bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB075: Development of SARS-CoV-2 neutralizing protein by ACE2 receptor engineering for severe infection and patients with underlying diseases
Authors: Kwon Byoung S. , Lee Seunghyun , Choi Jin-Kyung , Hwang Bora , Im Sun-Woo , Lim Yun-Sook , Kim Bumseok , Hwang Soon B. , Jeh HoonSung
Published: July 2021

Since the Spike protein on the surface of SARS coronavirus 2 (SARS-CoV-2) binds to the ACE2 receptor in human cells, the development of neutralizing proteins or antibodies targeting the receptor binding domain (RBD) of the spike protein is an important strategy for SARS-COV-2 therapy. We chose to develop molecularly-evolved soluble ACE2 protein on three grounds; 1) it can trap and neutralize the SARS-CoV-2 as neutralizing antibodies do, 2) it can supplement angiotensin II-converting enzyme activities that protect lung, heart, and kidneys of severe cases of infections and patients with underlying diseases, and 3) it may trap effectively SARS-CoV-2 mutants even though the mutations compromise the protection by neutralizing antibodies or vaccine. For the enhancement of ACE2 binding affinity to RBD, we used the 3D complex structure between ACE2 and RBD to select the major contributing ACE2 amino acids, a library targeting selected amino acids and random mutations were constructed and screened using yeast surface display. The engineered ACE2, EU129, was fused with the human IgG1 Fc for long half-life and viral clearance. The binding affinity of EU129 to RBD was increased by about 500-folds compared to ACE2 wild-type in SPR analysis, and the neutralizing activity was also increased by about 130-folds compared to ACE2 wild-type in surrogate virus neutralization test (sVNT). In addition, it was confirmed that the enzymatic activity of ACE2, which prevents organ damage due to SARS-CoV-2 infection in the human, is maintained at a level similar to that of ACE2 wild-type. In vitro assays using live SARS-CoV-2 virus and Vero E6 cells, EU129 was shown to be more effective in inhibiting viral infection and amplification than ACE2 wild-type, which was confirmed through protein and RNA level and cell morphology change of the live virus. In vivo stability assays using BALB/c mice, EU129 showed enhanced binding to the RBD and maintained enzymatic activity similar to ACE2 wild-type. We generated EU129 with the improved binding affinity and neutralizing activity through ACE2 receptor engineering. It has angiotensin II-converting enzymatic activity for organ protection, thus EU129 is a better therapeutic candidate for severe cases of SARS-CoV-2 infection and patients with underlying diseases such as cancers.

Citation Format: Byoung S. Kwon, Seunghyun Lee, Jin-Kyung Choi, Bora Hwang, Sun-Woo Im, Yun-Sook Lim, Bumseok Kim, Soon B. Hwang, HoonSung Jeh. Development of SARS-CoV-2 neutralizing protein by ACE2 receptor engineering for severe infection and patients with underlying diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB075.
Abstract LB099: The inhibitory effect of pemetrexed disodium heptahydrate on the growth of A549 cells
Authors: Mohiuddin Md , Kimura Hideharu , Sone Takashi , Watanabe Satoshi , Matsuoka Hiroki , Saeki Keigo , Terada Nanao , Abo Miki , Kasahara Kazuo
Published: July 2021

Background: Pemetrexed disodium heptahydrate (pemetrexed) is currently used for the treatment of patients with advanced non-small cell lung cancer (NSCLC). More than 30% of lung cancer cases involve KRAS mutations. In this study, we examined the anticancer ability of pemetrexed in KRAS-dependent A549 lung cancer cells. Methods: A549 cells were treated with pemetrexed for 72 h and then evaluated by a TUNEL assay, ROS assay and Western blotting. Results: The apoptotic effect of pemetrexed was observed through increased levels of cleaved caspase-9 (Asp 315), cleaved caspase-3 (Asp 175) and cleaved PARP. Furthermore, pemetrexed increased ROS-mediated DNA damage and enhanced the proapoptotic Bax protein expression, leading to the loss of mitochondrial membrane potential (MMP). The increased expression of the β-galactosidase protein was correlated with the induction of cellular senescence. Significantly, pemetrexed induced cytotoxicity without inhibiting the RAS/RAF/MEK/ERK signaling pathway, while pemetrexed induced autophagy, which activated the AMPK/mTOR signaling pathway. Conclusion: Taken together, these findings indicate that, in addition to apoptosis, pemetrexed-induced autophagy and cellular senescence, providing a promising direction for the development of KRAS-targeted therapy.

