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Exploring the complexity of the epithelial-to-mesenchymal transition (EMT) unveils a diversity of potential cell fates; however, the exact timing and mechanisms by which early cell states diverge into distinct EMT trajectories remain unclear. Studying these EMT trajectories through single-cell RNA sequencing is challenging due to the necessity of sacrificing cells for each measurement. In this study, we employed optimal-transport analysis to reconstruct the past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct trajectories leading to low EMT, partial EMT, and high EMT states. Cells along the partial EMT trajectory showed substantial variations in the EMT signature and exhibited pronounced stemness. Throughout this EMT trajectory, we observed a consistent downregulation of the EED and EZH2 genes. This finding was validated by recent inhibitor screens of EMT regulators and CRISPR screen studies. Moreover, we applied our analysis of early-phase differential gene expression to gene sets associated with stemness and proliferation, pinpointing ITGB4 , LAMA3 , and LAMB3 as genes differentially expressed in the initial stages of the partial versus high EMT trajectories. We also found that CENPF , CKS1B , and MKI67 showed significant upregulation in the high EMT trajectory. While the first group of genes aligns with findings from previous studies, our work uniquely pinpoints the precise timing of these upregulations. Finally, the identification of the latter group of genes sheds light on potential cell cycle targets for modulating EMT trajectories., Competing Interests: Competing interests statement:F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for both companies. She is also on the board of directors of Exscientia Plc. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. One of the authors (H.L.) was co-author on a review paper (in 2021) with one of the referees (Y.K.), and a different author (M.K.J.) was co-author on a different review paper (in 2023) with the other referee (Q.N.). All other authors declare no conflicts.

Genetically identical cells can respond heterogeneously to cancer therapy, with a subpopulation of cells often entering a temporarily arrested treatment-tolerant state before repopulating the tumor. To investigate how heterogeneity in the cell cycle arrest protein p21 arises, we imaged the dynamics of p21 transcription and protein expression along with those of p53, its transcriptional regulator, in single cells using live cell fluorescence microscopy. Surprisingly, we found that the rate of p21 transcription depends on the change in p53 rather than its absolute level. Through combined theoretical and experimental modeling, we determined that p21 transcription is governed by an incoherent feedforward loop mediated by MDM2. This network architecture facilitates rapid induction of p21 expression and variability in p21 transcription. Abrogating the feedforward loop overcomes rapid S-phase p21 degradation, with cells transitioning into a quiescent state that transcriptionally resembles a treatment-tolerant persister state. Our findings have important implications for therapeutic strategies based on activating p53.

Purpose: Identifying molecular and immune features to guide immune checkpoint inhibitor (ICI)-based regimens remains an unmet clinical need., Experimental Design: Tissue and longitudinal blood specimens from phase III trial S1400I in patients with metastatic squamous non-small cell carcinoma (SqNSCLC) treated with nivolumab monotherapy (nivo) or nivolumab plus ipilimumab (nivo+ipi) were subjected to multi-omics analyses including multiplex immunofluorescence (mIF), nCounter PanCancer Immune Profiling Panel, whole-exome sequencing, and Olink., Results: Higher immune scores from immune gene expression profiling or immune cell infiltration by mIF were associated with response to ICIs and improved survival, except regulatory T cells, which were associated with worse overall survival (OS) for patients receiving nivo+ipi. Immune cell density and closer proximity of CD8+GZB+ T cells to malignant cells were associated with superior progression-free survival and OS. The cold immune landscape of NSCLC was associated with a higher level of chromosomal copy-number variation (CNV) burden. Patients with LRP1B-mutant tumors had a shorter survival than patients with LRP1B-wild-type tumors. Olink assays revealed soluble proteins such as LAMP3 increased in responders while IL6 and CXCL13 increased in nonresponders. Upregulation of serum CXCL13, MMP12, CSF-1, and IL8 were associated with worse survival before radiologic progression., Conclusions: The frequency, distribution, and clustering of immune cells relative to malignant ones can impact ICI efficacy in patients with SqNSCLC. High CNV burden may contribute to the cold immune microenvironment. Soluble inflammation/immune-related proteins in the blood have the potential to monitor therapeutic benefit from ICI treatment in patients with SqNSCLC., (©2024 The Authors; Published by the American Association for Cancer Research.)

