Major Subtypes Research Articles

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

Association of Residential Exposure to Hazardous Air Pollutants with Risk of Non-Hodgkin Lymphoma and Multiple Myeloma.

Certain hazardous air pollutants (HAP) are known or suspected to pose immunological or cancer risk to humans, but evidence is limited from the general population. We assessed associations between residential exposure to HAPs at the census tract level and incident non-Hodgkin lymphoma (NHL) and multiple myeloma in the Nurses' Health Study (NHS, 1986-2012) and NHSII (1989-2019). We used the covariate-adjusted proportional hazards model to estimate hazard ratios (HR) of NHL, major NHL subtypes, and multiple myeloma per interquartile range increase in exposure to a given HAP and pooled the cohort-specific estimates using fixed-effects meta-analyses. There were 810 NHL and 158 multiple myeloma cases in NHS (1,700,707 person-years) and 379 NHL and 59 multiple myeloma cases in NHSII (2,820,772 person-years). Most HRs approximated unity. Meta-analyses did not show consistent evidence of associations between any HAP exposure and risk of NHL or multiple myeloma. Exposure to HAPs was not consistently associated with risks of NHL or multiple myeloma in these nationwide prospective cohorts of women. This is the first nationwide study assessing associations between residential HAP exposures and risk of lymphoid malignances in prospective cohorts and focuses on women, who have frequently been underrepresented in (primarily occupational) studies of exposure to HAPs.

Tissue-specific gene expression of genome-wide significant loci associated with major depressive disorder subtypes

Major depressive disorder (MDD) is a clinically and genetically heterogeneous disorder. To reduce heterogeneity, large-scale genome-wide association studies have recently identified genome-wide significant loci associated with seven MDD subtypes. However, it was unclear in which tissues the genes near those loci are specifically expressed. We investigated whether genes related to specific MDD subtypes would be preferably expressed in a specific tissue. At 14 novel subtype-specific loci related to seven MDD subtypes—(1) non-atypical-like features MDD, (2) early-onset MDD, (3) recurrent MDD, (4) MDD with suicidal thoughts, (5) MDD without suicidal thoughts, (6) MDD with moderate impairment, and (7) postpartum depression, we investigated whether 22 genome-wide significant genetic variant-mapped genes were tissue-specifically expressed in brain, female reproductive, male specific, cardiovascular, gastrointestinal, or urinary tissues in the Genotype-Tissue Expression (GTEx) subjects (n ≤ 948). To confirm the tissue-specific expression in the GTEx, we used independent Human Protein Atlas (HPA) RNA-seq subjects (n ≤ 95). Of 22 genes, nine and five genes were tissue-specifically expressed in brain and female reproductive tissues, respectively (p < 2.27 × 10−3). RTN1, ERBB4, and AMIGO1 related to early-onset MDD, recurrent MDD, or MDD with suicidal thoughts were highly expressed in brain tissues (d = 1.19–2.71), while OAS1, LRRC9, DHRS7, PSMA5, SYPL2, and GULP1 related to non-atypical-like features MDD, early-onset MDD, MDD with suicidal thoughts, or postpartum depression were expressed at low levels in brain tissues (d = −0.17−−1.48). DFNA5, CTBP2, PCNX4, SDCCAG8, and GULP1, which are related to early-onset MDD, MDD with moderate impairment, or postpartum depression, were highly expressed in female reproductive tissues (d = 0.80–2.08). Brain and female reproductive tissue-specific expression was confirmed in the HPA RNA-seq subjects. Our findings suggest that brain and female reproductive tissue-specific expression might contribute to the pathogenesis of MDD subtypes.

