Biology subjects

Levine, R. L.

Publications and source records attributed to Levine, R. L..

17 recordsLinked to original sources

bioRxiv2026

Generalist large language models complement tailor-made predictors for tumor genomics interpretation

General-purpose large language models (LLMs) are trained on large corpora to acquire broad knowledge, but whether LLMs can replace, or augment, task-specific models is unclear. We evaluated LLMs on three real-world, clinically important tumor genomic interpretation tasks, in order of increasing difficulty: (i) distinguishing tumor from non-tumor mutations (n=34,415 variants), (ii) distinguishing driver from passenger mutations (n=13,469 variants), and (iii) inferring cancer type from tumor sequencing reports across multiple assays and institutions (n=102,791 samples). The best general-purpose LLMs performed as well as the benchmark tailor-made predictor for task (i). Ensembling tailor-made models with zero-shot LLMs improved their performance for tasks (i) and (ii). For task (iii), LLMs outperformed or supplemented tailor-made models on out-of-distribution data. Without fine-tuning, current LLMs already can be useful in clinical genomic interpretation by adding complementary expertise to tailor-made, state-of-the-art predictors.

genomics↗

bioRxiv2025

scDNA: Single Cell DNA analysis software toolkit for subclonality discovery and assessment.

Advances in single cell multi-omics technologies have allowed for investigation into genotype-immunophenotype relationships and dynamic clonal changes in cancer patient samples. These technologies provide rich insights into genetic profiling, drivers of disease progression, and subclonal dynamics. Increased adoption and throughput of these technologies, necessitate accessible computational tools for processing and analysis. Toward developing easy to use computational tools, we introduce the Single Cell DNA (scDNA) package that allows for rapid analysis of single-cell molecular profiling. Our platform aims to provide a diagnostic summary tool for sample quality control, representative subclonal architecture within a sample, demultiplexing functionality for multi-sample processing, copy number variation, and mutation trajectory analysis to identify order of subclonal mutation acquisition. We showcase a series of vignettes on hematopoiesis datasets for each aim that reflects recently deployed uses of scDNA. Additionally, we show scDNA provides a modular, user-friendly framework that readily feeds into other standard software pipelines.

genomics↗

bioRxiv2025

CCAAT-enhancer binding protein delta functions as a tumor suppressor gene in acute myeloid leukemia

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

cancer biology↗

bioRxiv2025

Autocrine CXCL1-CXCR2 Signaling Mediates Leptomeningeal Resistance to Radiation Therapy

Leptomeningeal metastasis (LM) is a fatal neurological complication of cancer. Proton craniospinal irradiation (pCSI) has emerged as a promising life-prolonging intervention for LM patients, but the response to this treatment varies. Here, we aimed to characterize the molecular basis of pCSI resistance and response. Proteomic analysis of CSF collected from LM patients at baseline (before pCSI), and at multiple time points post-treatment, identified the CXC-motif chemokine, CXCL1, as associated with LM growth. Higher CXCL1 levels in the CSF prior to pCSI correlated with worse response to this treatment. To define the role of CXCL1 in LM, we established syngeneic mouse models of LM-CSI. We found that both metastatic cancer and host cells generate CXCL1. Genetic interruption of Cxcl1 expression in metastatic cancer, but not host cells, impaired cancer cell growth within the leptomeninges. Moreover, a subset of LM cancer cells expressed Cxcr2, the primary receptor for Cxcl1, and this population was enriched over time in the leptomeninges. Transcriptomic profiling of this rare population revealed an enrichment in pathways implicated in cell cycle progression. Finally, interruption of Cxcl1-Cxcr2 signaling with intrathecally-delivered Cxcr2 antagonist hampered LM growth and sensitized the cells to CSI. Our results demonstrate that the Cxcl1-Cxcr2 signaling axis mediates LM growth, and identifies a potential actionable intervention to improve response to pCSI and halt LM progression. One Sentence SummaryCXCL1-CXCR2 axis is a potential actionable therapeutic target to halt leptomeningeal metastasis progression and enhance response to craniospinal irradiation.

cancer biology↗

bioRxiv2024

Genotype-immunophenotype relationships in NPM1-mutant AML clonal evolution uncovered by single cell multiomic analysis

