bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-050: Memory sequencing reveals heritable single cell gene expression programs associated with therapy resistance
Authors: Shaffer Sydney , Emert Benjamin , Torre Eduardo , Raj Arjun
Published: August 2019

Single cancer cells can have substantial fluctuations in gene expression from non-genetic factors. However, it is unclear how long these fluctuations exist and whether they extend beyond the length of a single-cell division. Current technologies for gene expression measurements in single cells provide just one measurement of each cells’ transcriptome and fail to capture how these gene expression fluctuations change with time. We developed a new method combining Luria and Delbruck's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several cell divisions. MemorySeq revealed multiple gene modules that express together in rare cells within otherwise homogeneous clonal populations. Further, we found that these rare cell subpopulations are associated with biologically distinct phenotypes in multiple different cancer cell lines. In melanoma, we identified a rare subpopulation of cells expressing EGFR, NGFR, and AXL, and demonstrated that these cells are resistant to BRAF and MEK inhibitors. In triple-negative breast cancer cells, we identified a different gene expression signature consisting of CA9, and found that this subpopulation is resistant to paclitaxol. We validated these findings in melanoma cell lines by tagging the endogenous locus of MemorySeq gene, NGFR, with a fluorescent protein to directly visualize gene expression fluctuations in individual cells. Broadly, we believe that memory is an essential property of variability that will have phenotypic consequences for cancer cells. Thus, we anticipate that MemorySeq can be extended to uncover the transcriptional states of rare cells driving many phenotypes in cancer including drug resistance, differentiation, invasion, and metastasis.

Citation Format: Sydney Shaffer, Benjamin Emert, Eduardo Torre, Arjun Raj. Memory sequencing reveals heritable single cell gene expression programs associated with therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-050.
Abstract LB-052: Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS
Authors: Wong Wing Hing , Young Andrew , Druley Todd
Published: August 2019

While next-generation sequencing (NGS) has transformed our ability to characterize the genome, it is limited in detection of rare mutations due to a high error rate of ~0.5-2.0%. We have developed an error correction pipeline and previously demonstrated that our process is capable of detecting variants as rare as 1:10,000 or 0.0001 VAF. Using our error correction pipeline, we have recently identified leukemia-related hematopoietic clones in a cohort of young healthy bone marrow donors (median age 26). In the context of allogeneic hematopoietic stem cell transplantation (HSCT), we sought to determine whether these detected leukemia-related clones of donor origin selectively engraft and correlate with transplant-related morbidities in recipients post-HSCT. Genomic profiles of pre-transplant donor samples were compared to the genomic profiles of corresponding recipient samples post-HSCT. Results show that two-thirds of the healthy donor samples harbor deleterious hematopoietic clones and that these specific clones are significantly more likely to engraft compared to benign clones. We also found that 75% of recipients who had at least one persistently engrafted, deleterious mutation developed chronic GvHD versus 50% of those without persistently engrafted clones with deleterious mutations. In comparing the results from our error correction pipeline and conventional short tandem repeat (STR) testing for donor-patient chimerism, we showed that our method provides a highly sensitive approach to both screening for donors without deleterious clones and monitoring residual disease in recipients following hematopoietic stem cell transplants (HSCT). Furthermore, the clinical implications of these results warrant a large-scale study using our error correction pipeline to detect rare variants in HSCT.

Citation Format: Wing Hing Wong, Andrew Young, Todd Druley. Detection of rare, deleterious clonal mutations during unrelated allogeneic hematopoietic stem cell transplants using an error correction pipeline for NGS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-052.
Abstract LB-063: Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics
Authors: Avrutskaya Anya , Sonego Fabiane , Hauser Jacob , O’Koren Emily , Martin Gaëlle , Chaix Julie , Ball Robin , Bui Thi , Belle Ian , Hall Chassidy , Thiam Kader , Marval Paula L. Miliani De
Published: August 2019

Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents.

Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.
Abstract LB-071: Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology
Authors: Feng Qiang , Zhang Miao-yun , Lu Shi-Jiang
Published: August 2019