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1–10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

PPARγ attenuates cellular senescence of alveolar macrophages in asthma-COPD overlap

BackgroundAsthma-chronic obstructive pulmonary disease (COPD) overlap (ACO) represents a complex condition characterized by shared clinical and pathophysiological features of asthma and COPD in older individuals. However, the pathophysiology of ACO remains unexplored. We aimed to identify the major inflammatory cells in ACO, examine senescence within these cells, and elucidate the genes responsible for regulating senescence.MethodsBioinformatic analyses were performed to investigate major cell types and cellular senescence signatures in a public single-cell RNA sequencing (scRNA-Seq) dataset derived from the lung tissues of patients with ACO. Similar analyses were carried out in an independent cohort study Immune Mechanisms Severe Asthma (IMSA), which included bulk RNA-Seq and CyTOF data from bronchoalveolar lavage fluid (BALF) samples.ResultsThe analysis of the scRNA-Seq data revealed that monocytes/ macrophages were the predominant cell type in the lung tissues of ACO patients, constituting more than 50% of the cells analyzed. Lung monocytes/macrophages from patients with ACO exhibited a lower prevalence of senescence as defined by lower enrichment scores of SenMayo and expression levels of cellular senescence markers. Intriguingly, analysis of the IMSA dataset showed similar results in patients with severe asthma. They also exhibited a lower prevalence of senescence, particularly in airway CD206 + macrophages, along with increased cytokine expression (e.g., IL-4, IL-13, and IL-22). Further exploration identified alveolar macrophages as a major subtype of monocytes/macrophages driving cellular senescence in ACO. Differentially expressed genes related to oxidation-reduction, cytokines, and growth factors were implicated in regulating senescence in alveolar macrophages. PPARγ (Peroxisome Proliferator-Activated Receptor Gamma) emerged as one of the predominant regulators modulating the senescent signature of alveolar macrophages in ACO.ConclusionThe findings suggest that senescence in macrophages, particularly alveolar macrophages, plays a crucial role in the pathophysiology of ACO. Furthermore, PPARγ may represent a potential therapeutic target for interventions aimed at modulating senescence-associated processes in ACO.Key words ACO, Asthma, COPD, Macrophages, Senescence, PPARγ.

Evaluation of next-generation sequencing for measurable residual disease monitoring in three major fusion transcript subtypes of B-precursor acute lymphoblastic leukaemia

The use of next-generation sequencing (NGS) for monitoring measurable residual disease (MRD) in acute lymphoblastic leukaemia (ALL) has been gaining traction. This study aimed to investigate the utility of NGS in MRD monitoring for the three major fusion transcript (FT) subtypes of B-precursor ALL (B-ALL). The MRD results for 104 bone marrow samples from 56 patients were analysed through NGS and real time quantitative reverse transcription PCR (RT-qPCR) for the three major FTs: BCR::ABL1, TCF3::PBX1, and ETV6::RUNX1. To validate the NGS approach, NGS-MRD was initially compared with allele-specific oligonucleotide-qPCR-MRD, and the coefficient of determination was good (R2=0.8158). A subsequent comparison of NGS-MRD with FT-MRD yielded a good coefficient of determination (R2=0.7690), but the coefficient varied by subtype. Specifically, the R2 was excellent for TCF3::PBX1 ALL (R2=0.9157), good for ETV6::RUNX1 ALL (R2=0.8606), and subpar for BCR::ABL1 ALL (R2=0.5763). The overall concordance between the two methods was 83.7%, and an excellent concordance rate of 95.8% was achieved for TCF3::PBX1 ALL. Major discordance, which was defined as a >1 log difference between discordant NGS-MRD and FT-MRD, occurred in 6.7% of the samples, with all but one sample being BCR::ABL1 ALL. Among the four non-transplanted patients with BCR::ABL1-MRD (+)/NGS-MRD (-), three did not relapse after long-term follow-up. Our finding indicates that NGS-MRD has a better prognostic impact than RT-qPCR-MRD in ETV6::RUNX1 and BCR::ABL1 ALL, whereas in TCF3::PBX1 ALL, both methods exhibit comparable efficacy.

Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor.

The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.