Citation Format: Md Mohiuddin, Hideharu Kimura, Takashi Sone, Satoshi Watanabe, Hiroki Matsuoka, Keigo Saeki, Nanao Terada, Miki Abo, Kazuo Kasahara. The inhibitory effect of pemetrexed disodium heptahydrate on the growth of A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB099.
Abstract LB100: Pemetrexed disodium heptahydrate and cisplatin have distinctive growth inhibitory effects in monotherapy and combination therapy on A549 cells
Authors: Mohiuddin Md , Kimura Hideharu , Sone Takashi , Watanabe Satoshi , Matsuoka Hiroki , Saeki Keigo , Terada Nanao , Abo Miki , Kasahara Kazuo
Published: July 2021

Background: Cisplatin combined with pemetrexed disodium heptahydrate (pemetrexed) is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, its individual growth inhibitory effects on KRAS-dependent A549 human lung cancer cells as monotherapy and combination therapy are not well understood. The aim of this study was to compare the effects of cisplatin (CDDP) and pemetrexed (PEM) on A549 cells as monotherapy and combination therapy.Methods: For in vitro studies, A549 cells were exposed to cisplatin and pemetrexed as monotherapy and combination therapy for 72 h. The results were then evaluated by a cell viability assay, apoptosis assay, ROS assay, TUNEL assay and Western blotting.Results: Our results revealed that cisplatin monotherapy was the most potent growth inhibitor; cisplatin plus pemetrexed combination therapy had an intermediate effect; and pemetrexed monotherapy induced a minimal growth inhibitory effect in the A549 cell line, which was observed by the inhibition of the RAS/RAF/MEK/ERK signaling pathway. Furthermore, apoptotic effects were examined according to ROS-mediated DNA damage and increased levels of cleaved caspase-3, cleaved caspase-9 and cleaved PARP. Interestingly, cisplatin monotherapy was the primary eliminator of cellular senescence; combination therapy with cisplatin plus pemetrexed had an intermediate eliminator effect, while pemetrexed monotherapy increased cellular senescence in A549 cells, which was assessed by the expression of the β-galactosidase protein. The results of Western blotting of proteins in the AMPK/mTOR signaling pathway indicated that the highest inducer of autophagy was pemetrexed monotherapy, while combination therapy with cisplatin plus pemetrexed had an intermediate inhibitory effect on autophagy. Cisplatin monotherapy showed the strongest autophagy inhibitory activity.Conclusion: In light of these findings, cisplatin monotherapy showed the most potent anticancer effects, suggesting that cisplatin monotherapy may be more effective than pemetrexed monotherapy or cisplatin plus pemetrexed combination therapy in A549 cells.

Citation Format: Md Mohiuddin, Hideharu Kimura, Takashi Sone, Satoshi Watanabe, Hiroki Matsuoka, Keigo Saeki, Nanao Terada, Miki Abo, Kazuo Kasahara. Pemetrexed disodium heptahydrate and cisplatin have distinctive growth inhibitory effects in monotherapy and combination therapy on A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB100.
Abstract LB102: Effect of mistletoe extract on growth and proliferation of prostate cancer cells
Authors: Boyapati Kavya , Kumar Rajendra , Wilson Lillian , Yang Yuhan , Topiwala Deven , Soundappan Keerti , Panagopoulos Eleni , Page Ivelisse , Durham Jennifer , Carducci Michael , Paller Channing , Kachhap Sushant
Published: July 2021

Complementary and alternative medicines are explored for their role in improving the quality of life of patients with cancer. Many anti-cancer drugs have been derived from natural sources. Mistletoe extract, composed of lectins, viscotoxins, flavonoids, and other biologically active substances, has two mechanisms of action: tumor cytotoxicity and immunomodulation. The cytotoxic effects of mistletoe extract are a result of protein synthesis interference, cell cycle inhibition, and apoptosis induction. Further, mistletoe extract has been shown to have anti-angiogenic properties. In Europe, Helixor M, mistletoe extract from Viscum album mali, is used as an anthroposophic medicine in the treatment of malignant and non-malignant tumors, malignant disorders with accompanying impairment of the hematopoietic organs, stimulation of bone marrow activity, reducing the risk of tumor recurrence, and diagnosed pre-cancerous disorders. However, only limited evaluation of mistletoe extract has been conducted in the United States. We studied the effects of Helixor M in prostate cancer. To evaluate its effect on cell proliferation and survival, we treated androgen receptor (AR) positive (LNCaP, LAPC4 and VCaP) and AR negative (PC3 and DU-145) cell lines with Helixor M. Our data indicates that LNCaP and LAPC4 cells were most sensitive to Helixor M while PC3, DU-145, and VCaP cells were relatively resistant to treatment. While AR protein levels and NF-kappaB subunits remained unaffected by treatment, there was an induction of apoptosis along with a concomitant decrease in LC3B, an autophagy marker, in all the sensitive prostate cancer cell lines. In search of pathways that may synergize with growth inhibition by Helixor M, we utilized CRISPR knockouts of TBK1, a kinase involved in autophagy induction, in LNCaP cells and tested whether TBK1 knockouts are synthetically lethal with Helixor M. Helixor M induced increased apoptosis in TBK1 knockouts as compared to parental LNCaP cells, indicating that TBK1 loss might be synthetically lethal with Helixor M.

