Nat Commun Research Articles

Noise resistance of next-generation reservoir computing: a comparative study with high-order correlation computation

Reservoir computing (RC) methods have received more and more attention and applications in chaotic time series prediction with their simple structure and training method. Recently, the next-generation reservoir computing (NG-RC) method has been proposed by Gauthier et al. (Nat Commun 12:5564, 2021) with less training cost and better time series predictions. Nevertheless, in practice, available data on dynamic systems are contaminated with noise. Though NG-RC is shown highly efficient in learning and predicting, its noise resistance captivity is not clear yet, limiting its use in practical problems. In this paper, we study the noise resistance of the NG-RC method, taking the well-known denoising method, the high-order correlation computation (HOCC) method, as a reference. Both methods have similar procedures in respect of function bases and regression processes. With the simple ridge regression method, the NG-RC method has a strong noise resistance for white noise, even better than the HOCC method. Besides, the NG-RC method also shows a good prediction ability for small colored noise, while it does not provide correct reconstruct dynamics. In this paper, other than reconstruction parameters, four numerical indicators are used to check the noise resistance comprehensively, such as the training error, prediction error, prediction time, and auto-correlation prediction error, for both the short-time series and long climate predictions. Our results provide a systematic estimation of NG-RC’s noise resistance capacity, which is helpful for its applications in practical problems.

Organ Preservation in the 2020s: XVIVO Heart Box (Part 2)

The XVIVO Heart Box (XHB) is an emerging technology in cardiac transplantation that protects the donor heart via non-ischaemic heart preservation (NIHP). A first-in-human trial utilising XHB demonstrated its safe use in human cardiac transplantation [ [1] Nilsson J, Jernryd V, Qin G, Paskevicius A, Metzsch C, Sjöberg T, et al. A nonrandomized open-label phase 2 trial of nonischemic heart preservation for human heart transplantation. Nat Commun. 2020;11:2976. Google Scholar ]. In response, an Australian and New Zealand multicentre trial of extended (6–8 hours) NIHP of donor hearts for transplantation utilising XHB is underway. The current study is an open-labelled, single-arm, multicentre clinical trial. It aims to investigate the effect of NIHP on donor hearts with extended ischaemic time (6–8 hours) with respect to immediate and long-term post-transplant heart allograft function.

Multi‐omics characterization of brain‐based pseudotime estimates for Alzheimer’s disease progression

AbstractBackgroundThe cascade of molecular changes that leads to Alzheimer’s disease (AD) onset and progression remains incompletely understood. Recently, researchers have utilized manifold learning techniques to construct pseudo‐temporal models of the disease using brain transcriptomics data and quantify the progression of individuals along this trajectory using pseudotime (Mukherjee et al. Nat Commun 2020;11:5781). Downstream analyses provided insights into potentially disease‐driving pathways, including links to mitochondrial dysfunction. Here, we embedded these models into a multi‐omics context to enable a more comprehensive molecular characterization of disease progression.MethodThe AD Atlas (https://adatlas.org/) integrates multi‐scale molecular data from different studies and cohorts, including omics QTLs, correlation networks, differential expression data, as well as omics associations with AD and endophenotypes. Here, we used the AD Atlas to annotate metabolites that were significantly associated with brain‐based pseudotime estimates. Metabolite (n = 667) levels were measured in brain tissue samples (dorsolateral prefrontal cortex) from 154 female ROS/MAP participants using untargeted metabolomics and tested for association using linear regression. We used the resulting set of significant metabolites as input for the AD Atlas to extract a multi‐omics context network augmented with associations to AD. Subsequently, we applied pathway enrichment analysis to derive overrepresented biological processes potentially involved in disease progression.ResultIn total, 89 of the 667 metabolites showed a significant association with pseudotime after Bonferroni adjustment, 34 of which could be mapped to the AD Atlas database. The resulting multi‐omics network contained a total of 619 genes, 197 metabolites and links to 12 AD‐related phenotypes, including CSF amyloid pathology, brain glucose uptake measured by FDG‐PET and cognitive measures. Five of the 34 metabolites and nearly one‐third of the genes contained in the network showed significant associations with AD, with transcriptional changes most pronounced in the temporal cortex (n = 193). Enrichment analysis revealed functional links to neurotransmission and bioenergetics, pathways previously implicated in the pathogenesis of AD.ConclusionBy using metabolites as proxies for transcriptome‐derived pseudotime, we were able to investigate the molecular underpinnings of AD progression in a multi‐omics context. Our analysis provides further molecular evidence for pathways implicated in AD and emphasizes the potential of such an approach for future studies.

Novel RNA epigenetics-driven technologies for rapid prediction of cancer drug resistance.

e19001 Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells ( Statello, et al. Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study ( Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM & FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.

Exploring the prognostic and predictive impact of genomic loss of heterozygosity (LOH) in patients with metastatic colorectal cancer (mCRC).

