bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5684: Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress
Authors: Sannino Sara , Brodsky Jeffrey L.
Published: June 2022

Cancer cells survive under extreme environments that alter protein homeostasis, thereby provoking cellular stress responses, such as the unfolded protein response (UPR) and the integrated stress response (ISR). These pathways increase the expression of molecular chaperones to prevent the accumulation of toxic proteins in the cell. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor. We first showed that established breast cancer cell lines bin into classes based on their sensitivity to the drug. We next discovered that resistant cells have higher autophagy levels compared to the more sensitive lines. Moreover, gene and protein expression studies and imaging methods showed that autophagy was further induced by Hsp70 inhibition in resistant breast cancer cells. Consistent with the fact that Hsp70 normally targets aberrant proteins for degradation, our data indicated that resistance to compromised Hsp70 activity arose from elevated levels of autophagy. We next showed that Hsp70 inhibition specifically triggers the ISR pathway. Unexpectedly, protection against the Hsp70 inhibitor was dependent on the ISR sensor, GCN2, which is most commonly associated with the amino acid starvation response. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating the PERK pathway. These data reveal an unexpected route through which cancer cells can adapt to chaperone targeted-proteotoxicity and position GCN2 and autophagy as critical compensatory mechanisms for cancer cells when proteostasis is compromised.

Citation Format: Sara Sannino, Jeffrey L. Brodsky. Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress [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 5684.
Abstract 5692: Tissue specificity of chromosome aneuploidy correlates with BRCA-associated cancer risk
Authors: Wang Xinfeng , Jun Tomi , Sun Nan , He Jie , Huang Kuan-lin
Published: June 2022

Pathogenic germline variants of BRCA1 and BRCA2 disproportionally elevated the risk of specific cancer types (ex. breast [BRCA] and ovarian [OV] cancer) compared to tumors arising from other tissues. The reason underlying the strong tissue-specificity for BRCA-associated cancer risk remains largely unknown. Under the two-hit hypothesis, BRCA-mediated oncogenesis is thought to originate from a cell where the germline BRCA1/2 variant underwent loss of heterozygosity (LOH) of the wildtype BRCA allele, resulting in homologous recombination DNA repair deficiencies. Notably, tumor aneuploidy and loss of chromosome arms also show strong tissue specificity across cancer types. The apparent tissue specificities of BRCA-associated cancer risk and chromosome aneuploidy, together with deletion-induced LOH underlying the two-hit hypothesis, pose an intriguing possibility that they may be linked. Using genomic data from the TCGA PanCanAtlas and ICGC PCAWG projects, we calculated the frequencies of deletions of the chromosomal arms where the BRCA1/2 genes are located (17q for BRCA1; 13q for BRCA2) across different cancer types in tumors with no whole-genome doublings (WGD). Among TCGA cases, OV and BRCA showed significantly higher frequencies of 17q deletion (32.75% and 8.05%, respectively) than other cancer types (ranked 2/30 and 5/30, permutation p-value = 0.030 and 0.024), and relatively higher frequencies of 13q deletion (32.75% and 21.92%, respectively) compared to other cancers (ranked 5/32 and 8/32, p-value = 0.101 and 0.078). As expected, cancer types showing high fractions of BRCA1/2 deletions overlap with those showing 17q/13q deletions and often include BRCA and OV. Subsequently, we determined how often BRCA1/2 deletion co-occurred and thus may be caused by the corresponding 17q/13q deletions, showing that BRCA1 deletion carriers who also had chr17q deletion accounted for 40.76% in OV and 25.93% in BRCA patients, compared to 33.33% in all other cancer types while BRCA2 deletion carriers who also had chr13q deletion accounted for OV (53.97%) and BRCA (58.60%) patients, compared to 49.83% in all other cancer types. In addition, we validated our findings in non-overlapping cases of the ICGC PCAWG projects. Consistent with TCGA results, OV and BRCA showed higher frequencies of 17q and 13q deletion compared to other cancer types. To conclude, we identified a correlation between cross-cancer difference in arm-level and focal chromosome aneuploidy affecting BRCA1/2 and BRCA-associated cancer risk, and how chromosome aneuploid may give rise to tissue-specific risks of cancer remain to be investigated.

Citation Format: Xinfeng Wang, Tomi Jun, Nan Sun, Jie He, Kuan-lin Huang. Tissue specificity of chromosome aneuploidy correlates with BRCA-associated cancer risk [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 5692.
Abstract 5695: Roles of cGAS in DNA damage response
Authors: Chen Weidong , An Hyun-Jung , Lee Ga-Eun , Cho Yong-Yeon
Published: June 2022

