Citations

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Characterization of the recurrent genetic alterations in PDAC will yield improved understanding and therapies for this disease. Here, we report that PDAC patients with elevated expression of METTL16, one of the “writers” of RNA N6-methyladenosine (m6A) modification, may benefits from poly (ADP-ribose) polymerase (PARP) inhibitor treatment. Mechanistically, METTL16 interacts with MRE11 in an RNA-dependent manner; and, this interaction inhibits MRE11’s exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 at Ser419 within its C terminus, resulting in METTL16 conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression levels show increased sensitivity to PARP inhibitors, especially when combined with gemcitabine. Thus, our findings have revealed a role for METTL16 in homologous recombination repair and suggest that combination of PARP inhibitors with gemcitabine could be an effective treatment strategy for PDAC patients with high METTL16 expression.

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO 3 − ) in extracellular solutions is required for recording normal slow wave activity. HCO 3 − transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na + / HCO 3 − cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract. NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4 fl/fl mice were bred with ETV1 CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding Kit creERT2 with Rpl22 tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal Kit copGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion. NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice. NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the I P 3 R b inding protein released with i nositol 1, 4, 5‐ t risphosphate (IRBIT). Support or Funding Information Supported by NIH R01 DK57061. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Paraganglioma (PGL) is a rare form of neuroendocrine cancer for which the underlying molecular basis is poorly understood. A mysterious hallmark of many PGL tumors is the inactivation of succinate dehydrogenase (SDH), a mitochondrial enzyme of the TCA cycle. Why does SDH loss induce cancer in certain cells? As a result of SDH loss, PGL tumor cells accumulate succinate, which may have epigenetic consequences. For example, certain lysine residues on histone proteins in nucleosomes are subject to succinylation by succinyl‐CoA as a post translational modification (PTM) that converts the lysine cation to an anion. We hypothesize that the structure and stability of the nucleosome core particle (NCP) is affected by succinylation PTMs through weakening of electrostatic histone‐DNA and histone‐histone interactions. Proteomic analysis of an SDH‐loss cell line model of PGL previously demonstrated that the greatest increase in nucleosomal lysine succinylation maps to H4K79 when compared to a control cell line. The mammalian H4K79 residue lies within the nucleosome core near wrapped DNA. We specifically hypothesize that the charge inversion conferred by H4K79 succinylation, destabilizes the NCP. Point mutations at H4K79 have been generated as lysine succinylation mimics at this site. Biophysical and in vitro transcription assays are being utilized to validate the assembly of NCPs incorporating lysine succinylation mimics at this site and to interrogate their resulting stabilities. Analyses of alterations in NCP dynamics in variant NCP will help to elucidate the role of lysine succinylation of histones in the altered chromatin landscape observed during PGL oncogenesis. Support or Funding Information GM75148‐09‐10 PREP This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Abstract SET and MYND domain protein 2 (SMYD2) is a lysine methyltransferase that mediates histone H3 lysine 36 trimethylation (H3K36me3) and acts as a regulator of tumorgenesis and cystic growth. However, its role in renal fibrosis remains unknown. In this study, we found that SMYD2 was highly expressed in the murine kidney of renal fibrosis induced by unilateral ureteral obstruction, and primarily located in interstitial fibroblasts and renal tubular epithelial cells. Pharmacological inhibition of SMYD2 with AZ505, a highly selective inhibitor of SMYD2, protected against renal fibrosis and inhibited activation/proliferation of renal interstitial fibroblasts and conversion of epithelial cells to a profibrotic phenotype in this model. In cultured renal interstitial fibroblasts, treatment with AZ505 or silencing of SMYD2 by specific siRNA also inhibited serum‐ or TGF‐β1‐induced activation and proliferation of renal interstitial fibroblasts. Mechanistic studies showed that SMYD2 inhibition reduced phosphorylation of several profibrotic signaling molecules, including Smad3, extracellular signal‐regulated kinase 1/2, AKT, signal transducer and activator of transcription‐3 and nuclear factor‐κB in both injured kidney and cultured renal fibroblasts. AZ505 was also effective in suppressing renal expression of Snail and Twist, two transcriptional factors that mediate renal partial epithelial‐mesenchymal transition and fibrosis. Conversely, AZ505 treatment prevented downregulation of Smad7, a renoprotective factor in vivo and in vitro. These results indicate that SMYD2 plays a critical role in mediating conversion of epithelial cells to a profibrotic phenotype, renal fibroblast activation and renal fibrogenesis, and suggest that SMYD2 may be a potential target for the treatment of chronic fibrosis in kidney disease.