3576 Background: Genomic LOH consists in the loss of chromosomal regions and is associated with the homologous recombination repair (HRR) system deficiency (dHRR). In ovarian cancer, LOH-high predicts benefit from platinum-based chemotherapy and PARP inhibitors. In mCRC, the role of LOH has been poorly investigated. Methods: An NGS-based assay (FoundationOne CDx; Foundation Medicine, Inc. Cambrige, MA) was used to determine the percentage of genomic LOH and the presence of pathogenetic mutations in HRR-related genes in archival chemo-naïve tumor tissues of mCRC patients included in a real-world registry and in the AtezoTRIBE (NCT03721653) and AVETRIC (NCT04513951) studies. Both these trials assessed the combination of an anti-PDL1 (atezolizumab or avelumab) with the triplet FOLFOXIRI plus bevacizumab or cetuximab. The prespecified cut-off of ≥14.08 for LOH-high described for mCRC (Sokol et al., JCO Precis Oncol 2020) was adopted. Tumors with at least one biallelic alteration in any of the 27 genes involved in the HRR pathway (Riaz et al., Nat Commun 2017) included in the FoundationOne CDx panel (BARD1, BRCA1, BRCA2, BRIP1, MRE11A, NBN, PALB2, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RBBP8, XRCC2, ABL1, ATM, ATR, BAP1, CDK12, DNMT3A, ERCC4, FANCA, FANCC, FANCG, FANCL, PARP1) were defined dHRR. Results: Overall, 196 samples were analysed. None of 7 MSI-H tumors were classified as LOH-high. Fourteen (7%) and 6 (3%) of 189 MSS tumors were classified as LOH-high and dHRR, respectively. In particular, in LOH-high subgroup, 3 (21%) tumors were defined dHRR, while 3 (50%) tumors were classified LOH-high among dHRR tumors. LOH-high tumors were more frequently BRAF mutated (p = 0.019) and dHRR (p = 0.006) compared to LOH-low. Among patients receiving triplet chemotherapy +/- biologic agent alone (N = 55) or with an anti-PDL1 (N = 58), an interaction effect was shown between the effect of the addition of the checkpoint inhibitor and LOH with higher benefit in the LOH-high subgroup (N = 10) in terms of both PFS (pinteraction= 0.002) and OS (pinteraction< 0.001). In the cohort of patients not receiving the anti-PDL1, longer PFS was observed in patients with LOH-low (N = 125) respect to LOH-high (N = 6) tumors (12.1 vs 5.1 months, HR: 0.11, 95%CI: 0.04-0.26, p < 0.001). No differences in PFS or OS were reported between patients treated with first-line oxaliplatin- (N = 40) vs irinotecan-based doublets (N = 25) or with the triplet FOLFOXIRI (N = 55) vs doublets (N = 65) according to LOH status. No prognostic or predictive impact of HRR deficiency was shown. Conclusions: In MSS mCRC, LOH-high was associated with biallelic alterations in the HRR system, worse prognosis and higher benefit from the addition of anti-PDL1 agents to chemotherapy. Considering the low number of LOH-high tumors in our study, these results deserve confirmation in larger cohorts.

Guidance on Therapeutic Strategies for Metastatic Urothelial Cancer

Guidance on Therapeutic Strategies for Metastatic Urothelial Cancer

Abstract 424: Arachidonic Acid Synergizes With Aspirin In Cardioprotection Following Acute Myocardial Infarction

Introduction: Membrane-released arachidonic acid (AA) is metabolized via cyclooxygenase pathway to form prostanoids, including thromboxane, prostacyclin and prostaglandin E2. Aspirin suppresses platelet synthesis of thromboxane, rendering its first-line use for anti-thrombosis in patients with acute myocardial ischemia. We report that endogenous PGE2 protects against myocardial ischemia-reperfusion (MI/R) injury (Nat Commun. 2019;10(1):1888). The impact of AA administration while suppressing thromboxane on MI/R injury is unknown. Methods and Results: C57BL/6J mice were treated intravenously with vehicle, AA, aspirin, or their combination and then subjected to 30min coronary artery ligation and 24hr reperfusion. Cardiac function was assessed by ultrasonography and infraction size quantified by TTC staining. Cardiac microcirculation was assessed by laser doppler flow and vasomotor reactivity determined with isolated coronary arteries. Single treatment with AA at an optimized dose of 0.1mg/Kg, or combined with aspirin at a dose of 10mg/Kg that allowed sufficient suppressing of platelet activation over 24hr, decreased infarct size and improved heart function. Impressively, combination of AA with aspirin provided additional cardioprotection compared with either single treatment. While aspirin, not AA, ameliorated microvascular obstruction following MI/R injury, co-administration of AA and aspirin further increased coronary microcirculatory blood perfusion with improved vascular permeability compared with aspirin alone. MI/R impaired endothelium-dependent dilatation of coronary arteries, and the dual treatment, not AA or ASA alone, increased coronary artery relaxation. Cardiac infiltrated neutrophils and platelet-neutrophil aggregates were also reduced by the combination treatment, consistent with a suppressed leukocyte-ECs adhesion as assessed in vitro. Conclusions: AA and aspirin synergistically protect against MI/R injury and this is attributable to suppressed neutrophil inflammation and improved microvascular obstruction. AA might be used as an adjunct therapy to aspirin in patients with acute myocardial infarction.

