Inflammation-driven tumor implantation, such as port-site metastasis (PSM) following laparoscopic gynecologic surgery and peritoneal seeding during post-surgical recurrence, represents an aggressive clinical problem that remains poorly understood and lacks targeted therapies. To address this, we developed a non-surgical Mesothelium-Inflammation/Injury-Metastasis (MIM) model and investigated the role of the IL-1β/IL1R1/MYD88/IRAK1/4 axis and NLRP3 in epithelial ovarian cancer (EOC) seeding at inflamed or injured sites. This model created by a needle injury recapitulates inflammation-driven peritoneal seeding and mimics PSM and inflammation associated dissemination in peritoneum during recurrence. Seeding was dependent on Il1r1 but not Nlrp3, despite its role in regulating IL-1β production, as Il1ra-/- and Nlrp3-/- mice phenocopied wild-type C57BL/6 mice. Given the limited antitumor efficacy of IL-1β-targeting agents such as Anakinra and Canakinumab, we focused on IRAK4 as a therapeutic target. IRAK4 knockdown significantly prolonged survival, reduced tumor cell adhesion, downregulated E-cadherin and Wnt4, and induced S-phase/mitotic arrest. This led to the development of UR241-2, a small-molecule IRAK4 inhibitor, which was validated through molecular simulations, hotspot analysis, nanoBRET, global kinome profiling, and NF-κβ reporter assays. UR241-2 inhibited NF-κβ nuclear translocation and blocked IL-1β-induced IRAK4 phosphorylation. UR241-2 exhibited favorable drug-like properties, including absence of CYP or hERG inhibition, and acceptable CaCo-2 permeability, plasma protein binding, microsomal stability, and pharmacokinetics. In vivo, UR241-2 reduced SKOV3 xenograft growth, suppressed mesothelial seeding, and increased MHC-II+ macrophages and activated neutrophils in syngeneic high-grade epithelial ovarian HGS3 tumors. RNA-seq revealed enrichment of neutrophil activation signatures and suppression of extracellular matrix (ECM) gene programs. Together, these findings establish a role for the IL-1β/IL1R1/IRAK4 axis in inflammation-driven PSM and peritoneal seeding and ECM regulation in EOC, and demonstrate that IRAK4 inhibition activates antitumor immune responses, providing a therapeutic strategy to block metastatic seeding and improve tumor control.

Epithelial ovarian cancer (EOC) cells seed at mesothelial inflammation or injury sites. Lack of animal models recapitulating tumor cells seeding at inflamed sites in EOC hinders mechanistic studies and therapy developments. Here, we developed a non-surgical MIM (Mesothelium Inflammation/Injury Metastasis) model that recapitulates tumor cell seeding at inflamed sites. This model captures temporal changes in tumor immune microenvironment and tumor growth allowing for deeper mechanistic and preclinical therapeutic studies of EOC in-vivo. We show here that HGS-3 high-grade murine serous EOC cells seed at needle-induced injury sites in mesothelium/peritoneal wall, forming tumors both internally and protruding outward. Using MIM model, we found that deletion of IL1R1 in mice reduced EOC cell seeding at mesothelium injury/inflamed site in WT but not IL1ra-deficient mice. Treatment of MiM mice with a novel IRAK4 inhibitor we recently developed (UR241-2) revealed an essential role for IRAK4 signaling downstream IL-1β/IL-1R1 in fostering an anti-tumor inflammatory environment, and reduced tumor burden. We conclude that IRAK4 inhibitors can be more effective than IL-1/IL-1R1 targeting agents to control metastasis and peritoneal tumors, an unmet medical need in EOC recurrence. Downregulation of extracellular matrix (ECM), upregulation of neutrophil activation genes, reduced cell adhesion and migration exhibit how UR241-2 corrects ECM and immune disorders in EOC, making it less conducive to metastasis and tumorigenesis. ### Competing Interest Statement RKS, RGM. LMC and MWB are listed as the inventors on the patents related to UR241-2. Empire Discovery Institute (EDI) had licensed UR241-2 from the University of Rochester for cancer treatment. EDI had no roles in writing this manuscript. EDI and UR Ventures office of University of Rochester have cleared the contents of this manuscript for publication.