Introduction: There are many treatment options for patients who have osteonecrosis of the femoral head (ONFH) and management strategies vary widely both among and within individual countries. Although many researchers have attempted to elucidate the optimal strategies for managing this disease, the lack of large-scale randomized control trials and the lack of agreement on disease staging have curtailed the development of clear-cut guidelines. Materials and Methods: The Association Research Circulation Osseous (ARCO) group sought to address three questions for the management of patients who have ONFH: 1) What imaging studies are most sensitive and specific for the diagnostic evaluation of patients who have ONFH?; 2) What is the best treatment strategy for preventing disease progression in patients who have pre-collapse lesions?; and 3) What is the best treatment strategy for patients who have post-collapse disease? The Patient, Intervention, Comparison, and Outcome (PICO) format was used to formulate the search strategy for each research question. A systematic review will be performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. ARCO participants have been allocated to three groups, each representing one of the PICO questions. After qualitative and quantitative analysis of the data extracted from studies pertaining to each of the three research questions, a set of evidence-based clinical practice guidelines will be proposed for the management of patients who have ONFH. Discussion: It is not always clear which treatment method is optimal for the management of ONFH. Thus, many surgeons have developed and performed various procedures based on patient-specific factors. As there is no consensus on the optimal treatment for various stages of disease, it was clear that developing evidence-based clinical practice guidelines would provide more structure and uniformity to management of these patients. Therefore, the results of this systematic review will lead to the development guidelines that may improve patient-care strategies and result in better outcomes for patients who have ONFH.

Abstract Non‐small cell lung cancer patients with anaplastic lymphoma kinase or c‐ros oncogene 1 mutations who are treated with the tyrosine kinase inhibitor crizotinib rarely develop crizotinib‐associated renal cysts (CARCs). Here, we present a case report and review of the literature supporting the hypothesis that CARCs may correlate positively with progression‐free survival.

Abstract Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3′-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. Significance: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR–tyrosine kinase inhibitor resistant-related MET amplification. This article is highlighted in the In This Issue feature, p. 2659

AbstractFerroptosis is a newly identified cell death mechanism and potential biomarker for hepatocellular carcinoma (HCC) therapy; however, its clinical relevance and underlying mechanism remain unclear. In this study, transcriptome and methylome data from 374 HCC cases were investigated for 41 ferroptosis‐related genes to identify ferroptosis activity‐associated subtypes. These subtypes were further investigated for associations with clinical and pathological variables, gene mutation landscapes, deregulated pathways and tumour microenvironmental immunity. A gene expression signature and predictive model were developed and validated using an additional 232 HCC cases from another independent cohort. Two distinct ferroptosis phenotypes (Ferroptosis‐H and Ferroptosis‐L) were identified according to ferroptosis gene expression and methylation in the patients with HCC. Patients with the Ferroptosis‐H had worse overall and disease‐specific survival, and the molecular subtypes were significantly associated with different clinical characteristics, mRNA expression patterns, tumour mutation profiles and microenvironmental immune status. Furthermore, a 15‐gene ferroptosis‐related prognostic model (FPM) for HCC was developed and validated which demonstrated accurate risk stratification ability. A nomogram included the FPM risk score, ECOG PS and hepatitis B status was developed for eventual clinical translation. Our results suggest that HCC subtypes defined by ferroptosis gene expression and methylation may be used to stratify patients for clinical decision‐making.