Abstract A52: IDS and SETBP1 is highly prognostic in myelodysplastic neoplasms and is a candidate stem cell signature

Abstract Myelodysplastic neoplasms (MDS) are heterogenous blood disorders that arise from dysfunctional hematopoietic stem cells (HSCs) and progenitor cells (HSPCs). According to the cancer stem cell (CSC) model, MDS is organized as a cellular hierarchy that arises from the malignant transformation of HSPCs into rare CSCs. MDS-CSCs are thought to persist during treatment and regenerate disease during relapse. Prior studies have linked MDS-CSCs to MDS-HSPC (Woll et al. Cancer Cell 2014, Pang et al, PNAS 2013, Will et al, Blood 2012), however, a specific cell type has not been isolated and purified as the MDS-CSC. Prognostic gene expression signatures in MDS have also been linked to immature HSPCs (Shiozawa et al, Blood 2017), however, a cell specific signature has not been identified. There is a need to characterize a cell specific gene signature for MDS-CSCs in order to study these cells. To address this need, we performed iterative statistical analyses on MDS gene expression data in order to identify a candidate CSC signature. We hypothesized that by analyzing genes specifically up-regulated in MDS-HSPCs, we can derive a CSC specific gene signature that is not only associated with poor outcomes in MDS, but also marks a subset of cells in MDS with stem cell programs. Up-regulated genes in MDS-HSPCs compared to healthy controls were derived by re-analyzing 73 sorted samples (Woll et al, Cancer Cell 2014) using the limma (Ritchie et al, Nucleic Acids Res 2015). Using these genes, we subsequently analyzed their association with survival in a cohort of 244 MDS patients (Shiozawa et al, Blood 2017, Gerstung et al, Nat Commun 2015, Tyner et al, Nature 2018). We performed iterative Cox proportional hazard models on a training data (n=146), using single and multiple gene combinations. A 2 gene score (i.e., MDS2), comprising IDS and SETBP1, was identified as the most significantly associated feature with decreased survival in MDS compared to Age, Sex, Cytogenetic Risk, and an established MDS score (Shiozawa et al, Blood 2017). Single cell expression of MDS2 was evaluated in MDS scRNA-seq samples (Dussiau et al, BMC Biol2022), and rare cells were identified expressing high levels of MDS2, i.e., MDS2 cells. These cells were integrated with healthy HSPCs. MDS2 cells reside primarily between HSCs and MPPs on a diffusion map, following a differentiation trajectory towards GMPs and monocyte precursors. These observations are consistent with prior studies, as MDS-CSCs were shown to be enriched in HSCs and GMPs (Pang et al, PNAS 2013, Woll et al, Cancer Cell 2014). Analysis of upregulated genes in MDS2 cells revealed that antigen processing, assembly and presentation were the most enriched processes. This analysis supports our approach for identifying a cell specific gene signature, and future work will focus on further single cell analyses and evaluation of CSC content and function of MDS2 cells. Citation Format: Asiri Ediriwickrema, Andrew Gentles, Ravindra Majeti. IDS and SETBP1 is highly prognostic in myelodysplastic neoplasms and is a candidate stem cell signature [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A52.

Spatial cumulant models enable spatially informed treatment strategies and analysis of local interactions in cancer systems

Theoretical and applied cancer studies that use individual-based models (IBMs) have been limited by the lack of a mathematical formulation that enables rigorous analysis of these models. However, spatial cumulant models (SCMs), which have arisen from theoretical ecology, describe population dynamics generated by a specific family of IBMs, namely spatio-temporal point processes (STPPs). SCMs are spatially resolved population models formulated by a system of differential equations that approximate the dynamics of two STPP-generated summary statistics: first-order spatial cumulants (densities), and second-order spatial cumulants (spatial covariances). We exemplify how SCMs can be used in mathematical oncology by modelling theoretical cancer cell populations comprising interacting growth factor-producing and non-producing cells. To formulate model equations, we use computational tools that enable the generation of STPPs, SCMs and mean-field population models (MFPMs) from user-defined model descriptions (Cornell et al. Nat Commun 10:4716, 2019). To calculate and compare STPP, SCM and MFPM-generated summary statistics, we develop an application-agnostic computational pipeline. Our results demonstrate that SCMs can capture STPP-generated population density dynamics, even when MFPMs fail to do so. From both MFPM and SCM equations, we derive treatment-induced death rates required to achieve non-growing cell populations. When testing these treatment strategies in STPP-generated cell populations, our results demonstrate that SCM-informed strategies outperform MFPM-informed strategies in terms of inhibiting population growths. We thus demonstrate that SCMs provide a new framework in which to study cell-cell interactions, and can be used to describe and perturb STPP-generated cell population dynamics. We, therefore, argue that SCMs can be used to increase IBMs’ applicability in cancer research.