NK cells are cytotoxic cells critical for innate immune system. Early investigation using NK cells carrying CAR specific targeting tumor antigens have shown promising antitumor activities. CAR-T therapy requires either autologous or MHC matching cells to avoid graft-verse-host disease (GVHD), whereas allogeneic NK cells showed substantial anti-tumor activity without GVHD. Therefore, the “ready-to-kill” NK population is regarded as the ideal Off-The-Shelf immune cell therapy products. The development of NK-based immunotherapy, however, has been limited by unsatisfactory sources of human NK cells. Human induced pluripotent stem cells (iPSCs) offer unlimited source for manufacturing NK cells suitable for immunotherapy. Previously NK cell derivation methods from human iPS cells used feeder cells with multiple scale-up limited processes which are not suitable for industrial scale production. In this study we described a novel 3D-bioreactor platform that can continuously generate highly pure NK cells with strong cytotoxic activity for 2 weeks. First, undifferentiated human iPS cells were cultured as 3D-spheres with mesoderm inducing conditions in bioreactor, which converted 60-70% iPS cells into hemogenic endothelial progenitor (HEP, CD31+CD144+CD34+) in a week. HEP cells were then cultured under NK cell differentiation condition for up to 35 days. Starting from day 20, NK cells were started to release from these 3D-spheres which can be harvested daily by centrifugation. These iPS-NK cells can be cryopreserved for long time. Human iPS-NK cells display a distinctively homogenous morphology. Over 95% of collected cells express CD56, NKG2D, NKp44 and NKp46 markers; Approximately 30-50% of CD56+ iPS-NK cells express KIR2DL/DS1 and KIR2DS4 receptors; CD56+ PSC-NK cells do not express TCR and CD3, and CD19, which are specific markers for T and B cells, respectively. Surprisingly, we discovered that more than 80% CD56+ iPS-NK cells also expressed CD8 as compared to ≈30% of peripheral blood CD56+ NK cells, the increase of which is associated with disease regression in AIDS patients. More importantly, when iPS-NK cells and K562 leukemia cells were mixed together, even at a ratio of 1 to 1, more than 80% of leukemia cells were killed within hours as demonstrated both by flow cytometry analysis and time-lapse record. With the establishment of permanent master iPS-CAR cell lines, our novel technology platform will provide inexhaustible cell sources for the generation of truly off-the-shelf CAR-NK cells suitable for treatment of a large numbers of patients, which can revolutionize the field of immuno-oncology.

Citation Format: Qiang Feng, Miao-yun Zhang, Shi-Jiang Lu. Functional NK cells generated from human iPS cells with 3D-bioreactor for immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-071.
Abstract LB-074: Priming the tumor microenvironment with epigenetic modifiers to overcome resistance to immune checkpoint inhibitors
Authors: Knox Tessa J. , Sahakian Eva , Banik Debarati , Hadley Melissa , Palmer Erica , Kim Jennifer , Noonepalle Satish , Powers John , Gracia-Hernandez Maria , Oliveira Vasco , Cheng Fengdong , Chen Jie , Barinka Cyril , Pinilla-Ibarz Javier , Lee Norman , Kozikowski Alan , Villagra Alejandro
Published: August 2019

Histone deacetylases (HDACs) are involved in diverse cellular regulatory mechanisms including functions outside the chromatin environment. Several publications have demonstrated that selective HDAC inhibitors (HDACi) can influence tumor immunogenicity and the functional activity of specific immune cells. In particular, the selective inhibition of HDAC6 has been reported to decrease tumor growth in several malignancies. However, there is still no clarity about the cellular components mediating this effect. In this study, we evaluated the immunological modulation of the HDAC6i Nexturastat A in combination with anti-PD-1 checkpoint blockade therapy and the use of this HDAC6i as a priming agent to facilitate the transition of the tumor microenvironment from “cold” to “hot” in order to more specifically augment immune check-point blockade therapies. This combination of Nexturastat A and anti-PD-1 therapy demonstrated a significant reduction of tumor growth in syngeneic melanoma tumor animal models. Additionally, we observed a complete neutralization of the up-regulation of PD-L1 and other immunosuppressive pathways induced by the treatment with anti-PD-1 blockade. This combination also showed that the pre-treatment with selective HDAC6i induced major changes in the tumor microenvironment such as enhanced infiltration of immune cells, increased central and effector T cell memory, and a significant reduction of pro-tumorigenic M2 macrophages. The evaluation of the effect of HDAC6i on individual immune components suggest that the in vivo anti-tumor activity of HDAC6i is mediated by its effect on tumor cells and tumor associated macrophages, and not directly over T cells. Overall, our results indicate that selective HDAC6i could be used as immunological priming agents to sensitize immunologically “cold” tumors and subsequently improve ongoing immune check-point blockade therapies.

Note: This abstract was not presented at the meeting.