High-throughput sequencing (HTS) of clonal immunoglobulin (Ig) gene rearrangements has become a mainstay of B-lymphoblastic leukemia (B-ALL) measurable residual disease (MRD) monitoring; Ig HTS also provides insight into the widely documented leukemic clonal heterogeneity in Ig heavy chain (IgH) variable (V), diversity (D), and joining (J) gene rearrangements in B-ALL. B-ALL IgH clonal composition is canonically presumed to reflect the transformed cell's rearrangement state: B cells develop along a pathway with IgH D-J followed by IgH V gene rearrangement; thus, subclones comprised of a common DNJ sequence, but distinct alternative V gene involvement, would be assumed to derive from a cell stage at which only D-J recombination had taken place with persisting recombination machinery for subsequent V-DJ rearrangement. We and others have observed that this ‘V-DJ diversity’ may be associated with prognostic cytogenetic features, but its relevance as a supposed reflection of B cell developmental state has not been confirmed. Here, we define the biologic significance of IgH variable gene diversity in B-ALL and reveal its direct implications on HTS MRD and its potential impact on clinical outcome. We analyzed 148 B-ALL samples from patients enrolled on former Children's Oncology Group (COG) trials (N=99) and an institutional tissue bank (N=49) to test the functional implications of IgH clonal composition in B-ALL. We defined 3 cohorts: 1) No clonal IgH (N=7; 4.7%); 2) Diverse, with V-DJ ‘subclones’ containing unique V genes but a common DNJ stem (N=53; 35.8%), and 3) Non-diverse, with single VDJ rearrangement(s) per allele (N=88; 59.5%). We confirmed that the extent of V-DJ diversity in each B-ALL – quantified by Shannon entropy index – is inherently preserved across tissue sites (bone marrow (BM) vs. peripheral blood (PB) R2=0.99; P<.0001) and early induction therapy timepoints rather than merely a stochastic phenomenon. Chromosome microarray demonstrated no association between prognostic cytogenetics, V-DJ diversity, and other non-IgH breakpoint accumulation across 4 biologically distinct, prognostic cytogenetic categories. Using CyTOF, we found that regardless of the pattern of IgH rearrangement(s), all cases were enriched in pro-BII cell populations. However, by Gene Ontology and Hallmark gene set enrichment analysis (GSEA) and CyTOF, cases with V-DJ diversity had distinct transcriptional and intracellular proteomic features – including enriched gene and protein expression of pentose phosphate and mitochondrial metabolism genes and proteins, as well as activated mTOR signaling – compared to cases with non-diverse VDJ rearrangements. Further, by ATACseq, we observed distinct chromatin accessibility between diverse and non-diverse cohorts, including increased accessibility at the IgH locus in diverse cases. Integrated single-cell and clinical Ig HTS clonality data revealed that distinct V-DJ subclone sequences (derived from a similar progenitor population sharing a DNJ stem) comprise discrete cell populations over a variable range of abundances; therefore, all or some such sequences will not necessarily meet clinical HTS clonality/MRD assay criteria to be defined as ‘dominant/trackable’ for MRD determination. Both via single-cell RNA seq and custom Tapestri sequencing, we found that diverse V-DJ subclone sequences distinguish discrete cell subpopulations. Thus, we tested the impact of IgH rearrangement diversity on MRD detection and outcome using a dataset from 307 subjects with high risk (HR) pediatric B-ALL. Among the prognostically inferior cohort from COG AALL0232 which lacked any designated ‘trackable’ sequences (N=31/307) (Fries et al. Haematologica 2023), we found that 7 (of 31; 2% of the total 307) had subclonal IgH rearrangements with unique V/common DJ sequences of similar abundance such that none were designated 'trackable.' Despite treatment intensification based on positive end of induction (EOI) flow cytometry MRD on AALL0232, these patients had inferior 5-year EFS of 71.4% (95%CI 44.7-100%; P=.001). In 5 (of 7) cases, a subset of V-DJ 'subclones' were still detectable at the EOI timepoint, suggesting treatment resistance. In conclusion, IgH V-DJ diversity reflects distinct metabolic/transcriptional pathway activation in proB-like leukemia cells and defines discrete B-ALL cell populations with possibly distinct treatment responses, potentially impacting clinical outcome.