Cytosolic double strand DNAs released from the nucleus or mitochondria produce diverse cellular signals mediated by cyclic GAM-AMP synthase (cGAS) to modulate cell proliferation, metastasis, apoptosis, and necroptosis. However, the role of cGAS has not been fully elucidated when cells are stimulated with DNA damage response such as ultraviolet (UV). In this study, we found that DNA damage by treatment of cisplatin or UVB induced cGAS protein levels. We further found that cisplatin and UVB increased not only endogenous cGAS protein levels, but also ectopic expressed cGAS protein levels. The cGAS protein increase was showed positive correlation with H2AX phosphorylation at Ser139 (γH2AX). Moreover, although UVB stimulation suppressed cell proliferation in both stably expressing mock control (SK-MEL-2-mock) and cGAS in SK-MEL-2 (SK-MEL-2-cGAS) cells, suppression ratio in SK-MEL-2-cGAS cells was reduced compared to SK-MEL-2-mock cells. These results suggested that cGAS may have a potential to cell protection when the cells are stimulated with DNA damage. This hypothesis was supported by that cell proliferation of SK-MEL-2-cGAS cells were 2 folds faster than that of SK-MEL-2-mock cells after UVB stimulation. We further found that living cell population of SK-MEL-2-cGAS cells, which were 80% in no UVB stimulation, was reduced to 37% at 24 h and 34% at 48 h after 2 mJ/cm2 UVB stimulation, while SK-MEL-2-mock cells were reduced to 37% at 24 h and 20% at 48 h after 2 mJ/cm2 UVB stimulation. On the other hand, total cell death about 40% at 24 h and 65% at 48 h in SK-MEL-2-mock cells was reduced about 25% at 24 h and 36% at 48 h in SK-MEL-2-cGAS cells after 2 mJ/cm2 UVB stimulation. Taken together, we concluded that cGAS may play an important role to protect the cells from DNA damage.

Citation Format: Weidong Chen, Hyun-Jung An, Ga-Eun Lee, Yong-Yeon Cho. Roles of cGAS in DNA damage response [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 5695.
Abstract 5696: Effect of extracellular matrix stiffness on cancer cells radiation response
Authors: Lacombe Jerome , Muccio Melinda , Zenhausern Frederic
Published: June 2022

Over the past decade, it has become clear that the mechanical cues of extracellular matrix (ECM) are a major regulator of the cancer biology. In particular, ECM stiffness has shown an important role in chemoresistance modulation. However, despite the central role of radiotherapy in cancer management, little is known about the effect of ECM stiffness on cancer cells radiation response. In this study, we used polyacrylamide collagen-coated hydrogel model to reproduce a soft (0.7 kPa) and a stiff (70 kPa) matrix. The model was validated by atomic force microscopy force spectroscopy and biological analyses through cellular morphology, proliferation and YAP/TAZ mechanotransduction pathway that all confirmed the accuracy of our model. Then, lung cancer cells seeded on both soft and stiff hydrogels were irradiated, and the level of DNA damages were compared between the two conditions. First, alkaline comet assay revealed the total amount of DNA damages were higher in cells grown on a rigid ECM at 1 h post-irradiation than cells seeded on a soft matrix. Second, γH2AX/53BP1 foci assay showed that the number of DNA double strand breaks were also higher in cells grown on a rigid environment. However, the level of DNA damage was identical at 24 h post-irradiation for both stiffness, suggesting that a more efficient DNA damage repair response could take place for cells seeded in stiff matrix. In order to explain these observations, analyses on chromatin structure and nucleus shape were then performed. Data showed that cells cultured on stiff matrix displayed a larger nucleus, less condensed chromatin, and a decreased expression level of class I HDACs genes. Moreover, it was found that the level of total reactive oxygen species (ROS) and superoxide ion was also higher in cells grown on a rigid environment 24 h post-irradiation. Altogether these data suggest that a high stiffness may increase early DNA damages after irradiation, partially by remodeling chromatin that could expose DNA to radiation-induced genotoxic agents such as ROS. Additional investigations are required to understand better the mechanisms that could explain such differences, but these preliminary data highlighted the potential role of mechanical stress in cellular radiation response and paved the way to new therapeutic target for radiotherapy management.

Citation Format: Jerome Lacombe, Melinda Muccio, Frederic Zenhausern. Effect of extracellular matrix stiffness on cancer cells radiation response [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 5696.
Abstract 5708: The DNA methylation landscape of prostate cancer brain metastases
Authors: Gallon John , Rodriguez-Calero Antonio , Maletti Sina , Ng Charlotte , Rubin Mark , Piscuoglio Salvatore
Published: June 2022

Introduction: Improved survival in prostate cancer through more effective therapies has also led to an increase in the diagnosis of metastases to infrequent locations such as the brain. In the largest cohort of prostate cancer brain metastases (PCBM) yet studied, we investigated the DNA methylation landscape of PCBM.

Methods: Using Illumina EPIC arrays, we profiled the DNA methylation landscapes of 95 metastatic samples from 42 patients, with multi-regional analysis of the metastases from 31 patients. We compared DNA methylation profiles from metastases to the primary prostate cancer (PCa) samples of 18 of these patients. Multi-regional data was obtained for 15 of these. We performed unsupervised analyses of DNA methylation profiles across sample types, and identified differentially methylated CpG sites and regions (DMRs) between PCBM and PCa samples, and PCBM/PCa samples and normal prostate tissue. We integrated our data with targeted-RNA sequencing and whole exome sequencing (WES).

