bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4787: The cancer targeting synergy of tertbutylhydroquinone and a manganese porphyrin
Authors: LaMorte Joseph P. , Tamarin Sandra , Lalo Margueritte , Sokoloski David J. , Paul Jared J. , Eggler Aimee L.
Published: April 2023

Cancer cells utilize high basal levels of reactive oxygen species (ROS) to create conditions that facilitate rapid cell proliferation. This creates an Achilles heel, making cancer cells more susceptible than normal cells to cell death caused by a bolus dose of ROS. The common food preservative, tert-butylhydroquinone (tBHQ), generates ROS intracellularly via redox cycling. Herein, we tested the effect of combining tBHQ with a manganese porphyrin, compounds originally designed as super oxide dismutase mimetics which can also generate ROS intracellularly in cancer cells and have radioprotective effects under clinical investigation. We find that the combination treatment of tBHQ and the commercially available Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) is much more toxic to leukemic Jurkat lymphocytes than either compound alone, e.g., lowering the LC50 of tBHQ by two orders of magnitude to 1.0 μM. MnTMPyP acts as a catalyst to oxidize tBHQ, shown by UV-Vis spectroscopy. Flow cytometry analysis revealed that while 5 μM tBHQ decreases cell viability by less than 10% in 4 hours, the combination of 5 μM tBHQ and 12 μM MnTMPyP decreases viability by 60%, primarily by apoptosis. A clinically tested manganese porphyrin, Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin (Mn2BuOE), was as effective and more potent than MnTMPyP. We evaluated the cancer-targeting effect of tBHQ and Mn2BuOE in both Jurkats and a matched primary CD4 T cell line. The combination treatment induced significantly more cell death in cancerous Jurkats than in the normal CD4 cells. With the promise Mn2BuOE shows in pre-clinical studies and early clinical trials as adjunctive to radiotherapy, and the wide use of tBHQ in prepared food, the results shown here in CD4 cells suggest their combination may be an effective therapy to target cancer cells.

Citation Format: Joseph P. LaMorte, Sandra Tamarin, Margueritte Lalo, David J. Sokoloski, Jared J. Paul, Aimee L. Eggler. The cancer targeting synergy of tertbutylhydroquinone and a manganese porphyrin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4787.
Abstract 4790: Oxidation in the nucleotide pool as a novel targetable vulnerability in pancreatic ductal adenocarcinoma
Authors: Mateo-Victoriano Beatriz , Zhang Ling , Samaranayake Govindi J. , Due Cassie , Troccoli Clara , Zaias Julia , Nagathihalli Nagaraj , VanSaun Michael , Mohsen Michael G. , Kool Eric T. , Rai Priyamvada
Published: April 2023

Oncogenic RAS signaling is mediated by reactive oxygen species (ROS) signaling. However, oncogenic ROS also evoke tumor suppressive damage, necessitating adaptive redox protective mechanisms. We previously reported that MTH1, the main mammalian 8-oxodGTPase, comprises one such critical adaptation. High MTH1 expression significantly correlates with poor disease-free survival in several KRAS-driven tumors. We have shown in RAS-driven xenograft cancer models that MTH1 is critical for the maintenance of ROS-driven signaling and prevention of DNA damage-induced senescence. However, how MTH1 loss affects KRAS-driven spontaneous tumor formation is unknown. We focused on pancreatic ductal adenocarcinoma (PDAC) due to the high prevalence of driving oncogenic KRASG12D mutations, and the lack of MTH1 studies. Therefore, we depleted MTH1 via shRNA in MIA PacCa-2 and SU.86.86 cell lines and generated novel MTH1-null Pdx1Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ (KPC), Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) and corresponding MTH1-wildtype strains. We found MTH1 knockdown significantly reduced growth of KRAS-driven PDAC cells, and that germline MTH1 deficiency reduced tumor burden in the short latency PKT but not the long latency KPC animals. The PKT tumors exhibited higher overall 8-oxodGTPase activity and reduced total 8-oxo-dGTPase activity in the MTH1-deficient background, unlike KPC tumors. This finding suggests that in aggressive KRAS-driven tumors, redundant 8-oxodGTPase activity cannot compensate for MTH1 loss to prevent oncogenic stress-induced tumor suppression. Additionally, both MTH1-depleted PDAC cell lines and the smaller MTH1-null PKT tumors had decreased EGFR expression, suggesting MTH1 loss compromises this ROS-driven pathway. RNA-seq analysis revealed MTH1 deficiency altered MDSC gene expression in the PKT model, suggesting better anti-tumor response through improved immune infiltration in a typically immunologically cold tumor. Our results indicate MTH1 targeting could benefit aggressive PDAC stages. We further show PDAC tumors possess redundancy in MTH1 8-oxodGTPase function, which needs to be better understood in order to therapeutically leverage current MTH1 inhibitors, which do not target this redundancy.

Citation Format: Beatriz Mateo-Victoriano, Ling Zhang, Govindi J. Samaranayake, Cassie Due, Clara Troccoli, Julia Zaias, Nagaraj Nagathihalli, Michael VanSaun, Michael G. Mohsen, Eric T. Kool, Priyamvada Rai. Oxidation in the nucleotide pool as a novel targetable vulnerability in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4790.
Abstract 487: ITI-1020, a novel PDE type 1 inhibitor, reduces tumor growth in triple negative breast cancer (TNBC) mouse models
Authors: Voisin Maud , Kogan Aksinija A. , Goding Stephen , Snyder Gretchen L. , Davis Robert E.
Published: April 2023

Breast cancer is the most common cancer in women. Unfortunately, some patients do not benefit from most therapies and may even develop resistance to previously effective treatments leading to disease progression. Therefore, treatment resistance remains a significant challenge and new treatments are needed to overcome this problem.