The bone marrow microenvironment consists of diverse cell populations, including mesenchymal stromal cells (MSCs), osteolineage cells, fibroblasts, and endothelial cells, all of which support hematopoietic stem cells. The interaction of stromal populations with their microenvironment is essential for sustaining normal development and homeostasis. Aggressive myeloid leukemias such as acute myeloid leukemia (AML), often exploit these interactions to promote their progression. Even with notable progress in targeted therapies, the survival rate for patients with AML is approximately 30%, underscoring the need to discover new regulators of disease progression. Work by us, and others, has identified that signals from the bone marrow microenvironment can be actionable vulnerabilities for therapeutic targeting of AML. Our recent temporal single-cell RNA-sequencing based analyses of the AML microenvironment identified signals from the expanding MSC niche, such as apolipoproteins, that may play a role in AML progression. Apolipoproteins are complexes that transport lipids such as triglycerides and cholesterol in the bloodstream, and act as ligands for receptors involved in lipid metabolism. They can also regulate disease growth by interacting with cell surface receptors expressed on AML cells. However, the role that apolipoproteins play in leukemia progression is not well understood. We find that MSCs from murine and human bone marrow have the highest apolipoprotein E (ApoE) expression. Importantly, our immunohistochemistry-based assays indicate that APOE protein expression increases in the human bone marrow stroma with AML progression. Consistent with a functional role of niche-derived APOE in supporting AML cell growth, our experiments show that inhibiting APOE expression in MSCs from AML patients can block the growth of co-cultured patient-matched leukemia cells. To investigate the impact of ApoE on AML progression in vivo, we used loss-of-function murine models of ApoE. Our data showing that murine leukemia growth is delayed in ApoE knockout mice as compared to wild-type controls suggests that microenvironmental ApoE is essential for in vivo leukemia progression. Mechanistically, our data suggest that the effect of ApoE on leukemia cell growth may be due to its effect on MAPK signaling. Collectively, our studies establish a key role of leukemia tumor microenvironment derived apolipoproteins on AML progression.

Interactions of stem cells with their surrounding microenvironment are known to be essential for both normal development and for sustaining self-renewing adult stem cells, such as the hematopoietic stem cells (HSCs). Since cancers often hijack developmental signals for their progression, it is likely that niche-driven signals that sustain HSCs also influence the growth of leukemias arising from mutations in HSCs and early hematopoietic progenitors, such as acute myeloid leukemia (AML) and blast crisis chronic myeloid leukemia (bcCML). To identify candidate cell surface receptors essential for myeloid leukemia progression, we employed a comprehensive approach integrating our in vivo genome-wide CRISPR screen and our RNA-seq analysis of human LSCs from distinct myeloid leukemia subtypes. Of these, the taurine transporter (TauT), encoded by SLC6A6, was of particular interest since its expression is associated with poor prognosis in acute myeloid leukemia (AML) patients. Our experiments using TauT knockout mice indicate that TauT loss significantly impairs myeloid leukemia progression in vivo. An RNA-Seq analysis of freshly isolated TauT+/+ and TauT-/- leukemia cells identified a striking decrease in glycolysis associated genes in the absence of TauT. Consistent with this, Seahorse analysis showed significantly reduced basal glycolysis and glycolytic capacity in TauT-/- LSCs as compared to TauT+/+ controls. In addition, inhibiting TauT reduced the abundance of key glycolytic metabolites as measured by LC/MS spectrometry. Our experiments showing significant downregulation of mTOR pathway in the absence of TauT indicate that mTOR signaling may regulate glycolysis in leukemia cells. Consistent with a functional role of mTOR downstream of TauT function, our data shows that activating mTOR pathway in TauT-/- cells can rescue expression of glycolysis related genes and leukemic cell growth. Importantly, our experiments inhibiting TauT using shRNAs or small-molecule inhibitors identify a key role of this signal in the growth of patient-derived myeloid leukemia cells. Collectively, our data identify a critical requirement of the taurine-TauT axis in myeloid leukemia progression and indicate that the TauT inhibition may be of therapeutic value in myeloid leukemia. Sonali Sharma, Benjamin J. Rodems, Cameron D. Baker, Christina M. Kaszuba, Edgardo I. Franco, Takashi Ito, Laura M. Calvi, Michael W. Becker, Bradley R. Smith, Paul S. Brookes, Joshua C. Munger, Jane L. Liesveld, Craig T. Jordan, John M. Ashton, Jeevisha Bajaj. Taurine Transporter SLC6A6 promotes myeloid leukemia progression by regulating glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6653.

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.