Results: We highlight the epigenetic heterogeneity present in different regions of the PCBM from patients for which multi-regional data were available. While substantial DNA methylation changes were detected in PCa compared to normal prostate tissue, epigenetic dysregulation was strikingly more pronounced in PCBM samples. DNA methylation changes in PCBM involved striking hypermethylation particularly enriched at CpG islands, and associated with activity of the PRC2 complex. We observed a marked distinction of the methylation profiles of SPOP mutant and TMPRSS2-ERG samples. SPOP mutant samples acquired substantially more epigenetic aberrations, particularly enriched at CpG islands which were hypermethylated genome-wide compared to TMPRSS2-ERG PCBM samples, as well as SPOP mutant PCa samples. These changes were strongly associated with the activity of EZH2 and MYC, both of which were overexpressed specifically in SPOP mutant PCBM. TMPRSS2-ERG samples showed fewer DNA methylation changes, but those detected were associated with targets of the SP1 transcription factor.

Conclusion: We show that epigenetic heterogeneity is present in different regions of PCBM. While the distinct methylation landscapes of SPOP mutant and TMPRSS2-ERG PCa has been previously reported, we show that metastasis-specific DNA methylation changes emerge in each of these settings. These observations suggest that the acquisition of DNA methylation changes may be an important feature of PCBM, and may help to improve our understanding of the processes driving the occurrence of these rare metastases.

Citation Format: John Gallon, Antonio Rodriguez-Calero, Sina Maletti, Charlotte Ng, Mark Rubin, Salvatore Piscuoglio. The DNA methylation landscape of prostate cancer brain metastases [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 5708.
Abstract 5726: Splicing-based classifier for gastric cancer identifies epithelial-mesenchymal transition subtypes associated with survival
Authors: Jun Yukyung , Suh Yun-Suhk , Park SungHee , Lee Jieun , Kim Jong-Il , Lee Sanghyuk , Lee Wan-Ping , Anczuków Olga , Yang Han-Kwang , Lee Charles
Published: June 2022

Alternatively spliced RNA isoforms are a hallmark of tumors, but their nature, prevalence, and clinical implications in gastric cancer are unknown. We systematically profiled the splicing landscape of 83 gastric tumors and matched normal mucosa. We identified and experimentally validated eight splicing events that can classify all gastric cancers into three subtypes: Epithelial-splicing, Mesenchymal-splicing, and Hybrid-splicing. These subtypes were associated with distinct molecular signatures and epithelial-mesenchymal transition markers. Subtype-specific splicing events were enriched in motifs for splicing factors RBM24 and ESRP1, which were upregulated in Mesenchymal-splicing and Epithelial-splicing tumors, respectively. A simple classifier based only on RNA levels of RBM24 and ESRP1, and which is thus readily implementable in the clinic, is sufficient to distinguish gastric cancer subtypes and predict patient survival in multiple independent patient cohorts

Citation Format: Yukyung Jun, Yun-Suhk Suh, SungHee Park, Jieun Lee, Jong-Il Kim, Sanghyuk Lee, Wan-Ping Lee, Olga Anczuków, Han-Kwang Yang, Charles Lee. Splicing-based classifier for gastric cancer identifies epithelial-mesenchymal transition subtypes associated with survival [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 5726.
Abstract 5731: Structure-based development of a novel MYC inhibitor for neuroendocrine prostate cancer
Authors: Foo Jane , Ton Anh-Tien , Singh Kriti , Ban Fuqiang , Morin Helene , Lee Joseph , LeBlanc Eric , Lallous Nada , Cherkasov Artem
Published: June 2022

Background: MYC oncoproteins are important drivers of human cancers. More specifically, NMYC is amplified and overexpressed in neuroendocrine prostate cancer (NEPC). NMYC elicits its oncogenic effects by forming a heterodimer with MAX. This complex binds to regulating elements and activates the transcription of MYC target genes that play roles in tumor growth and progression. It is well known that members of the MYC family of proteins make compelling targets for cancer treatments. However, to this date, no tangible drug candidates have been developed into the clinics for MYC. In previous studies utilizing a rational computer-aided drug discovery (CADD) approach, we identified VPC-70551 as our most active hit. Subsequent optimization with the VPC-70551 scaffold has led to the identification of a new series of compounds. Using in silico drug screening followed by functional validation, we identified a small molecule inhibitor, VPC-70619, that exhibits higher microsomal stability and is better absorbed and tolerated orally than most MYC inhibitors described in the literature.

Method: We used a transcriptional reporter assay to determine the effect of VPC-70619 on MYC-mediated transcription. To validate on-target effect, we evaluated the effect of VPC-70619 treatment on MYC-positive (LASCPC-01, NCIH660, LNCaP N-MYC, 22Rv1 N-MYC) and MYC-negative (HO15.19) cancer cells. We also evaluated the effect of VPC-70619 on MYC/MAX interaction by PLA and on DNA binding by BLI. The direct binding between recombinant MYC/MAX protein complex and VPC-70619 was evaluated by MST.

Results: VPC-70619 inhibited MYC transcriptional activity in dose dependent manner and the proliferation of MYC-positive cell lines. VPC-70619 did not interfere with MYC/MAX interaction however it blocked the complex interaction with DNA. We confirmed the direct binding of VPC-70619 to the purified MYC/MAX complex by using MST.

Conclusion: This project presents the identification of a new MYC inhibitor that blocks the transcriptional activity of this oncogene and elucidates the molecular mechanism of action of this inhibitor. Our findings help prelude the development and discovery of more effective treatments for NEPC patients.