Sprouty genes regulate activated fibroblasts in mammary epithelial development and breast cancer

Stromal fibroblasts are a major stem cell niche component essential for organ formation and cancer development. Fibroblast heterogeneity, as revealed by recent advances in single-cell techniques, has raised important questions about the origin, differentiation, and function of fibroblast subtypes. In this study, we show in mammary stromal fibroblasts that loss of the receptor tyrosine kinase (RTK) negative feedback regulators encoded by Spry1, Spry2, and Spry4 causes upregulation of signaling in multiple RTK pathways and increased extracellular matrix remodeling, resulting in accelerated epithelial branching. Single-cell transcriptomic analysis demonstrated that increased production of FGF10 due to Sprouty (Spry) loss results from expansion of a functionally distinct subgroup of fibroblasts with the most potent branching-promoting ability. Compared to their three independent lineage precursors, fibroblasts in this subgroup are “activated,” as they are located immediately adjacent to the epithelium that is actively undergoing branching and invasion. Spry genes are downregulated, and activated fibroblasts are expanded, in all three of the major human breast cancer subtypes. Together, our data highlight the regulation of a functional subtype of mammary fibroblasts by Spry genes and their essential role in epithelial morphogenesis and cancer development.

Abstract LB312: Subtype- and race-specific differences in immune landscape of breast cancer

Abstract Tumor mass comprises of not just the tumor cells but a variety of stromal cells. Immune cells residing within the tumor microenvironment modulate the growth and behavior of the tumor cells through a bi-directional crosstalk and affect the outcomes of cancer therapies. Breast cancer (BC) is a highly heterogeneous disease having four major molecular subtypes. Triple-negative breast cancer (TNBC) is the most aggressive subtype and has limited available therapeutic options. Further, it disproportionately affects African American (AA) women, with nearly two times higher incidence as compared to Caucasian American (CA) women. It is associated with an earlier onset and overall higher mortality of BC. Here, we investigated the differences in the immune landscape between TNBC and non-TNBC subtypes and examined if there existed any race-specific patterns. A total of 81 breast cancer samples with a distribution of 55 non-TNBC (28 CA and 27 AA) and 26 TNBC (12 CA and 14 AA) were selected after histopathologic analysis. Tissues were analysis using the NanoString nCounter® pancancer immune panel to determine tissue-specific immune cell populations. We observed a significantly higher presence of tumor-infiltrating lymphocytes in TNBC compared to non-TNBC, with a higher immune score for CD45+ (total lymphocytes), T-cells, B-cells and tumor-associated macrophages. Specifically, among the T-cell population, Treg cells, Th1 cells, and CD8+ cells were higher in TNBC samples. The pathway prediction indicated 'primary immunodeficiency' and 'cancer immunotherapy by PD-1 blockade' pathways being significantly up-regulated in TNBC samples. Overexpression of CTLA4, IDO1, PDL1 and PDL2 as well as Treg marker genes, such as FOXP3 and LAG3, which are involved in immunosuppressive pathways, was observed in TNBC. In race-wise comparisons of non-TNBC and TNBC sample between CA and AA patients, we found that AA non-TNBC tumors had higher Treg cell scores. On the other hand, CA TNBC samples had higher Th1 and CD8+ T-cell scores compared to AA TNBC. The pathway prediction from differentially expressed genes between CA and AA non-TNBC samples showed complement activation pathway to be down-regulated in AA patients, which could be associated with an increased presence of Treg cells. Further, when comparing CA vs AA TNBC samples, we found significant up-regulation of oncogene BMI1 and down-regulation of genes involved in T-cell activation such as CD47, CD44, FOS, and S100A8 in AA TNBC, supporting their more aggressive nature. In conclusion, our data show that TNBC patients have a higher infiltration of immune cells, which remain suppressed due to the presence of Treg cells and PD1 signaling pathway. The presence of higher Treg cells in AA non-TNBC and lower Th1 and CD8+ T-cells in AA TNBC samples suggests an overall immune suppressed microenvironment in AA patients compared to CA patients. Overall, our data suggest that TNBC could be good candidate for immune therapy. Citation Format: Amod Sharma, Kunwar Somesh Vikramdeo, Sarabjeet Kour Sudan, Mohammad Aslam Khan, Mohammad Tahir, James Elliot Carter, Cindy Nelson, Ajay P Singh, Seema Singh. Subtype- and race-specific differences in immune landscape of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB312.