Citation Format: Tessa J. Knox, Eva Sahakian, Debarati Banik, Melissa Hadley, Erica Palmer, Jennifer Kim, Satish Noonepalle, John Powers, Maria Gracia-Hernandez, Vasco Oliveira, Fengdong Cheng, Jie Chen, Cyril Barinka, Javier Pinilla-Ibarz, Norman Lee, Alan Kozikowski, Alejandro Villagra. Priming the tumor microenvironment with epigenetic modifiers to overcome resistance to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-074.
Abstract LB-078: New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody
Authors: Zhou Grace , Ni Dongyao , Yan Runbin , Chen Xiaoqing , Liu Xianjie , Ma Jie , Tang Yuxin
Published: August 2019

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpes simplex viruses (oHSV) have a major impact on cancer therapy marred by rare failures of healthy organs. Here we report the properties of 3 families of oHSV expressing no immunomodulatory genes (T1 series), murine or human IL-12 (T2 series) and murine or human IL-12 and anti-PD-1 antibody (T3 series). Thus insertion of the genes encoding PD-1 Ab and IL-12 significantly augmented the oncolytic activity of oHSV bereft of immune-stimulatory genes (T1 series). In syngeneic mice the murine T3 oHSV induced significant intratumoral accumulation of IL-12, PD-1 Ab and IFN-γ and was effective against a variety of murine tumors. Thus T3 oHSV concentrates the immunomodulatory products in the environment of the tumors thereby reducing the risk of toxicity associated with systemic administration. T3 oHSV injected into tumors was more effective than systemic administration of anti PD-1 Ab and IL12 alone or in combination with intratumoral injection of T1 oHSV. T3 induces local and systemic immune response characterized by cytokine signature.

Citation Format: Grace Zhou, Dongyao Ni, Runbin Yan, Xiaoqing Chen, Xianjie Liu, Jie Ma, Yuxin Tang. New generation of oncolytic herpesviruses embodying immunotherapeutic genes encoding IL-12 and anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-078.
Abstract LB-085: PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas
Authors: Hill Kristen , Messina Jane , Wu Jie , Chalfant Charles E. , Kim Minjung
Published: August 2019

Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase (PTP) that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten and Cdkn2a null mice, tet-inducible and melanocyte-specific PTPN11 E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11 E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRAS Q61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

Citation Format: Kristen Hill, Jane Messina, Jie Wu, Charles E. Chalfant, Minjung Kim. PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-085.
Abstract LB-095: Aurora-A is elevated in cancer cells due to inactivation of EF1A2-PTEN-GSK3b regulated late S-G2 phase specific degradation pathway
Authors: Treekitkarnmongkol Warapen , Solis Luisa Maren , Kai Kazuharu , Sasai Kaori , Wistuba Ignacio I. , Sahin Aysegul A. , Wong Kwong K. , Thompson Alastair M. , Jltsumori Yoshimi , Lee Jonathan M. , Qin Lidong , Bawa-Khalfe Tasneem , Rad Roland , Abbott Catherine M. , Katayama Hiroshi , Sen Subrata
Published: August 2019

Elevated expression of Aurora kinase-A oncoprotein is detected in a subset of human cancers across different organ sites. The mechanism(s) underlying over expression of the protein in cancer cells have not been well elucidated. This study demonstrates that inactivation of a novel mechanism of EF1A2 regulated PTEN-GSK3β-SCF complex mediated Aurora-A protein degradation pathway in S-G2 phases of mitosis leads to enhanced stability/steady state level of Aurora-A in cancer cells. EEF1A2 regulates interaction of Aurora-A with activated PTEN, GSK3β, Neddylated CUL1 and FBXW7, promoting ubiquitination mediated degradation of Aurora-A that is rescued in EEF1A2 knockdown cells. Ectopic expression of EEF1A2, on the other hand, enhances formation of the complex with GSK3β mediated inactivating phosphorylation and reduction in Aurora-A protein, recapitulating the S-G2 phase events. Genetic ablation of EEF1A2 and PTEN in both in vitro cell lines and in vivo mouse models validated the results. Aurora-A over expressing mouse models of mammary carcinoma and human breast cancer tissue samples also corroborated the findings. Therapeutic targeting of PTEN-GSK3β-AKT pathway may, therefore, be effective in treating a subset Aurora-A over expressing cancers.

Citation Format: Warapen Treekitkarnmongkol, Luisa Maren Solis, Kazuharu Kai, Kaori Sasai, Ignacio I. Wistuba, Aysegul A. Sahin, Kwong K. Wong, Alastair M. Thompson, Yoshimi Jltsumori, Jonathan M. Lee, Lidong Qin, Tasneem Bawa-Khalfe, Roland Rad, Catherine M. Abbott, Hiroshi Katayama, Subrata Sen. Aurora-A is elevated in cancer cells due to inactivation of EF1A2-PTEN-GSK3b regulated late S-G2 phase specific degradation pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-095.
Abstract LB-101: Combined p53 dynamics and post-translational modifications determine subsequent protein expression and cell fate
Authors: Lu Dan , Holton Kristina , Hafner Antonina , DeHart Caroline , Sikora Jacek , Zhou Houjiang , Jambhekar Ashwini , Kelleher Neil , Gunawardena Jeremy , Lahav Galit
Published: August 2019