Citation Format: Jane Foo, Anh-Tien Ton, Kriti Singh, Fuqiang Ban, Helene Morin, Joseph Lee, Eric LeBlanc, Nada Lallous, Artem Cherkasov. Structure-based development of a novel MYC inhibitor for neuroendocrine 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 5731.
Abstract 5732: p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma development
Authors: Date Yuki , Ueno Tomoya , Ito Kosei
Published: June 2022

p53 deficiency and Myc dysregulation, driven by genetic and epigenetic alterations, respectively, are frequently associated with cancer. Previously, we reported that the combination of p53 inactivation and Myc overexpression, mediated by Runx transcription factors (TFs), leads to osteosarcoma (OS), the most common primary bone cancer. However, the mechanisms leading to Myc upregulation by Runx in a p53-deficient microenvironment remain unclear.

TGFβ is a main driver of the epithelial-to-mesenchymal transition and its signal-dependent TFs, Smads, are known to associate with lineage-determining TFs including Runx. As TGFβ paracrine signalling is reportedly increased during human OS development, we immunodetected increased co-expression of TGFβ and phospho-Smad2 in the bone marrow of osteoprogenitor-specific p53 knockout mice, Osterix (Osx)/Sp7-Cre; p53fl/fl mice, which are widely used as an animal model of human OS. Heterozygous deletion of the type II TGFβ receptor in those mice weakened their susceptibility to OS, phenocopying the overall life-extending effect of Myc depletion in the same model.

p53 disruption caused TGFβ to aberrantly induce Myc in osteoprogenitors, rendering the cells tumorigenic upon transplantation into immunocompromised mice. Within the 3-Mb Myc regulatory region, we identified a TGFβ-responsive element dubbed m340, which contains binding sites for multiple TFs including Runx. The m340 enhancer caused Myc upregulation upon p53 disruption by switching its associated promoter from the lncRNA Pvt1 to Myc. In support of this promoter switching mechanism, a specific disruption of the evolutionarily conserved p53 site within the Pvt1 promoter recapitulated TGFβ-induced Myc overexpression. At m340, TGFβ promoted its occupancy by a transcriptional complex consisting of Runx, its cooperative factors Smads and AP1, and CBP, further activating the enhancer by increasing H3K27ac deposition. Runx inhibition not only repressed the aberrant upregulation of Myc by reducing Smad occupancy at m340, but also suppressed OS development in vivo.

These results suggest that p53 deficiency causes Myc dysregulation through a novel promoter switching programme that is facilitated by TGFβ signalling and reveals a novel epigenetic mechanism underlying the interaction between cancer-predisposing genetic alterations (p53 deficiency) and environmental cues (TGFβ) in tumorigenesis. Considering the mediating role Runx plays in m340 activation, our study may provide a clinical rationale for targeting Runx in p53-deficient malignancies.

Citation Format: Yuki Date, Tomoya Ueno, Kosei Ito. p53 deficiency causes Myc dysregulation through a novel TGFβ-facilitated promoter switching mechanism to instigate osteosarcoma 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 5732.
Abstract 5784: Overcoming FFPE hurdles to enable high quality hybrid capture libraries and somatic mutation detection in matched tumor-normal patient samples
Authors: Heider Margaret R. , Sun Jian , Sexton Brittany S. , Langhorst Bradley W. , Gray Andrew , Higgins Lauren , Chen Lixin , Apone Lynne , Evans Thomas C. , Nichols Nicole M. , Dimalanta Eileen T. , Davis Theodore B. , Liu Pingfang
Published: June 2022

In cancer genomics, a common source of DNA is formalin-fixed, paraffin-embedded (FFPE) tissue from patient surgical samples, where in most cases high quality fresh or frozen tissue samples are not available. FFPE DNA poses many notable challenges for preparing NGS libraries, including low input amounts and highly variable damage from fixation, storage, and extraction methods. Due to the high cost of sequencing and variability of coverage, regions of interest are often specifically enriched using hybrid capture-based approaches, but these methods require a high input of diverse, uniform DNA library to achieve the coverage required for somatic mutation identification in tumor samples.We applied a multi-faceted approach to improving the yield, quality, and coverage from libraries generated from cancer patient sample sets. Using tumor-normal pairs, we evaluated the impact of our method on the sensitivity and accuracy of somatic variant detection. Matched frozen tissue provided a truth set of somatic variants for a particular tumor as well as a gold standard by which we could assess the quality of our FFPE hybrid capture libraries. Combining DNA damage repair and a novel enzymatic fragmentation mix upstream of library preparation not only reduced the false positive rate in somatic variant detection by repairing damage-derived mutations but also improved the library yield, complexity, coverage uniformity, and hybrid capture library quality metrics. The use of unique molecular identifier (UMI)-containing adaptors ensured accurate duplicate marking. Finally, a new PCR master mix boosts the library yield without compromising library quality in FFPE samples ranging from very poor quality to high quality. This combinatorial approach allows even the poorest quality FFPE samples to achieve high quality libraries with sufficient input for hybrid capture in our study.

