bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 804: Trapped PARP1 cytotoxicity is modulated by the ubiquitin-dependentsegregase p97
Authors: Krastev Dragomir B. , Lord Chris
Published: June 2022

Poly-(ADP-ribose) polymerase inhibitors (PARPi) elicit anti-tumor activity in homologous recombination defective cancers by promoting cytotoxic, chromatin-bound, “trapped” PARP1. Here we have elucidated a pathway that process trapped PARP1. By exploiting wild-type or trapping-resistant PARP1 transgenes combined with either a rapid immunoprecipitation mass-spectrometry of endogenous proteins (RIME)-based approach or PARP1 Apex2-proximity labelling linked to mass-spectrometry, we generated proteomic profiles of trapped and non-trapped PARP1 complexes. These experiments demonstrated an increase in the SUMO1/2 modifications upon trapping, as well as an interaction with the ubiquitin-regulated p97 ATPase (aka VCP). Subsequent experiments demonstrated that upon trapping, PARP1 is SUMOylated by the SUMO-ligase PIAS4 and subsequently ubiquitinated by the SUMO-targeted E3-ubiquitin ligase, RNF4, events that promote p97 recruitment and p97 ATPase-mediated removal of trapped-PARP1 from chromatin. Consistently, small molecule p97 complex inhibitors, including a metabolite of the clinically-used drug disulfiram, CuET that acts as a p97 sequestration agent, prolong PARP1 trapping and thus enhance PARPi-induced cytotoxicity in homologous recombination defective tumor cells and patient-derived tumor organoids. Taken together, these results suggest that p97 ATPase plays a key role in the processing of trapped PARP1 from chromatin and the response of homologous recombination defective tumor cells to PARPi.

Citation Format: Dragomir B. Krastev, Chris Lord. Trapped PARP1 cytotoxicity is modulated by the ubiquitin-dependentsegregase p97 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 804.
Abstract 819: ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors
Authors: McGill Michael P. , Rojas Carolina Mantilla , Threadgill David W.
Published: June 2022

ERBB4 is commonly over-expressed in human CRC, however the utility of ERBB4 as a target for colorectal cancer (CRC) therapeutics is largely unexplored. Our group recently identified a molecular subtype of CRC that arises independent of EGFR and displays a more aggressive growth phenotype than those dependent on EGFR. The robust growth phenotype of EGFR-independent tumors can be in part attributed to upregulation of IL10 signaling. In addition, EGFR-independent tumors display a significant upregulation of Erbb2 and Erbb4 transcripts in both spontaneous and sporadic CRC mouse models, perhaps compensating for the loss of EGFR. To investigate the importance of other ERBB receptors in EGFR-independent CRC tumor progression, we ablated Erbb4 to interrogate its influence on tumor development in vivo using a conditional allele of Erbb4tm1Fej (Erbb4f). ERBB4-deficient ApcMin/+ mice (ApcMin/+, ErbB4f/f, Tg(Vil1-Cre)) were established and used to show that ERBB4 ablation in the intestinal epithelia results in a significant decrease in the number of intestinal and colon tumors. Polyps lacking ERBB4 were also significantly reduced size, contrary to what is observed with loss of EGFR. We also recently developed ERBB2-deficient ApcMin/+ mice, and preliminary results suggest that Erbb2 ablation also results in a decrease in intestinal and colon tumor number and size. Consistent with data from EGFR-independent tumors, transcriptomic analysis of ERBB4-deficient intestinal tumors predicted down-regulation of IL10 signaling, which was validated through Il10 and Socs3 qPCR. To complement our findings, the observed down-regulation of Il10 and Socs3 at the transcript level corresponded with a significant decrease in IL10 levels in the serum of mice harboring ERBB4 deficient tumors when compared to mice without tumors. Furthermore, we found that transcript levels of Erbb4 significantly decreased after anti-IL10 treatment of EGFR-independent tumors in vivo. This illustrates a possible negative feedback loop whereby neutralizing IL10 also leads to a reduction of Erbb4 expression, further reducing IL10 signaling. Taken together, the data suggest that the absence of EGFR triggers Erbb4 upregulation, implicating an important role for ERBB4 in EGFR-independent colon tumor growth. These results suggest that therapeutic targeting of ERBB4 may lead to a reduction in IL10 signaling and reduced intestinal polyp multiplicity and size in EGFR-independent tumors and inform the development of novel approaches to treat CRC that exhibit aberrant expression of EGFR and ERBB4.