Immune checkpoint blockade (ICB) therapy targeting cytotoxic T-lymphocyte-associated protein 4, programmed death 1, and programmed death ligand 1 has shown durable remission and clinical success across different cancer types. However, patient outcomes vary among disease indications. Studies have identified prognostic biomarkers associated with immunotherapy response and patient outcomes derived from diverse data types, including next-generation bulk and single-cell DNA, RNA, T cell and B cell receptor sequencing data, liquid biopsies, and clinical imaging. Owing to inter- and intra-tumor heterogeneity and the immune system's complexity, these biomarkers have diverse efficacy in clinical trials of ICB. Here, we review the genetic and genomic signatures and image features of ICB studies for pan-cancer applications and specific indications. We discuss the advantages and disadvantages of computational approaches for predicting immunotherapy effectiveness and patient outcomes. We also elucidate the challenges of immunotherapy prognostication and the discovery of novel immunotherapy targets., Competing Interests: Declaration of interests C.J. Wu holds equity in BioNTech, Inc., and receives research support from Pharmacyclics. F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for both companies. She is also on the board of directors of Exscientia Plc. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript., (Copyright © 2023. Published by Elsevier Inc.)

Chimeric antigen receptor T cells (CAR-Ts) have remarkable efficacy in liquid tumors, but limited responses in solid tumors. We conducted a Phase I trial (NCT02107963) of GD2 CAR-Ts (GD2-CAR.OX40.28.z.iC9), demonstrating feasibility and safety of administration in children and young adults with osteosarcoma and neuroblastoma. Since CAR-T efficacy requires adequate CAR-T expansion, patients were grouped into good or poor expanders across dose levels. Patient samples were evaluated by multi-dimensional proteomic, transcriptomic, and epigenetic analyses. T cell assessments identified naive T cells in pre-treatment apheresis associated with good expansion, and exhausted T cells in CAR-T products with poor expansion. Myeloid cell assessment identified CXCR3 + monocytes in pre-treatment apheresis associated with good expansion. Longitudinal analysis of post-treatment samples identified increased CXCR3 - classical monocytes in all groups as CAR-T numbers waned. Together, our data uncover mediators of CAR-T biology and correlates of expansion that could be utilized to advance immunotherapies for solid tumor patients., Competing Interests: Declaration of interests C.J.W. receives research funding from Pharmacyclics and hold equity in BioNTech, Inc. F.M. is a cofounder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for Harbinger Health, Zephyr AI, and Red Cell Partners and Exscientia. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. E.S. consults for and holds equity in Lyell Immunopharma and consults for Lepton Pharmaceuticals and Galaria. M.S.M. is currently employed at Normunity and holds stock in AstraZeneca; her contributions to this work were made prior to these industry positions which are not relevant to the content of this manuscript. C.L.M. is an inventor on numerous patents and patents pending related to CAR-T cell therapies. C.L.M. holds equity in and receives research funding from Lyell Immunopharma and holds equity in and consults for CARGO Therapeutics and Link Cell Therapies. C.L.M. consults for Immatics, Mammoth, Ensoma, and Red Tree Venture Capital., (Published by Elsevier Inc.)