Citation Format: Margaret R. Heider, Jian Sun, Brittany S. Sexton, Bradley W. Langhorst, Andrew Gray, Lauren Higgins, Lixin Chen, Lynne Apone, Thomas C. Evans, Nicole M. Nichols, Eileen T. Dimalanta, Theodore B. Davis, Pingfang Liu. Overcoming FFPE hurdles to enable high quality hybrid capture libraries and somatic mutation detection in matched tumor-normal patient samples [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 5784.
Abstract 5791: Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples
Authors: Pastoret Cédric , Aliouat Amyra , Boulland Marie-Laure , Denoual Florent , Desmares Anne , De Tayrac Marie , Fest Thierry , Letellier Celeste
Published: June 2022

Introduction We developed a unique panel of 54 target genes dedicated to hematological malignancies (HematoPanel) and screened 1,108 clinical samples. We also performed transcriptomic analysis and evaluated its relevance for patients with acute lymphoblastic leukemia (ALL).

Methods DNA and RNA were extracted from cell pellets with the Maxwell® RSC (Promega®). Libraries for targeted high-throughput DNA sequencing (HTS) were generated in duplicate from 100 ng with the Advanta™ NGS Library Prep Assay kit using the LP 48.48 IFC (integrated fluidic circuit) on a Juno™ and sequenced on the NextSeq™ 550 (Illumina®) to a median depth of coverage of approximately 1,200X per sample. Transcriptomic libraries were generated from 50 ng RNA with the Advanta RNA-Seq XT NGS Library Prep Kit using the LP 48.Atlas™ IFC on Juno and sequenced on the NextSeq 550. A bioinformatics pipeline was developed for fusion transcript detection. Differential gene expression was analyzed with the DESeq2 package after STAR alignment.

Results In 2020 we sequenced 1,108 consecutive samples in routine practice with HematoPanel. The main indications were the suspicion of myeloproliferative neoplasms (32%), the evaluation of chronic lymphocytic leukemia (23%), and the diagnostic workup of cytopenias (15%). We detected 1,306 mutations in 654 patients (59%) (1 to 8 mutations per sample). Eight genes (TET2, SF3B1, TP53, DNMT3A, ASXL1, JAK2, SRSF2 and MYD88) were reported as mutated for more than 50 patients. We sequenced 22 samples including the MOLT-4 cell line, healthy donor samples, and 16 ALL samples for transcriptomic evaluation. From ALL samples, we detected 5 fusion transcripts previously identified by specific PCR (1 BCR-ABL, 2 ETV6-RUNX1, 1 KMT2A-AFF1, 1 TCF3-PBX1). Moreover, we detected 4 other fusion transcripts for 3 patients (EBF1-PDGFRB, FGFR1-MYO18A, P2RY8-CRLF2, and PAX5-NOL4L). Interestingly, these patients clustered with the BCR-ABL positive sample based on their expression profile. It confirms the diagnosis of Phi-like ALL and thus offers a therapeutic target for these patients.

Conclusion Microfluidics-based library prep using Juno offers a cost-effective strategy and straightforward workflows for both targeted HTS and RNA sequencing. We validated its use for hematological malignancy evaluation in routine practice.

Citation Format: Cédric Pastoret, Amyra Aliouat, Marie-Laure Boulland, Florent Denoual, Anne Desmares, Marie De Tayrac, Thierry Fest, Celeste Letellier. Microfluidics-based library preparation strategies for targeted high-throughput sequencing and RNA sequencing in hematological malignancies in clinical samples [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 5791.
Abstract 5806: Mieap forms membraneless organelles to compartmentalize and facilitate cardiolipin metabolism
Authors: Ikari Naoki , Nakamura Yasuyuki , Arakawa Hirofumi
Published: June 2022

Liquid droplets function as membraneless organelles that compartmentalize and facilitate efficient biological reactions. They are formed by proteins with intrinsically disordered regions (IDRs) via liquid-liquid phase separation. Mieap/SPATA18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of liquid droplets in mitochondria. Mieap liquid droplets specifically phase separate the mitochondrial phospholipid, cardiolipin. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions involved in cardiolipin metabolism, including biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1, and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap liquid droplets. Mieap-deficient mice exhibit altered crista structure in mitochondria of various tissues, including brown fat, and tend to become obese. These results suggest that Mieap drives formation of membraneless organelles to compartmentalize and promote cardiolipin metabolism at the inner mitochondrial membrane, thus potentially contributing to mitochondrial quality control.

Citation Format: Naoki Ikari, Yasuyuki Nakamura, Hirofumi Arakawa. Mieap forms membraneless organelles to compartmentalize and facilitate cardiolipin metabolism [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 5806.
Abstract 5812: Histone deacetylase 10 supports tumor growth under polyamine-limiting conditions
Authors: Stewart Tracy Murray , Steimbach Raphael R. , Foley Jackson R. , Miller Aubry K. , Casero Robert A.
Published: June 2022