ITI-1020 is a potent and highly selective inhibitor of the phosphodiesterase 1 (PDE1) enzyme. Given the role of PDE1 in the regulation of essential cellular functions, including among others the transition of monocytes to macrophages and potentially the infiltration of macrophages into the tumor microenvironment (TME), we hypothesize that ITI-1020 will alter macrophage infiltration and polarization, as well as improve T cell function and recruitment into the TME, thereby promoting antitumor immunity in murine cancer models.

We tested this hypothesis by studying the effect of ITI-1020 given, alone or in combination a PD-1 immune checkpoint inhibitor, on tumor growth, and TME composition of E0771 and 4T1 tumor-bearing mice, two syngeneic models of TNBC.

Mice treated with ITI-1020 alone or in combination with anti-PD-1 antibody showed significant inhibition of tumor growth compared to isotype controls in both type of tumors. Flow cytometry analysis of the tumor microenvironment showed a significant shift in the polarization of macrophages toward a more inflammatory phenotype, and/or a reduction in the number of exhausted T cells. Furthermore, scRNAseq analysis of tumors from mice treated with ITI-1020 in monotherapy showed altered expression of genes involved in inflammatory processes and cell proliferation within the tumor microenvironment and in the cancer cells.

These results suggest that ITI-1020 promotes antitumor immunity by a novel mechanism (PDE1 inhibition), leading to tumor growth inhibition in E0771 and 4T1 tumor models. Translating these findings to the clinic may provide a new hope for the treatment of refractory or resistant TNBC tumors.

Citation Format: Maud Voisin, Aksinija A. Kogan, Stephen Goding, Gretchen L. Snyder, Robert E. Davis. ITI-1020, a novel PDE type 1 inhibitor, reduces tumor growth in triple negative breast cancer (TNBC) mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 487.
Abstract 4914: Role of ClpP in the anti-cancer effects of imipridone ONC201 and ONC206
Authors: Lee Andrew , Maranto Cristina , Foster Scott , Morrow Sara , Allen Joshua E. , Lanier Randall , Sethna Phiroze , Prabhu Varun V.
Published: April 2023

ONC201 is the first bitopic dopamine receptor D2 (DRD2) antagonist and allosteric mitochondrial protease ClpP agonist. Downstream of target engagement, ONC201 activates the integrated stress response pathway resulting in selective tumor cell death. ONC201 is well tolerated and induces durable tumor regressions in H3 K27M-mutant glioma patients. ONC206, a derivative of ONC201 currently in Phase I trials for adult and pediatric brain tumors, is also a bitopic DRD2 antagonist and ClpP agonist with nanomolar potency. We further evaluated in vitro efficacy and mechanism of action specific to ClpP for ONC206 and investigated acquired resistance to ONC201 and ONC206. FITC-casein and peptide degradation assays confirmed that ONC206 acts as an agonist of human ClpP with enhanced potency relative to ONC201. Broad efficacy in the nanomolar range for ONC206 (GI50 <78-889nM, 72h) was observed in 1,088 Genomics of Drug Sensitivity in Cancer (GDSC) cancer cell lines with increased sensitivity in cell lines exhibiting high ClpP mRNA expression. Among solid tumors, nervous system-related cell lines were particularly sensitive. Gene Expression Profiling Interactive Analysis (GEPIA) database analysis showed ClpP mRNA was overexpressed in glioblastoma cells relative to normal cells. GDSC observations were confirmed in glioblastoma (GBM) (T98G, A172, U87MG), H3.3 K27M mutant diffuse intrinsic pontine glioma (DIPG) (SF8628, SF7761) and astrocytoma (H4, U118MG) cell lines. ONC206 was more potent than ONC201 in cell viability assays for all glioma lines tested. SF8628 cells with a CRISPR-mediated ClpP knockout were shown to demonstrate resistance to both ONC201 and ONC206. Polyclonal resistant cell lines were generated through prolonged passaging in increasing concentrations of ONC201 or ONC206 (SF8628 and T98G cells). Cells with acquired resistance to ONC201 (ONC201Res) or ONC206 (ONC206Res) showed cross resistance to both imipridones. Time-stability of resistance was confirmed by removing drug for 2 to 4 weeks and retesting. RNAseq analysis revealed upregulation of the integrated stress response with imipridone treatment in T98G parental cells but not resistant clones. Whole genome sequencing revealed ClpP mutations in both ONC201Res and ONC206Res cells. Our results confirm the role of ClpP in the mechanism of action of ONC201 and ONC206 in tumor cells.