Triple-negative breast cancer (TNBC) is a heterogeneous disease with limited treatment options. To characterize TNBC heterogeneity, we defined transcriptional, epigenetic, and metabolic subtypes and subtype-driving super-enhancers and transcription factors by combining functional and molecular profiling with computational analyses. Single-cell RNA sequencing revealed relative homogeneity of the major transcriptional subtypes (luminal, basal, and mesenchymal) within samples. We found that mesenchymal TNBCs share features with mesenchymal neuroblastoma and rhabdoid tumors and that the PRRX1 transcription factor is a key driver of these tumors. PRRX1 is sufficient for inducing mesenchymal features in basal but not in luminal TNBC cells via reprogramming super-enhancer landscapes, but it is not required for mesenchymal state maintenance or for cellular viability. Our comprehensive, large-scale, multiplatform, multiomics study of both experimental and clinical TNBC is an important resource for the scientific and clinical research communities and opens venues for future investigation., Competing Interests: Declaration of interests The following authors report current employment: Eli Lilly (B.J.), Shasqi, Inc (M.A.), GenieUsGenomics (A.T.), Morrison & Foerster LLP (A.G.), AstraZeneca (M.B.E. and L.E.S.), Odyssey Therapeutics (J.D.J.). K.P. serves on the Scientific Advisory Boards (SABs) of Novartis, Ideaya Biosciences, and Scorpion Therapeutics; holds equity options in Scorpion Therapeutics and Ideaya Biosciences; and receives sponsored research funding from Novartis, where she consults. F.M. is a cofounder of and has equity in Harbinger Health, has equity in Zephyr AI, and consults for Harbinger Health and Zephyr AI. She is on the board of directors of Exscientia Plc. She declares that none of these relationships are directly or indirectly related to the content of this manuscript. P.S. is a consultant for Novartis, Genovis, Guidepoint, The Planning Shop, ORIC Pharmaceuticals, Cedilla Therapeutics, Syros Pharmaceuticals, Blueprint Medicines, Curie Bio, Differentiated Therapeutics, Excientia, Ligature Therapeutics, Merck, Redesign Science, Sibylla Biotech, and Exo Therapeutics; he receives research funding from Novartis. A.G.L. serves on the SAB of Flash Therapeutics, Zentalis Pharmaceuticals, and Trueline Therapeutics and consults for AbbVie. M.B. receives research funding from Novartis, where he also serves on the SAB and acts as a consultant. He is a member of the SAB for Kronos Bio and GV20 Therapeutics and holds equity in both companies. He also serves on the SAB for FibroGen and is a consultant for Belharra Therapeutics. K.W.W. serves on the SAB of TScan Therapeutics, SQZ Biotech, Bisou Bioscience Company, DEM BioPharma, and Nextechinvest; receives sponsored research funding from Novartis; and is a co-founder, stockholder, and advisory board member of Immunitas Therapeutics. D.D. receives research support from Canon, Inc. H.W.L. receives research funding from Novartis., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Researchers are leveraging what we have learned from model organisms to understand if the same principles arise in human physiology, development, and disease. In this collection of Voices, we asked researchers from different fields to discuss what tools and insights they are using to answer fundamental questions in human biology., Competing Interests: Declaration of interests V.G.S. serves as an advisor to and/or has equity in Branch Biosciences, Ensoma, and Cellarity, all unrelated to the present work. F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, serves as a consultant for Harbinger Health and Zephyr AI, and is on the board of directors of Exscientia Plc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Immune escape is a prerequisite for tumor growth. We previously described a decline in intratumor activated cytotoxic T cells and T cell receptor (TCR) clonotype diversity in invasive breast carcinomas compared to ductal carcinoma in situ (DCIS), implying a central role of decreasing T cell responses in tumor progression. To determine potential associations between peripheral immunity and breast tumor progression, here, we assessed the peripheral blood TCR clonotype of 485 breast cancer patients diagnosed with either DCIS or de novo stage IV disease at younger (<45) or older (≥45) age. TCR clonotype diversity was significantly lower in older compared to younger breast cancer patients regardless of tumor stage at diagnosis. In the younger age group, TCR-α clonotype diversity was lower in patients diagnosed with de novo stage IV breast cancer compared to those diagnosed with DCIS. In the older age group, DCIS patients with higher TCR-α clonotype diversity were more likely to have a recurrence compared to those with lower diversity. Whole blood transcriptome profiles were distinct depending on the TCR-α Chao1 diversity score. There were more CD8 + T cells and a more active immune environment in DCIS tumors of young patients with higher peripheral blood TCR-α Chao1 diversity than in those with lower diversity. These results provide insights into the role that host immunity plays in breast cancer development across different age groups., Competing Interests: Competing interests statement:K.P. serves on the Scientific Advisory Boards of Novartis, Ideaya Biosciences, and Scorpion Therapeutics, holds equity options in Scorpion Therapeutics and Ideaya Biosciences, and receives sponsored research funding through Dana-Farber from Novartis. F.M. is a cofounder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for Harbinger Health, and Zephyr AI. She is also on the board of directors of Exscientia Plc. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. D.D. receives research funding from Canon, Inc. J.P. is currently an employee of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. J.P. contributed to this work prior to their employment at Merck Sharp & Dohme LLC. The opinions or perspectives expressed herein do not represent the opinions or perspectives of Merck Sharp & Dohme LLC.