Cytosolic histone deacetylase 10 (HDAC10) is specifically deacetylates the modified polyamine N8-acetylspermidine (N8-AcSpd). Although intracellular concentrations of N8-AcSpd are low, extracellular sources can be abundant, particularly in the colonic lumen. Extracellular polyamines, including those from the diet and microbiota, can support tumor growth both locally and at distant sites. However, the contribution of N8-AcSpd is unknown. We hypothesized that HDAC10, by converting N8-AcSpd to spermidine, may provide a source of this growth-supporting polyamine in circumstances of reduced polyamine biosynthesis, such as in polyamine-targeting, anticancer therapies. Inhibitors of polyamine biosynthesis, such as difluoromethylornithine (DFMO), inhibit tumor growth but with compensatory uptake of extracellular polyamines that have limited their clinical success. Combining DFMO with inhibitors of polyamine uptake have improved the antitumor response. However, acetylated polyamines may use different transport machinery than the parent molecules. The current study uses CRISPR/Cas9-mediated HDAC10-knockout cell lines and recently developed, selective HDAC10 inhibitors to investigate the contribution of HDAC10 in maintaining tumor cell proliferation. Inhibition of cell growth by DFMO-associated polyamine depletion is successfully rescued by the provision of physiological concentrations of exogenous N8-AcSpd, which is converted to spermidine and spermine, only in cell lines with HDAC10 activity. Loss of HDAC10 prevents both restoration of polyamine levels and growth rescue, implicating HDAC10 in supporting polyamine-associated tumor growth. These data suggest the utility of HDAC10-specific inhibitors as an antitumor strategy that may have particular value in improving the response to polyamine-blocking therapies.

Citation Format: Tracy Murray Stewart, Raphael R. Steimbach, Jackson R. Foley, Aubry K. Miller, Robert A. Casero. Histone deacetylase 10 supports tumor growth under polyamine-limiting conditions [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 5812.
Abstract 5815: Metabolomics in an Apc-mutant genetic background: Mechanistic insights from acute vs. chronic spinach intake
Authors: Neja Sultan , Dashwood Roderick H.
Published: June 2022

There is growing interest in the crosstalk between the gut microbiome, metabolomic features, and disease pathogenesis. Colorectal cancer is a major health burden worldwide, linked in part to modifiable risk factors associated with diet and lifestyle (1). The current investigation compared long-term (26 week) and acute (3 day) dietary spinach intake in a genetic model of colorectal cancer. Metabolomic analyses in the polyposis in rat colon (Pirc) model and in wildtype animals corroborated key contributions to anticancer outcomes by spinach-derived linoleate bioactives and a butanoate metabolite linked to increased α-diversity of the gut microbiome (2). Combining linoleate and butanoate metabolites in human colon cancer cells revealed enhanced apoptosis and reduced cell viability, paralleling the apoptosis induction observed in colon tumors from rats given long-term spinach treatment. Mechanistic studies indicated reactivation of immune-associated major histocompatibility complex genes in cell-based assays and in vivo, plus differential roles of the metabolites in targeting Wnt/β-catenin signaling (3). Clinical translation of the findings from this investigation to at-risk patients might provide valuable quality-of-life benefits by delaying surgical interventions and drug therapies with adverse side effects (4,5).

References1.Xi, Y., Xu, P. Global colorectal cancer burden in 2020 and projections to 2040. Transl Oncol 2021;14: 1011741.Chen, Y.-S. et al. Dietary spinach reshapes the gut microbiome in an Apc-mutant genetic background: mechanistic insights from integrated multi-omics. Gut Microbes 2021;13:1972756. 1.Liu, F. et al. Suppression of membranous LPR5 recycling, Wnt/β-catenin signaling, and colon carcinogenesis by 15-LOX-1 peroxidation of linoleic acid in PI3P. Cell Rep 2020;32:108049.1.Ulusan, A.M. et al. Optimization of erlotinib plus sulindac dosing regimens for intestinal cancer prevention in an Apc-mutant model of Familial Adenomatous Polyposis (FAP). Cancer Prev Res (Phila) 2021;14:325-336.1.Samadder, N.J. et al. Association of sulindac and erlotinib vs placebo with colorectal neoplasia in Familial Adenomatous Polyposis: secondary analysis of a randomized clinical trial. JAMA Oncol 2018:4:671-677.

Citation Format: Sultan Neja, Roderick H. Dashwood. Metabolomics in an Apc-mutant genetic background: Mechanistic insights from acute vs. chronic spinach intake [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 5815.
Abstract 5816: Serum metabolomic profile of exposure to hair dye
Authors: Lim Jungeun , Huang Jiaqi , Weinstein Stephanie J. , Albanes Demetrius
Published: June 2022

Hair dye products now represent one of the most rapidly growing beauty and personal care industries. The association between hair dye use and the development of cancer has been a research focus, including for breast, prostate, and bladder cancer, yet biological mechanisms have not been established. In this first examination of metabolomic response to hair dye exposure, we compared the serum metabolite profiles of male hair dye users and nonusers to identify differences related to biological mechanisms relevant to how exposure may influence cancer risk. The Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study was a randomized, controlled cancer prevention trial of vitamin supplementation that enrolled 50–69-year-old Finnish male smokers of whom 75 hair dye users and 125 nonusers were studied. Ultrahigh performance liquid chromatography-tandem mass spectrometry was used to assay baseline serum samples, and the difference in log-transformed, batch-normalized metabolite signals between the exposed and unexposed men was estimated using linear regression, adjusting for age, body mass index, and smoking. We measured 1,401 metabolites, of which 11 compounds, including 4 amino acids and 3 xenobiotics, differed significantly between the two groups after correction for multiple comparisons (FDR<0.1). Redox-related glutathione metabolism was heavily represented, including cysteineglutathione disulfide which showed the strongest association with hair dye use (effect size (β) = -0.685; p<0.0001), along with oxidized cysteine-glycine (β = -0.325; p<0.0001) and several compounds relevant to antioxidation/ROS pathways. Metabolites previously associated with prostate cancer risk also differed significantly between hair dye user and non-users. Our findings point to biological mechanisms through which exposure to hair dyes may impact risk of prostate and other cancers.