Citation Format: Kavya Boyapati, Rajendra Kumar, Lillian Wilson, Yuhan Yang, Deven Topiwala, Keerti Soundappan, Eleni Panagopoulos, Ivelisse Page, Jennifer Durham, Michael Carducci, Channing Paller, Sushant Kachhap. Effect of mistletoe extract on growth and proliferation of prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB102.
Abstract LB116: Prostaglandin F2 Receptor Negative Regulator (PTGFRN) is a novel target to inhibit tumor growth via antibody drug conjugate
Authors: Marquez Jorge , Dong Jianping , Dong Chun , Serrero Ginette
Published: July 2021

Introduction: Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for a growing number of cancers, particularly hematopoietic and lymphoid tumors. There is need to identify new targets for ADCs, particularly for cancers with unmet needs. Methods: Our strategy has been to develop hybridoma libraries developed against cancer cells surface proteins and select antibody able to bind and internalize in cancer cell lines. Selected antibody can then be used for identification of the cancer cell surface target by immunoprecipitation (IP) and mass spectrometric analysis (Mass-spec), followed by target validation. After target validation, binding and target positivity can be tested by flow cytometry and western blot analysis on several cancer cell lines. The in vitro and in vivo efficacy of the antibody conjugated to a cytotoxic payload as ADC can then be evaluated with the target positive cancer cell lines.Results: Using the strategy described above, we have selected a monoclonal antibody named 33B7 able to bind and internalize in several cancer cell lines. The cell surface target binding 33B7 was identified by IP/Mass-spec as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was then validated by showing that cells transfected with PTGFRN cDNA bound and internalized the antibody. Screening by Flow bidning and by western blot identified several human cancer cells lines expressing PTGFRN. In vitro treatment of these cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. Conclusion: These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers. The elucidation of PTGFRN role in cancer cells, particularly in possibly mediating metastasis is presently being carried out.

Citation Format: Jorge Marquez, Jianping Dong, Chun Dong, Ginette Serrero. Prostaglandin F2 Receptor Negative Regulator (PTGFRN) is a novel target to inhibit tumor growth via antibody drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB116.
Abstract LB122: The complete estrogen receptor antagonist (CERAN) OP-1250 shrinks ER+ brain metastases in an intracranial xenograft tumor model expressing mutant ESR1
Authors: Hodges-Gallagher Leslie , Parisian Alison D. , Sun Richard , Myles David C. , Klein Pamela M. , Harmon Cyrus L. , Kushner Peter J.
Published: July 2021

Estrogen receptor-positive (ER+) breast cancer can undergo metastatic spread to the brain, which occurs in up to 15% of HER2-/ER+ metastatic breast cancer patients. Although endocrine therapy is not explicitly approved for brain metastasis and clinical trials are lacking, anecdotal reports of clinical responses by endocrine agents motivate further investigation. One such agent is the ER antagonist tamoxifen, which has previously been shown to reach high levels in brain tissue and ER+ brain metastases in patients with breast cancer*. We previously reported that OP-1250, a novel, oral complete ER antagonist (CERAN), penetrates the brain and is efficacious at shrinking tumors in a xenograft brain metastasis model in which patient derived human ER+ tumors were intracranially transplanted into immunocompromised mice**. Here we compare the ability of OP-1250 to shrink brain tumors in this model with tamoxifen and other agents. Whereas tamoxifen indeed had therapeutic effects in this brain xenograft model, the effect was not as profound as that of OP-1250, which was able to completely clear the tumors in many animals, as determined by MRI.

* Lien et al. Distribution of tamoxifen and metabolites into brain tissue and brain metastases in breast cancer patients. Br. J. Cancer, 1991, 63, 641-645.**Hodges-Gallagher, et al. OP-1250 a complete estrogen receptor antagonist that penetrates the brain and prevents lethality from an intracranial xenograft tumors expressing mutant ESR1, abstract, AACR 2020

Citation Format: Leslie Hodges-Gallagher, Alison D. Parisian, Richard Sun, David C. Myles, Pamela M. Klein, Cyrus L. Harmon, Peter J. Kushner. The complete estrogen receptor antagonist (CERAN) OP-1250 shrinks ER+ brain metastases in an intracranial xenograft tumor model expressing mutant ESR1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB122.
Abstract LB158: Implications of Targeting Novel Pathways in Exhausted T Cells for Immuno-Oncology Therapeutic Intervention
Authors: McPherson Rhoanne , Acklam Francis , Hay Joanne , Barbour Mark , Grant Courtney , Singh Preeti , Gambardella Adriana , Rzepecka Justyna , Anderton Stephen , Gooding Hayley
Published: July 2021

The induction of CD8+ T cell exhaustion, driven by the tumor microenvironment, is thought to be a major mechanism by which tumors can escape immune surveillance and are able to persist and progress. T cell exhaustion is typically characterized by the expression of co-inhibitory markers such as PD-1, LAG-3 and TIM-3 and a reduced capacity for pro-inflammatory cytokine production. Whilst treatment with therapeutics targeted at checkpoint inhibitors, such as Nivolumab (anti-PD-1), have shown great promise in the treatment of a subset of individuals and tumor types, resistance to these approaches is prevalent. As such, novel approaches are required in order to provide alternative options for the rescue of T cell exhaustion and cancer treatment.

Here we describe the characteristics of an in vitro human T cell exhaustion assay whereby in vitro stimulated T cells phenotypically and functionally recapitulate the exhausted T cells found within the tumor microenvironment and where exhaustion can be partially rescued by treatment with anti-PD-1. Furthermore, we demonstrate the effect of treatment with an inhibitor of a novel target identified in exhausted T cells on T cell function; both through analysis of pro-inflammatory cytokine production and the ability to kill tumor cells. These assays and approaches enable investigation into the ability of compounds to influence reversal of T cell exhaustion where anti-PD-1 treatment does not fully reverse the exhausted phenotype and offers the ability to test combination therapy approaches.