Cyclin-dependent kinases 4/6 (CDK4/6) inhibitors such as palbociclib are approved for the treatment of metastatic estrogen receptor-positive (ER+) breast cancer in combination with endocrine therapies and significantly improve outcomes in patients with this disease. However, given the large number of possible pairwise drug combinations and administration schedules, it remains unclear which clinical strategy would lead to best survival. Here, we developed a computational, cell cycle-explicit model to characterize the pharmacodynamic response to palbociclib-fulvestrant combination therapy. This pharmacodynamic model was parameterized, in a Bayesian statistical inference approach, using in vitro data from cells with wild-type estrogen receptor (WT-ER) and cells expressing the activating missense ER mutation, Y537S, which confers resistance to fulvestrant. We then incorporated pharmacokinetic models derived from clinical data into our computational modeling platform. To systematically compare dose administration schedules, we performed in silico clinical trials based on integrating our pharmacodynamic and pharmacokinetic models as well as considering clinical toxicity constraints. We found that continuous dosing of palbociclib is more effective for lowering overall tumor burden than the standard, pulsed-dose palbociclib treatment. Importantly, our mathematical modeling and statistical analysis platform provides a rational method for comparing treatment strategies in search of optimal combination dosing strategies of other cell-cycle inhibitors in ER+ breast cancer., Significance: We created a computational modeling platform to predict the effects of fulvestrant/palbocilib treatment on WT-ER and Y537S-mutant breast cancer cells, and found that continuous treatment schedules are more effective than the standard, pulsed-dose palbociclib treatment schedule., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Monocytes and monocyte-derived macrophages (MDMs) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here, we show that PDGFB-driven GBM cells induce the expression of the potent proinflammatory cytokine IL-1β in MDM, which engages IL-1R1 in tumor cells, activates the NF-κB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1β/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1β/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, and reduced exhausted CD8+ T cells and thereby extends the survival of tumor-bearing mice. In contrast to IL-1β, IL-1α exhibits antitumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss-of-interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively active NF-κB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1β could be considered as an effective therapy specifically for proneural GBM.

The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells. At a cellular level, H3K36M facilitates epithelial plasticity by rendering fibroblasts insensitive to TGFβ signals. At a molecular level, H3K36M enables the decommissioning of mesenchymal enhancers and the parallel activation of epithelial/stem cell enhancers. This enhancer rewiring is Tet dependent and redirects Sox2 from promiscuous somatic to pluripotency targets. Our findings reveal a previously unappreciated dual role for H3K36 methylation in the maintenance of cell identity by integrating a crucial developmental pathway into sustained expression of cell-type-specific programmes, and by opposing the expression of alternative lineage programmes through enhancer methylation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

RNA-sequencing (RNA-seq) has become an increasingly cost-effective technique for molecular profiling and immune characterization of tumors. In the past decade, many computational tools have been developed to characterize tumor immunity from gene expression data. However, the analysis of large-scale RNA-seq data requires bioinformatics proficiency, large computational resources and cancer genomics and immunology knowledge. In this tutorial, we provide an overview of computational analysis of bulk RNA-seq data for immune characterization of tumors and introduce commonly used computational tools with relevance to cancer immunology and immunotherapy. These tools have diverse functions such as evaluation of expression signatures, estimation of immune infiltration, inference of the immune repertoire, prediction of immunotherapy response, neoantigen detection and microbiome quantification. We describe the RNA-seq IMmune Analysis (RIMA) pipeline integrating many of these tools to streamline RNA-seq analysis. We also developed a comprehensive and user-friendly guide in the form of a GitBook with text and video demos to assist users in analyzing bulk RNA-seq data for immune characterization at both individual sample and cohort levels by using RIMA., (© 2023. Springer Nature Limited.)