Citation Format: Jungeun Lim, Jiaqi Huang, Stephanie J. Weinstein, Demetrius Albanes. Serum metabolomic profile of exposure to hair dye [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 5816.
Abstract 5817: Exosomal miR-4466 from nicotine-activated neutrophils promotes tumor cell stemness & metabolism in brain metastasis
Authors: Tyagi Abhishek , Wu Shih-Ying , Sharma Sambad , Wu Kerui , Zhao Dan , Deshpande Ravindra , Watabe Kounosuke
Published: June 2022

Smoking is associated with lung cancer and has a profound impact on tumor immunity. Nicotine, the addictive and non-carcinogenic smoke component, influences various brain cells and the immune system. However, little is known how long-term use of nicotine affects clinical outcomes in patients with brain metastasis. To address this question, we conducted a retrospective analysis of 810 lung cancer patients with smoking history and assessed brain metastasis. We found that lung cancer patients of current smokers have significantly higher brain metastatic incidence compared to the never-smokers. Immune profiling of the brain metastatic lesions among smokers showed predominant infiltration of neutrophils compared to never-smokers. Notably, mice pre-exposed to nicotine showed a significantly increased number of neutrophils in the brains even before implantation of cancer cells (pre-metastatic niche), and their numbers progressively increased during the metastatic stage. Neutrophil depletion during metastatic colonization caused a remarkable decrease in brain metastasis. Furthermore, we found that chronic exposure of nicotine recruited STAT3-activated N2-neutrophils that secreted exosomal miR-4466 in the brain. This pre-metastatic niche promoted stemness and metabolic switching via SKI/SOX2/CPT1A axis in the tumor cells thereby enabling brain metastasis. Importantly, exosomal miR-4466 levels were found to be elevated in serum/urine of cancer-free subjects with a smoking history and promote tumor growth in vivo, suggesting that exosomal miR-4466 may serve as a promising prognostic biomarker for predicting increased risk of metastatic disease among smoker(s). Our findings suggest a novel pro-metastatic role of nicotine-induced N2-neutrophils in the progression of brain metastasis. We also demonstrated that inhibiting nicotine-induced STAT3-mediated neutrophil polarization effectively abrogated brain metastasis in vivo. Our finding for the first time provides compelling evidence related to the mechanism(s) of neutrophil-mediated brain metastasis that arises from nicotine use. Our results also identified a potential prognostic biomarker for the onset of metastasis as a risk-reduction measure in lung cancer patients and implicated the risk of using nicotine gateway for smoking cessation in cancer patients with a smoking history.

Citation Format: Abhishek Tyagi, Shih-Ying Wu, Sambad Sharma, Kerui Wu, Dan Zhao, Ravindra Deshpande, Kounosuke Watabe. Exosomal miR-4466 from nicotine-activated neutrophils promotes tumor cell stemness & metabolism in brain metastasis [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 5817.
Abstract 5820: NF-κB signaling regulates miRNA expression in ovarian cancer
Authors: Kamdar Rahul D. , Harrington Brittney S. , Korrapati Soumya , Wong Nathan , House Carrie D. , Annunziata Christina M.
Published: June 2022

Epithelial ovarian cancer (EOC) remains the fifth leading cause of cancer related death in women worldwide, partly due to the survival of chemoresistant, stem-like tumor-initiating cells (TICs) that promote disease relapse. Previous studies described the role of the NF-kB pathway in promoting TIC chemoresistance and survival through the action of key transcription factors (TFs) like RelA and RelB. These TFs serve as key master regulators of genes important for the inflammatory response, which also overlap with the regulation of targets associated with cancer promotion. We previously showed that RelA and RelB exert different effects in ovarian cancer cells, with RelB relatively more important for tumor initiation. In this study, we hypothesize that NF-kB signaling regulates the expression of several microRNAs (miRNAs) differentially through RelA and RelB to promote TIC survival and proliferation. miRNAs comprise a subset of small, noncoding RNAs that regulate gene expression and present potential therapeutic targets for clinical use. Inducible shRNA stably was expressed in OV90 EOC cells to knockdown RelA or RelB; cells were subsequently analyzed by miR-seq to identify differentially expressed miRNAs in cells grown in TIC vs adherent (adh) conditions. To validate the miR-seq findings, we performed qPCR assays of the candidate miRNAs differentially expressed in TIC or adh conditions with RelA or RelB knocked down. We confirmed the decreased expression of oncogenic miRNAs hsa-miR-105-5p and hsa-miR-452-5p, while also confirming the increased expression of tumor suppressive miRNA hsa-miR-34a-5p observed in the miR-seq using OV90 cells. Ongoing work will validate these expression changes in additional cell lines and conduct functional assays to characterize the effect of mimicking or inhibiting these miRNAs of interest. The identification of miRNAs differentially expressed in TICs under the control of RelA or RelB will provide key insights in designing treatments against them to prevent disease relapse in patients with EOC.