Citation Format: Rhoanne McPherson, Francis Acklam, Joanne Hay, Mark Barbour, Courtney Grant, Preeti Singh, Adriana Gambardella, Justyna Rzepecka, Stephen Anderton, Hayley Gooding. Implications of Targeting Novel Pathways in Exhausted T Cells for Immuno-Oncology Therapeutic Intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB158.
Abstract LB176: Anti-4-1BB x PD-1, a bispecific anti-cancer therapeutics
Authors: Kwon Byoung S. , Lee Hanna , Park Jin Sung , Oh Yeonji , Han SeungHee , Choi Jin Kyung , Hwang Bora , Im Sun Woo , Lee Seunghyun , Jeh HoonSung
Published: July 2021

4-1BB and PD-1 are both T cell costimulatory receptors/immune checkpoint regulators. 4-1BB/4-1BBL provides stimulatory signals while PD-1/PD-L1, suppressive ones to T lymphocytes. Various forms of agonists to 4-1BB or blockers to PD-1 have shown a potent anti-cancer activity by modulating mainly CD8+ T cells. Thus, synergistic or additive anti-cancer effect may be achieved by combining the agonists to 4-1BB and blockers to PD-1. We have tested various combinations of antibodies and proteins with consideration of overall size of the therapeutic candidates. The best anti-cancer activities were obtained by combination of an agonistic anti-4-1BB antibody and soluble PD-1 that binds to PD-L1 with high affinity. To obtain molecularly-evolved PD-1 we first used the 3D complex between PD-1 and PD-L1 to select the major contributing PD-1 amino acids, a library targeting selected amino acids and random mutations were constructed and screened using yeast surface display. The affinity-matured resulting PD-1 is called euPD-1. We finally constructed EU505 that composed of a scFv of anti-4-1BB and euPD-1. Through cell binding and dual antigen assays, we confirmed that EU505 binds both targets independently at the same time. The result of 4-1BB bioassays, a biologically relevant MOA-based assay, showed that potent PD-L1-dependent T cell activation with EU505. In-vitro killing assays showed that EU505 selectively activated T cells which in turn killed high PD-L1-expressing cells, but not low-PD-L1-expressing ones. EU505 demonstrated much stronger tumor-killing effect compared with each component alone when we tested against human PD-L1-expressing tumor cells in a 4-1BB Knock-in mouse model. Furthermore, it was observed that number of effector CD8+ T cells increased in the peripheral blood upon EU505 injection and consequently tumor size was reduced. EU505, a promising anti-cancer drug, appears to enhance CD8+ T cell infiltration and activate T cells in situ at the tumor sites by binding two different targets of 4-1BB and PD-L1 simultaneously.

Citation Format: Byoung S. Kwon, Hanna Lee, Jin Sung Park, Yeonji Oh, SeungHee Han, Jin Kyung Choi, Bora Hwang, Sun Woo Im, Seunghyun Lee, HoonSung Jeh. Anti-4-1BB x PD-1, a bispecific anti-cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB176.
Abstract LB177: Targeting SIRPα with APX701, a Novel Myeloid Checkpoint Inhibitor
Authors: Alvarado Ryan , Krishnan Sushma , Srivastava Minu K. , Tan Christine , Huang George , Jalgaonkar Swati , Bahjat Frances R. , Filbert Erin L. , Yang Xiaodong
Published: July 2021

Therapeutically targeting tumor myeloid cells has emerged as a complementary strategy to existing cancer immunotherapy approaches. The interaction of tumor-expressed CD47 with SIRPα (Signal Regulatory Protein-α, SIRPA) on macrophages, dendritic cells, and neutrophils inhibit key immune effector mechanisms. Targeting the SIRPα-CD47 axis represents a novel approach to enhance anti-tumor immunity by augmenting or reactivating critical tumor clearance mechanisms. While agents that target CD47 have shown promise in clinical trials, they present hematological toxicities and antigen sink issues that narrow their therapeutic application. Our approach targets SIRPα to focus on relevant mechanisms related to myeloid cell activation and increase of tumor cell phagocytosis while simultaneously avoiding the undesired effects of targeting CD47. Furthermore, SIRPγ, a close relative of SIRPα, is expressed on T cells and potentially co-stimulates T cell proliferation and is required for trans-endothelial T cell migration. We sought to develop an anti-SIRPα therapeutic that does not cross-react with SIRPγ and avoids potentially deleterious effects on T cell functions. Here, we describe APX701, a novel anti-SIRPα antibody generated using Apexigen's APXiMAB™ proprietary antibody discovery platform, and its differentiated properties as compared to other approaches targeting the SIRPα-CD47 interaction. APX701 binds to human SIRPα at a unique epitope with high affinity (Kd = 0.95nM) and with no detectable binding to SIRPγ expressed on primary human T cells. APX701 strongly binds to the major SIRPα variants—V1 and V2—and potently blocks their interaction with CD47, thereby allowing therapeutic targeting of SIRPα in a majority of patients. APX701 reverses SIRPα-mediated suppression of macrophage phagocytosis, resulting in potent destruction of a variety of tumor cells both as a single agent and in combination with anti-TAA (tumor associated antigen) antibodies including cetuximab, rituximab, and trastuzumab. Moreover, targeting SIRPα with APX701 does not interfere with T cell function, an effect previously observed following anti-CD47 treatment. Together, these data demonstrate the unique binding profile of APX701 and its ability to potently reactivate myeloid cell-mediated anti-tumor immunity. Our data support the development of APX701, a promising myeloid checkpoint inhibitor, for the treatment of cancer.