Abstract 295: Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer

Abstract Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC). Our recent work demonstrates that spermine synthase (SMS), a polyamine biosynthetic enzyme that converts spermidine to spermine, is overexpressed in CRC, which is required for balancing cellular spermidine levels to facilitate CRC tumorigenesis (Nat Commun 11:3243, 2020). Our findings reveal SMS as an attractive therapeutic target in CRC; yet, genetic depletion of SMS expression only shows a limited antitumor effect. Using unbiased metabolomics and transcriptomics analyses, we identified a lipid metabolism reprograming as among the most impacted metabolic change by SMS depletion in CRC cells. Specifically, SMS depletion significantly altered long-chain fatty acid, triacylglycerol and phospholipid metabolism. Furthermore, targeted inhibition of SMS significantly increased the number of lipid droplets and the levels of long-chain fatty acid acylcarnitines for oxidative phosphorylation in mitochondria, and upregulated expression of genes associated with increased mobilization of polyunsaturated fatty acids and the genes associated with lipid peroxidation for induction of ferroptosis, an iron-dependent form of nonapoptotic cell death. The glutathione peroxidase 4 (GPX4) is a key negative regulator of ferroptosis by neutralizing lipid peroxides. Notably, pharmacological inhibition of GPX4 or its upstream regulator system xc − in combination with genetic depletion or pharmacologic inhibition of SMS synergistically caused lipid peroxidation leading to ferroptosis induction and marked suppression of CRC cell growth. Collectively, these findings highlight lipid metabolism reprograming as an adaptive response to targeted inhibition of SMS to enable CRC cell survival, which represents an Achilles’ heel that can be exploited for potential effective therapy for CRC. Citation Format: Murong Ma, Yubin Guo, Qing Ye, Pan Deng, Daheng He, Chi Wang, Qing-Bai She. Targeting spermine synthase triggers lipid metabolism reprogramming as a new therapeutic option to combat colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 295.

Abstract 3580: Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245

Abstract SAR444245 (formerly THOR-707) is a site-specific pegylated recombinant human IL-2 molecule which blocks IL-2R alpha-binding while retains near-native affinity for beta/gamma IL-2 receptor subunits. SAR444245 reduces B16-F10 tumor proliferation in C57BL/6 mice and induces a short transient migration of CD8+ T and NK cells from peripheral blood to lymphoid organs before a strong proliferation (Ptacin et al, Nat Commun 2021). Here we report CD8+ T cells imaging post SAR444245 as monotherapy in naive and tumor bearing mice using nuclear imaging. An anti-murine CD8+ PET probe was injected in mice 24 hours post SAR444245 administration and nuclear imaging was performed at different times over 6 days. In C57BL/6 naive and in poorly immunogenic B16-F10 tumor bearing animals, images reveal a decreased Standardized Uptake Value (SUV) within blood in treated animals at 3mg/kg compared to control ones. In parallel, blood SUV Ratio (SUVR) increases within spleen, lymph nodes and thymus after SAR444245 injection compared to control mice. On tumor site, a stronger SUVR is measured at 144 hours post injection in SAR444245 treated group than in the control group. A dose escalation ranging from 1 to 6 mg/kg of SAR444245 was performed in highly immunogenic CT26 tumor bearing animal. Blood SUV decreases in the treated groups compared to the control group. In lymphoid organs, all SUVR increase at 144 hours post injection in the treated groups compared to the control one. In CT26 tumors, a slight increase of the SUVR is observed for groups treated at 3 and 6 mg/kg. In parallel, a pronounced reduction of tumor growth is observed in the treated group at 6mg/kg. These studies non-invasively confirm the mode of action of SAR444245 with a rapid relocation of CD8+ T cells from peripheral blood to lymphoid organs and tumor site. Citation Format: Sebastion d'Heilly, Fabrice Tirode, Maxime Gaulin, Dudzicki Anne, Guillaume Bluet, Stéphane Guerif, Francis Descamps, Peter Casteels, Xiangming Li, Rui Wang, Robin Meng, Erwan Jouannot. Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3580.

Abstract 5585: Tissue based next generation sequence is more accurate than ctDNA to detected pathogenic variants in MTAP wild type tumors

Abstract Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) prevents invasive biopsies and captures tumor heterogeneity. In a prior report by Zhang et al (Nat Commun 2021), certain actionable mutations were more prevalent in ctDNA than tissue-based databases (e.g., EGFR 48% versus 25.2%). Our group investigated if discrepancies in patterns of pathogenic variants between liquid biopsy and tissue specimens in a subtype of metastatic urothelial carcinoma (MTAP deleted) can inform the best approach of utilizing tissue or blood. MTAP (methyl-adenosine phosphorylase) is an enzyme involved in salvage purine biosynthesis and its deletion confers sensitivity to pemetrexed and taxanes, but resistance to immune checkpoint inhibitors. The MTAP gene is deleted in 25% of urothelial carcinomas. We report herein the pattern of pathogenic variants from ctDNA in 17 patients with metastatic urothelial carcinoma, 10 with MTAP-deletion and 17 with MTAP wild-type. Among 10 patients with MTAP-deleted urothelial carcinoma with collected ctDNA, 3 had no detected pathogenic variant. The most common pathogenic variants in 7 patients were TP53 (4/7, 57%), TERT (4/7, 57%), ERBB2 (2/7, 28%); PIK3CA (2/7, 28%), and NFE2L2 (2/7, 28%). The mutations found in paired ctDNA and tissue based NGS were TP53 in 26.6 % (4/15), TERT in 21.0 % (4/19), ERBB2 in 66.6 % (2/3), PIK3CA in 50 % (2/4), and NFE2L2 in 100% (1/1) of patients. Three of the 10 patients with collected ctDNA had ELF3 frameshift loss-of- function alterations on tissue not found on ctDNA. Among 54 patients with MTAP wild-type urothelial carcinoma, 26 had collected ctDNA. Among these 26 patients, 9 had no detected pathogenic variants. Among the 17 patients with pathogenic variants, the most common mutations were TP53 (76%, 13/17), TERT (47%, 8/17), ERBB2 (24%, 4/17), RB1 (24%, 4/17), and BRCA1 or BRCA2 (24%, 4/17). Three of these 17 patients had no or insufficient tissue for NGS. The mutations found in paired ctDNA and tissue based NGS were TP53 in 100% (14/14), TERT in 64% (9/14), ERBB2 in 43% (6/14), RB1 in 29% (5/14), and BRCA1 or BRCA2 in 36% (5/14) of patients. MTAP wild-type patients had a higher frequency of TERT alterations (p=0.012), but not ERBB2, (p=0.453) than patients with MTAP-deleted type tumors. Ongoing evaluation of timing and location of pathogenic variants will inform where (primary tumor, metastatic site) and when (after chemotherapy, immune checkpoint inhibitors, antibody drug conjugates) to biopsy patients on progression after cancer therapies. Citation Format: Andre Luiz De Souza. Tissue based next generation sequence is more accurate than ctDNA to detected pathogenic variants in MTAP wild type tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5585.