Citation Format: Andrew Lee, Cristina Maranto, Scott Foster, Sara Morrow, Joshua E. Allen, Randall Lanier, Phiroze Sethna, Varun V. Prabhu. Role of ClpP in the anti-cancer effects of imipridone ONC201 and ONC206. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4914.
Abstract 4941: Development of small molecule RPN13 inhibitors for treatment of glioblastoma
Authors: Chang Yung-Nien , Anchoori Ravi K. , Yamashita Alex Shimura , Rosa Marina da Costa
Published: April 2023

Glioblastoma (GBM) is an aggressive and deadly brain cancer with a one-year median survival due to the lack of effective targeted treatments. Our goal is to develop a small molecule inhibitor targeting RPN13 protein in the 19S proteasome complex as a new drug for GBM. RPN13 is a proteasome ubiquitin receptor that recognizes ubiquitin-conjugated proteins that are thus tagged for degradation via the 20S proteasome enzymatic complex. GBM is highly sensitive to proteasome inhibition due to its aggressive growth and accumulation of misfolded polyubiquitinated proteins that is insufficiently managed despite increased proteasome levels and enhanced proteasome activity (i.e. proteotoxic stress). Up Therapeutics judiciously designed drug-like RPN13 inhibitors to overcome limitations in the prototype RPN13 inhibitor RA190 identified at Johns Hopkins University. We selected Up284 as lead compound based upon our preliminary studies showing drug-like characteristics, specificity (binding to Cys88 of RPN13), and proteasome inhibition that produces a cytotoxic accumulation of polyUb proteins, ER stress, and reactive oxygen species. Up284 treatment selectively triggers apoptosis in solid tumor types including GBM cell lines and oncospheres. A suitable Up284 formulation was selected by measuring aqueous solubility, stability and on-target activity in mice imaged using a proteasome-dependent in vivo reporter. Up284 can be administered i.v. or orally, and it penetrates through the BBB. As a proof of concept for treatment of GBM, Up284 demonstrated on-target activity in intracranial syngeneic GL261 tumor model by inhibiting proteasome function in tumor tissues. Up284 also well tolerated and regressed tumors in syngeneic, xenograft and spontaneous models of ovarian and breast cancers with acceptable safety profile. Up284 also synergize with current frontline chemotherapeutics and can be used as a single agent or in combination for the treatment of wide range of cancers.

Citation Format: Yung-Nien Chang, Ravi K. Anchoori, Alex Shimura Yamashita, Marina da Costa Rosa. Development of small molecule RPN13 inhibitors for treatment of glioblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4941.
Abstract 4942: Heme targeting and its mechanistic role in triple-negative breast cancer suppression
Authors: Berisha Eranda , del Carmen Chacon Castro Maria , Ashrafi Adnin , Salamat Narges , Alemi Parinaz Sadat , Zhang Li
Published: April 2023

Breast cancer is the second leading cause of cancer-related deaths and comprises of approximately 30% of all cancer cases affecting women in the United States. There are three subtypes of breast cancer based on receptor profiles, with the third subtype being triple-negative breast cancer. TNBC accounts for about 10-15% of all breast cancers. The TNBC subtype lacks estrogen, progesterone, and HER2 receptors; it is associated with BRCA1 and BRCA2 mutations. This type of cancer has poor prognosis, tends to be more aggressive, and has limited treatment options compared to the other two subtypes. Heme is an essential molecule in the oxidative phosphorylation pathway and in the generation of reactive oxygen species (ROS). ROS generation causes oxidative stress and induces DNA damage in breast cancer. Therefore, targeting its activity potentially suppresses TNBC tumors. Heme-sequestering protein 2 (HeSP2) binds and sequesters heme. It has been shown that HeSP2 treatment of MDA-MB-231 cells inhibited cell proliferation and colony formation. Moreover, HeSP2 suppresses tumor growth in subcutaneously implanted TNBC MDA-MB-231 xenografts in NOD/SCID mice. Furthermore, in vitro studies using murine TNBC cell models 4T1-fluc Neo and EMT6-fluc Puro cells have also shown that cell proliferation and colony formation were inhibited after HeSP2 treatments of increasing concentrations. Thus, these results encourage the potential use of HeSP2 for breast tumor suppression. To further explore the effects of heme targeting and sequestration, we will determine heme flux in TNBC cell models by measuring the levels of heme uptake, export, synthesis, and degradation. Moreover, we will examine mitochondrial markers for OXPHOS complexes in HeSP2 treated TNBC xenografts using immunohistochemical analysis. Further studies are still underway to understand the role of heme in breast cancer, with preliminary results indicating that heme targeting, and sequestration effectively suppress triple-negative breast cancer and its progression.

Citation Format: Eranda Berisha, Maria del Carmen Chacon Castro, Adnin Ashrafi, Narges Salamat, Parinaz Sadat Alemi, Li Zhang. Heme targeting and its mechanistic role in triple-negative breast cancer suppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4942.
Abstract 4982: The somatic mutation signature detected in gynecological mucosal melanoma
Authors: Li Zhongwu , Shen Lianyi , Liu Siyao
Published: April 2023

Background: Gynecological Mucosal Melanoma(GMM) is aggressive disease with a poor prognosis, where few studies focusing on its target drug and mutation signatures. Cancer as outcome of DNA mutagenesis process and can be inferred from somatic mutations signature by analyzing the genome sequence. Due to the rapid development of next-generation sequencing (NGS) and molecularly driven targeted therapy, discovering different mutation signature in cancer plays an important role in precision medicine.