Background: Glioblastomas comprise heterogeneous cell populations with dynamic, bidirectional plasticity between treatment-resistant stem-like and treatment-sensitive differentiated states, with treatment influencing this process. However, current treatment protocols do not account for this plasticity. Previously, we generated a mathematical model based on preclinical experiments to describe this process and optimize a radiation therapy fractionation schedule that substantially increased survival relative to standard fractionation in a murine glioblastoma model., Methods: We developed statistical models to predict the survival benefit of interventions to glioblastoma patients based on the corresponding survival benefit in the mouse model used in our preclinical study. We applied our mathematical model of glioblastoma radiation response to optimize a radiation therapy fractionation schedule for patients undergoing re-irradiation for glioblastoma and developed a first-in-human trial (NCT03557372) to assess the feasibility and safety of administering our schedule., Results: Our statistical modeling predicted that the hazard ratio when comparing our novel radiation schedule with a standard schedule would be 0.74. Our mathematical modeling suggested that a practical, near-optimal schedule for re-irradiation of recurrent glioblastoma patients was 3.96 Gy × 7 (1 fraction/day) followed by 1.0 Gy × 9 (3 fractions/day). Our optimized schedule was successfully administered to 14/14 (100%) patients., Conclusions: A novel radiation therapy schedule based on mathematical modeling of cell-state plasticity is feasible and safe to administer to glioblastoma patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Purpose: Primary and acquired resistance to osimertinib remain significant challenges for patients with EGFR-mutant lung cancers. Acquired EGFR alterations such as EGFR T790M or C797S mediate resistance to EGFR tyrosine kinase inhibitors (TKI) and combination therapy with dual EGFR TKIs may prevent or reverse on-target resistance., Patients and Methods: We conducted two prospective, phase I/II trials assessing combination osimertinib and dacomitinib to address on-target resistance in the primary and acquired resistance settings. In the initial therapy study, patients received dacomitinib and osimertinib in combination as initial therapy. In the acquired resistance trial, dacomitinib with or without osimertinib was administered to patients with EGFR-mutant lung cancers with disease progression on osimertinib alone and evidence of an acquired EGFR second-site mutation., Results: Cutaneous toxicities occurred in 93% (any grade) of patients and diarrhea in 72% (any grade) with the combination. As initial therapy, the overall response to the combination was 73% [95% confidence interval (CI), 50%-88%]. No acquired secondary alterations in EGFR were observed in any patients at progression. In the acquired resistance setting, the overall response was 14% (95% CI, 1%-58%)., Conclusions: We observed no acquired secondary EGFR alterations with dual inhibition of EGFR as up-front treatment, but this regimen was associated with greater toxicity. The combination was not effective in reversing acquired resistance after development of a second-site acquired EGFR alteration. Our study highlights the need to develop better strategies to address on-target resistance in patients with EGFR-mutant lung cancers., (©2023 American Association for Cancer Research.)

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329., (© 2023 by The American Society of Hematology.)

Glioblastoma (GBM) is the most aggressive brain tumor, with a median survival of ∼15 months. Targeted approaches have not been successful in this tumor type due to the large extent of intratumor heterogeneity. Mosaic amplification of oncogenes suggests that multiple genetically distinct clones are present in each tumor. To uncover the relationships between genetically diverse subpopulations of GBM cells and their native tumor microenvironment, we employ highly multiplexed spatial protein profiling coupled with single-cell spatial mapping of fluorescence in situ hybridization (FISH) for EGFR, CDK4, and PDGFRA. Single-cell FISH analysis of a total of 35,843 single nuclei reveals that tumors in which amplifications of EGFR and CDK4 more frequently co-occur in the same cell exhibit higher infiltration of CD163 + immunosuppressive macrophages. Our results suggest that high-throughput assessment of genomic alterations at the single-cell level could provide a measure for predicting the immune state of GBM., Competing Interests: Declaration of interests M.J. is a member of a scientific advisory board at ResistanceBio (formerly Viosera Therapeutics)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 ( T ranscript R egulating E ndoderm and activated by so X17 ) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation., Competing Interests: AL, AB, HK, CM, RB, AR, HW, SM, BB, PG, RT, KP, JH, TM, DM, RM, DH, FM, JR, AM No competing interests declared, (© 2023, Landshammer, Bolondi et al.)