Abstract P3-06-03: Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer

Abstract Background: In early-stage triple-negative breast cancer (TNBC), there is accumulating evidence of a correlation between tumor-infiltrating lymphocytes in tumor tissue and favorable clinical outcomes, with a high CD8+/regulatory T-cell (Treg) ratio after neoadjuvant chemotherapy being predictive of overall survival and associated with pathologic complete response (Ladoire S, et al. Br J Cancer. 2011; Park YH, et al. Nat Commun. 2020). Trilaciclib is a transient inhibitor of cyclin-dependent kinase 4/6 that is administered intravenously prior to chemotherapy. In preclinical studies, trilaciclib has been shown to have immune-enhancing effects by differentially arresting CD8+ T-cell and Treg subsets, which is followed by the faster recovery of CD8+ T cells than Tregs in the tumor microenvironment. Methods: This phase 2, single-arm, open-label study aims to evaluate neoadjuvant, single-dose trilaciclib followed by trilaciclib plus dose-dense anthracycline/cyclophosphamide and taxane in patients with early-stage TNBC (NCT05112536). Patients with previously untreated, non-metastatic, confirmed TNBC and a primary tumor ≥ 1.5 cm of any nodal status receive a single dose of trilaciclib 240 mg/m2 during the lead-in phase, followed by 4 cycles of doxorubicin 60 mg/m2 plus cyclophosphamide 600 mg/m2, and 12 weekly cycles of paclitaxel 80 mg/m2. Trilaciclib 240 mg/m2 is administered prior to the first chemotherapy dose of each cycle. Pembrolizumab 400 mg every 6 weeks starting on day 1, cycle 1, and/or carboplatin AUC 1.5 every week starting on day 1, cycle 5, is allowed per investigator discretion. Tumor biopsies and peripheral blood samples are collected prior to any treatment, 7 days ± 1 day post administration of trilaciclib, and during surgery, with an additional blood sample collection on day 1, cycle 2. The primary objective is to evaluate the immune-based mechanism of action of trilaciclib after a single dose of trilaciclib, as measured by changes in the CD8+/Treg ratio in tumor tissue. Pathologic complete response, safety and tolerability, and additional exploratory immune biomarker endpoints will also be assessed. Results: As of June 3, 2022, 9 patients with early-stage TNBC had been enrolled and 8 patients had received the trilaciclib lead-in dose and initiated doxorubicin/cyclophosphamide. Patients had a median age of 53.0 years, and all had stage II tumors at diagnosis, with 7 having ductal carcinoma. The median number of chemotherapy cycles received was 3 (range 1–6), and all 8 patients received pembrolizumab. Seven patients continue study treatment; 1 patient discontinued due to disease progression. Five patients had an adverse event (AE) related to any study treatment, including 4 patients with ≥ 1 trilaciclib-related AE. There were no grade ≥ 3 treatment-related AEs or serious AEs. On-treatment, post-trilaciclib monotherapy biopsies were available for 4 patients. Following neoadjuvant trilaciclib treatment, the median density of stromal CD8+ T cells increased from 103.1/mm2 at baseline to 229.8/mm2 at day 7. The median CD8+/Treg ratio increased in 2 patients from 1.85 at baseline to 1.90 at day 7. Conclusions: Preliminary analysis of on-treatment tumor biopsies from 4 patients suggests that a single dose of trilaciclib may modulate the immune cell composition in the tumor microenvironment to support antitumor immune responses. The increase in CD8+ T cells following 7-day neoadjuvant treatment with trilaciclib supports previous data suggesting a role in T-cell infiltration. The complete dataset from all patients (estimated enrollment: N ≈ 24) and additional biomarker analyses will be presented. Citation Format: Michael Danso, Joyce O’Shaughnessy, Lisa S. Wang, Kailash Mosalpuria, Sara Hurvitz, Shom Goel, Sarah Ahn, Subing Cao, John S. Yi, Taofik Oyekunle, Amanda Jacobson, Andrew Beelen, Jeremy Force. Trilaciclib induces immune changes within the tumor microenvironment in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-03.