Methods: The study was conducted at a high-volume melanoma center, the Peking University Cancer Hospital. We enrolled female patients with melanoma (n = 22) from 2017 to 2021. Subsequently, whole-exome sequencing performed on the Formalin-fixed paraffin-embedded (FFPE) specimens from 22 GMM patients and match normal tissues of patients. Tumor mutational burden (TMB) was measured as the number of nonsynonymous somatic mutations of whole-exome sequence coding region. Mutation signature detection base pair of six substitution subtypes: C>A, C>G, C>T, T>A, T>C, and T>G. NMF can be used to decompose matrix A into two nonnegative matrices.

Results: In all mutations of 22 GMM patients, we screen two mutation signatures. Signature 1 showed a strong correlation [0.77;cosine similarity value (CSV)] to the signature 3 from the COSMIC database, which represents failure of DNA double-strand break-repair by homologous recombination, which predominated in 55%(12/22) GMM patients. Signature 2 showed a strong correlation to Cosmic signature 1 (CSV = 0.791), which is has been found in all cancer types correlated with age cancer diagnosis. Patient-18 did not any treatment in our hospital, follow-up was lost. Kaplan-Meier revealed that Signature 2 was associated with longer overall survival in GMM patients and later recurrence or progression.

Conclusions: This study aimed to identify prognostic markers and the development of targeted therapy for the treatment of GMM.

Citation Format: Zhongwu Li, Lianyi Shen, Siyao Liu. The somatic mutation signature detected in gynecological mucosal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4982.
Abstract 5022: Atypical protein kinase C inhibitors abrogate malignant breast cancer
Authors: Oishee Nuzhat Nowshin , Khalid Khandker Mohammad , Acevedo-Duncan Dr. Mildred
Published: April 2023

Breast cancer is the most common cancer type with expected cases of approximately 284,200 according to the National Cancer Institute. Atypical Protein Kinase C (aPKC) plays a significant role in many cancer types. A positive correlation between elevated PKC levels and both the invasive and chemotactic potential of human breast cancer cell lines has been suggested in in-vitro studies. In addition, the atypical protein kinase C isoforms (aPKC), PKC- and PKC- are involved in the increased proliferation of several cancer types, including breast cancer and ovarian cancer. Hence, this project focused on utilizing aPKC inhibitors, ICA-1S (5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide and - Stat (8-hydroxy-1,3,6- naphthalenetrisulfonic acid), on two malignant breast cancer cell lines (T47D and BT549) to observe the effect on cell proliferation. To observe the effects of the aPKC inhibitors on the malignant breast cancer cells, cell proliferation assays were conducted, where cells were treated with ICA-1S, and - Stat for 72 hours (about 3 days). Furthermore, to observe if ICA-1S induces apoptosis in the malignant breast cancer cells, both the cell lines were treated with ICA-1S for 120 hours (about 5 days), and western blotting techniques were utilized. The results indicated that in T47D cell line, treatment with ICA-1S (50uM) and - Stat (10uM) caused 52% and 75% reduction in cell proliferation, respectively. Moreover, with BT549 cell line, treatment with - Stat (1uM) decreased the cell proliferation by 40%. Additionally, it was observed by western blotting that ICA-1S induces apoptosis in BT549 cell line. This was shown by an increase in the cleavage of caspase 3 and PARP, along with a decrease in survivin, and phospho-BAD. However, ICA-1S did not induce apoptosis in T47D cell line. Further investigation will be conducted on determining the effect of aPKC inhibitors on the proliferation, invasion, and migration of the malignant breast cancer cell lines. In addition, siRNA treatment will be applied to knockdown PKC- and PKC- and subsequent effect will be observed. In conclusion, PKC- and PKC- plays a significant role on breast cancer, and further investigation will be conducted to analyze these roles, which will lead to the determination of a signaling pathway via which atypical protein Kinase C functions in breast cancer.

Citation Format: Nuzhat Nowshin Oishee, Khandker Mohammad Khalid, Dr. Mildred Acevedo-Duncan. Atypical protein kinase C inhibitors abrogate malignant breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5022.
Abstract 5030: Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death
Authors: Lazarte Jassy Mary S. , Tilghman Syreeta L. , Lamango Nazarius S.
Published: April 2023

Overexpression and hyperactivity of the estrogen receptor drives 67-80% and 90% of breast cancer in women and men, respectively. Resistance to aromatase inhibitor (AI) therapies necessitates the continuous search for novel therapies. Previous studies demonstrate AI-resistance is associated with hormone-independence, enhanced motility, and increased growth factor signaling. Here, long-term letrozole-treated (LTLT-Ca) breast cancer cells were used to evaluate polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential alternative therapies for the treatment of aromatase inhibitor-resistant breast cancer. We determined the potency of PCAIs as anticancer agents by evaluating their effects on cell viability, phosphorylation of MAPK pathway enzymes, G-proteins levels, cell migration and apoptosis. Among the PCAIs tested, NSL-YHJ-2-27 showed significant potency against cell viability with an EC50 of 3.6 µM. MEK (p-MEK1/2), ERK (p-ERK1/2), and p90RSK (p-p90RSK) phosphorylation were significantly increased by 178, 119 and 125%, respectively, over controls. In addition, of the seven G-proteins evaluated, only KRAS and NRAS showed significant increases of 49% and 97%, respectively. Cell proliferation and colony formation were impeded by 82% and 74%, respectively, after PCAIs treatment. Migration of the LTLT-Ca cells was inhibited by 80% following treatment with 5 µM of NSL-YHJ-2-27. Lastly, the PCAIs also caused the degeneration of the spheroids after 10 µM PCAIs treatment, dead cells were very apparent after AO/EB staining. Our findings suggest that the PCAIs’ suppression of cell viability and activation of the MAPK pathway causes apoptosis possibly through the activation of the proapoptotic p-p90RSK isoforms. The results reveal the ability of the PCAIs to effectively cause a negative impact on several cancer hallmarks suggesting their potency as anticancer agents. These findings also support a potential role for PCAIs’ use in treating breast cancers that have become resistant to aromatase inhibitor therapies.