Inflammatory breast cancer (IBC) is a difficult-to-treat disease with poor clinical outcomes due to high risk of metastasis and resistance to treatment. In breast cancer, CD44+CD24- cells possess stem cell-like features and contribute to disease progression, and we previously described a CD44+CD24-pSTAT3+ breast cancer cell subpopulation that is dependent on JAK2/STAT3 signaling. Here we report that CD44+CD24- cells are the most frequent cell type in IBC and are commonly pSTAT3+. Combination of JAK2/STAT3 inhibition with paclitaxel decreased IBC xenograft growth more than either agent alone. IBC cell lines resistant to paclitaxel and doxorubicin were developed and characterized to mimic therapeutic resistance in patients. Multi-omic profiling of parental and resistant cells revealed enrichment of genes associated with lineage identity and inflammation in chemotherapy-resistant derivatives. Integrated pSTAT3 chromatin immunoprecipitation sequencing and RNA sequencing (RNA-seq) analyses showed pSTAT3 regulates genes related to inflammation and epithelial-to-mesenchymal transition (EMT) in resistant cells, as well as PDE4A, a cAMP-specific phosphodiesterase. Metabolomic characterization identified elevated cAMP signaling and CREB as a candidate therapeutic target in IBC. Investigation of cellular dynamics and heterogeneity at the single cell level during chemotherapy and acquired resistance by CyTOF and single cell RNA-seq identified mechanisms of resistance including a shift from luminal to basal/mesenchymal cell states through selection for rare preexisting subpopulations or an acquired change. Finally, combination treatment with paclitaxel and JAK2/STAT3 inhibition prevented the emergence of the mesenchymal chemo-resistant subpopulation. These results provide mechanistic rational for combination of chemotherapy with inhibition of JAK2/STAT3 signaling as a more effective therapeutic strategy in IBC., Significance: Chemotherapy resistance in inflammatory breast cancer is driven by the JAK2/STAT3 pathway, in part via cAMP/PKA signaling and a cell state switch, which can be overcome using paclitaxel combined with JAK2 inhibitors., (©2022 The Authors; Published by the American Association for Cancer Research.)

Cancer prevention has a profound impact on cancer-associated mortality and morbidity. We previously identified TGFβ signaling as a candidate regulator of mammary epithelial cells associated with breast cancer risk. Here, we show that short-term TGFBR inhibitor (TGFBRi) treatment of peripubertal ACI inbred and Sprague Dawley outbred rats induces lasting changes and prevents estrogen- and carcinogen-induced mammary tumors, respectively. We identify TGFBRi-responsive cell populations by single cell RNA-sequencing, including a unique epithelial subpopulation designated secretory basal cells (SBCs) with progenitor features. We detect SBCs in normal human breast tissues and find them to be associated with breast cancer risk. Interactome analysis identifies SBCs as the most interactive cell population and the main source of insulin-IGF signaling. Accordingly, inhibition of TGFBR and IGF1R decrease proliferation of organoid cultures. Our results reveal a critical role for TGFβ in regulating mammary epithelial cells relevant to breast cancer and serve as a proof-of-principle cancer prevention strategy., (© 2022. The Author(s).)

Patients with non-small cell lung cancer (NSCLC) who have distant metastases have a poor prognosis. To determine which genomic factors of the primary tumor are associated with metastasis, we analyzed data from 759 patients originally diagnosed with stage I-III NSCLC as part of the AACR Project GENIE Biopharma Collaborative consortium. We found that TP53 mutations were significantly associated with the development of new distant metastases. TP53 mutations were also more prevalent in patients with a history of smoking, suggesting that these patients may be at increased risk for distant metastasis. Our results suggest that additional investigation of the optimal management of patients with early-stage NSCLC harboring TP53 mutations at diagnosis is warranted in light of their higher likelihood of developing new distant metastases., (© 2022. The Author(s).)

Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the β-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Intra-tumor heterogeneity (ITH) of human tumors is important for tumor progression, treatment response, and drug resistance. However, the spatial distribution of ITH remains incompletely understood. Here, we present spatial analysis of ITH in lung adenocarcinomas from 147 patients using multi-region mass spectrometry of >5,000 regions, single-cell copy number sequencing of ~2,000 single cells, and cyclic immunofluorescence of >10 million cells. We identified two distinct spatial patterns among tumors, termed clustered and random geographic diversification (GD). These patterns were observed in the same samples using both proteomic and genomic data. The random proteomic GD pattern, which is characterized by decreased cell adhesion and lower levels of tumor-interacting endothelial cells, was significantly associated with increased risk of recurrence or death in two independent patient cohorts. Our study presents comprehensive spatial mapping of ITH in lung adenocarcinoma and provides insights into the mechanisms and clinical consequences of GD., Competing Interests: DECLARATION OF INTERESTS The other authors declare no competing financial interests.

Cancer is one of the leading causes of death, but mortality can be reduced by detecting tumors earlier so that treatment is initiated at a less aggressive stage. The tradeoff between costs associated with screening and its benefit makes the decision of whom to screen and when a challenge. To enable comparisons across screening strategies for any cancer type, we demonstrate a mathematical modeling platform based on the theory of queuing networks designed for quantifying the benefits of screening strategies. Our methodology can be used to design optimal screening protocols and to estimate their benefits for specific patient populations. Our method is amenable to exact analysis, thus circumventing the need for simulations, and is capable of exactly quantifying outcomes given variability in the age of diagnosis, rate of progression, and screening sensitivity and intervention outcomes. We demonstrate the power of this methodology by applying it to data from the Surveillance, Epidemiology and End Results (SEER) program. Our approach estimates the benefits that various novel screening programs would confer to different patient populations, thus enabling us to formulate an optimal screening allocation and quantify its potential effects for any cancer type and intervention., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: F.M. is a co-founder and consultant of Harbinger Health and is a consultant for Red Cell Partners and Zephyr AI. No other conflicts to declare.

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34 + -enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34 - bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells., Competing Interests: Conflict of interest disclosure A.M. has consulted for Janssen, PharmaEssentia, Constellation and receives research funding from Relay Therapeutics. E.S.W. reports personal fees from Jazz Pharmaceuticals, Takeda Pharmaceutical Company, Novartis, and Pfizer. F.M. is the co-founder of an oncology company. J.S.G. has consulted for AbbVie, Takeda, and Astellas and receives research support from AbbVie, Genentech, Prelude, AstraZeneca, and Eli Lilly. D.J.D. receives research support from Glycomimetics, Novartis, AbbVie, and Blueprint Medicines and has consulted for Incyte, Jazz, Novartis, Pfizer, Shire, Takeda, Amgen, Forty-Seven, Agios, Autolos, and Blueprint Medicines. G.S.H. has received research support from Bayer, Merck, Incyte, and Constellation and has received honoraria from Constellation, Jazz, Novartis, and Celgene/BMS. R.M.S. has advisory board, DSMB, and/or steering committee membership at Syntrix/ACI Clinical, Takeda, Elevate Bio, Syndax Pharma, AbbVie, Syros, Gemoab, BerGenBio, Foghorn Thera, GSK, Aprea, Innate, Actinium, and OncoNova. M.R.L. has received research support from AbbVie and Novartis., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Mapping the genetic basis of complex traits is critical to uncovering the biological mechanisms that underlie disease and other phenotypes. Genome-wide association studies (GWAS) in humans and quantitative trait locus (QTL) mapping in model organisms can now explain much of the observed heritability in many traits, allowing us to predict phenotype from genotype. However, constraints on power due to statistical confounders in large GWAS and smaller sample sizes in QTL studies still limit our ability to resolve numerous small-effect variants, map them to causal genes, identify pleiotropic effects across multiple traits, and infer non-additive interactions between loci (epistasis). Here, we introduce barcoded bulk quantitative trait locus (BB-QTL) mapping, which allows us to construct, genotype, and phenotype 100,000 offspring of a budding yeast cross, two orders of magnitude larger than the previous state of the art. We use this panel to map the genetic basis of eighteen complex traits, finding that the genetic architecture of these traits involves hundreds of small-effect loci densely spaced throughout the genome, many with widespread pleiotropic effects across multiple traits. Epistasis plays a central role, with thousands of interactions that provide insight into genetic networks. By dramatically increasing sample size, BB-QTL mapping demonstrates the potential of natural variants in high-powered QTL studies to reveal the highly polygenic, pleiotropic, and epistatic architecture of complex traits., Competing Interests: AN, KL, AR, SG, DT, FM, MD No competing interests declared, (© 2022, Nguyen Ba et al.)