Abstract P4-02-13: Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials

Abstract Background: Hormone-receptor (HR)+/HER2- breast cancer (BC) is a biologically heterogeneous disease. Homologous recombination deficiency (HRD) and BRCA mutations have been previously reported to be associated with worse outcomes in HR+/HER2- metastatic BC patients receiving CDK4/6 inhibitors and endocrine therapy. Here, we assess the relation between HRD and RB-loss signatures, intrinsic subtyping, the PAM50-based chemo-endocrine score, and response to chemotherapy-based therapy and endocrine treatment in HR+/HER2- early BC. Methods: GIADA is a multicentric neoadjuvant phase II trial that treated premenopausal patients with Luminal B (LumB)-like BC (HR-positive, HER2-negative, with Ki67>20% and/or histologic Grade 3) with a combination of chemotherapy, immunotherapy and endocrine treatment. Expression of 758 genes on baseline tumor samples from all 43 patients was quantified by nCounter platform. The LETLOB phase II trial randomized postmenopausal women with clinical stage II-IIIA HR+/HER2- BC to neoadjuvant letrozole + lapatinib or letrozole + placebo for 6 months (Guarneri, JCO 2014). Gene-expression data (Affymetrix platform) from pre-treatment frozen core-biopsies was available from 66 out of 92 pts enrolled. Intrinsic subtype was assigned using a research-based PAM50 subtype predictor. A published HRD signature (Peng, Nat Commun 2014) and a signature of RB loss (RBsig), previously reported to potentially predict resistance to CDK4/6 inhibitors in HR+/HER2- BC (Malorni, Oncotarget 2016) were computed. The PAM50 based chemo-endocrine score (CES) was calculated using published definition (Prat, CCR 2017). Higher values of CES indicate increased endocrine sensitivity, while lower values indicate chemosensitivity. Association between genomic signatures was assessed through Pearson’s correlation coefficient. Association of genomic signatures with pCR was assessed through logistic regression and association with PEPI scores was assessed through Kruskal-Wallis test. Results: HRD signature levels were significantly higher in non-luminal (Basal-like and HER2-enriched) tumors as compared to Luminal (A or B) tumors (p>0.001 in the GIADA trial, p=0.021 in the LETLOB trial). Moreover, higher levels of HRD signature were associated with higher levels of RB-loss signature (Pearson correlation 0.355, p=0.020 in the GIADA trial; Pearson correlation 0.942, p< 0.001 in the LETLOB trial), higher levels of Basal-like signature (Pearson correlation 0.502, p< 0.001 in the GIADA trial; Pearson correlation 0.373, p=0.002 in the LETLOB trial) and lower levels of CES (Pearson correlation -0.422, p=0.005 in the GIADA trial; Pearson correlation -0.763, p< 0.001 in the LETLOB trial), indicative of higher chemosensitivity. In the GIADA trial, higher levels of HRD signature (p=0.018) and RBloss signature (p=0.073) and lower levels of CES (p=0.007) were associated with higher pCR rates after chemo, endocrine and immunotherapy. In the LETLOB trial, lower levels of HRD signature (p=0.068) and RBloss signature (p=0.042) and higher levels of CES (p=0.050) were associated with higher sensitivity to endocrine treatment (lower PEPI scores, 0 vs 1-3 vs 4 or more, after neoadjuvant letrozole). Conclusions: In HR+/HER2- early BC, HRD gene signatures, RB-loss gene signatures and non-luminal (especially Basal-like) intrinsic subtyping are associated with each other and associated with higher sensitivity to chemotherapy-based therapy and lower sensitivity to endocrine treatment. These observations might help correctly tailor systemic therapy, including biologic agents, in patients with HR+/HER2- early and advanced BC. Citation Format: Gaia Griguolo, Federica Miglietta, Laia Paré, Daniele G. Generali, Antonio Frassoldati, Antonino Musolino, Simon Spazzapan, Grazia Vernaci, Tommaso Giarratano, Marcello Lo Mele, Giancarlo Bisagni, Federico Piacentini, Enrico Tagliafico, Katia Cagossi, Francesca Schiavi, Claudia Pinato, Aleix Prat, Valentina Guarneri, Maria Vittoria Dieci. Homologous recombination deficiency, RB-loss gene signatures, intrinsic subtype and response to neoadjuvant treatment in HR+/HER2- early breast cancer: a correlative analysis of two phase II trials [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-13.