Acute myeloid leukemia (AML) is a multi-clonal disease, existing as a milieu of clones with unique but related genotypes as initiating clones acquire subsequent mutations. However, bulk sequencing cannot fully capture AML clonal architecture or the clonal evolution that occurs as patients undergo therapy. To interrogate clonal evolution, we performed simultaneous single cell molecular profiling and immunophenotyping on 43 samples from 32 NPM1-mutant AML patients at different stages of disease. Here we show that diagnosis and relapsed AML samples display similar clonal architecture patterns, but signaling mutations can drive increased clonal diversity specifically at relapse. We uncovered unique genotype-immunophenotype relationships regardless of disease state, suggesting leukemic lineage trajectories can be hard-wired by the mutations present. Analysis of longitudinal samples from patients on therapy identified dynamic clonal, transcriptomic, and immunophenotypic changes. Our studies provide resolved understanding of leukemic clonal evolution and the relationships between genotype and cell state in leukemia biology.

cancer biology↗

bioRxiv2024

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well- characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long- term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

bioengineering↗

bioRxiv2023

Deep generative model deciphers derailed trajectories in acute myeloid leukemia

Biological insights often depend on comparing conditions such as disease and health, yet we lack effective computational tools for integrating single-cell genomics data across conditions or characterizing transitions from normal to deviant cell states. Here, we present Decipher, a deep generative model that characterizes derailed cell-state trajectories. Decipher jointly models and visualizes gene expression and cell state from normal and perturbed single-cell RNA-seq data, revealing shared and disrupted dynamics. We demonstrate its superior performance across diverse contexts, including in pancreatitis with oncogene mutation, acute myeloid leukemia, and gastric cancer.

bioinformatics↗

bioRxiv2023

Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives. Key Points1.Sphingolipidomics separates acute myeloid leukemia (AML) patients and cell lines into two subtypes. 2.The subtype with low hexosylceramide and high sphingomyelin defines a new high-risk subtype with poor clinical outcomes.

systems biology↗

bioRxiv2022

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer with experimental CH driven by Dnmt3a+/{Delta}, we found higher tumor penetrance and increased tumor burden compared to controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency. This study provides conceptually novel insights into non-tumor-cell-autonomous effect of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies. SUMMARYA pre-clinical mouse model demonstrates that genetic alterations in the blood system characteristic of clonal hematopoiesis (CH) contribute to an aggressive solid tumor phenotype. It further identifies cancer angiogenesis as a potential therapeutic target to mitigate adverse CH effects.

cancer biology↗

bioRxiv2022

Jak2V617F Reversible Activation Shows an Essential Requirement for Jak2V617F in Myeloproliferative Neoplasms

Janus kinases (JAKs) mediate cytokine signaling, cell growth and hematopoietic differentiation.1 Gain-of-function mutations activating JAK2 signaling are seen in the majority of myeloproliferative neoplasm (MPN) patients, most commonly due to the JAK2V617F driver allele.2 While clinically-approved JAK inhibitors improve symptoms and outcomes in MPNs, remissions are rare, and mutant allele burden does not substantively change with chronic JAK inhibitor therapy in most patients.3, 4 This has been postulated to be due to incomplete dependence on constitutive JAK/STAT signaling, alternative signaling pathways, and/or the presence of cooperating disease alleles;5 however we hypothesize this is due to the inability of current JAK inhibitors to potently and specifically abrogate mutant JAK2 signaling. We therefore developed a conditionally inducible mouse model allowing for sequential activation, and then inactivation, of Jak2V617F from its endogenous locus using a Dre-rox/Cre-lox dual orthogonal recombinase system. Deletion of oncogenic Jak2V617Fabrogates the MPN disease phenotype, induces mutant-specific cell loss including in hematopoietic stem/progenitor cells, and extends overall survival to an extent not observed with pharmacologic JAK inhibition. Furthermore, reversal of Jak2V617F in MPN cells with antecedent loss of Tet26, 7 abrogates the MPN phenotype and inhibits mutant stem cell persistence suggesting cooperating epigenetic-modifying alleles do not alter dependence on mutant JAK/STAT signaling. Our results suggest that mutant-specific inhibition of JAK2V617F represents the best therapeutic approach for JAK2V617F-mutant MPN and demonstrate the therapeutic relevance of a dual-recombinase system to assess mutant-specific oncogenic dependencies in vivo.

cancer biology↗

bioRxiv2022

Modeling clonal evolution and oncogenic dependency in vivo in the context of hematopoietic transformation