Citation Format: Michael P. McGill, Carolina Mantilla Rojas, David W. Threadgill. ERBB4 mediates IL10-induced growth of EGFR-independent colon tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 819.
Abstract 820: Nuclear receptor TLX inhibits cancer cell intrinsic properties required for triple-negative breast cancer progression
Authors: Nelczyk Adam T. , Gamage Hashni E. Vidana , Ma Liqian , McHenry Michael T. , Henn Madeline A. , Kadiri Mohammed , Wang Yu , Gupta Anasuya Das , Krawczynska Natalia , He Sisi , Spinella Michael J. , Nelson Erik R.
Published: June 2022

The primary purpose of our work was to characterize the effects of the nuclear receptor TLX (NR2E1) in triple-negative breast cancer (TNBC) in order to evaluate its potential therapeutic value in a subtype of breast cancer that has proven particularly challenging to treat, primarily due to the lack of targeted modes of intervention. Unfortunately, breast cancer continues to be the second deadliest form of cancer among women in the United States. Considering the substantial public health implications, development of new therapeutic strategies for highly aggressive breast cancer subtypes, such as TNBC, is imperative. While the function of nuclear receptors such as the estrogen and androgen receptors has been extensively characterized in cancers of the breast and prostate respectively, due in part to their amenable nature to ligand modulation, it is likely that other nuclear receptors may represent therapeutic targets for these malignancies. Indeed, probing of available clinical datasets demonstrated that nuclear receptor TLX is most highly expressed in basal and estrogen receptor (ER)-negative breast cancer patients, and that ER-negative patients with higher TLX expression had increased relapse-free and overall survival. Therefore, we hypothesized that TLX could influence the pathophysiology of TNBC. Utilizing a stable overexpression model in the TNBC cell lines, MDA-MB-231 and MDA-MB-468, we have shown that TLX can inhibit critical oncogenic properties in the in vitro setting, including proliferation, migration and invasion. Furthermore, xenograft and lung colonization studies demonstrated that TLX continued to exert anti-oncogenic effects in the in vivo environment. In agreement with these results, transcriptomic analysis of TLX-overexpressing cells and xenograft tumors showed that TLX can regulate genes and pathways that are known to play crucial roles in the growth and metastatic dissemination of cancer. As previously published works have identified several putative TLX ligands, our findings demonstrating that TLX functions as an anti-cancer factor in TNBC provides a strong rationale for future research aimed at therapeutically targeting this nuclear receptor.

Citation Format: Adam T. Nelczyk, Hashni E. Vidana Gamage, Liqian Ma, Michael T. McHenry, Madeline A. Henn, Mohammed Kadiri, Yu Wang, Anasuya Das Gupta, Natalia Krawczynska, Sisi He, Michael J. Spinella, Erik R. Nelson. Nuclear receptor TLX inhibits cancer cell intrinsic properties required for triple-negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 820.
Abstract 829: p53 dimers elicit tumor suppressive activities through an altered metabolic program
Authors: Gencel-Augusto Jovanka , Su Xiaoping , Lozano Guillermina
Published: June 2022

p53 is the most frequently mutated gene in human cancer. As a tetrameric transcription factor, mutation of the p53 Tetramerization Domain (TD) is a mechanism by which cancers abrogate wild-type (WT) p53 function. p53 TD mutations result in a protein that preferentially forms monomers or dimers. Although it is accepted that tetrameric p53 is required for full tumor suppressive activities, the physiological relevance of monomeric and dimeric states of p53 is not well understood. We have established the first in vivo models for hot-spot p53 TD mutants, p53R339P and p53A344D (murine), that result in monomeric and dimeric p53, respectively. These models closely resemble Li-Fraumeni Syndrome (LFS) patients with germline p53 TD alterations and will be used to characterize their physiological activities.

p53R339P and p53A344D proteins failed to form tetramers. These mutants were deficient in their transactivation ability for canonical p53 target genes, and unable to induce apoptosis following IR. Both p53 TD mutant mice rescued the p53-dependent embryo lethality caused by genetic deletion of Mdm2, suggesting these are loss-of-function alleles. Monomeric p53 bearing mice succumbed mainly to thymic lymphomas (~60%), with a median survival similar to p53-/- mice (~130 days). Dimeric p53 bearing mice displayed lower incidence of thymic lymphomas (~25%) and longer survival (~180 days). Analyses of the transcriptomes elicited after DNA damage in normal thymus revealed that the p53 signaling pathway is dampened in dimeric p53 thymi. However, under basal conditions, p53 dimers upregulate a lipid metabolic program that is associated with PPAR activation. Further RNA sequencing of common metabolic tissues of the mouse (pancreas, liver, and intestine) revealed that they upregulate lipid-related metabolic programs in p53 dimer samples. This study describes, for the first time, the consequences of altering p53 tetramerization in vivo. Monomer and dimer forms of p53 do not retain most WT p53 features. p53 dimers may exert tumor suppression as seen by extended survival and tumor spectra shift. p53 dimer-specific activities on PPAR signaling and lipid metabolism may explain these phenotypes. These activities are possible therapeutic targets for LFS patients.

Citation Format: Jovanka Gencel-Augusto, Xiaoping Su, Guillermina Lozano. p53 dimers elicit tumor suppressive activities through an altered metabolic program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 829.
Abstract 842: ADAR and DDX54 promote genomic stability in breast cancer cells
Authors: Ryu Sua , Cottrell Kyle A. , Soto-Torres Luisángely , Schabb Angela , Weber Jason D.
Published: June 2022

Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme that binds dsRNA and converts adenosine to inosine. ADAR promotes cell survival and proliferation in multiple types of tumors, including breast cancer. However, the mechanism of ADAR-driven oncogenicity is not fully established. We focused on ADAR p110, the constitutively expressed and nuclear-localized isoform, and its role in managing genotoxic stress in cancer. ADAR p110 is involved in resolving R-loops, the DNA-RNA hybrids produced during transcription, and DNA damage response (DDR). We sought to elucidate the mechanism of ADAR-associated R-loop resolution and DDR that can be targeted in breast cancer. To identify ADAR-interacting proteins, proximity labeling was performed using ADAR p110 fused to ascorbate peroxidase (APEX2), followed by mass spectrometry. Among the positively enriched hits, known ADAR-interacting proteins associated with R-loop and DDR were validated by immunoprecipitation and immunofluorescence. DEAD-box helicase DDX54 was among the nine RNA helicases interacting with ADAR. DDX54 and ADAR showed an additive effect on preventing γH2AX signal. Additionally, knockdown (KD) of DDX54 increased ADAR expression, whereas ADAR KD did not affect DDX54 expression. These results suggest that DDX54 coordinates with ADAR to prevent genotoxic stress in breast cancer cells. As DDX54 and ADAR are both associated with R-loop resolution and DDR, we hypothesize that DDX54 and ADAR suppress R-loop accumulation and/or other types of genotoxic stress. This may be through independent pathways despite the physical interaction between ADAR and DDX54.

Citation Format: Sua Ryu, Kyle A. Cottrell, Luisángely Soto-Torres, Angela Schabb, Jason D. Weber. ADAR and DDX54 promote genomic stability in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 842.
Abstract 843: Isoform-specific disruption of the TP73 gene reveals a critical role for TAp73gamma in tumorigenesis and obesity via leptin
Authors: Kong Xiangmudong , Sun Wenqiang , Zhang Jin , Chen xinbin
Published: June 2022

TP73, a member of the p53 family, is expressed as TAp73 and ΔNp73 along with multiple C-terminal isoforms (α-η). Interestingly, TAp73 is shown to have opposing functions in tumorigenesis but the mechanism remains unclear. Here, we showed that deletion of exon 11 (E11) switches p73α to p73γ, the latter of which has oncogenic activities. Moreover, E11-deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, chronic inflammation and tumor incidence, indicating that p73γ cannot compensate p73α for tumor suppression and fertility. E11-deficient mice were also prone to obesity and B-cell lymphomas, indicating a unique role of p73γ in lipid metabolism and tumorigenesis. Mechanistically, we showed that Leptin, a pleiotropic adipocytokine, was highly induced by p73γ and necessary for p73γ-mediated oncogenic activity. Importantly, p73α-γ isoform switch was detected in dog lymphomas and human prostate carcinomas along with elevated expression of Leptin. Our study indicates that the p73γ-Leptin pathway promotes tumorigenesis and alters lipid metabolism.

Citation Format: Xiangmudong Kong, Wenqiang Sun, Jin Zhang, xinbin Chen. Isoform-specific disruption of the TP73 gene reveals a critical role for TAp73gamma in tumorigenesis and obesity via leptin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 843.
Abstract 844: Germline TP53 missense mutations at codon338 confer adult-onset cancer risk through weakened p53 protein oligomeric stability that impairs its tumor suppressive mechanisms
Authors: Fischer Nicholas W. , Turk Jaime , Ong Noel , Duan Shili , Kaustov Lilia , Kratz Christian , Pajtler Kristian , Kim Raymond , Arrowsmith Cheryl , Malkin David
Published: June 2022

Pathogenic TP53 germline variants predispose carriers to a high lifelong cancer risk in Li-Fraumeni syndrome (LFS). LFS presents as a broad phenotypic spectrum with highly variable age-of-onset and tumor types. The mechanisms leading to these phenotypic differences remain largely unknown, and many less frequent variants are not clinically defined as pathogenic or benign. Two missense mutations in the tetramerization domain of the p53 protein, F338S and F338C, have recently been of clinical interest; however, they are currently classified as variants of uncertain significance. Normally, p53 assembles as a tetrameric complex to bind DNA and transcriptionally regulate the expression of target genes involved in the cellular response to stress, acting as a crucial barrier to malignant transformation. Alterations to p53 structure can therefore lead to malfunction and ultimately tumor formation. Our structural analysis of the tetramerization domain demonstrates that F338S and F338C have significantly lower thermal stability compared to the wildtype protein. Furthermore, our functional assessments of apoptosis and the suppression of colony formation indicate that both variants operate with reduced tumor suppressor activity, although they maintain near wildtype functionality. We anticipate that our results will help to better understand why these mutations cause a history of predominantly adult-onset cancers in the affected families and could justify refining surveillance measures for these individuals.