Effect of bulky anions on the liquid-liquid phase transition in phosphonium ionic liquids: Ambient and high-pressure dielectric studies

Although the first-order liquid–liquid phase transition (LLT) has been reported to exist in various systems (i.e., phosphorus, silicon, water, triphenyl phosphite, etc.), it is still one of the most challenging problems in the field of physical science. Recently, we found that this phenomenon occurs in the family of trihexyl(tetradecyl)phosphonium [P666,14]+ based ionic liquids (ILs) with different anions (Wojnarowska et al in Nat Commun 13:1342, 2022). To understand the molecular structure–property relationships governing LLT, herein, we examine ion dynamics of two other quaternary phosphonium ILs containing long alkyl chains in cation and anion. We found that IL with the anion containing branched –O–(CH2)5–CH3 side chains does not reveal any signs of LLT, while IL with shorter alkyl chains in the anion brings a hidden LLT, i.e., it overlaps with the liquid-glass transition. Ambient pressure dielectric and viscosity measurements revealed a peculiar behavior of ion dynamics near Tg for IL with hidden LLT. Moreover, high-pressure studies have shown that IL with hidden LLT has relatively strong pressure sensitivity compared to the one without first-order phase transition. At the same time, the former exposes the inflection point indicating the concave-convex character of logτσ(P) dependences.

Chronic heart failure following hemorrhagic myocardial infarction: mechanism, treatment and outlook.

Myocardial infarction (MI), the blockage of arterial blood supply of the heart, is among the most common causes of death worldwide. Even when patients receive immediate treatment by re-opening blocked arteries, they often develop chronic heart failure (CHF) in the aftermath of MI events. Yet, the factors that contribute to the development of MI-associated CHF are poorly understood. In our recent study (Nat Commun 13:6394), we link intramyocardial hemorrhage, an injury which can occur during reperfusion of areas affected by MI, to an increased risk of CHF. Mechanistically, our data suggest that an iron-induced adverse cascade of events after hemorrhagic MI drives fatty degeneration of infarcted tissue, which ultimately contributes to negative cardiac remodeling. In this Microreview, we discuss the implications of our findings regarding the molecular mechanism, more targeted treatment options as well as perspectives in the clinical care of CHF after hemorrhagic MI.

Editors' Selections from Relevant Scientific Publications.

Highlights from the Literature| February 06 2023 Editors' Selections from Relevant Scientific Publications Author & Article Information Online Issn: 1940-6215 Print Issn: 1940-6207 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Prev Res (Phila) (2023) 16 (2): 63. https://doi.org/10.1158/1940-6207.CAPR-16-2-HFL Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record February 6 2023 Citation Editors' Selections from Relevant Scientific Publications. Cancer Prev Res (Phila) 1 February 2023; 16 (2): 63. https://doi.org/10.1158/1940-6207.CAPR-16-2-HFL Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Multiple Myeloma (from Scientific Animations via Wikimedia Commons) Gene expression signatures associated with precursor states for multiple myeloma could enable earlier diagnosis and intervention with targeted therapies. Multiple myeloma is typically preceded by the appearance of well-defined abnormalities in the plasma cell population, but the scarcity of these abnormal cells has confounded efforts to understand which patients exhibiting those phenotypes will ultimately progress. Boiarsky et al. overcame this challenge by performing single-cell RNA sequencing analysis of tens of thousands of plasma cells from dozens of patients. By comparing pre-malignant cells to healthy controls, they identified distinctive patterns of gene activity that correlate with onset of multiple myeloma. Some of these include pathways that can potentially be targeted with already-available drugs to prevent onset of malignancy. Boiarsky R, … Getz G. Nat Commun. 2022 Nov 17;13(1):7040. cfDNA for multi cancer early detection (from Racheljunewong via Wikimedia Commons) Liquid biopsy tests... You do not currently have access to this content.