Cancer evolution is a multifaceted process involving the acquisition of somatic mutations and progressive epigenetic dysregulation of cellular fate. Both cell-intrinsic mechanisms and environmental interactions provide selective pressures capable of promoting clonal evolution and expansion, with single-cell and bulk DNA sequencing offering increased resolution into this process1-4. Advances in genome editing, single-cell biology and expressed lentiviral barcoding have enabled new insights into how transcriptional/epigenetic states change with clonal evolution5,6. Despite the extensive catalog of genomic alterations revealed by resequencing studies7,8, there remain limited means to functionally model and perturb this evolutionary process in experimental systems9. Here we integrated multi-recombinase (Cre, Flp, and Dre) tools for modeling reversible, sequential mutagenesis from premalignant clonal hematopoiesis to acute myeloid leukemia. We demonstrate that somatic acquisition of Flt3 activating mutations elicits distinct phases of acute and chronic activation resulting in differential cooperativity with Npm1 and Dnmt3a disease alleles. We next developed a generalizable allelic framework allowing for the reversible expression of oncogenic mutations at their endogenous loci. We found that reversal of mutant Flt3 resulted in rapid leukemic regression with distinct alterations in cellular compartments depending upon co-occurring mutations. These studies provide a path to model sequential mutagenesis and deterministically investigate mechanisms of transformation and oncogenic dependency in the context of clonal evolution.

cancer biology↗

bioRxiv2022

Integrated Single-Cell Genotyping and Chromatin Accessibility Charts JAK2V617F Human Hematopoietic Differentiation

In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wildtype cells. To chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed Genotyping of Targeted loci with single-cell Chromatin Accessibility (GoT-ChA). This high-throughput, broadly accessible platform links genotypes to chromatin accessibility at single-cell resolution, across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from myeloproliferative neoplasm (MPN) patients with JAK2V617F-mutated hematopoiesis, where the JAK2 mutation is known to perturb hematopoietic differentiation. Differential accessibility analysis between wildtype and JAK2V617F mutant progenitors revealed both cell-intrinsic and cell state-specific shifts within mutant hematopoietic precursors. An early subset of mutant hematopoietic stem and progenitor cells (HSPCs) exhibited a cell-intrinsic pro-inflammatory signature characterized by increased NF-{kappa}B and JUN/FOS transcription factor motif accessibility. In addition, mutant HSPCs showed increased myeloid/erythroid epigenetic priming, preceding increased erythroid and megakaryocytic cellular output. Erythroid progenitors displayed aberrant regulation of the {gamma}-globin locus, providing an intrinsic epigenetic basis for the dysregulated fetal hemoglobin expression observed in MPNs. In contrast, megakaryocytic progenitors exhibited a more specialized inflammatory chromatin landscape relative to early HSPCs, with increased accessibility of pro-fibrotic JUN/FOS transcription factors. Notably, analysis of myelofibrosis patients treated with JAK inhibitors revealed an overall loss of mutant-specific phenotypes without modifying clonal burden, consistent with clinical responses. Finally, expansion of the multi-modality capability of GoT-ChA to integrate mitochondrial genome profiling and cell surface protein expression measurement enabled genotyping imputation and discovery of aberrant cellular phenotypes. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner. We envision that GoT-ChA will thus serve as a foundation for broad future explorations to uncover the critical link between mutated somatic genotypes and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

cancer biology↗

bioRxiv2020

Leukemia cell of origin influences apoptotic priming and sensitivity to LSD1 inhibition

Previous studies have established that the cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity. However, the mechanisms governing cell-of-origin-driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to cytotoxic chemotherapy and express high levels of the transcription factor Evi1 compared to leukemias derived from myeloid progenitors. Here, we compared drug sensitivity and expression profiles of murine and human leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for Evi1 in modulating p53 protein stability and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 inhibitors in addition to classical genotoxic stresses. p53 loss-of-function in Evi1low progenitor-derived leukemias induces resistance to LSD1 inhibition. By contrast, Evi1high leukemias are sensitized to LSD1 inhibition by the BH3 mimetic venetoclax, resulting in enhanced apoptosis and greater reductions in disease burden. Our findings demonstrate a cell-of-origin determined novel role for EVI1 in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in high-risk, chemoresistant EVI1high AML.