Citation Format: Nicholas W. Fischer, Jaime Turk, Noel Ong, Shili Duan, Lilia Kaustov, Christian Kratz, Kristian Pajtler, Raymond Kim, Cheryl Arrowsmith, David Malkin. Germline TP53 missense mutations at codon338 confer adult-onset cancer risk through weakened p53 protein oligomeric stability that impairs its tumor suppressive mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 844.
Abstract 847: Role of RHOA in diffuse gastric cancer tumors
Authors: Martinez-Barriocanal Agueda , Dopeso Higinio , Jiménez-Flores Lizbeth M. , Santos Juliana C. , Anguita Estefania , Bilic Josipa , Nieto Rocio , García-Vidal Elia , Martín Manuel Sánchez , Landolfi Stefania , Kobayashi Kazuto , Hernandez-Losa Javier , Schwartz Simo , Cajal Santiago Ramón y , Arango Diego
Published: June 2022

Gastric cancer constitutes the third leading cause of cancer mortality worldwide and tumors with diffuse histology have poor prognosis and low response rates to therapy. Missense mutations in the RHOA (Ras homolog family member A) GTPase have recently been identified in 24% of patients with diffuse gastric cancer. RHOA mutations are not randomly distributed along the coding sequence, but enriched in specific and recurrent hotspots. Substitution in codon 42 (Y42C) occurs in approximately 50% of the patients in which RHOA is mutated. RHOA orchestrates cellular processes such as proliferation, differentiation, migration and invasion, whose deregulation is essential for the onset, maintenance and progression of tumors. However, the functional role of wild type RHOA and RHOA mutants remains poorly characterized in gastric cancer. We used isogenic cell lines systems and animal models to study the impact of RHOA in the gastric tumorigenesis. RHOA loss strongly increased the proliferation, migration and invasion of diffuse gastric cancer cells in vitro, but also when growing as subcutaneous xenografts in immunodeficient mice, and in experimental mouse models of peritoneal and lung metastasis. Stomach tumor burden was also increased in transgenic mice conditionally expressing a dominant negative form of RHOA in the gastrointestinal tract (RHOA T19N). Moreover, cells overexpressing wild-type RHOA or G14V RHOA (a constitutive active form of the GTPase) exhibited reduced tumorigenic features both in vitro and in vivo. Conversely, diffuse gastric cancer cells engineered for overexpressing RHOA Y42C mutant, displayed enhanced growth and invasion in vitro. Importantly, stomach cancer tumors formed more efficiently in a transgenic mouse model in which we targeted the expression of Y42C RHOA mutant to the gastric epithelium. Collectively, these results demonstrate that RHOA has tumor-suppressive activity in diffuse gastric cancer, while the recurrent Y42C hotspot RHOA mutation found in these tumors is oncogenic.

Citation Format: Agueda Martinez-Barriocanal, Higinio Dopeso, Lizbeth M. Jiménez-Flores, Juliana C. Santos, Estefania Anguita, Josipa Bilic, Rocio Nieto, Elia García-Vidal, Manuel Sánchez Martín, Stefania Landolfi, Kazuto Kobayashi, Javier Hernandez-Losa, Simo Schwartz, Santiago Ramón y Cajal, Diego Arango. Role of RHOA in diffuse gastric cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 847.
Abstract 848: Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance
Authors: Gonzalez-Martinez David , Roth Lee , Mumford Thomas , Mosse Yael , Tulpule Asmin , Bivona Trever , Bugaj Lukasz Jan
Published: June 2022

Drug resistance remains a significant obstacle in the successful treatment of cancer, highlighting the critical need to understand how oncogenes and cancer drugs impact cell physiology and resistance development. EML4-ALK is a receptor tyrosine kinase (RTK) fusion oncogene that drives 3-7% of lung cancer. Despite potent ALK inhibitors, EML4-ALK+ cancers frequently develop resistance during therapy. Recently, it was discovered that EML4-ALK and other RTK fusions form cytoplasmic protein condensates, and that condensate formation was required for oncogenic signaling. However, whether oncogenic condensates play a role in drug responses is unclear. In this study, we applied an optogenetic technique called ‘functional profiling’ to understand how EML4-ALK condensates impact cell signal transmission and drug response. Using light-stimulated RTKs, we found that EML4-ALK condensates strongly suppress signaling through transmembrane RTKs, including through EGFR, a central receptor in resistance development. Strikingly, treatment with ALK inhibitors (ALKi) rapidly restored and hypersensitized RTK signaling. We found that EML4-ALK condensates suppress RTK signals through sequestration of the downstream adapter Grb2, which is essential for signaling through EGFR and other RTKs. The release of Grb2 from condensates resensitized RTKs within 10s of minutes of ALKi addition. Resensitized RTKs, in turn, caused sporadic RTK activation pulses throughout the cell population, and pulses originated from paracrine RTK signals released by apoptotic neighbors. We found that these paracrine signals counteracted ALK inhibitor therapy and promoted survival and drug tolerance. Blocking paracrine signals through co-treatment of ALKi with inhibitors of either EGFR or matrix metalloproteases enhanced cell killing and minimized long-term drug tolerance. Our study uncovers a role for oncogenic condensates in drug resistance signaling, reveals a novel mechanism for oncogene-induced suppression of RTK signaling, and suggests novel co-therapies to more effectively treat cancers driven by EML4-ALK and possibly other RTK fusions. Our work also demonstrates the potential of functional optogenetic profiling for drug discovery to promote cancer therapy.