Citation Format: Ryan Alvarado, Sushma Krishnan, Minu K. Srivastava, Christine Tan, George Huang, Swati Jalgaonkar, Frances R. Bahjat, Erin L. Filbert, Xiaodong Yang. Targeting SIRPα with APX701, a Novel Myeloid Checkpoint Inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB177.
Abstract LB183: Erythritol acts as tumor enhancer and suppressor depending on concentrations in brain tumor cell lines
Authors: Nassani Rayan , AlAmri Hassan , Alrfaei Bahauddeen M.
Published: July 2021

Erythritol (ERT) is a simple four-carbon sugar alcohol, that is produced endogenously via pentose phosphate pathway from glucose in all human cells mainly erythrocytes with a plasma level of ∼9.8 μM. It can be obtained exogenously from diets as an FDA-artificial sweetener. It possesses hydroxyl groups that are responsible for hydroxyl radical scavenging effect (antioxidant property). Recent studies have pointed out an ERT protection based on concentration dependant effect on endothelial cells. The protection includes surviving death under high glucose conditions in diabetic patients. In addition, ERT has been reported in pre-diagnostic serum samples of glioblastoma patients, which was elevated. It was not known whether ERT elevation was defensive or harmful. We studied the cytotoxic activity of ERT on brain tumor cell lines (U87, Daoy) using metabolic activity assay (MTT) with different erythritol concentrations. We observed that on high concentrations (2.87 M, 1.43 M, 0.72 M), there was a significant growth inhibition (P<0.05). On the other hand, lower concentrations (0.36 M, 8 mM, 4 mM, 2 mM, 0.8 mM, 36 µM, 8.2 µM, 4.1µM, 8.2nM), showed growth enhancement. ERT is known with its antioxidant effect in a concentration dependent manner. Therefore, we studied the antioxidant activity of ERT using ROS-Glo H2O2 Assay. We observed that on the high concentrations of ERT there was a significant decrease in hydrogen peroxide concentration to less than 50%, this suggests that ERT has antioxidant effect against hydrogen peroxide. In contrast, low ERT concentrations (around physiological level) showed significant enhancement to tumor growth (P<0.05). In conclusion, we found that erythritol plays an important role in brain cancer development and hydrogen peroxide regulation through a concentration dependant manner. Since ERT is found normally in human plasma, controlling ERT concentration will grant ERT the ability to work as therapeutic enhancer for brain cancer treatment or even a biomarker for prognosis.

Citation Format: Rayan Nassani, Hassan AlAmri, Bahauddeen M. Alrfaei. Erythritol acts as tumor enhancer and suppressor depending on concentrations in brain tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB183.
Abstract LB196: Comparison of whole exome sequencing and targeted sequencing in patients with advanced bladder cancer
Authors: Chawla Neal Shiv , Zengin Zeynep , Govindarajan Ameish , Pal Sumanta K.
Published: July 2021

Background: Next generation sequencing (NGS) to identify genomic alterations (GAs) in tumor samples are increasingly used in clinical practice. Targeted sequencing (TS) or whole exome sequencing (WES) are used by various commercially available platforms. Differing methods often result in poor inter-reproducibility, even when testing the same tissue. Bladder cancer has substantial genetic heterogeneity as compared to many other malignancies. The purpose of this study is to compare WES and TS in bladder cancer. Methods: Patients with advanced bladder cancer who had undergone tissue WES (GEM ExTra®) or TS (FoundationOne CDx® [FMI]) in the course of routine clinical care were assessed. Genes with at least one detected GA were selected, with others excluded. All detected GAs were classified as being detected on TS only, WES only, concordant (detected on both platforms), or partially concordant (different GAs detected within the same gene). Variants of uncertain significance were excluded for this study. Results: In our series, 25 patients had WES and 42 patients had TS - 14 patients (21%) had both tests performed. A total of 88 genes were detected to have at least one GA across all samples. Of all detected GAs, 66/162 (41%) were concordant, with 45/162 (28%) and 51/162 (31%) of GAs detected exclusively by WES and TS, respectively. The genes with the highest proportion of concordance when detected, were TERT (70%), TP53 (85%) and ARIDA1A (50%). SOX4 and E2F3 were detected on WES only, while RBM and CHEK2 were detected on TS only. Notably, six TP53 GAs were detected exclusively on TS. TP53, TERT, MLL2/KMT2D, ERBB2 were each partially concordant on one occasion. The difference in the rate of concordant GAs in samples from the same collection site and date (41%), as compared to those in which both differed (35%) were not statistically significant (p=0.54). Conclusions: Although the majority of GAs identified were concordant, each platform exclusively identified GAs with varying clinical significance. SOX4 and E2F3 mutation, shown to correlate with invasive potential in bladder cancer, were detected exclusively on WES. In contrast, RBM and CHEK2 (of known significance in melanoma and breast cancer) were found exclusively on TS. Larger series are needed to further establish the concordance of these approaches.