The tumor suppressor protein p53 is activated by a diverse repertoire of cellular stresses (e.g. oncogenic signalling, DNA damage) and as a transcription factor induces the expression of response pathways genes (including cell cycle arrest, senescence, apoptosis). While p53 is frequently lost during tumorigenesis, approximately 50% of human tumors retain intact p53, potentially rendering them sensitive to p53-induced cell death during radiotherapy. Recent single cell analysis from our lab has shown that irradiation of cells does not cause a simple sustained accumulation of p53, but rather, induces pulsatile p53 dynamic that oscillates at 5-hour periods resulting from a p53-Mdm2 negative feedback loop, leading to reversible cell cycle arrest. Disrupting this feedback loop by small molecules results in sustained p53 dynamics, leading to irreversible apoptosis or senescence. Moreover, extensive work by others has shown that post-translational modifications (PTM) on p53 also govern the cell fate decision process.

Here we show that aside from differences in p53 dynamics following irradiation, multiple PTMs on p53 also differ, with the latter causing quantitative changes of p53 binding to interactions proteins and subsequent alterations in promoter binding. In contrast, p53 dynamics govern broad transcription dynamics of target genes. For example, pulsatile p53 dynamics result in several distinct patterns of mRNA transcription including a cluster of oscillating p53 target genes. However, these oscillatory mRNA dynamics are lost at the protein level. The only p53 target protein that oscillates is the Mdm2 negative regulator of p53, while all other targets gradually accumulate over time. Furthermore, sustained p53 dynamics over 24h results in far greater accumulation of nearly all target proteins compared to pulsatile p53 dynamics, which likely explains the differences in the reversibility of cell fate decisions. The results described here uncover a link between p53 dynamics and p53 PTMs providing an overall mechanistic cell fate selection process by p53, which have important implications both for understanding of cell signalling processing as well as improving p53-mediated therapies.

Citation Format: Dan Lu, Kristina Holton, Antonina Hafner, Caroline DeHart, Jacek Sikora, Houjiang Zhou, Ashwini Jambhekar, Neil Kelleher, Jeremy Gunawardena, Galit Lahav. Combined p53 dynamics and post-translational modifications determine subsequent protein expression and cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-101.
Abstract LB-111: Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology
Authors: O'Connor Matthew , Nicolaides Theodore , Zhang Jie , Flohr Alexander , Iacone Roberto , Mayweg Alexander V. , Epstein David M. , Buck Elizabeth
Published: August 2019

Mutation of either the intracellular catalytic domain or the extracellular domain of the receptor for epidermal growth factor (EGFR) drives oncogenicity. Extracellular domain EGFR mutations are highly expressed in patients with glioblastoma. Despite clinical success with targeting EGFR catalytic site mutants, no drugs have proven effective in glioblastoma patients expressing extracellular EGFR mutations. Herein, we define the molecular mechanism for oncogenic activation of families of extracellular EGFR mutations and reveal how this mechanism renders current generation small molecule ATP-site inhibitors ineffective. We demonstrate that a group of the most commonly expressed extracellular domain EGFR mutants expressed in glioblastomas is activated by disulfide-bond mediated covalent homodimerization, collectively referred to as locked dimerization (LoDi-EGFR oncogenes). Strikingly, current generation small molecules binding to the active kinase conformation potently inhibit catalytic site mutants, but induce covalent dimerization and activate LoDi-EGFR receptors, manifesting in paradoxical acceleration of proliferation. These data demonstrate how the locked-dimer mechanism of EGFR oncogenesis has profound impact on the activity of small molecules acting at the distal catalytic site, providing further evidence for “inside-out” allosteric signaling in EGFR. This provides a mechanistic understanding for the failure of current generation EGFR inhibitors to effectively treat LoDi-EGFR mutants in GBM and sets guidelines for discovery of selective LoDi-EGFR inhibitors.