Identification and characterization of novel genetic variants in the first Chinese family of mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis type IIIC (MPS IIIC) is one of inherited lysosomal storage disorders, caused by deficiencies in lysosomal hydrolases degrading acidic mucopolysaccharides. The gene responsible for MPS IIIC is HGSNAT, which encodes an enzyme that catalyses the acetylation of the terminal glucosamine residues of heparan sulfate. So far, few studies have focused on the genetic landscape of MPS IIIC in China, where IIIA and IIIB were the major subtypes. In this study, we utilized whole-exome sequencing (WES) to identify novel compound heterozygous variants in the HGSNAT gene from a Chinese patient with typical MPS IIIC symptoms: c.743G>A; p.Gly248Glu and c.1030C>T; p.Arg344Cys. We performed in silico analysis and experimental validation, which confirmed the deleterious pathogenic nature of both variants, as evidenced by the loss of HGSNAT activity and failure of lysosomal localization. To the best of our knowledge, the MPS IIIC is first confirmed by clinical, biochemical and molecular genetic findings in China. Our study thus expands the spectrum of MPS IIIC pathogenic variants, which is of importance to dissect the pathogenesis and to carry out clinical diagnosis of MPS IIIC. Moreover, this study helps to depict the natural history of Chinese MPS IIIC populations.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Retracted and republished from: "The current state of research on influenza antiviral drug development: drugs in clinical trial and licensed drugs".

Influenza viruses (IVs) threaten global human health due to the high morbidity, infection, and mortality rates. Currently, the influenza drugs recommended by the Food and Drug Administration are oseltamivir, zanamivir, peramivir, and baloxavir marboxil. These recommended antivirals are currently effective for major subtypes of IVs as the compounds target conserved domains in neuraminidase or polymerase acidic (PA) protein. However, this trend may gradually change due to the selection of antiviral drugs and the natural evolution of IVs. Therefore, there is an urgent need to develop drugs related to the treatment of influenza to deal with the next pandemic. Here, we summarized the cutting-edge research in mechanism of action, inhibitory activity, and clinical efficacy of drugs that have been approved and drugs that are still in clinical trials for influenza treatment. We hope this review will provide up-to-date and comprehensive information on influenza antivirals and generate hypotheses for screens and development of new broad-spectrum influenza drugs in the near future.

The contracts word list: Integral vocabulary for reading and writing English contracts

Both Business English as a Lingua Franca (BELF) and Legal English as a Lingua Franca (LELF) users engage with legal contracts regularly. However, contractual language is notoriously complex, due in part to the vocabulary it contains. To help mitigate the challenges related to learning the vocabulary of contracts, this paper introduces a word list of American English contracts called the Contracts Word List (CWL). We describe the compilation of the Corpus of English Business Contracts (CEBC) that represents six major contract types in 48 million words. The extraction of prevalent and specialized words from the CEBC is described, following validation procedures in terms of the word list's reliability and stability. The final word lists contains 684 lemmas along with definitions, parts of speech, illustrative concordance lines, and adjusted frequencies from the full corpus and each major contract sub-type. We provide recommendations to ELF professionals and teachers on using the CWL.

Exploring the causal relationship between Takayasu arteritis and inflammatory bowel disease using Mendelian randomization.