Macrophages are prime therapeutic targets due to their pro-tumorigenic and immunosuppressive functions in tumors, but the varying efficacy of therapeutic approaches targeting macrophages highlights our incomplete understanding of how the tumor microenvironment (TME) can influence regulation of macrophages. The circadian clock is a key internal regulator of macrophage function, but how circadian rhythms of macrophages may be influenced by the tumor microenvironment remains unknown. We found that conditions associated with the TME such as polarizing stimuli, acidic pH, and elevated lactate concentrations can each alter circadian rhythms in macrophages. Circadian rhythms were enhanced in pro-resolution macrophages but suppressed in pro-inflammatory macrophages, and acidic pH had divergent effects on circadian rhythms depending on macrophage phenotype. While cyclic AMP (cAMP) has been reported to play a role in macrophage response to acidic pH, our results indicate that pH-driven changes in circadian rhythms are not mediated solely by the cAMP signaling pathway. Remarkably, clock correlation distance analysis of tumor-associated macrophages (TAMs) revealed evidence of circadian disorder in TAMs. This is the first report providing evidence that circadian rhythms of macrophages are altered within the TME. Our data further suggest that heterogeneity in circadian rhythms at the population level may underlie this circadian disorder. Finally, we sought to determine how circadian regulation of macrophages impacts tumorigenesis, and found that tumor growth was suppressed when macrophages had a functional circadian clock. Our work demonstrates a novel mechanism by which the tumor microenvironment can influence macrophage biology through altering circadian rhythms, and the contribution of circadian rhythms in macrophages to suppressing tumor growth.

Riboswitches are structured RNAs that sense small molecules to control expression. Prequeuosine1 (preQ1)-sensing riboswitches comprise three classes (I, II and III) that adopt distinct folds. Despite this difference, class II and III riboswitches each use 10 identical nucleotides to bind the preQ1 metabolite. Previous class II studies showed high sensitivity to binding-pocket mutations, which reduced preQ1 affinity and impaired function. Here, we introduced four equivalent mutations into a class III riboswitch, which maintained remarkably tight preQ1 binding. Co-crystal structures of each class III mutant showed compensatory interactions that preserve the fold. Chemical modification analysis revealed localized RNA flexibility changes for each mutant, but molecular dynamics (MD) simulations suggested that each mutation was not overtly destabilizing. Although impaired, class III mutants retained tangible gene-regulatory activity in bacteria compared to equivalent preQ1-II variants; mutations in the preQ1-pocket floor were tolerated better than wall mutations. Principal component analysis of MD trajectories suggested that the most functionally deleterious wall mutation samples different motions compared to wildtype. Overall, the results reveal that formation of compensatory interactions depends on the context of mutations within the overall fold and that functionally deleterious mutations can alter long-range correlated motions that link the riboswitch binding pocket with distal gene-regulatory sequences.

Background: Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. Methods: We used the Tcf21 (transcription factor 21) MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Results: Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21- lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. Conclusions: This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

[This corrects the article DOI: 10.3389/fncel.2022.1096872.].

The LungMAP program has been working for over a decade to provide the lung community with a comprehensive resource for the lung research community, including reference data sets and protocols [1]. The University of Rochester has served as the Human Tissue Core and worked extensively with Research Center lab groups. We have established protocols for procuring human tissue including, but not limited to lung, and processing them for a variety of downstream analysis modalities including the generation of single cell suspensions [2, 3, 4]. We have successfully utilized singe cells suspensions to generate single cell CITESeq data utilizing the 10X Genomics Single Cell 3' (v3.1) RNA sequencing platform [5-7]. This protocol describes the steps necessary for preparing generating sequencing data following staining single cells with ADT-tagged antibodies (including library preparation, sequencing steps, and mapping steps). References 1. https://www.lungmap.net/about-lungmap/lungmap-objectives/ 2. 602.2 Donor Acceptance Criteria for URMC HTC SenNet Inclusion dx.doi.org/10.17504/protocols.io.biz7kf9n. 3. 603.3 & 604.5_URMC_HTC_Lung and Lobe Processing for SenNet dx.doi.org/10.17504/protocols.io.n2bvj395plk5/v1 4. 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820 dx.doi.org/10.17504/protocols.io.biz5kf86 5. 709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing) V.2 dx.doi.org/10.17504/protocols.io.e6nvw1jk7lmk/v2 6. TotalSeq‱-A Antibodies and Cell Hashing with 10x Single Cell3' Reagent Kit v3 3.1 Protocol dx.doi.org/10.17504/protocols.io.8aahsae 7. Chromium Next GEM Single Cell 3’ Reagent Kits v3.1(Dual Index) User Guide. Document Number: CG000315. October 2022.