Citation Format: Matthew O'Connor, Theodore Nicolaides, Jie Zhang, Alexander Flohr, Roberto Iacone, Alexander V. Mayweg, David M. Epstein, Elizabeth Buck. Epidermal growth factor receptor oncogenes expressed in glioblastoma are activated as covalent dimers and exhibit unique pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-111.
Abstract LB-125: KDM5D as a novel gender-specific gene target for metastatic colorectal cancer
Authors: Li Jiexi , Lan Zhengdao , Wang Alan Y. , DePinho Ron
Published: August 2019

Male colorectal cancer (CRC) patients have higher rates of incidence, mortality and poorer responsiveness to therapy compared to female patients, making it critical to identify the significant factors contributing to the gender differences and to explore gender-specific therapeutic interventions. Here, combining unbiased expression profiling and xenograft mouse model discovered the Y chromosome gene KDM5D as a male-specific pro-metastasis gene and its upregulation contributes to poorer survival in both human and mouse. Knockout of Kdm5d in male metastatic CRC cell lines aborted the liver metastasis occurrence while overexpressing Kdm5d in female primary CRC cell lines enabled their metastasis potential. Mechanistically, ChIP-seq analysis showed KDM5D primarily works as a demethylase for histone modification H3K4me2/3, downregulating metastasis-suppressing genes. KDM5D also regulates the chromosome accessibility, indicated by H3K27Ac level change and validated with ATAC-seq. Kras G12D mutation regulates Kdm5d expression and is correlated with poorer survival in male mice, which is validated in silico in human CRC data in TCGA. KDM5 family inhibitor "compound 48" showed significant power to inhibit the migration capability of human metastatic CRC cell line LoVo. This study provided a new understanding of the effect of gender on CRC and a novel gender specific therapeutic target for metastatic CRC.

Citation Format: Jiexi Li, Zhengdao Lan, Alan Y. Wang, Ron DePinho. KDM5D as a novel gender-specific gene target for metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-125.
Abstract LB-127: Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression
Authors: Dong Zhiwei , Garcia Gonzalo Lepez , Zhang Cheng , Dankert Erin N. , Harenza Jo Lynne , Hou Xiaonan , Li Hu , Maris John M. , Zhu Shizhen (Jane)
Published: August 2019

Neuroblastoma is an embryonal tumor arising in the peripheral sympathetic nervous system (PSNS), and often presents with widespread metastasis at diagnosis. We recently demonstrated that overexpression of LMO1 can synergize with MYCN to promote tumor metastasis in our zebrafish model system. Here, we identify a “metastasis suppressor” gene – growth arrest specific 7 (GAS7) – whose loss of function or reduced expression can promote dissemination and migration of neuroblastoma cells with MYCN overexpression, both in vitro and in vivo. GAS7 resides on chromosome 17p13.1, a region that is deleted in high-risk neuroblastomas with higher frequency in MYCN-amplified cases. Expression levels of GAS7 are also significantly downregulated in MYCN-amplified or otherwise MYCN-overexpressing human neuroblastoma cells, as well as MYCN-overexpressing zebrafish PSNS cells, suggesting an inverse relationship between the oncogenic activities of MYCN and GAS7downregulation. To determine whether loss of function or reduced expression of GAS7 could contribute to neuroblastoma pathogenesis in the context of MYCN overexpression, we developed a gas7 knockout zebrafish line using a transcription activator-like effector nucleases (TALEN) method. Loss of function of gas7 promoted hematogenous metastasis of MYCN-overexpressing tumor cells to tissues and organs that paralleled the common sites of metastasis in neuroblastoma patients. Further RNA-sequencing and qRT-PCR analyses showed that representative signature genes encoding proteins involved in tumor cell-cell or cell-extracellular matrix interactions were significantly downregulated in fish tumors with loss of function of gas7, consistent with our electron microscopic finding in the fish model showing a loss of contact among tumor cells in fish lacking functional gas7, suggesting that GAS7 may physically act as a metastasis suppressor. Thus, in high-risk neuroblastoma with MYCNamplification or overexpression, we propose that deletion or reduced expression of GAS7 cooperates with MYCN to promote neuroblastoma cell dissemination. Although the precise mechanism of this interaction remains unclear, we suggest that it relies, in part, on the downregulation of pivotal genes involved in tumor cell-cell or cell-extracellular matrix contacts.

Citation Format: Zhiwei Dong, Gonzalo Lepez Garcia, Cheng Zhang, Erin N. Dankert, Jo Lynne Harenza, Xiaonan Hou, Hu Li, John M. Maris, Shizhen (Jane) Zhu. Loss of function of GAS7 promotes neuroblastoma metastasis in the context of MYCN overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-127.
Abstract LB-135: Targeting the myeloid-cell specific SRC-kinase HCK improves anti-tumor immunity
Authors: Ernst Matthias R. , Chand Ashwini , Huntington Nicholas , O'Donoghue Robert , Poh Ashleigh
Published: August 2019

Elevated expression of the myeloid cell-specific Src-family kinase HCK occurs in a majority of solid malignancies and correlates with poor patient survival [Poh et al., Frontiers Oncol 2018]. We have recently shown that genetic ablation, or therapeutic inhibition, of HCK suppresses the growth of primary tumors arising in the colon, stomach, lung and other mutagenized epithelia of mice. Mechanistically, we have attributed this to the capacity of HCK activity to support and retain the polarization of a wound-healing, Tie2+ (i.e. alternative activated) endotype of tumor-associated macrophages and concomitant tumor vascularization [Poh et al., Cancer Cell2017].