Takayasu arteritis (TA) and inflammatory bowel disease (IBD) are two distinct diseases; however, previous studies have reported many cases of IBD-TA coexistence. Additionally, the incidence of IBD in patients with TA is estimated to be significantly higher than the incidence in the general population. Therefore, the two diseases are anticipated to be linked. Mendelian randomization (MR) analysis assesses whether an exposure might causally affect an outcome by using genetic variants inherited randomly at conception, thereby reducing the impact of confounding and reverse causality. The present study aimed to investigate the potential causal relationship between TA and IBD using MR analysis. Two-sample MR analysis, in which TA and IBD were regarded as the exposure and outcome, respectively, was conducted to investigate whether the two diseases are causally related using the R TwoSampleMR package. Summary GWAS data of TA consisted of 516 Turkish cohorts and 462 controls, and 119 patients and 993 controls of European ancestry. Summary data of IBD was from a sub-study of the International Inflammatory Bowel Disease Genetics Consortium (IIBDGC) that comprised 31,665 cases and 33,977 controls of European ancestry. Additionally, separate MR analyses stratified by the two major subtypes of IBD, Crohn's disease (CD) and ulcerative colitis (UC), were performed. Various statistical tests, including the intercept of MR-Egger regression, funnel plots, Cochran's Q tests, and leave-one-out sensitivity analyses, were employed to assess the presence of heterogeneity and horizontal pleiotropy among single nucleotide polymorphisms (SNPs). In the primary analysis using the inverse-variance weighted (IVW) method, the risk of developing IBD for a patient with TA compared to a non-TA control increased 1.053 times (Odds Ratio (OR) = 1.053, P = 0.065). The MR-Egger method (OR = 1.025, P = 0.470) yielded results consistent with this null finding. However, both the weighted median method (OR = 1.038, P = 0.002) and the weighted mode method (OR = 1.051, P = 0.009) identified a significant harmful causal effect. The MR outcomes from separate subgroup analyses slightly diverged from those of IBD and TA. Specifically, for CD, three methods indicated that TA is a risk factor: IVW estimated the OR as 1.045 (P = 0.032), MR-Egger as 0.997 (P = 0.957), weighed median as 1.028 (P = 0.021), and weighted mode as 1.031 (P = 0.022), respectively. This study represents one of the initial investigations into the potential causal association between TA and IBD. With three MR methods, including the primary IVW approach, indicating a notable effect on TA on CD, our analysis findings offer some indication that TA could be a contributing risk factor for CD.

Circadian rhythm mechanism in the suprachiasmatic nucleus and its relation to the olfactory system.

Animals need sleep, and the suprachiasmatic nucleus, the center of the circadian rhythm, plays an important role in determining the timing of sleep. The main input to the suprachiasmatic nucleus is the retinohypothalamic tract, with additional inputs from the intergeniculate leaflet pathway, the serotonergic afferent from the raphe, and other hypothalamic regions. Within the suprachiasmatic nucleus, two of the major subtypes are vasoactive intestinal polypeptide (VIP)-positive neurons and arginine-vasopressin (AVP)-positive neurons. VIP neurons are important for light entrainment and synchronization of suprachiasmatic nucleus neurons, whereas AVP neurons are important for circadian period determination. Output targets of the suprachiasmatic nucleus include the hypothalamus (subparaventricular zone, paraventricular hypothalamic nucleus, preoptic area, and medial hypothalamus), the thalamus (paraventricular thalamic nuclei), and lateral septum. The suprachiasmatic nucleus also sends information through several brain regions to the pineal gland. The olfactory bulb is thought to be able to generate a circadian rhythm without the suprachiasmatic nucleus. Some reports indicate that circadian rhythms of the olfactory bulb and olfactory cortex exist in the absence of the suprachiasmatic nucleus, but another report claims the influence of the suprachiasmatic nucleus. The regulation of circadian rhythms by sensory inputs other than light stimuli, including olfaction, has not been well studied and further progress is expected.