Citation Format: Neal Shiv Chawla, Zeynep Zengin, Ameish Govindarajan, Sumanta K. Pal. Comparison of whole exome sequencing and targeted sequencing in patients with advanced bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB196.
Abstract LB198: Inhibition of the RAC Activator VAV3 by the small molecule IODVA1 impedes RAC signaling & overcomes resistance to tyrosine kinase inhibition in lymphoblastic leukemia
Authors: Nassar Nicolas N. , Hegde Shailaja , Wunderlich Kark , Lin Yuan , Ahmadian Reza , Seibel William , Zheng Yi , Mizukawa Benjamin E. , Vinnedge Lisa Privette , Cancelas Jose A.
Published: July 2021

Aberrant activation of RHO guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in transformation and tumorigenesis. Consequently, a small molecule inhibitor of RhoGEF activities can be used as an anti-cancer drug. Herein, we used cellular, mouse, and humanized models of RAC-dependent BCR-ABL1-driven and Ph-like lymphoblastic leukemia to identify VAV3, a tyrosine phosphorylation-dependent RacGEF, as the target of the small molecule IODVA1. We show that IODVA1 binds tightly to VAV3, inhibits RAC activation and signaling, and increases pro-apoptotic activity in BCR-ABL1-transformed cells only. Consistent with this mechanism of action, both VAV3-deficient leukemic cells and mouse models of BCR-ABL1 leukemia do not respond to IODVA1. IODVA1 eradicates leukemic propagating activity of TKI-resistant BCR-ABL1(T315I) B-ALL cells after treatment withdrawal by decreasing RAC signaling in vivo. Importantly, IODVA1 is superior to standard of care dasatinib and ponatinib at prolonging the survival of PDX models of relapsed pediatric Ph+ and TKI-resistant Ph+ B-ALL with commonly found genetic mutations especially after treatment withdrawal. Cells representing pediatric ALL patients with diverse genetic lesions are highly sensitive to IODVA1 ex vivo and this sensitivity is VAV3-dependent. IODVA1 thus spearheads a novel class of drugs that inhibits a RacGEF and holds promise as an anti-tumor therapeutic agent.

Citation Format: Nicolas N. Nassar, Shailaja Hegde, Kark Wunderlich, Yuan Lin, Reza Ahmadian, William Seibel, Yi Zheng, Benjamin E. Mizukawa, Lisa Privette Vinnedge, Jose A. Cancelas. Inhibition of the RAC Activator VAV3 by the small molecule IODVA1 impedes RAC signaling & overcomes resistance to tyrosine kinase inhibition in lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB198.
Abstract LB200: A transcriptionally distinct subpopulation of healthy acinar cells exhibit features of pancreatic progenitors and pancreatic ductal adeno carcinoma
Authors: Gopalan Vishaka , Singh Arashdeep , Rashidi Farid , Wang Li , Ruppin Eytan , Arda Efsun , Hannenhalli Sridhar
Published: July 2021

The stochasticity of transcription leads to phenotypic variation within a clonal population. This means that within a healthy tissue, a fraction of cells could assume an “edge” transcriptional state that is primed for a transition toward malignancy. We find such an edge transcriptional state amongst acinar cells in a publicly available PDAC single-cell RNA-seq dataset. These acinar edge cells (AE) cells have transcriptomes that are highly diverged from a typical normal acinar cell, and show transcriptional similarities to pancreatic ductal adenocarcinoma (PDAC).

The AE state can be found in several single-cell datasets from healthy human pancreatic tissue. The AE state is not mutationally-driven as AE cells in healthy tissues do not possess any known oncogenic driver mutations, nor are they clonally derived. The transition from a non-edge acinar to an AE state recapitulates key aspects of pancreatic biology; genes associated with this transition are enriched for known markers of acinar de-differentiation and acinar-ductal metaplasia. Further, the AE state represents a partial reversion to a SOX9+PTF1A+ multi-potent embryonic pancreatic progenitor state, likely through the activation of KLF5, HES1 and RBPJ targets. Remarkably, the promoters of genes upregulated in the AE state are significantly hypomethylated in PDAC relative to normal pancreas, while the genes up-regulated in the AE state are up-regulated in PDAC samples in the Cancer Genome Atlas (TCGA) database. It is thus likely that transcriptomic changes in the edge state are fixed epigenetically during tumor initiation and progression. A gene signature derived from the AE cells shows strong association with survival among PDAC patients. Remarkably, we also find that the fraction of AE cells in the healthy pancreas increases with age, which points to a strong non-genetic and transcriptomic component to the known increase in PDAC risk with age.

We also observed edge-like transcriptomic heterogeneity among alveolar, ciliated and club cells in the human lung, and amongst hepatocytes in liver tissues. This suggests that, across tissues, gene expression heterogeneity can lead to sub-populations of healthy cells across tissues existing in pre-malignant states.