Here we extend these observations to MC38 colon cancer, B6/F10 melanoma and EO771 breast cancer allograft model, where the growth of the primary lesion and its metastatic spread is suppressed in HCK knockout hosts. More importantly, HCK gene deletion or therapeutic HCK inhibition with a small kinase inhibitor in mouse models further enhances the anti-tumor effects conferred by either chemotherapy or single agent immunotherapies based on anti-PD-L1, anti-CTLA4 or agonistic-CD40 antibodies. These observations are underpinned by increased tumour cell apoptosis that coincides with enhanced expression of ifng, gzmb, prf1 and other effector proteins in NK and infiltrating CD8+ cells. Likewise, HCK inhibition confers increased expression of il12 and ifng in tumor-associated macrophages, which adopt a more conventional activated endotype. Because HCK-deficiency of the host also associated with increased abundance of tumor infiltrating effectors cells, we propose that therapeutic targeting of HCK not only “makes tumors hotter”, but also improves host anti-tumor immunity. We are therefore developing novel HCK-specific small molecule inhibitors.

Citation Format: Matthias R. Ernst, Ashwini Chand, Nicholas Huntington, Robert O'Donoghue, Ashleigh Poh. Targeting the myeloid-cell specific SRC-kinase HCK improves anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-135.
Abstract LB-139: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer
Authors: Qu Yang , Zhao Gang , Li Hui
Published: August 2019

Background: Regulatory T cells (Tregs) are potent immunosuppressive cells of the immune system. They facilitate the survival and progression of tumor through their ability to suppress the antitumor immunity. The expression of tumor necrosis factor receptor type II (TNFR2) on Tregs is reported to identify the maximally suppressive Treg population in both mice and human. According to our previous research, expression of TNFR2 appeared to correlate better than CD25+ and CD127− with FoxP3 expression.

Aim: The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer are needed to be studied.

Methods: The expression and phenotype of TNFR2 + Tregs in peripheral blood and tumor tissues of 33 cases of gastric cancer patients and in peripheral blood of 15 normal controls were detected by flow cytometry. The ratio of Foxp3+Tregs and TNFR2+Tregs of 145 cases of gastric cancer patients was analyzed by multicolor immunofluorescence assay. We analyzed their relationship with gastric cancer stage, metastasis and other clinical indicators by clinical and pathological data.

Results: Flow cytometry showed that the expression of TNFR2 in human peripheral blood Tregs was significantly higher than that in conventional T cells (Tconvs) in peripheral blood CD4 + T cell subsets of gastric cancer patients and healthy individuals (P<0.05). The proportion of TNFR2 + cells in peripheral blood Tregs cells was significantly higher in patients with gastric cancer than in normal subjects (P <0.05). Tregs and Tconvs cells expressed very low level of CTLA-4 and TIM-3 in both normal controls and gastric cancer patients. Multicolour immunofluorescence showed that the ratio of Foxp3 + Tregs and TNFR2 + Tregs was increased in the tumor tissue, and TNFR2 + Tregs cells showed a tendency to increase with tumor progression and lymphatic metastasis. Higher levels of infiltration of TNFR2+ Tregs cells are significantly associated with patients’ OS (P<0.05) and DFS (P<0.05).

Conclusion: Our findings revealed that TNFR2 could be a promising molecular marker associated with poor prognosis. The mechanism by which TNFR2+Tregs promote tumor survival and progression in gastric cancer is still needed further research.

Citation Format: Yang Qu, Gang Zhao, Hui Li. The effects and mechanisms of TNF-α/TNFR2 in regulatory T cells on microenvironment and progression in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-139.
Abstract LB-171: Global variation in prostate cancer incidence and mortality rates, 1980-2013
Authors: Freeman MaryBeth , Jemal Ahmedin
Published: August 2019

Context: Previous studies documented significant international variation in prostate cancer rates due to differences in detection practices, availability of treatment, and genetic factors.

Objective: To provide updated contemporary prostate cancer incidence and mortality patterns across five continents using the most recent cancer incidence data from the International Agency for Research on Cancer and mortality data from the World Health Organization (WHO).