Artesunate induces ferroptosis by regulating MT1G and has an additive effect with doxorubicin in diffuse large B-cell lymphoma cells

Diffuse Large B-cell lymphoma (DLBCL) is a highly aggressive disease with heterogeneous outcomes and marked variability in the response to chemotherapy. DLBCL comprises two major subtypes: germinal centre B-cell-like (GCB) and activated B-cell-like (ABC). Our study highlights the extensive antitumour activity of artesunate (ART) against both major DLBCL subtypes. Transcriptome analysis suggests the potential involvement of ferroptosis in artesunate-induced cell death. Because of low glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels, along with the accumulation of free iron (Fe2+), artesunate induces the excessive production of reactive oxygen species (ROS), ultimately leading to ferroptosis, a form of cell death driven by phospholipid peroxidation. A putative target of artesunate, metallothionein 1G (MT1G), was selected for further analysis. Subsequent studies revealed that inhibiting MT1G expression in vitro significantly impedes the ferroptosis-promoting activity of artesunate by reducing lipid peroxidation and iron accumulation. We also showed that the combination of artesunate and doxorubicin had a marked additive inhibitory effect on GCB and ABC DLBCL cells. In conclusion, artesunate induces ferroptotic death in GCB and ABC DLBCL cells by attenuating the GPX4/GSH antioxidant defence system and increasing intracellular iron levels, indicating its therapeutic potential for relapsed or refractory DLBCL.

Abstract 4334: Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing

Abstract Cervical cancer is caused by human papillomavirus (HPV) infection and is the most common cause of cancer death in low-resource countries. We characterized 18 cervical and 4 head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, and cancer driver mutations. This cell line panel represents the major molecular subtypes of cervical cancer. Structural variation analysis revealed recurrent chromosomal alteration and telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alteration in cancer that has not been studied using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease deletion of the DNA after breakage. Some BFB events are complex, involving interchromosomal or intrachromosomal insertions or rearrangements. In total, there were three different types of BFB events: Type I, II, and III. Type I BFB events have staggered ends but no additional insertion at the fusion site. Type II BFB events have an insertion of DNA from another chromosome at the junction, and Type III has an insertion of sequences from the same chromosome as the BFB. BFB cycles are thought to resolve with the addition of a telomere. However, a search for telomeric sequences did not identify any on chromosomes impacted by BFB. In many cancers, BFB events co-occur with chromothripsis; however, in our cervical cancers, only 1 out of 13 had chromosomal shattering near the BFB event, indicating that the mechanism of chromosome rearrangement may be different in cervical cancers. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Analysis of 911 publicly available cervical cancer exomes showed that YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer (x2, p&amp;lt;0.0001) and is three times more common in African Americans (x2=12, p=0.003). Therefore, cervical cancer patients with YAP1/BIRC2/BIRC3 amplification, especially those of African American ancestry, might benefit from anti-YAP1/BIRC2/BIRC3 therapies. Our panel provides models for all the major HPV types and subtypes of cervical cancer that will aid in developing new therapeutic approaches. In summary, we uncovered novel insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing. We are expanding long-read sequencing to other cancer cell lines to gain knowledge of events associated with BFB. Citation Format: Isabel Rodriguez, Nicole Rossi, Ayse Gokce Kesus, Yi Xie, Tanveer Ahmad, Asher Bryant, Hong Lou, Jesica Godinez Paredes, Rose Milano, Mikhail Kolmogorov, Sonam Tulsyan, Jazmyn Bess, Vera Mukhina, Daria Gaykalova, Laksh Malik, Kimberley J. Billingsley, Cornelis Blauwendraat, Lisa Mirabello, Michael Dean. Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4334.