Citation Format: David Gonzalez-Martinez, Lee Roth, Thomas Mumford, Yael Mosse, Asmin Tulpule, Trever Bivona, Lukasz Jan Bugaj. Inhibition of RTK fusion condensates enhances signal perception and promotes drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 848.
Abstract 855: Analysis of canonical uveal melanoma mutations reveals novel signaling effects
Authors: Owens Dawn Alexandra , Kurtenbach Stefan , Kuznetsoff Jeffim N. , Rodriguez Daniel A. , Cruz Anthony , Harbour J.
Published: June 2022

Uveal Melanoma (UM) is the most common primary ocular cancer in adults. Virtually all UM have activating mutations in the Gq signaling pathway. Metastatic risk in patients correlates with additional secondary mutations, where the lowest metastatic risk (class 1) is associated with EIF1AX and SF3B1, and the highest metastatic risk (class 2) is associated with BAP1 mutations. The interplay between the initiating Gq pathway mutations, and the secondary mutations found in all UM has not been well understood yet. Here, we utilize a multi-omics approach, to describe gene expression changes, the proteome, and phosphatome, following GNAQ mutation and BAP1 knockout in primary human uveal melanocytes. We generated human uveal melanocyte cell lines with inducible and specific mutations and analyzed via RNAseq, ATACseq, and mass spectrometry. Results reveal significant changes in pathways related to p53, chromatin regulation/modification, gene expression, and RNA processing with noted dysregulation of genes JUN, HDAC4, YAP, and SOX10, which cause senescence in melanocytes following GNAQ mutation, and are relieved through additional mutation in BAP1.

Citation Format: Dawn Alexandra Owens, Stefan Kurtenbach, Jeffim N. Kuznetsoff, Daniel A. Rodriguez, Anthony Cruz, J. Harbour. Analysis of canonical uveal melanoma mutations reveals novel signaling effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 855.
Abstract 860: Identifying mechanisms associated with slow proliferation of BAP1 mutant uveal melanoma cells
Authors: Chua Vivian , Baqai Usman , Purwin Timothy J. , Jeon Angela , Han Anna , Aplin Andrew
Published: June 2022

Uveal melanoma (UM) is the deadliest form of eye cancer in adults. Primary tumors in the uveal tract are often treated successfully by enucleation or radiotherapy but 50% of cases develop metastases which occur primarily in the liver. Patients diagnosed with metastatic UM usually have a poor prognosis with a short median survival of 5-18 months due to the lack of effective treatment options. Metastatic UM responds poorly to many therapeutic options. Inactivating mutations in BRCA1-associated protein 1 (BAP1), a tumor suppressor gene and de-ubiquitinating (DUB) enzyme, have been reported in >80% metastasizing UM and thus are associated with elevated risks of UM metastasis. However, the role of BAP1 in UM metastasis is unclear. Interestingly, silencing of BAP1 in UM cell lines was shown to inhibit UM cell proliferation and cell cycle progression. Consistent with this finding, we show that BAP1-deficient or BAP1 mutant UM cells proliferated slower than BAP1 wild-type (WT) cell lines. Re-expression of BAP1 in a BAP1-deficient or mutant UM cell line, MP65, also increased the proliferation rate of cells. Analysis of reverse phase protein array (RPPA) identified that phosphorylation of the S6 ribosomal protein is elevated in BAP1 proficient cells. Re-expression of BAP1 WT also increased phosphorylation of S6 and its upstream regulator, p70S6K1. Additionally, BAP1 mutant UM cells were more viable compared to isogenic cells expressing BAP1 WT in response to amino acid deprivation. These findings suggest that loss of BAP1 is associated with better survival under environmental stress, a critical step during metastasis. Positivity for pS6 was detected in all metastatic UM specimen tested regardless of BAP1 status. This suggests a role of the tumor microenvironment in regulating pS6 levels in UM cells at the metastatic site. Our findings show BAP1-dependent regulation of UM cell proliferation and response to stress, and uncover S6 and its associated pathway as an opportunity for therapeutic target in BAP1 mutant UM.

Citation Format: Vivian Chua, Usman Baqai, Timothy J. Purwin, Angela Jeon, Anna Han, Andrew Aplin. Identifying mechanisms associated with slow proliferation of BAP1 mutant uveal melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 860.
Abstract 867: Velcrin-induced cleavage of tRNA-Leu-TAA by SLFN12 RNase causes cancer cell death
Authors: Lee Sooncheol , Hoyt Stephanie , Wu Xiaoyun , Garvie Colin , McGaunn Joseph , Cherniack Andrew D. , Meyerson Matthew , Greulich Heidi
Published: June 2022