Citation Format: Vishaka Gopalan, Arashdeep Singh, Farid Rashidi, Li Wang, Eytan Ruppin, Efsun Arda, Sridhar Hannenhalli. A transcriptionally distinct subpopulation of healthy acinar cells exhibit features of pancreatic progenitors and pancreatic ductal adeno carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB200.
Abstract LB201: Understanding genetic variation in cancer using nanopore targeted sequencing
Authors: Iyer Shruti V. , Kramer Melissa , Goodwin Sara , McCombie W. Richard
Published: July 2021

Structural variations (SV), a hallmark of genomic instability in cancer can either activate oncogenes or inactivate tumor suppressor genes. SVs tend to be recurrent and have been associated with several cancer types. Next-generation sequencing (NGS) is mostly blind to large SVs, lacking sensitivity with false positive rates up to 89% in SV detection. Long-read, single-molecule sequencing platforms can address larger variations as they typically generate read lengths of tens of thousands of bases and have helped identify thousands of genomic features pertinent to cancer that were previously missed by short-read sequencing. However, the throughput and coverage offered by whole genome long read sequencing makes it infeasible to conduct large-scale genomic studies.Targeted sequencing significantly improves accuracy and coverage by offering the depth necessary to detect rare alleles in a heterogenous population of cells. Using the nanopore Cas9-targeted sequencing (nCATS), a PCR-free enrichment system from Oxford Nanopore Technologies, we targeted 10 prominent cancer genes in MCF 10A and SK-BR-3 breast cell lines. However, we observed that the number of reads generated for targets longer than 30kb were not sufficient to accurately call SVs. To further enhance this approach, we developed an Affinity-based Cas-9-Mediated Enrichment (ACME) step, that uses HisTag DynabeadsTM to pulldown non-target reads. With ACME we achieved ~ 75-fold enrichment of the BRCA2 region and 35-65x coverage of this ~90 kb target on our panel. We observed an increase in enrichment and coverage of other genes on the panel as well, with enrichment as high as 5000-fold for some genes. While ACME is a biochemical approach, recently, adaptive sequencing or “Read Until”, a computational approach that ‘rejects' non-target reads has been used by a couple of groups for target enrichment. Using UNCALLED, a variation of Read Until, we observed an ~10x coverage of our targets. Recognizing the potential and limitations of both, biochemical and computational approaches, our efforts now are directed towards a combined approach where we expect to achieve a minimum enrichment of 200-fold across all targets. With a 200-fold enrichment of a 3.5Mb target, we should get between 200x - 400x coverage of the target, which should be high enough to detect SVs in as low as 10% of the cells in the sample. This would support large scale SV analysis to help define the landscape of such variants in the population and identify regions of therapeutic or diagnostic interest.

Citation Format: Shruti V. Iyer, Melissa Kramer, Sara Goodwin, W. Richard McCombie. Understanding genetic variation in cancer using nanopore targeted sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB201.
Abstract LB215: Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype
Authors: Chen Xi , Adhikary Gautam , Shrestha Suruchi , Xu Wen , Eckert Richard
Published: July 2021

Epidermal squamous cell carcinoma (SCC) is one of the most common cancers. Ultraviolet radiation exposure leads to DNA damage in keratinocyte stem cells leading to transformation. Epidermal cancer stem cells (ECS cells) display aggressive spheroid formation, invasion, and migration, and form rapidly growing and highly vascularized tumors. The transglutaminase 2 (TG2) cancer stem cell maintenance proteins is present at high levels in ECS cells and is required for maintenance of the ECS cell phenotype. Identifying how TG2 maintains the ECS cell phenotype is an important goal. RNA transcriptomic analysis, comparing gene expression in wild-type and TG2 knockout cells, reveals that hepatocyte growth factor (HGF) signaling is markedly reduced in TG2 knockout cells, a finding that was confirmed by showing that TG2 knockdown/knockout reduces HGF and MET mRNA and protein. HGF/MET signaling appears to play an important role in maintaining the cancer phenotype, as inhibition of HGF/MET reduces ECS cell spheroid formation, invasion and migration. Stem cell and EMT marker expression and MEK1/2 and ERK1/2 activity are also reduced. These changes phenocopy the response to TG2 knockdown. Treatment with MEK1/2 and ERK1/2 inhibitors suppresses the ECS cell phenotype indicating that MAPK signaling is a major HGF/MET target, and MET knockout reduces ECS cell tumor formation which is associated with reduced MEK1/2 and ERK1/2 activity. These findings suggest that TG2 maintains HGF/MET signaling which stimulates MEK1.2 and ERK1/2 activity to enhance the aggressive ECS cell cancer phenotype and drive tumor formation.