Abstract 4738: Overcoming therapy resistance in rhabdomyosarcoma by targeting the PI3K-MAPK pathway

Abstract Rhabdomyosarcoma (RMS) is a devastating pediatric cancer. There has not been any significant change in the treatment options for the last 4 decades, especially for relapsed or advanced disease. There is an urgent need to identify alternative treatment options to improve survival outcomes of RMS patients. RMS is comprised of two major subtypes as defined by the presence or absence of PAX3/PAX7-FOXO1 gene fusion. Fusion-negative RMS (FN RMS) is characterized by alterations in the RAS-PI3K-MAPK pathway in &amp;gt;90% of the cases. However, the role of the PI3K-MAPK pathway in therapy resistance and disease relapse remains poorly characterized. We have generated two FN-RMS lines, RD and SMS-CTR, with resistance to the standard-of-care chemotherapeutic agent, vincristine, through long-term culturing in incrementally increased concentrations of the drug. By RNA sequencing, vincristine-resistant RD and SMS-CTR cells demonstrated increased expression of genes in the RAS-PI3K-MAPK pathway compared to the parental lines. We hypothesize that increased activity of the PI3K-MAPK pathway contributes to therapy resistance in FN RMS. To test the hypothesis, we first showed that targeted disruption of PIK3CA by shRNA inhibited the cell growth of vincristine-resistant FN RMS cells. The same cells also showed increased sensitivity to the MEK inhibitor, trametinib, as well as the PIK3Cα inhibitor, alpelisib, compared to the parental cells. We then showed that zebrafish FN-RMS tumors treated with the combination of vincristine and alpelisib significantly inhibited tumor growth compared to the treatment with each agent alone. Through integrative analysis of RNA sequencing and ATAC-seq data, we identified MYOD1 and NFATC2 as among the top candidate upstream regulators. Targeted disruption of either MYOD1 or NFATC2 by CRISPR/Cas9 reduced cell growth of vincristine-resistant FN RMS cells and altered expression of the genes in the PI3K-MAPK pathway, implicating MYOD1 and NFATC2 in driving abnormal activity of this pathway. To assess the relevance of our findings in the clinical settings, we showed that selective components in the PI3K-MAPK pathways are up-regulated in 3 matched primary-recurrence/metastasis paraffinized tissue samples of human FN RMS by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Our findings indicate that targeting the PI3K-MAPK pathway is a promising targeted therapy option for overcoming therapy resistance in RMS. Citation Format: Yadong Wang, Eleanor Chen. Overcoming therapy resistance in rhabdomyosarcoma by targeting the PI3K-MAPK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4738.

Abstract 3499: Non-small cell lung cancer (NSCLC) histology classification using DNA methylation data captured from liquid biopsies

Abstract Background: Non-small cell lung cancer (NSCLC) makes up ~85% of lung cancers, and its major subtypes are adenocarcinoma (AD) and squamous cell carcinoma (SC). The histology classification of NSCLC has significant therapeutic and prognostic implications. As liquid biopsies are rapid and non-invasive, computational methods to classify NSCLC subtypes using cell-free DNA (cfDNA) are desired. Methods: Using The Cancer Genome Atlas (TCGA) NSCLC methylation data, we developed a method to classify AD and SC subtypes from cfDNA samples. We mapped TCGA methylation data to Guardant Infinity’s promoter regions and transformed them to allow for the transfer of the TCGA trained model to Infinity cfDNA epigenomic data. An advantage of this approach is that TCGA training data originates from the primary tumor, while cfDNA samples have low tumor fraction (TF) and may also contain signals from metastatic tumors. We trained a penalized Logistic Regression model with LASSO. To better fit the model to Infinity cfDNA data, we performed region filtering. Results: Our model effectively classified AD and SC subtypes from TCGA samples (AUC 0.97) (10-fold cross validation; Table 1). When evaluating on cfDNA samples with epigenomic TF ≥ 1%, the final model shows reasonable performance (AUC 0.94) (Table 1); the performance decreases when reduced to TF &amp;gt; 0.05% (AUC 0.89) as expected (Table 1). When evaluating cfDNA from uncategorized lung samples, the predicted AD probabilities have significant association with KRAS mutations (p-value = 0.027). Conclusion: Our results show that the penalized Logistic Regression model trained with tissue TCGA methylation data through mapping, transformation, and region selection can classify NSCLC subtypes from cfDNA samples. This method may offer a fast, non-invasive tool to inform subtype-based clinical decisions, especially in the setting of poorly-differentiated carcinomas, and to identify subtype switches upon resistance to targeted therapies. Table 1: Model evaluation on tissue TCGA samples and cfDNA Infinity samples (different TF cutoffs) Sample type N samples AUC Accuracy Sensitivity Specificity Precision F1 score tissue TCGA 907 0.97 0.93 0.94 +/- 0.02 0.93 +/- 0.02 0.94 +/- 0.02 0.94 +/- 0.01 cfDNA Infinity 17 (TF ≥ 1%) 0.94 0.88 0.83 +/- 0.3 0.91 +/- 0.17 0.83 +/- 0.3 0.83 +/- 0.21 cfDNA Infinity 29 (TF &amp;gt; 0.05%) 0.89 0.83 0.88 +/- 0.23 0.81 +/- 0.17 0.64 +/- 0.28 0.74 +/- 0.2 Citation Format: Laura Tung, Denis Tolkunov, Catalin Barbacioru, Deepika Kale, Scott Shell, Jill Tsai, Leylah Drusbosky, Kaushal Parikh, Han-Yu Chuang. Non-small cell lung cancer (NSCLC) histology classification using DNA methylation data captured from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3499.

