Biology subjects

Stegmaier, K.

Publications and source records attributed to Stegmaier, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Unbiased, systematic surfaceome profiling of Acute Leukemia to identify novel immunotherapy targets

Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A-rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets.

molecular biology↗

bioRxiv2026

Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit

Rhabdomyosarcoma (RMS) is a multi-subtype, high-risk pediatric sarcoma with a low mutational burden. The mutations found in RMS often alter genes involved in transcriptional control. Approaches to target dysregulated RMS transcription have remained elusive. Here, we develop a novel approach to target RMS transcription comprising simultaneous targeting of two distinctly acting transcriptional co-activators. We discover a common identity-controlling pan-RMS core regulatory circuit (CRC) composed of oncogenic and lineage-specific myogenic master transcription factors (mTFs). Using a super-enhancer-based reporter screen, we identify the EP300/CBP inhibitor A485 as a potent inhibitor of the pan-RMS CRC, though with efficacy-limiting toxicities. To enhance efficacy, we identify the mTF-binding co-activator EYA2 as a co-factor of this pan-RMS CRC and exploit a new second-generation EYA1/2 inhibitor, LG1-34, to disrupt its function. Combined co-activator inhibition inactivates the CRC and synergistically reduces RMS growth. This strategy dually targets CRC-associated co-activators to cooperatively suppress the RMS transcriptome and enforce cell death.

cancer biology↗

bioRxiv2026

Tabular foundation model predicts alternative lengthening of telomeres (ALT) and identifies SMARCAL1 as a target in ALT-driven cancers

Alternative lengthening of telomeres (ALT) is a telomerase-independent pathway used by aggressive cancers to maintain their replicative immortality. Because ALT is absent from normal human cells, it is an appealing target for cancer therapy, but the lack of ability to determine ALT status at scale has hindered discovery. Here, we developed ALTitude, a machine learning method from a tabular foundation model that infers ALT from cell line whole genome sequencing data, without need for paired germline analysis. We deployed ALTitude across the DepMap, doubling the number of known ALT+ cancer models. Systematic integration of ALTitude with CRISPR-Cas9 screens yielded the selective dependency on SMARCAL1 in ALT+ cell lines, where we show it stabilizes the ALT phenotype. Acute depletion of SMARCAL1 leads to G2/M arrest, mitotic catastrophe, and cell death. These data provide a valuable resource for studying ALT-related genomic features and present SMARCAL1 as a therapeutic target for ALT+ malignancies.

cancer biology↗

bioRxiv2025

Chromatin Perturbation Promotes Susceptibility to Hypomethylating Agents

Cancer-directed drugs are often clinically deployed without definitive understanding of their molecular mechanisms of action (MOA). Hypomethylating agents (HMAs), which result in the degradation of the DNA methyltransferase 1 (DNMT1), have been deployed for decades in the treatment of haematological malignancies1,2. The precise mechanism of action of these drugs, however, has been debated, rendering the design of rational combination therapies challenging. Here, we identified the deubiquitinating enzyme USP48 as a crucial regulator of posttranslational histone modification in the context of DNA demethylation. USP48 loss selectively enhances response to DNMT1 inhibition, leading to a rapid induction of cell death. We demonstrate that USP48 is localized at sites of DNA damage and deubiquitinates H2A variants and proteins important for DNA damage repair. Functionally, loss of USP48 triggers an increase in chromatin accessibility upon HMA treatment, rendering AML cells more susceptible to DNA damage. Our results support USP48 as a posttranslational histone modifier for chromatin stability and DNA damage in response to HMA-related DNA demethylation. These findings propose USP48 as a new target for combination therapy with HMAs for acute myeloid leukaemia (AML).

