMEM/F12 media with 10% fetal bovine serum and gentamicin. For the TMZ experiment, cells were plated on plastic coverslips (1 × 104cells/coverslip; n=4-6/group) and incubated overnight. TTFields (200 kHz; field intensity of ~1.6 V/cm). were applied with or without 50 µM TMZ for 5 days, followed by the XTT proliferation assay. For the radiation experiment, cells were plated on plastic coverslips (5 × 104cells/coverslip; n=3/group) and incubated overnight. TTFields (200 kHz; field intensity of ~1.6 V/cm). were applied for 3 days followed by irradiation with 0, 2, 4, or 6 Gy. Cells were harvested and plated to assess clonogenic potential. After 3 days, cells were stained with crystal violet and plates were scanned to determine cell counts. Data were analyzed by one-way ANOVA with Tukey's post-hoc test.

Results: In the TTFields/TMZ experiment, control XTT values were 0.665 ± 0.052, TMZ only were 0.480±0.024, TTFields only were 0.550±0.080 and TTFields + TMZ were 0.164 ± 0.062 (mean ± SD; ANOVA p<0.0001). In the TTFields/radiation experiment, there was no statistical difference in cell counts across the radiation doses in the control cells. In the TTFields treated groups, the mean cell counts were 62146, 53324, 41070 and 28680 in the control, 2 Gy, 4Gy, and 6 Gy groups (ANOVA p=0.002).

Conclusions: In GBM cells generated from a newly diagnosed patient's tumor. TTFields application in combination with TMZ reduced cell proliferation by 75%, compared to only 20-30% for either treatment alone. Also, TTFields application prior to radiation significantly reduced cell counts. These results suggest that newly diagnosed patients would benefit from TTFields therapy earlier in the course of treatment.

Citation Format: Sharon K. Michelhaugh, Sandeep Mittal. In vitro TTFields enhance the response to temozolomide or radiation in GBM cells from a newly diagnosed patient [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 574.