Citation Format: Jassy Mary S. Lazarte, Syreeta L. Tilghman, Nazarius S. Lamango. Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5030.
Abstract 505: Small molecule circadian clock compounds display therapeutic potential in targeting glioblastoma stem cells
Authors: Chan Priscilla , Wu Lian , Hovsepyan Anahit , Mkhitaryan Seda , Karapetyan Gevorg , Shah Khalid , Wakimoto Hiroaki , Kamenecka Theodore , Solt Laura A. , Cope Jamie , Moats Rex A. , Hirota Tsuyoshi , Rich Jeremy N. , Kay Steve A.
Published: April 2023

Glioblastoma multiforme (GBM) is the most common primary brain tumor, claiming the lives of roughly 10,000 Americans each year. Despite being the first cancer analyzed through the Cancer Genome Atlas, treatment success remains minimal, resulting average survival time of patients is 15 months following diagnosis and a five-year survival rate is 6.8%. One of the major challenges in treating GBM is the presence of GBM stem cells (GSCs) that are resistant to temozolomide (TMZ) chemotherapy and radiation, which is part of the current standard of care for GBM following maximal surgical resection. We found, however, that GSCs have a unique dependence on core circadian clock proteins, Brain and Muscle ARNTL-Like 1 (BMAL1) and Circadian Locomoter Output Cycles Protein Kaput (CLOCK), which is not observed in differentiated GBM cells or normal neuronal stem cells. Here we explore the use of novel small molecule circadian clock compounds that either lower BMAL1 transcription (REV-ERB agonists) or inhibit BMAL1::CLOCK heterodimer transcriptional activity (Cryptochrome (CRY) stabilizers or Casein Kinase (CK) 1/2 inhibitors) in targeting GSCs in in vitro patient derived cell lines and in in vivo GBM patient-derived xenograft (PDX) models. GSCs display increased sensitivity to clock compounds at single agent and combinations of clock compounds compared to non-cancerous cells, U2OS human osteosarcoma cells, and differentiated GSCs. Additionally, clock compounds are significantly more effective in targeting GSCs than TMZ. The clock compound SHP1705 increased over survival and delayed tumor growth in GBM PDX models. These results highlight the therapeutic potential small molecule circadian clock compounds have against GBM as both a single agent and adjuvant to existing therapies by specifically targeting the GSC population.

Citation Format: Priscilla Chan, Lian Wu, Anahit Hovsepyan, Seda Mkhitaryan, Gevorg Karapetyan, Khalid Shah, Hiroaki Wakimoto, Theodore Kamenecka, Laura A. Solt, Jamie Cope, Rex A. Moats, Tsuyoshi Hirota, Jeremy N. Rich, Steve A. Kay. Small molecule circadian clock compounds display therapeutic potential in targeting glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 505.
Abstract 5192: NCG-MHC-dKO mice: an excellent model for PBMC reconstitution and pharmacodynamic evaluation in the absence of GvHD
Authors: Wang Huiyi , Xing Jun , Fan Jialu , Hou Huanhuan , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang , Ju Cunxiang
Published: April 2023

Human immune cell reconstitution (PBMC or CD34+ hematopoietic stem cell; HSC) is commonly performed in immunodeficient mouse strains such as NCG, NSG, or NOG. The use of PBMC is preferred for multiple reasons, including faster engraftment, lower cost, and the presence of mature immune cell populations. Although there are donor-to-donor variations in GvHD occurrence, and specific donors can be screened, the symptoms of GvHD typically occur within 4 weeks. GvHD significantly limits the study window required to evaluate the efficacy of therapeutic agents, such as cancer immunotherapies.

GvHD is an immune reaction triggered mainly by donor T cells identifying the mouse MHC class I and class II as foreign and attacking the host cells and organs. Deletion of β2m (NCG- β2m-KO) reduces the occurrence of GvHD; however, β2m is not only in the MHC class I subunit but also in the FcRn subunit, of which deletion shortens the half-life of IgG, making it unsuitable for IgG antibody agent evaluation. In addition, the deletion of MHC class I or class II alone results in an imbalanced CD4/CD8 ratio. To solve these problems, we developed the NCG-MHC-dKO mouse model by knocking out the H2K1, H2D1, and H2Ab1 genes. Compared with the existing NCG- β2m-KO or other β2m null mouse models, the NCG-MHC-dKO significantly prolonged survival, reduced GvHD occurrence, and did not affect the CD4/CD8 ratio when reconstituted with PBMC. Furthermore, treatment with anti-PD-L1 antibodies significantly inhibited MDA-MB-231 tumor cell growth in PBMC-reconstituted NCG-MHC-dKO mice, similar to NCG mice. Based on our preliminary data, the NCG-MHC-dKO mouse is a promising model for antibody and cell therapy agent evaluation, assessing treatment-related cytokine release syndrome, and evaluating long-term toxicities of cell therapies.