cancer biology↗

bioRxiv2025

CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

Alveolar rhabdomyosarcoma (aRMS) is a fusion-driven pediatric cancer with poor survival and limited therapeutic options. To uncover novel vulnerabilities, we employed complex-based analysis of the DepMap functional genomic data, identifying CDK8 as a dependency in aRMS. Both CDK8 knockout and pharmacologic inhibition impaired tumor cell growth and induced myogenic differentiation in vitro and in vivo. Compared to genetic loss, CDK8 inhibition induced more dynamic transcriptional changes. With a genome-scale CRISPR-Cas9 drug modifier screen, we determined that the maximal anti-tumor activity of the CDK8 inhibitor requires the presence of the Mediator kinase module and transcriptional cooperation with the SAGA complex. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. These findings suggest a distinct gain-of-function mechanism of the CDK8 inhibitor and establish a strong rationale for CDK8 inhibition as a differentiation-inducing therapeutic strategy in aRMS. STATEMENT OF SIGNIFICANCEWe provide a framework for uncovering therapeutic targets by network-based analysis of functional genomic screens. We identify CDK8 as a druggable target in aRMS and determine that CDK8 inhibition drives myogenic differentiation and impairs tumor progression via a collaborative mechanism involving the Mediator kinase module, SAGA complex, and SIX4.

cancer biology↗

bioRxiv2025

KAT6A/B inhibition synergizes with retinoic acid and enhances the efficacy of GD2-targeted immunotherapy in neuroblastoma

High-risk neuroblastoma accounts for about 15% of childhood cancer deaths and arises from precursors of the peripheral sympathetic nervous system. Retinoids are clinically used to inhibit growth of neuroblastoma cells through reconfiguration of the regulatory enhancer landscape. Its effects, however, are completely reversible after drug withdrawal, leading to rapid tumor cell proliferation. Here, we sought to identify epigenetic modifiers that potentiate the antiproliferative effects of retinoids in neuroblastoma. We identified PF-9363, an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, as synergistically inhibiting neuroblastoma growth in combination with retinoids. PF-9363 plus retinoids induces durable growth arrest, which persists beyond retinoid withdrawal in vitro and in vivo with sustained Polycomb-mediated repression of oncogenic transcription factors MYCN, PHOX2B and GATA3. Moreover, PF-9363 plus retinoids increases GD2 expression, rendering neuroblastoma cells more sensitive to anti-GD2 immunotherapy. Overall, our studies demonstrate that KAT6A/B inhibition increases the effectiveness of retinoids and GD2-targeted immunotherapy in neuroblastoma.

cancer biology↗

bioRxiv2025

Riboflavin drives nucleotide biosynthesis and iron-sulfur metabolism to promote acute myeloid leukemia

Riboflavin is a diet-derived vitamin in higher organisms that serves as a precursor for flavin mononucleotide and flavin adenine dinucleotide, key cofactors that participate in oxidoreductase reactions. Here, using proteomic, metabolomic and functional genomics approaches, we describe a specific riboflavin dependency in acute myeloid leukemia and demonstrate that, in addition to energy production via oxidative phosphorylation, a key biological role of riboflavin is to enable nucleotide biosynthesis and iron-sulfur cluster metabolism. Genetic perturbation of riboflavin metabolism pathways or exogenous depletion in physiological culture medium induce nucleotide imbalance and DNA damage responses, as well as impair the stability and activity of proteins which utilize [4Fe-4S] iron-sulfur clusters as cofactors. We identify a window of therapeutic opportunity upon riboflavin starvation or chemical riboflavin metabolism perturbation and demonstrate that this strongly synergizes with BCL-2 inhibition. Our work identifies riboflavin as a critical metabolic dependency in leukemia, with functions beyond energy production.

cancer biology↗

bioRxiv2025

Therapy resistance in AML is mediated by cytoplasmic sequestration of the transcriptional repressor IRF2BP2

While the development of venetoclax with azacitidine (ven/aza) has improved AML therapy, drug resistance remains a major challenge. Notably, primary ven/aza-resistant AML are frequently reliant on MCL1, however, the underlying mechanisms remain unclear. Co-immunoprecipitation of MCL1 from ven/aza-resistant AML samples coupled with mass spectrometry analysis identified the transcriptional repressor Interferon Regulatory Factor 2 Binding Protein 2 (IRF2BP2) as an MCL1 binding partner. This interaction results in cytoplasmic IRF2BP2 localization and loss of transcriptional repression within ven/aza-resistant leukemic stem cells (LSC). Consequently, ven/aza-resistant LSC have increased IRF2BP2 target gene expression, including acyl-CoA synthetase long-chain family member 1 (ACSL1), an essential rate-limiting enzyme for fatty acid oxidation (FAO). Inhibition of ACSL1 functionally impaired ven/aza-resistant LSC through a depletion of long-chain acyl-carnitine metabolites and FAO. Collectively, these data provide evidence for a previously undescribed mechanism by which MCL1 mediates IRF2BP2 cytoplasmic sequestration and consequent de-repression of ACSL1, thereby promoting ven/aza-resistance in AML.