Citation Format: Rahul D. Kamdar, Brittney S. Harrington, Soumya Korrapati, Nathan Wong, Carrie D. House, Christina M. Annunziata. NF-κB signaling regulates miRNA expression in ovarian 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 5820.
Abstract 5823: Studying the role of microRNAs that target stem cell genes in the emergence of tumor heterogeneity in development of human colorectal cancers
Authors: Stark Victoria A. , Facey Caroline O. B. , Opdenaker Lynn M. , Boman Bruce M.
Published: June 2022

We investigated miRNAs that are implicated to target genes that encode different stem cell markers (SC) during tumorigenesis of colorectal cancer (CRC), one of the leading causes of cancer deaths in the US. One reason behind the lack of efficacy in cancer drug therapeutics is due to the development of tumor heterogeneity. We hypothesize that different miRNAs target different SC genes, which leads to the emergence of multiple cancer SC (CSC) subpopulations and tumor heterogeneity. Flow cytometry helped evaluate the expression of multiple SC markers including ALDH1, CD166, LGR5, and LRIG1 in the HT29 CRC cell line. CSC sub-populations were isolated using fluorescence activated cell sorting (FACS) and the expression of miRNAs in each CSC subpopulation was measured using NanoString profiling. The differential expression of miRNAs between CSC subpopulations helped rank the different levels of miRNA upregulation in each of the CSC subpopulations. The mRNAs predicted to be targeted by the most significantly upregulated miRNAs were identified using bioinformatics analysis via NanoString and mRNAs were classified according to their predicted function by David functional annotation analysis. Altogether, our results indicate that multiple CSC subpopulations exist in the HT29 cell-line, and we identified unique miRNA signatures for each CSC subpopulation. Moreover, the mRNAs predicted to be targeted by the miRNAs in each CSC subpopulation were found to have different functional classifications. We conclude that multiple CSC subpopulations emerge in HT29 cells due to the expression of unique miRNAs that target different SC genes (and their co-expressed genes), resulting in the emergence of multiple CSC subpopulations and tumor heterogeneity.

Citation Format: Victoria A. Stark, Caroline O. B. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the role of microRNAs that target stem cell genes in the emergence of tumor heterogeneity in development of human colorectal 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 5823.
Abstract 5828: Functional role of long non-coding RNA YTHDF3-AS1 in breast cancer
Authors: Bera Alakesh , Radhakrishnan Surya , Russ Eric , Biswas Diya , Subramanian Madhan , Pollard Harvey B. , Hu Hai , Shriver Craig D. , Biswas Roopa , Srivastava Meera
Published: June 2022

Breast cancer (BrCa) is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Importantly, current treatment options for metastatic breast cancer fail to elicit an anti-tumor response, leading to a 5-year survival rate below 30%. While most classes of therapy focus on inhibiting or targeting specific proteins, long non-coding RNAs (lncRNAs) offer a unique, relatively untapped, source of potential therapeutic targets. Ongoing experiments are evaluating the regulatory role of the lncRNA YTHDF3-AS1 (ENSG00000270673), which has the potential to develop into a novel therapeutic intervention of BrCa. Through genome-wide analysis, we found that YTHDF3-AS1 expression correlates with essential developmental regulatory genes such as CA3, VPS28, EXOSC4, and TM2D2 and according to pathway analysis is likely linked with the Wnt pathway. Additionally, TCGA data reveals that over 5% of patients with BrCa have an increased copy number of YTHDF3-AS1. Although the role of YTHDF3-AS1 has yet to be explored, YTHDF3 expression was recently shown to be a critical driver of breast cancer metastasis. Of note, YTHDF3-AS1 is upstream of the protein-coding region, but within the promoter region of the YTHDF3 gene. Mechanistically, this allows the lncRNA YTHDF3-AS1 to potentially upregulate the expression of the protein-coding YTHDF3 gene, as two known functions of antisense lncRNAs describe the ability to 1) induce nearby gene expression through binding to the promoter region and 2) stabilize the RNA product of the nearby gene through binding to the 5’UTR. Interestingly, this manner of relationship between antisense (or "divergent") lncRNAs and their protein-coding partner was shown to be highly prevalent in pluripotent stem cells, with the downregulation of both the antisense and protein-coding transcripts following differentiation. Together, our preliminary analysis and previous literature suggest that the lncRNA YTHDF3-AS1 may contribute to the upregulation of YTHDF3, which is a known metastatic promoting protein. Our current study aims to determine the functional role of YTHDF3-AS1 in BrCa oncogenesis, with an emphasis on the mechanistic actions of this lncRNA for the development of future therapeutic approaches.

Citation Format: Alakesh Bera, Surya Radhakrishnan, Eric Russ, Diya Biswas, Madhan Subramanian, Harvey B. Pollard, Hai Hu, Craig D. Shriver, Roopa Biswas, Meera Srivastava. Functional role of long non-coding RNA YTHDF3-AS1 in breast 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 5828.