Further analysis of SARS-CoV-2 genome sequencing data identifies several highly recurrent genetic variants with low allele frequencies, which, if filtered out, provide estimates consistent with tighter transmission bottlenecks.

Intratumor heterogeneity is postulated to cause therapeutic resistance. To prospectively assess the impact of HER2 ( ERBB2 ) heterogeneity on response to HER2-targeted therapy, we treated 164 patients with centrally confirmed HER2-positive early-stage breast cancer with neoadjuvant trastuzumab emtansine plus pertuzumab. HER2 heterogeneity was assessed on pretreatment biopsies from two locations of each tumor. HER2 heterogeneity, defined as an area with ERBB2 amplification in >5% but <50% of tumor cells, or a HER2-negative area by FISH, was detected in 10% (16/157) of evaluable cases. The pathologic complete response rate was 55% in the nonheterogeneous subgroup and 0% in the heterogeneous group ( P < 0.0001, adjusted for hormone receptor status). Single-cell ERBB2 FISH analysis of cellular heterogeneity identified the fraction of ERBB2 nonamplified cells as a driver of therapeutic resistance. These data suggest HER2 heterogeneity is associated with resistance to HER2-targeted therapy and should be considered in efforts to optimize treatment strategies. SIGNIFICANCE: HER2-targeted therapies improve cure rates in HER2-positive breast cancer, suggesting chemotherapy can be avoided in a subset of patients. We show that HER2 heterogeneity, particularly the fraction of ERBB2 nonamplified cancer cells, is a strong predictor of resistance to HER2 therapies and could potentially be used to optimize treatment selection. See related commentary by Okines and Turner, p. 2369 . This article is highlighted in the In This Issue feature, p. 2355 ., (©2021 American Association for Cancer Research.)

Systematic collection of fresh tissues for research at the time of diagnostic image-guided breast biopsy has the potential to fuel a wide variety of innovative studies. Here we report the initial experience, including safety, feasibility, and laboratory proof-of-principle, with the collection and analysis of research specimens obtained via breast core needle biopsy immediately following routine clinical biopsy at a single institution over a 14-month period. Patients underwent one or two additional core biopsies following collection of all necessary clinical specimens. In total, 395 patients were approached and 270 consented to the research study, yielding a 68.4% consent rate. Among consenting patients, 238 lesions were biopsied for research, resulting in 446 research specimens collected. No immediate complications were observed. Representative research core specimens showed high diagnostic concordance with clinical core biopsies. Flow cytometry demonstrated consistent recovery of hundreds to thousands of viable cells per research core. Among a group of HER2 + tumor research specimens, HER2 assessment by flow cytometry correlated highly with immunohistochemistry (IHC) staining, and in addition revealed extensive inter- and intra-tumoral variation in HER2 levels of potential clinical relevance. Suitability for single-cell transcriptomic analysis was demonstrated for a triple-negative tumor core biopsy, revealing substantial cellular diversity in the tumor immune microenvironment, including a prognostically relevant T cell subpopulation. Thus, collection of fresh tissues for research purposes at the time of diagnostic breast biopsy is safe, feasible and efficient, and may provide a high-yield mechanism to generate a rich tissue repository for a wide variety of cross-disciplinary research., (© 2021. The Author(s).)