cancer biology↗

bioRxiv2025

Rewiring the fusion oncoprotein EWS/FLI1 in Ewing sarcoma with bivalent small molecules

Deregulated transcription is a defining hallmark of cancer, especially pediatric malignancies, which are frequently driven by fusion transcription factors. Targeting transcription factors directly has been challenging as they lack druggable pockets. Recently, chemically induced proximity has enabled the rewiring of transcriptional activators to drive expression of pro-apoptotic genes using bivalent small molecules. Targeting fusion transcription factors, such as EWS/FLI1 in Ewing sarcoma, with these compounds, may open new therapeutic avenues. Here, we develop a small molecule, EB-TCIP, that recruits FKBP12F36V-tagged EWS/FLI1 to DNA sites bound by the transcriptional regulator BCL6, leading to rapid expression of BCL6 target genes. EB-TCIP activity is dependent on ternary complex formation and specific to cells that express FKBP-EWS/FLI1. This proof-of-concept study demonstrates that EWS/FLI1 can be relocalized on chromatin to induce genes that are ordinarily regulated by a transcriptional repressor. Insights herein will guide the development of bivalent molecules that rewire fusion transcription factors.

cancer biology↗

bioRxiv2024

CEBPA repression by MECOM blocks differentiation to drive aggressive leukemias

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, yet the mechanisms through which this occurs remain poorly understood. Increased expression of the stem cell transcription factor, MECOM, underlies one key driver mechanism in largely incurable AMLs. How MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem cell-like states by directly repressing pro-differentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kb downstream of the myeloid differentiation regulator CEBPA, is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo, suggesting a broadly applicable differentiation-based approach for improving therapy.

cancer biology↗

bioRxiv2024

Lineage-dependence of the neuroblastoma surfaceome defines tumor cell state-dependent and independent immunotherapeutic targets

BackgroundNeuroblastoma is a heterogeneous disease with adrenergic (ADRN)- and therapy resistant mesenchymal (MES)-like cells driven by distinct transcription factor networks. Here, we investigate the expression of immunotherapeutic targets in each neuroblastoma subtype and propose pan-neuroblastoma and cell state specific targetable cell-surface proteins. MethodsWe characterized cell lines, patient-derived xenografts, and patient samples as ADRN-dominant or MES- dominant to define subtype-specific and pan-neuroblastoma gene sets. Targets were validated with ChIP- sequencing, immunoblotting, and flow cytometry in neuroblastoma cell lines and isogenic ADRN-to-MES transition cell line models. Finally, we evaluated the activity of MES-specific agents in vivo and in vitro. ResultsMost immunotherapeutic targets being developed for neuroblastoma showed significantly higher expression in the ADRN subtype with limited expression in MES-like tumor cells. In contrast, CD276 (B7-H3) and L1CAM maintained expression across both ADRN and MES states. We identified several receptor tyrosine kinases (RTKs) enriched in MES-dominant samples and showed that AXL targeting with ADCT-601 was potently cytotoxic in MES-dominant cell lines and showed specific anti-tumor activity in a MES cell line-derived xenograft. ConclusionsImmunotherapeutic strategies for neuroblastoma must address the potential of epigenetic downregulation of antigen density as a mechanism for immune evasion. We identified several RTKs as candidate MES-specific immunotherapeutic target proteins for the elimination of therapy-resistant cells. We hypothesize that the phenomena of immune escape will be less likely when targeting pan-neuroblastoma cell surface proteins such as B7-H3 and L1CAM, and/or dual targeting strategies that consider both the ADRN- and MES-cell states. Key PointsO_LICellular plasticity influences the abundance of immunotherapeutic targets. C_LIO_LISubtype-specific targets may be susceptible to epigenetically-mediated downregulation. C_LIO_LIImmunotherapeutic targets in development, B7-H3 and L1CAM, show "pan-subtype" expression. C_LI Importance of StudyNeuroblastoma is a lethal childhood malignancy that shows cellular plasticity in response to anti-cancer therapies. Several plasma membrane proteins are being developed as immunotherapeutic targets in this disease. Here we define which cell surface proteins are susceptible to epigenetically regulated downregulation during an adrenergic to mesenchymal cell state switch and propose immunotherapeutic strategies to anticipate and circumvent acquired immunotherapeutic resistance.