Velcrins are small molecules that induce apoptosis of cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) by inducing PDE3A-SLFN12 complex formation. Recently, we found that SLFN12 is an RNase and PDE3A binding dramatically activates SLFN12 RNase activity, resulting in velcrin-mediated apoptosis. However, the mechanism of cancer cell death by the PDE3A-SLFN12 complex is poorly understood. In this study, we found that SLFN12 specifically recognizes tRNA-Leu-TAA and induces its cleavage in velcrin-sensitive cell lines. According to tRNA-sequencing, tRNA-Leu-TAA isodecoders decreases by 2-fold to 5-fold in Hela cells treated with DNMDP compared to DMSO control, while other tRNAs are not differentially expressed. Down-regulation of tRNA-Leu-TAA by velcrin treatment is observed only in velcrin-sensitive cell lines but not insensitive cell lines. Furthermore, we tested whether SLFN12 digests tRNA-Leu-TAA in vitro. Purified wild-type SLFN12 cleaves tRNA-Leu-TAA but not heat-denatured or catalytically inactive SLFN12. Even though tRNA-Leu-TAA is weakly digested by low concentrations of SLFN12, co-incubation with purified PDE3A increases SLFN12 RNase activity, which is even further upregulated by velcrin treatment. To verify specificity for tRNA-Leu-TAA, we assessed activity of SLFN12 against a panel of in vitro transcribed tRNAs. We found that purified SLFN12 is significantly more efficient at degrading tRNA-Leu-TAA when compared with tRNA-Leu-TAG or tRNAs for other amino acids. Digestion of tRNA-leu-TAA by SLFN12 could inhibit mRNA translation due to the delivery of leucine amino acid to elongating ribosomes. To test this hypothesis, we designed the mutant tRNA-Leu-[CAG:TAA] in which the anticodon of tRNA-Leu-CAG was replaced by TAA anticodon. Ectopic expression of wild-type tRNA-Leu-TAA or tRNA-Leu-CAG has no effect on viability of DNMDP-treated cells, whereas expression of tRNA-Leu-[CAG:TAA] genes partially rescues the cells from DNMDP-induced cytotoxicity. These data confirm that impaired leucine delivery is responsible at least in part for the observed effects of DNMDP treatment.

Citation Format: Sooncheol Lee, Stephanie Hoyt, Xiaoyun Wu, Colin Garvie, Joseph McGaunn, Andrew D. Cherniack, Matthew Meyerson, Heidi Greulich. Velcrin-induced cleavage of tRNA-Leu-TAA by SLFN12 RNase causes cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 867.
Abstract 882: A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway
Authors: Laubach Kyra , Yan Wenshen , Kong Xiangmudong , Chen Mingyi , Zhang Jin , Chen Xinbin
Published: June 2022

p73, a member of the p53 family, is expressed as two N-terminal isoforms: TA, a tumor suppressor and ΔN, an oncoprotein. Alternative RNA splicing at the 3` end gives rise to multiple C-terminal isoforms, but their function is largely unknown. To address this, CRISPR was used to knock out exon 12 (E12) in several cell lines and mice, leading to isoform switching from p73α to p73α1. p73α1 is a novel isoform whose expression we detected in multiple cancer cell lines. We found that knockout of E12 inhibits the cell proliferation and migration abilities of H1299 and MIA PaCa-2 cancer cells and E12-/- MEFs exhibit increased cellular senescence. Similarly, ectopic expression of p73α1 in H1299 cells suppresses cell proliferation and knockdown of p73α1 restores the cell proliferative and migratory capacities of H1299 E12-/- cells. Consistently, we found that E12+/- mice are not prone to spontaneous tumors, unlike Trp73+/- mice, but instead are prone to chronic systemic inflammation and show elevated IL-1β and IL-6 expression in inflamed tissues. We then identified Notch1, a master regulator of the inflammatory response, as a mediator of p73α1-induced inflammation and tumor suppression, and found it to be elevated in inflamed E12+/- mouse tissues. Furthermore, we found that knockdown of Notch1 enhances cell proliferation and migration in H1299 E12-/- cells. Together, these data suggest that p73α1 plays a role in the inflammatory response and tumor suppression via Notch1.

Citation Format: Kyra Laubach, Wenshen Yan, Xiangmudong Kong, Mingyi Chen, Jin Zhang, Xinbin Chen. A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 882.
Abstract 883: Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer
Authors: Bergman Riley , Park Ben , Croessmann Sarah
Published: June 2022

SF3B1 is the most commonly mutated splicing factor in cancer, occurring in a broad variety of hematologic and solid malignancies, including: breast, melanoma, pancreatic, bladder, colorectal, leukemia, and more. SF3B1 is a protein component of the spliceosome responsible for recognizing intronic splice sites and stabilizing the interaction between the spliceosome and pre-mRNA transcripts. Hotspot mutations in SF3B1 result in a neomorphic protein that causes aberrant splicing of hundreds of transcripts. These transcriptional alterations have several proposed impacts on known cancer pathways; however, the mechanisms by which SF3B1 mutations contribute to tumorigenesis are incompletely understood. Interestingly, systematic analysis of online cancer databases shows SF3B1 mutations and TP53 alterations are mutually exclusive in cancers with predominant SF3B1 hotspots, suggesting either synthetic lethality or overlapping roles in promoting tumorigenesis. To address this question, TP53 was knocked-out using CRISPR-Cas9 in AAV-generated SF3B1 K700E mutant knock-in and wild type MCF-10A breast epithelial cells. Successful generation of TP53 knock-outs in both the SF3B1 mutant and wild type groups suggests the mutations are unlikely to be synthetic lethal. Instead, this finding may direct us to novel mechanisms by which SF3B1 mutations contribute to cancer development and progression. Two overarching areas of interest based on preliminary data include changes in mitochondrial metabolism and metabolic reprogramming and alterations in DNA damage, repair, and genomic stability. Further defining the relationship between mutant SF3B1 and TP53 may reveal new mechanisms of tumorigenesis and reveal therapeutic vulnerabilities that can be additionally leveraged against the large subset of cancers with TP53 mutations.