Citation Format: Xi Chen, Gautam Adhikary, Suruchi Shrestha, Wen Xu, Richard Eckert. Transglutaminase 2 maintains hepatocyte growth factor signaling to enhance the epidermal cancer stem cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB215.
Abstract LB234: The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy
Authors: Barber Alison G. , Xu Zhiwen , Rahman Justin , Goel Hira Lal , Mercurio Arthur M. , Burkart Christoph , Nangle Leslie A.
Published: July 2021

Lung cancer remains one of the leading cancer types and is the foremost cause of cancer-related deaths, yet treatment options for advanced lung cancer remain limited. Novel therapies targeting factors critical to the progression of lung cancer are essential. Neuropilin-2 (NRP2) acts as a co-receptor for VEGF and has been correlated with aggressive cancer growth and metastasis. The expression of NRP2 has been shown to be upregulated in several cancers, including non-small cell lung cancer (NSCLC), and the expression of NRP2 in NSCLC is associated with increased invasive tumor growth in vitro as well as a significant decrease in patient survival. As shown previously, aTyr has developed ATYR2810, a domain-specific anti-human monoclonal antibody to NRP2 which blocks its binding to VEGF. Importantly, ATYR2810 blocks VEGF binding in a highly specific manner and does not affect the binding of Semaphorin-3F or subsequent Semaphorin-3F-induced dimerization of NRP2 and PlexinA1. In this study, we have used ATYR2810 to examine the effects of blocking VEGF-mediated NRP2 signaling both as monotherapy and in combination therapy. Using in vitro 3D colony formation assays, we found that ATYR2810 used as monotherapy led to a significant reduction in colony formation in the A549 NSCLC cell line. The effects of ATYR2810 were also examined in NSCLC xenografts, where the use of ATYR2810 as a single agent was found to result in the inhibition of tumor growth. Interestingly, the tumor growth inhibition observed with ATYR2810 as monotherapy was comparable to that of cisplatin monotherapy, suggesting that ATYR2810 may be a potent inhibitor of tumor growth in the A549 xenograft model. The effects of ATYR2810 combination therapy in NSCLC xenografts was also examined, and we found that the use of ATYR2810 in combination with either 5-FU or cisplatin enhanced tumor growth inhibition as compared to the use of either chemotherapeutic agent alone. Taken together, these results suggest that NRP2 may be an important target in aggressive NSCLC and that the use of our novel ATYR2810 antibody to block VEGF-mediated NRP2 signaling may serve as a valuable therapeutic agent both as monotherapy as well as in combination therapy.

Citation Format: Alison G. Barber, Zhiwen Xu, Justin Rahman, Hira Lal Goel, Arthur M. Mercurio, Christoph Burkart, Leslie A. Nangle. The Neuropilin-2 targeting antibody ATYR2810 inhibits non-small cell lung cancer tumor growth in monotherapy and combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB234.
Abstract LB252: Evaluation of the pan-class I phosphoinositide 3-kinase (PI3K) inhibitor copanlisib in the Pediatric Preclinical Testing Consortium osteosarcoma in vivo models
Authors: Harrison Douglas , Gill Jonathan , Hingorani Pooja , Roth Michael , Zhang Wendong , Teicher Beverly , Earley Eric J. , Erickson Stephen W. , Gatto Gregory , Kurmasheva Raushan T. , Houghton Peter J. , Smith Malcolm A. , Kolb Edward A. , Gorlick Richard
Published: July 2021

Background. Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor with activity against all four PI3K class I isoforms (PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ). Copanlisib was approved for use by the FDA in the treatment of refractory follicular lymphoma in 2017. Preclinical data have suggested that deregulation of the PI3K pathway is a key driver of multiple malignancies. The relevance of this pathway in osteosarcoma tumorigenesis remains a subject of debate, however, whole genome and RNA sequencing data have revealed several PI3K aberrations in osteosarcoma tumor samples. The in vivo effects of copanlisib were studied in the Pediatric Preclinical Testing Consortium osteosarcoma xenograft models. Methods. The in vivo anticancer effects of copanlisib were assessed in a panel of 6 osteosarcoma models (OS-2, OS-9, OS-31, OS-33, OS-36, and OS-60). Copanlisib was administered by oral gavage at a dose of 10 mg/kg/day, two days on and five days off, repeated weekly for 4 weeks. Time to event and tumor volume responses were defined and analyzed utilizing standard PPTC statistical methods. Results. Copanlisib was well tolerated in the models with minimal weight loss and no treatment related mortality as compared to controls. Copanlisib induced prolonged event-free survival (EFS) in 5/6 osteosarcoma models (p<0.05, Gehan-Wilcoxon). Tumor regression (mean minimum attained relative tumor volume (minRTV) < 1.0) was not observed for any of the tested models. 3/6 models exhibited lower min RTV compared to untreated controls (p < 0.05, Wilcoxon rank sum). All osteosarcoma models showed progressive disease as their objective response measure. Conclusions. While copanlisib induced prolonged EFS in 5/6 osteosarcoma models, no tumor regression was seen, with all models developing progressive disease suggesting minimal activity. While copanlisib did not result in tumor regression, further study is needed to fully explore the role of the PI3K pathway in the pathogenesis of osteosarcoma.

Citation Format: Douglas Harrison, Jonathan Gill, Pooja Hingorani, Michael Roth, Wendong Zhang, Beverly Teicher, Eric J. Earley, Stephen W. Erickson, Gregory Gatto, Raushan T. Kurmasheva, Peter J. Houghton, Malcolm A. Smith, Edward A. Kolb, Richard Gorlick. Evaluation of the pan-class I phosphoinositide 3-kinase (PI3K) inhibitor copanlisib in the Pediatric Preclinical Testing Consortium osteosarcoma in vivo models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB252.