Citation Format: Huiyi Wang, Jun Xing, Jialu Fan, Huanhuan Hou, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao, Cunxiang Ju. NCG-MHC-dKO mice: an excellent model for PBMC reconstitution and pharmacodynamic evaluation in the absence of GvHD. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5192.
Abstract 5198: Humanized mice for preclinical efficacy evaluation of drugs targeting GARP
Authors: Liu Xing , Wu Meirong , Wang Huiyi , Yu Weiwei , Xu Jianming , Sun Hongyan , Ju Cunxiang , Wang Hongyu , Chen Santi Suryani , Li Zhiying , Moore Mark Wade , Zhao Jing , Gao Xiang
Published: April 2023

Introduction: GARP (Glycoprotein-A repetitions predominant) is a cell surface receptor which is coded by Lrrc32 (Leucine rich repeat containing protein 32) and involved in the activation of TGF-beta. The expression of GARP has been widely found in various cells including Treg, activated B cells, mesenchymal stromal cells and many types of cancer cells. In the tumor microenvironment (TME), GARP from Treg promotes the growth of tumor via secretion of immune suppressive TGF-beta.

Result: We established GARP humanized mouse model (hGARP) on C57BL/6 background in which the signal peptide and extracellular domain of mouse GARP (mGARP) was replaced by human GARP (hGARP). Similar expression level of hGARP on Treg cells in splenocytes from C57BL/6-hGARP mice was observed compared with mGARP in wildtype mice. After treating with anti-hGARP antibody, tumor growth was inhibited in the cohort engrafted with MC38 colorectal cancer cell line. Flow cytometry analysis of the tumor infiltrating lymphocytes revealed an abundant accumulation of hGARP on the Treg cells suggesting that blocking of hGARP in Treg cells contributed to the therapeutic efficiency. In addition, hGARP expression was also found on platelet cells from C57BL/6-hGARP mice, which is similar to human. After treating with anti-hGARP, we also found a significant reduction of platelet cells in C57BL/6-hGARP mice. In vivo assay suggested that the reduction of platelet cells from C57BL/6-hGARP mice correlated well with the binding of anti-hGARP.

Conclusion: C57BL/6-hGARP is a well-established mouse model for the pre-clinical evaluation of both therapeutic effect and toxicity of anti-hGARP therapy.

Citation Format: Xing Liu, Meirong Wu, Huiyi Wang, Weiwei Yu, Jianming Xu, Hongyan Sun, Cunxiang Ju, Hongyu Wang, Santi Suryani Chen, Zhiying Li, Mark Wade Moore, Jing Zhao, Xiang Gao. Humanized mice for preclinical efficacy evaluation of drugs targeting GARP. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5198.
Abstract 5205: B6-KrasLSL-G12C mouse model for assessing the occurrence and development of lung and pancreatic cancer tumors
Authors: Yang Xiaoliu , Jiang Yunlong , Wu Xue , Zhang Mingkun , Zhao Jing , Chen Santi Suryani , Li Zhiying , Trujillo Erica , Moore Mark Wade , Gao Xiang , Ju Cunxiang
Published: April 2023

In May 2021, the FDA approved sotorasib (Lumakras™) as the first treatment for adult patients with non-small cell lung cancer (NSCLC) harboring the KRASG12C genetic mutation. To study the relationship between the mutational activation of KRAS and tumorigenesis, and promote the development of KRAS-G12C inhibitors, we established the B6-Loxp-Stop-Loxp Kras G12C (B6-KrasLSL-G12C) strain. Upon induction of Cre recombinase, floxed Stop sequences will be deleted in the mouse genome, and the oncogenic KRAS-G12C protein will be expressed at endogenous levels allowing for control of the timing and location of tumor initiation. The C57BL/6-KrasLSL-G12C mice were crossed with the Lyz2-cre mice to develop a spontaneous lung cancer model. KRAS G12C mutant could be detected in type II alveolar epithelial cells (ATII). Furthermore, these mice develop lung cancer at 8-10 weeks of age. In addition, when B6-KrasLSL-G12C mice were crossed with the B6-P53LSL-R172H and Ptf1/p48-Cre mice, the offspring developed pancreatic cancer after 10-12 weeks of tamoxifen treatment.

In conclusion, B6-KrasLSL-G12C mice can be used to study the occurrence and development of pancreatic and lung cancer, as well as the potential applications of cancer therapies.

Citation Format: Xiaoliu Yang, Yunlong Jiang, Xue Wu, Mingkun Zhang, Jing Zhao, Santi Suryani Chen, Zhiying Li, Erica Trujillo, Mark Wade Moore, Xiang Gao, Cunxiang Ju. B6-KrasLSL-G12C mouse model for assessing the occurrence and development of lung and pancreatic cancer tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5205.
Abstract 5218: Genome-wide association study of African Americans reveals new susceptibility loci contributing to lung cancer and mechanisms of population-specific etiology
Authors: Byun Jinyoung , Han Younghun , Xiao Xiangjun , Lusk Christine , Badr Hoda J. , Hung Rayjean , Schwartz Ann G. , Amos Christopher I. ,   INTEGRAL Consortium
Published: April 2023