A Sampling of Highlights from the Literature

The timing of immune interactions modulates antitumor responses (from LetsgomusicStyle via Wikimedia Commons)Immune cell interactions are critical to mounting effective antitumor responses. Wang et al. show that antitumor responses are coordinated by dendritic cell (DC)–CD8+ T-cell interactions and that expression of key circadian rhythm genes governs immune function, which is also affected by the time of day (day vs. night). Cervantes-Silva et al. reveal that DC antigen presentation is also dependent on expression of circadian rhythm genes, which regulate DC mitochondrial morphology and metabolism, and thus, affect T-cell priming. These studies highlight that induction of antitumor responses is a coordinated, rhythmic process, and a deeper understanding of immune rhythmicity can improve therapeutic strategies.Wang C, …, Scheiermann C. Nature 2022 December 5. DOI:10.1038/s41586-022-05605-0.Cervantes-Silva MP, …, Curtis AM. Nat Commun 2022 December 5;13:7217.IL2 is less active in the acidic tumor microenvironment (from BQUB17-ONadal via Wikimedia Commons)High-dose IL2 is approved for treating melanoma and renal cell carcinoma but is used infrequently because of limited efficacy and systemic on-target toxicity. Gaggero et al. find that the acidic pH of the tumor microenvironment (TME), created as a result of tumor production of lactic acid, reduces IL2 signaling in tumor-infiltrating CD8+ T cells, impairing the efficacy of high-dose IL2 therapy in preclinical models. A mutated IL2 variant (Switch-2) that binds IL2Rα with higher affinity at acidic pH than neutral pH shows enhanced antitumor activity with reduced on-target toxicity in mice. The data provide new insight into physicochemical properties of the TME that can impact immunotherapy efficacy and suggest ways to overcome this.Gaggero S, …, Mitra S. Sci Immuol 2022 December 2;7:eade5686.Eosinophils contrib-ute to the efficacy of ICB (from Blausen Medical via Wikimedia Commons)Understanding the mechanisms behind why immune checkpoint blockade (ICB) is not effective in many patients is key to improving treatment. Blomberg et al. find that eosinophils are enriched in ICB-responding patients with breast cancer. This observation was confirmed in a mouse model, and CD4+ T cells, IL5, and IL33 were identified as the driving factors for eosinophil enrichment. Increased eosinophil infiltration boosts eosinophil-dependent activation of CD8+ T cells, which in turn enhances efficacy of ICB. The data emphasize how immune cells other than T cells are affected by ICB and how they can be critical determinants of treatment efficacy.Blomberg OS, …, Kok M. Cancer Cell 2022 December 15;41:106–123.E10.Antitumor T-cell responses require two steps (from DENI070688 via Wikimedia Commons)Understanding T-cell differentiation can reveal insights into the mechanisms behind effective antitumor responses. Prokhnevska et al. demonstrate that T cells are activated via two key steps: first, initial priming and differentiation into a TCF-1+ stemlike phenotype in tumor-draining lymph nodes (TDLN); and second, acquisition of an effector program after infiltrating the tumor microenvironment (TME). Effector programming of CD8+ T cells is dependent on co-stimulation from antigen-presenting cells in the TME, as opposed to in the TDLNs. This process differs from canonical priming/activation of CD8+ T cells in other contexts and adds to our understanding of how T cells mount responses to cancer.Prokhnevska N, …, Kissick H. Immunity 2022 December 28;56:107–124.Eosinophils contribute to the efficacy of ICB (from Boghog2 via Wikimedia Commons)IL22 is known to facilitate tumorigenesis. Two new studies identify mechanisms by which IL22 promotes tumor metastasis and suggest the cytokine as a therapeutic target for preventing metastasis. Giannou et al. find that mice lacking IL22 and mice treated with an IL22-specific blocking antibody are protected from liver metastasis in a colorectal cancer model. Mechanistically, IL2 from liver-resident iNKT17 cells acts on endothelial cells, promoting cancer cell extravasation into the liver. In mouse models of lung and breast cancer, Briukhovetska et al. show that T helper–cell production of IL22 induces overexpression of CD155 on tumor cells. Binding of CD155 to CD226 on natural killer (NK) cells causes CD266 internalization and impaired NK-cell function, promoting metastasis.Giannou AD, …, Huber S. Immunity 2023 January 10;56:125–142.E12.Briukhovetska D, …, Kobold S. Immunity 2023 January 10;56:143–161.E11.Engineered live cancer cells can promote effective antitumor immunity (from Rita Elena Serda via NIH Flickr)The use of inactivated tumor cells as a therapeutic vaccine has been shown to provide limited clinical benefit. Chen et al. show that engineering live cancer cells to secrete IFNβ and GM-CSF, to be resistant to IFNβ, and to have a double kill switch (HSV1 thymidine kinase and rapamycin-activated caspase 9) yields therapeutic cancer cell–based vaccines that induce long-term antitumor immunity and improve survival in several preclinical models. The translatability of this approach is highlighted by the observation that similar engineering of an established human glioblastoma cell line yields a vaccine that reduces tumor growth and improves survival in a humanized model.Chen K-S, …, Shah K. Sci Trans Med 2023 January 4;14:eabo4778.

Dynamics and thermodynamics of bimolecular reactions at heterogeneous biological interfaces.

The behaviors of biomolecules at heterogeneous biological aqueous-nonaqueous interfaces markedly differ from those in bulk water. We found that various biomolecular reactions, including protein unfolding, protein-ligan binding, chlorophyll demetallation, redox reactions, and crystallization, are accelerated by a factor of 103 or higher at the interface of micron-sized water droplets (microdroplets) (PNAS, 2015; QRevPhys, 2015 and 2017). The FRET analysis revealed that molecules are highly localized at the nanoscale regime near the water surface, which is in part responsible for the observed accelerated dynamics (JPCB, 2020). Besides, the thermodynamics of biomolecular reactions are also altered near the water surface. We found that molecules are in highly ordered states near the aqueous-nonaqueous interface (Sci Adv, 2020, Sci Rep, 2020). This minimized entropy lowers entropic barriers so that thermodynamically unfavorable reactions such as phosphorylation and RNA formation can proceed without any enzyme or ATP energy source at room temperature (PNAS 2017, 2018). Moreover, the charge exchange between water molecules and biomolecules spontaneously occurs at the water interface, leading to the oxidation of water molecules and the reduction of biomolecules in the Krebs cycle (JACS, 2019; Nat Commun, 2018; PNAS, 2018, 2019; Chem Sci, 2019). Vibrational Stark spectroscopy coupled with stimulated Raman excited fluorescence microscopy showed the presence of a strong intrinsic interfacial electric field formed at the surface of the microdroplet (JPCL, 2020). This strong electric field, combined with the partial desolvation effect, can be responsible for the unusual behaviors of biomolecules at the aqueous-nonaqueous interface (Annu Rev Phys Chem, 2020). These findings suggest that the dynamics and thermodynamics of biomolecular reactions occurring in cells with various aqueous-nonaqueous interfaces and compartments may significantly differ from those in bulk water.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).