Evidence acquisition: We present estimated age-standardized prostate cancer incidence and mortality rates by country and WHO regions for 2012 based on GLOBOCAN. We examined long-term (1980 onwards) trends in prostate cancer incidence and mortality rates for 38 countries with high quality population-based incidence and mortality data. Trends were expressed as annual percent change using Joinpoint model. We also examined short-term (most recent 5 years) trends in prostate cancer among 44 countries with available incidence data and 71 countries with available mortality data.

Evidence synthesis: The highest incidence rates during the most recent 5 years are found in Brazil, Lithuania, and Australia, whereas the lowest incidence rates are found in Asia (India, Thailand, and Bahrain). The highest mortality rates are found in the Caribbean (Barbados, Trinidad and Tobago, and Cuba), sub-Saharan Africa (South Africa), parts of former Soviet Union (Lithuania, Estonia, and Latvia), whereas the lowest rates are found in Asia (Thailand and Turkmenistan). Of the 44 countries with high quality incidence data, prostate cancer incidence rates during the most recent five data years increased in 4 countries (with Bulgaria showing the largest increase), decreased in 7 countries (with the biggest decrease in the United States), and stabilized in the remaining 31 countries. During the same time period, in contrast, among the 71 countries considered for the mortality trend, rates decreased in 14 countries, increased in 3 countries, and remained stable in 54 countries.

Conclusions: In 2012, prostate cancer was the most commonly diagnosed cancer among men in 96 countries and the leading cause of death in 51 countries. In the most recent 5 years of data examined, prostate cancer incidence and mortality rates are decreasing or stabilizing in most parts of the world. Future studies should monitor trends in mortality rates and late-stage disease to assess the impact of reduction in PSA testing in several countries.

Citation Format: MaryBeth Freeman, Ahmedin Jemal. Global variation in prostate cancer incidence and mortality rates, 1980-2013 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-171.
Abstract LB-172: Understanding the mechanism underlying vitamin D and prostate cancer: Exploring telomere length
Authors: Major Jacqueline M. , Graubard Barry I. , Weinstein Stephanie J. , Kim Jeeyun , Vivo Immaculata De , Virtamo Jarmo , Savage Sharon A. , Albanes Demetrius
Published: August 2019

Background: In an effort to understand the molecular mechanism behind the association between circulating vitamin D and prostate cancer, we measured relative telomere length and 25(OH)D levels in a cohort of older Finnish men.

Methods: Using a nested study design, we examined quantitative PCR measured telomere length, season-adjusted serum 25-hydroxyvitamin D [25(OH)D], and subsequent prostate cancer risk in 1000 men (n=500 cases, 500 matched controls) participating in the extension study of the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Trial. Conditional regression models were used to explore mediation and effect modification of telomere length on the relation between serum 25(OH)D and prostate cancer risk.

Results: An increased risk of prostate cancer was observed among men with longer (>median) relative telomere length (OR = 1.41, 95% CI: 1.06 to 1.90; continuous: OR = 2.36, 95%CI: 0.96 to 5.83) as well as men with higher 25(OH)D concentrations (OR = 1.67, 95% CI: 1.08 to 2.57 for highest vs. lowest quintile, P-trend = 0.03). No significant association between 25(OH)D levels and telomere length was observed. A suggested joint effect of 25(OH)D and telomere length on prostate cancer risk was observed.

Conclusion: Serum 25(OH)D and relative telomere length are independent predictors of prostate cancer risk in this sample of older male Finnish smokers. No evidence of mediation between circulating vitamin D concentrations and telomere length was observed. Future studies are needed to further clarify the underlying biologic mechanisms of the 25(OH)D-prostate cancer association.

Citation Format: Jacqueline M. Major, Barry I. Graubard, Stephanie J. Weinstein, Jeeyun Kim, Immaculata De Vivo, Jarmo Virtamo, Sharon A. Savage, Demetrius Albanes. Understanding the mechanism underlying vitamin D and prostate cancer: Exploring telomere length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-172.
Abstract LB-174: Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population
Authors: Lin Yingsong , Nakatochi Masahiro , Ito Hidemi , Kamatani Yoichiro , Sakamoto Hiromi , Ishii Hiroshi , Sasahira Naoki , Ueno Makoto , Egawa Naoto , Mori Mitsuru , Nakao Haruhisa , Adachi Yasushi , Wakai Kenji , Tsugane Shoichiro , Yamamoto Masayuki , Shimizu Atsushi , Kadowaki Takashi , Yoshida Teruhiko , Matsuda Fumihiko , Kubo Michiaki , Kikuchi Shogo , Matsuo Keitaro
Published: August 2019