Lung cancer incidence and mortality in the U.S. have substantial ethnic disparities. African Americans of both sexes have the high incidence rate at 56.8 and the highest death rate at 38.1 per 100,000 people from 2015 to 2019. Non-Hispanic black men have the highest incidence at 73.9 and the highest death rate at 56.3 per 100,000 people. While many genome-wide association studies (GWAS) of lung cancer have discovered ~45 putative risk loci, a large proportion of the heritability of lung cancer remains unexplained. To date, most single population-based GWAS have been performed in Europeans while there are a few Asian ancestry-specific genetic studies and two cross-ancestry studies. Non-European GWAS of lung cancer has been underrepresented. Although African Americans have higher lung cancer incidence and poorer lung cancer survival rate, and smoke fewer cigarettes per day compared to Europeans, few comprehensive GWAS of lung cancer in African Americans has been conducted. This study aims to decipher the genetic component of predisposition to lung cancer among African Americans. This study aims to decipher the genetic component of predisposition to lung cancer among African Americans. We conducted an African-ancestry GWAS comprising 2,280 lung cancer cases and 4,301 controls to comprehensively characterize common and low-frequency lung cancer genetic susceptibility loci using HRC imputed lung cancer data. The novel variants in or near VWF on 12p13.31 (OR=1.29, P=6.93 × 10−9) for overall lung cancer and GACAT3 on 2p24.3 (0.65, 1.24 × 10−9), TRIP13 on 5p15.33 (3.49, 1.00 × 10−8), ERC1 on 12p13.33 (4.64, 6.08 × 10−9), LMAN1L (near CYP1A1) on 15q24.1 (4.25, 8.71 × 10−9) for lung squamous cell cancer were identified at a genome-wide significance level. Our GWAS of lung cancer identified five novel genetic susceptibility associations and confirmed several variants on 15q25 and 19q13. Further works are required to elucidate the possible biological mechanisms underlying these associations among lung cancer and histological subtypes in African Americans.

Citation Format: Jinyoung Byun, Younghun Han, Xiangjun Xiao, Christine Lusk, Hoda J. Badr, Rayjean Hung, Ann G. Schwartz, Christopher I. Amos, INTEGRAL Consortium. Genome-wide association study of African Americans reveals new susceptibility loci contributing to lung cancer and mechanisms of population-specific etiology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5218.
Abstract 523: Targeting myc-amplified cancers with a novel prodrug inhibiting aurora A kinase
Authors: Chi Ya-Hui , Chang Chun-Ping , Yeh Teng-Kuang , Wang Wan-Ping , Chen Yen-Ting , Tsai Chia-Hua , Chen Yan-Fu , Ke Yi-Yu , Wang Jing-Ya , Chen Ching-Ping , Hsieh Tsung-Chih , Wu Mine-Hsine , Chen Chiung-Tong
Published: April 2023

Upregulation of Aurora kinases has been associated with increased tumor progression, and thus they are appealing targets for the development of anti-cancer therapies. In addition to have a critical role in cell cycle regulation, Aurora kinases have been shown to stabilize MYC-family oncoproteins for the maintenance of malignancy. Aurora kinase inhibitors such as Alisertib has demonstrated compelling anti-tumor efficacies; however, the reported side effects including serious haematological disturbances have limited its risk-benefit ratio in clinical use. In this study we discovered a novel pyrimidine-based Aurora kinase inhibitor compound A that degraded MYC- and MYCN- oncoproteins with better potency than Alisertib. The acyl-based prodrug design leads to the discovery of compound B which was able to regress MYC- or MYCN- overexpressing tumor xenografts including small cell lung cancer, neuroblastoma, hepatocellular carcinoma and medulloblastoma using a once-a-week (QW) oral dosing regimen. Pharmacokinetic studies revealed that the tumor/plasma ratio of compound B was about 20 at 24 h post drug administration, and the active compound remained detectable in the tumors after 7 days. No significant haematological or liver/kidney biochemical aberration was observed in mice treated with up to 500 mg/kg of DBPR728 on a QW dosing regimen in a 21-day cycle. The unique pharmacokinetic and molecular properties of compound B hold potential clinical promise for treating MYC- and MYCN- amplified tumors with manageable on-target haematological adverse effects caused by Aurora kinase inhibition.

Citation Format: Ya-Hui Chi, Chun-Ping Chang, Teng-Kuang Yeh, Wan-Ping Wang, Yen-Ting Chen, Chia-Hua Tsai, Yan-Fu Chen, Yi-Yu Ke, Jing-Ya Wang, Ching-Ping Chen, Tsung-Chih Hsieh, Mine-Hsine Wu, Chiung-Tong Chen. Targeting myc-amplified cancers with a novel prodrug inhibiting aurora A kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 523.
Abstract 5236: Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene
Authors: Szabo Elizabeth , Blackburn Emily , Pagano Patrick , Rath Abhijit , Heinen Christopher
Published: April 2023