Abstract 282: SFRP4+ cancer associated fibroblasts activate epithelial to mesenchymal transitions in malignant cells and dictate adverse clinical outcomes in stomach cancer

Abstract Cancer-associated fibroblasts (CAFs), a pivotal component in the tumor microenvironment (TME) of gastric cancer (GC), play a crucial role in tumor progression and resistance to treatment. In this study, we performed analyses to build a comprehensive cellular landscape of fibroblasts in GC EMT using three single-cell RNA cohorts including ours (GSE183904, GSE134520 and GSE167297). We observed distinct fibroblast subtypes (CXCL14+/POSTN+, CXCL14+/APOE+, SFRP4+, and SFRP2+ fibroblasts). Among the subtypes, we observed that SFRP4+ fibroblasts sharing cellular origins with normal tissue-residing SFRP2+ myofibroblasts. Notably, SFRP4+ fibroblasts showed a regional prediction for tumor and deep tumor layers compared to normal and superficial tumor layers, respectively. To further understand the role of these fibroblasts, we employed spatial transcriptome data. Spatially resolving the cellular interactions within the TME, the adjacency of SFRP4+ CAFs to malignant cells undergoing epithelial-to-mesenchymal transition (EMT), was observed. Ligand-receptor analyses demonstrated SFRP4+CAFs regulate key genes of EMT in malignant cells (TGFBI, COL1A1, SPARC, COL3A1, COL1A2, and BGN). The regulatory roles of SFRP4+ CAFs in driving the epithelial-mesenchymal transition (EMT) of malignant cells were further validated in vitro. Clinical relevance was also demonstrated with the correlation between the abundance of SFRP4+ CAF and clinical outcomes, i.e., high SFRP4+CAF abundance was consistently associated with shorter survival of GC patients across cohorts. Thus, we present SFRP4+ CAFs as major fibroblast subtypes in GC TME. Spatial cellular architectures and in vitro evidence support that SFRP4+fibroblasts activate the EMT of malignant cells along with their association with patient prognosis. This study advances our understanding into the mechanistic roles of SFRP4+ fibroblasts in GC pathogenesis and progression, paving the way for potential targeted therapeutic strategies. Citation Format: Seungho Lee, Austin Cho, Hyoung-Il Kim, Hoon Hur, Jae-Ho Cheong, Tae-Min Kim. SFRP4+ cancer associated fibroblasts activate epithelial to mesenchymal transitions in malignant cells and dictate adverse clinical outcomes in stomach cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 282.