Citation Format: Riley Bergman, Ben Park, Sarah Croessmann. Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 883.
Abstract 888: Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells
Authors: Song Jingwen , Chen Xiaozhuo
Published: June 2022

The tumor microenvironment (TME) has concentrations of extracellular ATP (eATP) 103-104 times higher than normal healthy tissues, implying that eATP may have important roles in tumor development and progression. Our previous study, cited by a Nature Reviews Cancer paper, showed that the extracellular ATP (eATP) is internalized by tumors via macropinocytosis, a type of endocytosis, to increase intracellular ATP (iATP) levels and support tumor cells growth, drug resistance, and cancer cell movement by initiating epithelial to mesenchymal transition (EMT). Recently, our lab found that eATP functions in promoting cancer cell growth, metastasis are similar to those of TGF-β, a well-known EMT and cancer stem cells (CSCs) inducer. Increasing reports state that the EMT and CSCs share some molecular links, but the detailed mechanisms and inductor of them are still far from clear. In this report, eATP’s function in CSCs induction was investigated first. Result shows that eATP treatment significantly induces the protein expression level of CSC related makers, increases cell colony numbers, and cell populations with CSC markers expressed in human lung cancer A549 cells. In addition, RNA-sequencing analysis reveals the gene Stanniocalcin 1 (STC1) is significantly upregulated in both eATP and TGF-β treatments. STC1 gene has been reported to be a competitive biomarker in the tracking of cancers’ progressing. Besides, STC1 exhibits concentration-dependent stimulation effects on electron transportation chain (ETC) and respiration. However, the detail function of STC1 gene in eATP induced cell growth is unknown. To find out the mechanism, CRISPR-Cas9 technique was used to generate STC1 gene targeted knockout (STC1 KO) A549 cells. STC1 KO cells show slower cell growth rate, lower intracellular ATP level and lower oxygen consumption rate (OCR) when compared with normal A549 cells in vitro. These results strongly indicate STC1 gene involved in eATP induced cell growth by alteration iATP level and metabolic profile. Overall, this study not only unravels new role of eATP in CSC induction, but also provides new mechanism of eATP induced cell growth by altering the protein expression level of STC1.

Citation Format: Jingwen Song, Xiaozhuo Chen. Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 888.
Abstract 89: The microRNA-3622b-3p contributes to tumor progression and metastasis through an miR-3622-AIFM2 axis in prostate cancer
Authors: Wang Lizhong , Zhang Chao , Ye Ping
Published: June 2022

microRNA (miR)-3622 family is composed of two members, miR-3622a/b, but their roles remain debatable. In the present study, we found that expression of miR-3622a was lower, whereas expression of miR-3622b-3p was higher in human prostate cancer tissues than in normal prostate tissues. miR-3622a-3p inhibited cell migration and invasion of human prostate cancer cells, whereas miR-3622b-3p facilitated cell proliferation, migration, and invasion. To address the opposing roles of miR-3622 family members in various human prostate cancer cell lines, we knocked out endogenous miR-3622, including both miR-3622a/b. Our results showed that miR-3622 knockout reduced cell proliferation, migration, and invasion in vitro and tumor growth and metastasis in vivo. Furthermore, we found that AIFM2, a direct target of miR-3622b-3p, is responsible for miR-3622 knockout-induced apoptosis, identifying an miR-3622-AIFM2 axis that contributes to oncogenic function during tumor progression. The results show that miR-3622b-3p is upregulated in human prostate cancers and has an oncogenic function in tumor progression and metastasis through an miR-3622-AIFM2 axis.

Citation Format: Lizhong Wang, Chao Zhang, Ping Ye. The microRNA-3622b-3p contributes to tumor progression and metastasis through an miR-3622-AIFM2 axis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 89.
Abstract 912: A novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of PRC2 target genes
Authors: Abdallah Aalaa , Cardona Herminio J. , Picketts David J. , Brat Daniel J. , Li Xiao-Nan , Becher Oren J.
Published: June 2022

Pediatric High Grade Glioma (pHGG) is a collection of molecularly distinct subtypes with different prognoses depending on the genetic drivers. One particularly aggressive subtype is H3.3G34R mutant gliomas, which are currently incurable and in need of improved therapies. Interestingly, H3.3G34R mutant gliomas commonly harbor TP53mutations, ATRX mutations, and alterations in PDGFRA signaling. The mechanism by which H3.3G34R promotes gliomagenesis is still somewhat unclear and additional models are needed to dissect its role in tumorigenesis. We used the RCAS Tv-a system to model H3.3G34R pHGG in mice. Nestin expressing progenitors in the frontal cortex of Nestin-Tva (Ntva); p53 fl/fl; ATRX fl/fl or Ntva; p53 fl/fl mice were infected with H3.3G34R or H3.3WT, PDGFA, and Cre, and monitored for signs and symptoms of tumor formation. RNAseq analysis and immunophenotype were used to characterize the tumors. We observed that H3.3G34R did not significantly impact tumor latency independent of ATRX status. ATRX loss significantly increased tumor latency independent of H3.3G34R (P < 0.01) from 90 days to 143 days. H&E analysis revealed that the majority of tumors in all groups were high grade and that mice with ATRX loss were more likely to develop low grade tumors though this trend did not reach significance (P = 0.075). Expression of Ki67, GFAP and Olig2 was present in all groups, as shown by Immunophenotypic analysis. RNAseq analysis of murine tumor tissue revealed that ATRX loss in the context of G34R led to significant differential expression of 113 genes (78 upregulated and 35 downregulated) including upregulation of PRC2 target genes including HoxA genes. These findings highlight the cooperation between ATRX loss and H3.3G34R in gliomagenesis.