cancer biology↗

bioRxiv2023

A compendium of Amplification-Related Gain Of Sensitivity (ARGOS) genes in human cancer

Chromosomal gains are among the most frequent somatic genetic alterations occurring in cancer. While the effect of sustained oncogene expression has been characterized, the impact of copy-number gains affecting collaterally-amplified "bystander" genes on cellular fitness remains less understood. To investigate this, we built a comprehensive map of dosage compensations across human cancers by integrating expression and copy number profiles from over 8,000 TCGA tumors and CCLE cell lines. Further, we analyzed the effect of gene overexpression across 17 human cancer ORF screens to provide an overview of genes that prove toxic to cancer cells when overexpressed. Combining these two independent approaches we propose a class of Amplification-Related Gain Of Sensitivity (ARGOS) genes. These genes are located in commonly amplified regions of the genome, have lower expression levels than expected by their copy-number status, and are toxic to cancer cells when overexpressed. We experimentally validated CDKN1A and RBM14 as high-confidence pan-cancer ARGOS genes in lung and breast cancer cell line models. We additionally suggest that RBM14s mechanism of toxicity involves altered DNA damage response and innate immune signaling processes following gene overexpression. Finally, we provide a comprehensive catalog of compensated, toxic, and ARGOS genes as a community resource.

cancer biology↗

bioRxiv2023

EZH2 synergizes with BRD4-NUT to drive NUT carcinoma growth through silencing of key tumor suppressor genes

NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUTs ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCEIdentification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.

cancer biology↗

bioRxiv2023

Rapid-kinetics degron benchmarking reveals off-target activities and mixed agonism-antagonism of MYB inhibitors

Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.

molecular biology↗

bioRxiv2023

Leukemia core transcriptional circuitry is a sparsely interconnected hierarchy stabilized by incoherent feed-forward loops

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.

systems biology↗

bioRxiv2022

STAT3 cooperates with the core transcriptional regulatory circuitry to drive MYC expression and oncogenesis in anaplastic large cell lymphoma

Anaplastic large cell lymphoma (ALCL) is an aggressive, CD30+ T-cell lymphoma of children and adults. ALK fusion transcripts or mutations in the JAK-STAT pathway are observed in most ALCL tumors, but the mechanisms underlying tumorigenesis are not fully understood. Here we show that dysregulated STAT3, together with a core transcriptional regulatory circuit consisting of BATF3-IRF4- IKZF1, co-occupies gene enhancers to establish an oncogenic transcription program and maintain the malignant state of ALCL. Critical downstream targets of this network in ALCL cells include the proto-oncogene MYC, which requires active STAT3 to facilitate high levels of MYC transcription. The activity of this auto-regulatory transcription loop is reinforced by MYC binding to the enhancer regions associated with STAT3 and each of the core regulatory transcription factors. These findings provide new insights for understanding how dysregulated signaling pathways hijack cell-type-specific transcriptional machinery to drive tumorigenesis and create therapeutic vulnerabilities in genetically defined tumors.

cancer biology↗

bioRxiv2022

Targeting TRIP13 in Wilms Tumor with Nuclear Export Inhibitors

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

cancer biology↗

bioRxiv2021

VRK1 is required in VRK2-methylated cancers of the nervous system

Collateral lethality occurs when loss of one paralog renders cancer cells dependent on the remaining paralog. Combining genome scale CRISPR/Cas9 screens coupled with RNA-sequencing in over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase Vaccinia-Related Kinase 1 (VRK1) for their survival. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout (KO) sensitized cells to VRK1 suppression, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 suppression. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas, and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced Barrier-to-Autointegration Factor (BAF) phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas. Statement of SignificanceWe credential VRK1 as a target in adult and pediatric gliomas, and neuroblastomas with VRK2 promoter methylation. This demonstrates the utility of paralog-driven synthetic lethal interactions for biomarker-linked, targeted therapeutics.

cancer biology↗