Lynch syndrome (LS) is a hereditary condition that increases patients’ lifetime risk of cancer, primarily colorectal cancer. LS is caused by germline mutations in mismatch repair (MMR) genes, MLH1, MSH2, MSH6, and PMS2. Identification of these mutations is important in LS diagnosis in order to guide treatment plans and preventative care for patients’ and their families. However, the consequences of some variants in these genes are not immediately obvious, granting them classification as variants of uncertain significance (VUS). Half of known variants in the MMR gene MSH6 are VUS and, thus, the goal of my project is to provide evidence to help better determine their pathogenic significance. Laboratory functional analysis to determine whether these variants disrupt MMR function in human cells can provide such evidence toward this goal. We have previously used CRISPR gene editing to recreate variants in the endogenous loci for the MMR genes MSH2 and MLH1 in human embryonic stem cells (hESC) in order to test their effects on MMR function in a cellular environment. We will now use test whether this approach can be used to study MSH6 VUS. Using these edited hESC lines we are examining the impact of the variants on RNA and protein stability, repair of DNA microsatellites, which is a hallmark of normal MMR function, and induction of the MMR-dependent DNA damage response. Using a panel of known MSH6 pathogenic and benign controls, we will calibrate these functional assays in order to associate functional performance with likelihood of pathogenicity. We will next test a separate panel of known controls to validate these assays. Finally, we will examine up to 35 VUS. With this compilation of data, we will convert the results into a quantitative Odds of Pathogenicity value. This Odds of Pathogenicity score can be used by expert variant interpretation committees to help readily reclassify these variants, and provide a clearer diagnosis for these suspected Lynch syndrome patients.

Citation Format: Elizabeth Szabo, Emily Blackburn, Patrick Pagano, Abhijit Rath, Christopher Heinen. Functional analysis of variants of uncertain significance of the MSH6 mismatch repair gene. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5236.
Abstract 5309: Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells
Authors: Quaglietta Paula R. , Kissoondoyal Ashby , Malkin Ethan , Mejia Salvador , Gong Ann , Malkin David , Baertschiger Reto M.
Published: April 2023

Extracellular vesicles (EVs) are nano-sized (30-1000 nm) membranous particles released by nearly all cell types. EV biology is a rapidly growing field, however its relative novelty and the intrinsic heterogeneity of EVs result in a lack of standardized and reproducible protocols. We sought to develop and validate an EV isolation method using rhabdomyosarcoma cell lines, with multiple characterization/validation methods, to improve the reproducibility and reliability of EV research in pediatric cancer research. EVs were isolated and analyzed in triplicate from 4 rhabdomyosarcoma cell lines (RH4, RH18, RH30, RD), of varying clinical characteristics including subtype, TP53 status and FOXO1 fusion status. Cells were grown to 70% confluency in six 175cm2 flasks, to ensure sufficient EV content for all downstream analyses, then incubated for 48 hours in media containing 10% EV-depleted FBS. The resultant 120mL of conditioned media (CM) per cell line was collected and subject to differential ultracentrifugation to isolate EVs. CM was centrifuged twice, then concentrated to less than 10mL using 3kDa MWCO filter tubes. Concentrated CM was centrifuged twice before passing through a 0.22μm pore-sized syringe filter prior to the final ultracentrifugations. The sample was ultracentrifuged and the EV pellet was carefully washed, re-centrifuged and resuspended in 100μL of PBS. Protein analysis by Western blot confirmed the presence of EV markers (syntenin-1, TSG101) and absence of non-EV markers (calnexin, histone H2Ax). EV quantification and size distributions were evaluated by NanoSight nanoparticle tracking analysis and visualized by transmission electron microscopy. All isolations were positive for EV markers and negative for non-EV markers by Western Blotting. All isolations showed similar size distributions by nanoparticle tracking analysis, within the range of 30-250nm. EVs in each sample with the typical cup-like shape were observed in electron microscope images of EVs. Overall, our method demonstrates good reproducibility of EV isolations from multiple rhabdomyosarcoma cell lines. This methodology can be used as a baseline for other cell lines to provide greater consensus within the field, allowing further discoveries analyzing the secretome of patient-derived cell lines with the potential to translate into more specific diagnostic tools.

Citation Format: Paula R. Quaglietta, Ashby Kissoondoyal, Ethan Malkin, Salvador Mejia, Ann Gong, David Malkin, Reto M. Baertschiger. Isolation and validation of small extracellular vesicles from rhabdomyosarcoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5309.
Abstract 5331: DNA-encoded macrocyclic compound libraries for challenging targets
Authors: Zhang Ying , Keefe Anthony , Centrella Paolo , Cuozzo John , Soutter Holly , Resnicow Daniel , Habeshian Sevan , Clark Matt
Published: April 2023

Increasingly drug discovery programs require the development of orally available and cell permeable classes of drugs derived from the chemical space beyond Lipinski’s rule of 5 (bRO5). The chameleon-like physicochemical properties and differentiated binding modes of bRO5 compounds make them suitable starting points, especially for difficult-to-drug targets beyond traditional small molecule drug discovery. The synthesis of macrocyclic (MC) peptide DNA-Encoded Libraries (DEL) aligns with these efforts and has been an important addition to the rapidly growing field of DNA-encoded small molecule library screening. DEL screening of de novo designed bRO5 compounds will aid the exploration of bRO5 chemical space. X-Chem’s macrocyclic compound libraries apply chemistries beyond traditional peptide formation and extend to the inclusion of diverse building block classes. A case study of a successful screening campaign utilizing these MC DEL for Bcl2 will be presented.

Citation Format: Ying Zhang, Anthony Keefe, Paolo Centrella, John Cuozzo, Holly Soutter, Daniel Resnicow, Sevan Habeshian, Matt Clark. DNA-encoded macrocyclic compound libraries for challenging targets. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5331.