Citation Format: Aalaa Abdallah, Herminio J. Cardona, David J. Picketts, Daniel J. Brat, Xiao-Nan Li, Oren J. Becher. A novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of PRC2 target genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 912.
Abstract 913: Tert mutant promoter mouse model induces cancer progression in BrafV600E-driven thyroid tumors: A novel tool to understand the biology of telomerase-reactivated cancers
Authors: Landa Inigo , Hao Jingzhu , Xu Bin , Giacalone Joseph , Herbert Zach , Blasco Maria A. , Knauf Jeffrey A. , Ghossein Ronald , Fagin James A.
Published: June 2022

Hotspot mutations in the proximal promoter of the telomerase reverse transcriptase (TERT) gene are the first cross-cancer alterations lying in a gene regulatory region. TERT promoter mutations (TPMs) are enriched in advanced thyroid tumors and constitute markers of disease severity. TPMs enhance TERT transcription, which is otherwise silenced in adult tissues, thus reactivating a bone fide oncoprotein. To study TERT deregulation and its downstream consequences in a biologically accurate model, we generated a Tert-mutant promoter mouse model via CRISPR/Cas9 editing of the equivalent murine locus and crossed these animals with thyroid-specific BrafV600E-mutant mice. BrafV600E animals develop highly penetrant papillary thyroid tumors (PTC) by week 5, but do not progress. In contrast, BrafV600E+TertMUT animals showed an increased incidence of poorly differentiated thyroid cancers (PDTC) by 20 weeks (30% vs. 0% in BrafV600E; chi-squared P= 0.03), mimicking those exhibited by an alternative transgenic model of Tert overexpression (BrafV600E+K5-Tert; 36% PDTCs). Mouse Tert promoter mutation increased Tert transcription in vitro and in vivo, as reported in patients’ tumors carrying TPMs. Braf+Tert animals partially responded to MAPK pathway inhibition (dabrafenib plus trametinib), showing that MAPK signaling remains relevant in these specimens. Interestingly, RNA sequencing of Tert-reactivated murine thyroid tumors showed unique transcriptomic profiles (compared to BrafV600E alone), suggesting that downstream effects, other than the canonical Tert-mediated telomere maintenance, operate in cancers harboring TPMs. These cancer models of telomerase reactivation provide excellent pre-clinical settings to understand the regulatory mechanisms and biological underpinnings of TPM-induced progression of thyroid and other tumors, and to explore novel therapeutic strategies.

Citation Format: Inigo Landa, Jingzhu Hao, Bin Xu, Joseph Giacalone, Zach Herbert, Maria A. Blasco, Jeffrey A. Knauf, Ronald Ghossein, James A. Fagin. Tert mutant promoter mouse model induces cancer progression in BrafV600E-driven thyroid tumors: A novel tool to understand the biology of telomerase-reactivated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 913.
Abstract 926: IIKKa deletion amplifies renal transitional epithelial stem cells and initiates lethal invasive papillary urothelial cell carcinoma development
Authors: Li Xin , Zhu Feng , Jiang Chengfei , Zhao Yongmei , Tran Bao , Butcher Donna , Wu Xiaolin , Debra Tross , Hu Yinling
Published: June 2022

Urothelial cell carcinoma is the most common cancer of the renal pelvis or ureter in humans. Due to a lack of animal models, its pathogenic causes remain elusive. Here, we generated IkkαΔKsp mice lacking Ikkα specifically in epithelial cells of the developing kidney and genitourinary tract as well as renal tubules. The mutant mice developed invasive papillary urothelial cell carcinomas initiated from the pelvis, leading to hydronephrosis, and the animals eventually died. Depleting neutrophils or antibiotic treatment slowed down the development of phenotypes but did not rescue them, suggesting that epithelial-cell-autonomous aberrant alterations induced by Ikkα loss are critical for the initiation of urothelial cancer. Moreover, We found that the urothelial cell carcinomas in the mutant mice highly expressed cancer stem-cell markers such as Sox2, K15, p63, Nanog, and CD44, as well as FGFR3 and osteopontin (OPN). Thus, we hypothesize that Ikkα ablation-mediated OPN and FGFR3 overexpression regulates and amplifies CD44+ renal cancer-stem cells for tumor initiation. These alterations are shared with human papillary urothelial cell carcinomas.

Citation Format: Xin Li, Feng Zhu, Chengfei Jiang, Yongmei Zhao, Bao Tran, Donna Butcher, Xiaolin Wu, Tross Debra, Yinling Hu. IIKKa deletion amplifies renal transitional epithelial stem cells and initiates lethal invasive papillary urothelial cell carcinoma development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 926.