Abstract Purpose: We recently reported the development of a cell-free DNA (cfDNA) targeted methylation (TM)-based sequencing approach for a multi-cancer early detection (MCED) test that includes cancer signal origin prediction. Here, we evaluated the prognostic significance of cancer detection by the MCED test using longitudinal follow-up data. Experimental Design: As part of a Circulating Cell-free Genome Atlas (CCGA) substudy, plasma cfDNA samples were sequenced using a TM approach, and machine learning classifiers predicted cancer status and cancer signal origin. Overall survival (OS) of cancer participants in the first 3 years of follow-up was evaluated in relation to cancer detection by the MCED test and clinical characteristics. Results: Cancers not detected by the MCED test had significantly better OS (P < 0.0001) than cancers detected, even after accounting for other covariates, including clinical stage and method of clinical diagnosis (i.e., standard-of-care screening or clinical presentation with signs/symptoms). Additionally, cancers not detected by the MCED test had better OS than was expected when data were adjusted for age, stage, and cancer type from the Surveillance, Epidemiology, and End Results (SEER) program. In cancers with current screening options, the MCED test also differentiated more aggressive cancers from less aggressive cancers (P < 0.0001). Conclusions: Cancer detection by the MCED test was prognostic beyond clinical stage and method of diagnosis. Cancers not detected by the MCED test had better prognosis than cancers detected and SEER-based expected survival. Cancer detection and prognosis may be linked by the underlying biological factor of tumor fraction in cfDNA.

12102 Background: Allostatic load (AL) conceptualizes the effects of chronic psychosocial adversity on physiologic dysregulation. To date, studies have shown an association between elevated AL and higher disease-specific and overall mortality among cancer patients; however, none have focused on multiple myeloma (MM) patients. We aim to understand the relationship between baseline AL, symptom burden, and mortality among patients enrolled in the E1A11 therapeutic trial in MM. Methods: ECOG-ACRIN E1A11 was a phase III RCT comparing induction with Bortezomib (Arm A) versus Carfilzomib (Arm B) in conjunction with Lenalidomide +Dexamethasone. AL included 7 biomarkers: BMI, alkaline phosphatase, creatinine, C-reactive protein, white blood cell count, albumin and creatinine clearance. AL7 was a composite summary score with a point was assigned for each biomarker value in the highest quartile, except for albumin and creatinine clearance, where a point was assigned for values in the lowest quartile. Endpoints included symptom burden at baseline and ̃1 month, non-completion of induction therapy, and overall survival (OS). Functional Assessment of Cancer Therapy Multiple Myeloma (FACT-MM) items assessed patient-reported symptom burden, including fatigue (item HI7), pain (GP4), and bother by side effects of treatment (GP5) on a 5-point Likert scale. Multivariable logistic regressions assessed the effect of AL7 (ranging 0-7) on high-pain, -fatigue, and -bother (QOL score > = 3 vs < 3), and non-completion of induction therapy. The effect of AL7 on OS was assessed using multivariable Cox regression. Regression covariates included study arm, age, sex, race, ECOG performance status, and the target symptom burden score at baseline. Results: The study cohort included 1087 patients. Mean baseline AL7 was 1.8 (±1.4). In adjusted analysis, a unit increase in AL7 was associated with a greater odds of high pain (OR 1.15, 95%CI [1.04-1.27]) and high fatigue (OR 1.19, 95%CI [1.07-1.32]) at baseline, which did not persist at ̃1 month (pain OR 0.96, 95%CI [0.84-1.10]; fatigue OR 1.03, 95%CI [0.91-1.16]). There was no association between AL7 and high side effect bother at baseline (OR 1.06, 95% CI [0.83-1.35]) or at ̃1 month (OR 1.06, 95%CI [0.90-1.24]). There was no association between AL7 and induction non-completion (OR 1.07, 95%CI [0.96-1.18]). Notably, each unit increase in AL7 was associated with higher mortality (HR 1.26, 95%CI [1.14-1.39]). Conclusions: Despite its association with fatigue and pain at baseline, AL7 was not associated with these symptoms at ̃1 month nor induction non-completion. However, elevated baseline AL7 was associated with poorer OS. AL composite score at baseline, which we interpret as a measure of physiological dysregulation associated with adverse social factors, may have implications on clinical outcomes within clinical trials despite presumed equal treatment access.