cancer biology↗

bioRxiv2020

Single cell mutational profiling delineates clonal trajectories in myeloid malignancies

Myeloid malignancies, including acute myeloid leukemia (AML), arise from the proliferation and expansion of hematopoietic stem and progenitor cells which acquire somatic mutations. Bulk molecular profiling studies on patient samples have suggested that somatic mutations are obtained in a step-wise fashion, where mutant genes with high variant allele frequencies (VAFs) are proposed to occur early in disease development and mutations with lower VAFs are thought to be acquired later in disease progression1-3. Although bulk sequencing informs leukemia biology and prognostication, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity and clone size, or offer definitive evidence of mutational order. To elucidate the clonal framework of myeloid malignancies, we performed single cell mutational profiling on 146 samples from 123 patients. We found AML is most commonly comprised of a small number of dominant clones, which in many cases harbor co-occurring mutations in epigenetic regulators. Conversely, mutations in signaling genes often occur more than once in distinct subclones consistent with increasing clonal diversity. We also used these data to map the clonal trajectory of each patient and found that specific mutation combinations (FLT3-ITD + NPM1c) synergize to promote clonal expansion and dominance. We combined cell surface protein expression with single cell mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our studies of clonal architecture at a single cell level provide novel insights into the pathogenesis of myeloid transformation and how clonal complexity contributes to disease progression.

cancer biology↗

bioRxiv2019

UVA and UVB elicit distinct mutational signatures in melanoma

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single, narrow band ultraviolet-A (UVA; 320-400nm) or ultraviolet-B (UVB; 280-320nm) exposure in mouse models predisposed to Braf- or Nras-mutant melanoma. Exposures approximated the amount of UVA or UVB energy contained in ~40 minutes of summer sunlight. Tumor onset was accelerated in all UVB-, but only half of UVA- irradiated mice as compared to unirradiated controls. Melanomas from both mouse models, harbored recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A irrespective of UV status. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many SNVs (1,025 vs. 435) and five times as many dipyrimidine variants (33.3 vs. 5.7) than tumors from similarly irradiated Nras-mutant mice. We identified a mutational signature enriched in UVB-accelerated tumors which mirrored COSMIC signatures associated with human skin cancer. Notably, this signature was enriched to a greater extent in Braf- than Nras-mutant murine melanomas. These data suggest that oncogenic BRAF may enhance UVB carcinogenesis to promote melanoma formation at anatomic sites with low or intermittent sun exposure.

cancer biology↗

bioRxiv2019

Stag1 and Stag2 regulate cell fate decisions in hematopoiesis through non-redundant topological control

Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains unresolved. We show Stag2 deletion in hematopoietic stem/progenitor cells (HSPC) results in altered hematopoietic function, increased self-renewal, and impaired differentiation. ChIP-sequencing revealed that while Stag2 and Stag1 can bind the same loci, a component of Stag2 binding sites are unoccupied by Stag1 even in Stag2-deficient HSPCs. While concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to blunted HSPC commitment to the B-cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.\n\nOne Sentence SummaryStag1 rescues topologically associated domains in the absence of Stag2, but cannot restore chromatin architecture required for hematopoietic lineage commitment

cell biology↗

bioRxiv2018

Isoform switching as a mechanism of acquired resistance to isocitrate dehydrogenase inhibition

Somatic mutations in cytosolic or mitochondrial isoforms of isocitrate dehydrogenase (IDH1 or IDH2, respectively) contribute to oncogenesis via production of the metabolite 2-hydroxyglutarate (2HG). Isoform-selective IDH inhibitors suppress 2HG production and induce clinical responses in patients with IDH1- and IDH2-mutant malignancies. Despite the promising activity of IDH inhibitors, the mechanisms that mediate resistance to IDH inhibition are poorly understood. Here, we describe four clinical cases that identify mutant IDH isoform switching, either from mutant IDH1 to mutant IDH2 or vice versa, as a mechanism of acquired clinical resistance to IDH inhibition in solid and liquid tumors.\n\nSignificanceIDH-mutant cancers can develop resistance to isoform-selective IDH inhibition by \"isoform switching\" from mutant IDH1 to mutant IDH2 or vice versa, thereby restoring 2-hydroxyglutarate (2HG) production by the tumor. These findings underscore a role for continued 2HG production in tumor progression and suggest therapeutic strategies to prevent or overcome resistance.

cancer biology↗