Instructions for delivery of investigator data via the SMDNAS02 drive.

This study presents postnatal transcriptomic changes in major cell populations in human lung, namely endothelial, epithelial, mesenchymal cells, and leukocytes. Although human postnatal lung development continues through early adulthood, our results demonstrate that greatest transcriptional changes occur in first few months of life during neonate to infant transition. These early transcriptional changes in lung parenchyma are particularly notable for functional maturation and activation of alveolar type II cell genes.

Signals from the microenvironment are known to be critical for development, sustaining adult stem cells, and for oncogenic progression. While candidate niche-driven signals that can promote cancer progression have been identified1-6, concerted efforts to comprehensively map microenvironmental ligands for cancer stem cell specific surface receptors have been lacking. Here, we use temporal single cell RNA-sequencing to identify molecular cues from the bone marrow stromal niche that engage leukemia stem cells (LSC) during oncogenic progression. We integrate these data with our RNA-seq analysis of human LSCs from distinct aggressive myeloid cancer subtypes and our CRISPR based in vivo LSC dependency map7 to develop a temporal receptor-ligand interactome essential for disease progression. These analyses identify the taurine transporter (TauT)-taurine axis as a critical dependency of myeloid malignancies. We show that taurine production is restricted to the osteolineage population during cancer initiation and expansion. Inhibiting taurine synthesis in osteolineage cells impairs LSC growth and survival. Our experiments with the TauT genetic loss of function murine model indicate that its loss significantly impairs the progression of aggressive myeloid leukemias in vivo by downregulating glycolysis. Further, TauT inhibition using a small molecule strongly impairs the growth and survival of patient derived myeloid leukemia cells. Finally, we show that TauT inhibition can synergize with the clinically approved oxidative phosphorylation inhibitor venetoclax8, 9 to block the growth of primary human leukemia cells. Given that aggressive myeloid leukemias continue to be refractory to current therapies and have poor prognosis, our work indicates targeting the taurine transporter may be of therapeutic significance. Collectively, our data establishes a temporal landscape of stromal signals during cancer progression and identifies taurine-taurine transporter signaling as an important new regulator of myeloid malignancies.

Detailed characterization of the B-lymphoblastic leukemia (B-ALL) cells which invade the central nervous system (CNS) has been limited by practical challenges. To test whether the clonal composition of the cerebrospinal fluid (CSF) reflects the primary B-ALL tissue, we applied immunoglobulin (Ig) high-throughput sequencing (HTS) of archival CSF cytospin preparations from six patients with morphologically defined CNS involvement. We discovered that most CSF clones are detectable at some timepoint in the primary tissue, but that shifting clonal abundance is prevalent across tissue sites between diagnosis and relapse. Ig HTS of CSF cytospins may improve understanding of sanctuary site dissemination in B-ALL.