Pancreatic cancer is one of the most challenging malignancies because of its dismal prognosis, largely unknown etiology, and absence of early detection method. Here we report the results of a meta-analysis of three genome-wide association studies (GWAS) involving 2039 pancreatic cancer cases and 32592 control subjects, which represent the largest sample size in the Japanese population. To complement the SNP-based GWAS, we performed a gene-based GWAS using MAGMA. Given the pleiotropic effects observed for the top variants, we performed a Mendelian randomization (MR) analysis to address the possible causal relationship between type-2 diabetes (T2D) and pancreatic cancer risk, using 82 T2D-related SNPs and 25 hemoglobin A1c (HbA1c)-related SNPs as instrumental variables. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0×10-8), as well as 4 loci with suggestive evidence of association (P<1.0×10-6) for pancreatic cancer. Of the identified risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (OR=1.46, 95%CI=1.29-1.66, P=4.28×10-9), an Asian-specific, non-synonymous variant of glycoprotein 2 (GP2) predicted to be highly deleterious. Additionally, gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified variants in the GP2 gene exhibited pleiotropic effects on multiple traits, including type 2 diabetes, Hemoglobin A1c (HbA1c) levels, and pancreatic cancer. MR analysis suggested that genetically increased HbA1c levels may be causally associated with increased pancreatic cancer risk. Further fine mapping and functional characterization are required to elucidate the effects of common GP2 gene variants on pancreatic cancer susceptibility. Moreover, our findings highlight genetic susceptibility factors shared between T2D and pancreatic cancer.

Citation Format: Yingsong Lin, Masahiro Nakatochi, Hidemi Ito, Yoichiro Kamatani, Hiromi Sakamoto, Hiroshi Ishii, Naoki Sasahira, Makoto Ueno, Naoto Egawa, Mitsuru Mori, Haruhisa Nakao, Yasushi Adachi, Kenji Wakai, Shoichiro Tsugane, Masayuki Yamamoto, Atsushi Shimizu, Takashi Kadowaki, Teruhiko Yoshida, Fumihiko Matsuda, Michiaki Kubo, Shogo Kikuchi, Keitaro Matsuo. Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-174.
Abstract LB-197: NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer
Authors: Beerli Roger R. , Waldmeier Lorenz , Gébleux Rémy , Pretto Francesca , Grawunder Ulf
Published: August 2019

Introduction: We report on the development and preclinical validation of NBE-002, a next-generation antibody drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC). NBE-002 consists of a humanized monoclonal antibody directed against the receptor tyrosine kinase ROR1, expressed on the surface of numerous solid cancers including TNBC, and is site-specifically conjugated to a derivative of the highly potent anthracycline PNU-159682.

Results: Therapeutic efficacy of NBE-002 was evaluated in ROR1-low/-intermediate/-high patient-derived xenograft (PDX) models of TNBC, lung adenocarcinoma, ovarian carcinoma, and a variety of sarcomas. NBE-002 was found to display significant anti-tumor activity in each indication. The most pronounced effect was achieved in TNBC, where complete tumor regression was observed already at the lowest ADC dose tested (0.33 mg/kg), even in models expressing low levels of ROR1 (H-score ≤70). Since these PDX studies were performed in immune-compromised hosts, and anthracyclines are known inducers of immunogenic cell death, anti-tumor activity was also investigated in syngeneic tumor models in immune-competent hosts. Anti-tumor activity of PNU-ADCs involved activation of the immune system, as shown by evaluation of NBE-002 or a Trastuzumab-PNU conjugate (T-PNU) in ROR1- or HER2-positive syngeneic breast cancer models, respectively. Depletion of CD8 T cells severely reduced anti-tumor activity, demonstrating an important role for T cells in driving tumor regression. Furthermore, when tumor free animals were re-challenged with the same tumor, tumor growth was inhibited in the absence of any further ADC administration, indicating the development of an immunological memory. Notably, combination of ADC and checkpoint inhibition, such as α-PD1 or α-CTLA4, significantly enhanced tumor eradication following the treatment.

Conclusion: Our results demonstrate that NBE-002 is a highly effective and promising targeted therapeutic for the treatment of ROR1 positive TNBC and potentially other solid tumor indications that warrants clinical development. Considering the pronounced immune-modulatory functions of the PNU payload, NBE-002 may be particularly well suited for combination therapy with immune checkpoint inhibitors. NBE-002 is currently undergoing GMP manufacturing and initiation of clinical studies is expected in mid-2020.

Citation Format: Roger R. Beerli, Lorenz Waldmeier, Rémy Gébleux, Francesca Pretto, Ulf Grawunder. NBE-002, an anthracycline-based immune-stimulatory antibody drug conjugate (iADC) targeting ROR1 for the treatment of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-197.