Background: Intraclonal genetic variation in the immunoglobulin heavy chain variable gene (IGHV) has been widely documented in B-lymphoblastic leukemia (B-ALL), but its biologic significance as a presumed reflection of the B cell stage of leukemic transformation has not been systematically evaluated. Using ultradeep, targeted sequencing of the rearranged IgH gene in 22 patients with B-ALL, we previously demonstrated that there are subsets of patients with either extensive or minimal diversity in recombination activating gene (RAG)-mediated variable (V), diversity (D), and joining (J) gene rearrangements and that gene expression profiling (GEP) reveals enrichment of oxidative phosphorylation (OXPHOS), glycolysis, and fatty acid metabolism Hallmark pathways among cases with V-DJ rearrangement diversity (Fries et al. ASH 2022). We observed enrichment in these same metabolic signatures among leukemia cells with a pre-B cell phenotypic signature found to be predictive of relapse (Good et al. Nat Med 2018; Liu et al. ASH 2021). To understand the functional implications of this metabolic activation observed among cases with IgH diversity, we applied cytometry by time of flight (CyTOF) phenotyping to determine if IgH clonal composition reflects the metabolic and proliferative activation inherent to B cell stage of origin which may have relevance to disease behavior and outcome. Methods: Using viably-frozen, pre-treatment bone marrow (BM) and/or peripheral blood (PB) biospecimens from a cohort of 18 patients with B-ALL with known IgH clonal composition (7 with extensive V-DJ diversity, 8 without diversity, and 3 lacking any dominant clonal sequence), we performed deep phenotyping by mass cytometry and compared signaling pathway activation patterns between cohorts and compared to healthy controls. Samples were analyzed by mass cytometry with a 40-marker panel including B cell developmental phenotypic and metabolic proteins. Developmental classification was performed as previously described (Good et al. 2018). To test statistical significance in frequency of each subpopulation between two groups, we applied a multiple unpaired t-test with correction by Holm-Sidak method. Results: We discovered that all leukemia samples - regardless of IgH clonal composition - were significantly enriched for phenotypic features reflecting a pro-BII cell state, whereas enrichment of other more or less mature cell stages varied according to IgH composition. Developmental classification demonstrated that cases lacking a clonal IgH rearrangement (N=3) - presumed to have derived from a B cell stage preceding an initial DJ joining event - were enriched at the pro-BII population (P<0.0001), as were cases with extensive V-DJ diversity (N=7; P=0.019), and those lacking in diversity (N=8; P=0.0007) compared to healthy BM. However, cases with V-DJ diversity were uniformly enriched for OXPHOS, glycolytic, and pentose phosphate pathway (PPP)-associated protein expression compared to those without V-DJ diversity. In cells with a pro-BII phenotype, differentially expressed proteins between diverse and non-diverse cases included proteins essential for glycolysis (GLUT1, ENO1, PKM1), PPP (PGD, TKT), and OXPHOS (SDHA, CS, ATPA5, CytC). Conclusions: Cellular metabolism is a known hallmark of cancer proliferation, leukemia stem cell maintenance, and therapeutic escape, just as genetic diversification is integral to tumor evolution. Using primary B-ALL specimens, we show that metabolic pathway activation coincides with extensive RAG-mediated IGHV gene diversification across B cell developmental phenotypes. Contrary to the canonical assumption in B-ALL that V-DJ rearrangement and diversity is tied to developmental state, our analysis suggests that all cases are enriched at the proB-preB transition, but with differences in cellular metabolism that distinguish the diverse from homogeneous rearrangement state. These data suggest that IgH composition is not merely a passive genetic signature exploitable for tracking purposes in B-ALL, but may also inherently reflect underlying biology relevant to clinical outcomes.

Riboswitches regulate downstream gene expression by binding cellular metabolites. Regulation of translation initiation by riboswitches is posited to occur by metabolite-mediated sequestration of the Shine-Dalgarno sequence (SDS), causing bypass by the ribosome. Recently, we solved a co-crystal structure of a prequeuosine1-sensing riboswitch from Carnobacterium antarcticum that binds two metabolites in a single pocket. The structure revealed that the second nucleotide within the gene-regulatory SDS, G34, engages in a crystal contact, obscuring the molecular basis of gene regulation. Here, we report a co-crystal structure wherein C10 pairs with G34. However, molecular dynamics simulations reveal quick dissolution of the pair, which fails to reform. Functional and chemical probing assays inside live bacterial cells corroborate the dispensability of the C10-G34 pair in gene regulation, leading to the hypothesis that the compact pseudoknot fold is sufficient for translation attenuation. Remarkably, the C. antarcticum aptamer retained significant gene-regulatory activity when uncoupled from the SDS using unstructured spacers up to 10 nucleotides away from the riboswitch-akin to steric-blocking employed by sRNAs. Accordingly, our work reveals that the RNA fold regulates translation without SDS sequestration, expanding known riboswitch-mediated gene-regulatory mechanisms. The results infer that riboswitches exist wherein the SDS is not embedded inside a stable fold.

Hearing loss caused by the death of cochlear hair cells (HCs) might be restored through regeneration from supporting cells (SCs) via dedifferentiation and proliferation, as observed in birds. In a previous report, ERBB2 activation in a subset of cochlear SCs promoted widespread down-regulation of SOX2 in neighboring cells, proliferation, and the differentiation of HC-like cells. Here we analyze single cell transcriptomes from neonatal mouse cochlear SCs with activated ERBB2, with the goal of identifying potential secreted effectors. ERBB2 induction in vivo generated a new population of cells with de novo expression of a gene network. Called small integrin-binding ligand n-linked glycoproteins (SIBLINGs), these ligands and their regulators can alter NOTCH signaling and promote cell survival, proliferation, and differentiation in other systems. We validated mRNA expression of network members, and then extended our analysis to older stages. ERBB2 signaling in young adult SCs also promoted protein expression of gene network members. Furthermore, we found proliferating cochlear cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear SCs can alter the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.