Biology subjects

Robinson, M. E.

Publications and source records attributed to Robinson, M. E..

7 recordsLinked to original sources

bioRxiv2025

Single-cell transcriptomics uncovers that cell-to-cell communication controls p53 activation

Conventional chemotherapy remains a cornerstone of cancer treatment and is highly effective in the adjuvant and neoadjuvant settings. However, therapeutic resistance, especially in the metastatic context, limits its long-term efficacy. While significant progress has been made in elucidating mechanisms of chemotherapy resistance, the role of cell-to-cell communication in modulating treatment response remains largely unexplored. Here, we combined microfluidic technology with single-cell RNA sequencing (scRNA-seq) to directly compare the transcriptional responses of cells in communication versus those in isolation. Upon dox treatment, we observed robust activation of the p53 pathway in communicating cells, whereas this response was markedly impaired in isolated cells. Moreover, pharmacological inhibition of caveolar-mediated endocytosis mimics the loss of p53 activation, further implicating this pathway in the communication-dependent regulation of chemotherapy response. This finding uncovers a previously unrecognized and essential role for paracrine signalling in mediating p53-dependent responses to chemotherapy. Our study highlights the critical importance of cell-to-cell communication in shaping therapeutic outcomes and opens new avenues for understanding and potentially overcoming chemotherapy resistance.

cancer biology↗

bioRxiv2023

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

immunology↗

bioRxiv2023

Targeted engagement of β-catenin-Ikaros complexes in refractory B-cell malignancies

In most cell types, nuclear {beta}-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of {beta}-catenin but critically depended on GSK3{beta} for effective {beta}-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that {beta}-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, {beta}-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive {beta}-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3{beta} small molecule inhibitors to subvert {beta}-catenin degradation. Clinically approved GSK3{beta}-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of {beta}-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3{beta}-inhibitors for targeted engagement of lymphoid-specific {beta}-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTSO_LIUnlike other cell lineages, B-cells express nuclear {beta}-catenin protein at low baseline levels and depend on GSK3{beta} for its degradation. C_LIO_LIIn B-cells, {beta}-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. C_LIO_LICRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed {beta}-catenin-dependent Myc repression and induction of cell death. C_LIO_LIThe discovery of GSK3{beta}-dependent degradation of {beta}-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3{beta}-inhibitors for the treatment of refractory B-cell malignancies. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/532152v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1b70e4dorg.highwire.dtl.DTLVardef@10bfcc8org.highwire.dtl.DTLVardef@b10eorg.highwire.dtl.DTLVardef@1683a3e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAbundant nuclear {beta}-catenin C_LIO_LI{beta}-catenin pairs with TCF7 factors for transcriptional activation of MYC C_LIO_LIB-cells rely on efficient degradation of {beta}-catenin by GSK3{beta} C_LIO_LIB-cell-specific expression of Ikaros factors C_LI Unique vulnerability in B-cell tumors: O_LIGSK3{beta}-inhibitors induce nuclear accumulation of {beta}-catenin. C_LIO_LI{beta}-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC C_LI

cancer biology↗

bioRxiv2022

Frontotemporal network engagement by human intracranial stimulation in limbic hubs

BackgroundPrefrontal circuits in the human brain play an important role in cognitive and affective processing. Neuromodulation therapies delivered to certain key hubs within these circuits are being used with increasing frequency to treat a host of neuropsychiatric disorders. However, the detailed neurophysiological effects of stimulation to these hubs are largely unknown. MethodsHere, we performed intracranial recordings across prefrontal networks while delivering electrical stimulation to two well-established white matter hubs involved in cognitive regulation and depression: the subcallosal cingulate (SCC) and ventral capsule/ventral striatum (VC/VS). ResultsWe demonstrate a shared frontotemporal circuit consisting of the ventromedial PFC, amygdala, and lateral orbitofrontal cortex where gamma oscillations are differentially modulated by stimulation target. Additionally, we found subject-specific responses to stimulation in the dorsal anterior cingulate cortex and demonstrate the capacity for further tuning of neural activity using current-steered stimulation. ConclusionsOur findings indicate a potential neurophysiological mechanism for the dissociable therapeutic effects seen across the SCC and VC/VS DBS targets for psychiatric neuromodulation and our results lay the groundwork for personalized, network-guided neurostimulation therapy.

neuroscience↗

bioRxiv2020

The innate sensor ZBP1-IRF3 axis regulates cell proliferation in multiple myeloma

ZBP1 is an inducible, non-constitutively expressed cellular nucleic acid sensor that triggers type I interferon (IFN) responses via phosphorylation and activation of the transcription factor (TF) IRF3 by TBK1. However, the role of the ZBP1-IRF3 axis in cancer is not known. Here we show that ZBP1 is selectively and constitutively expressed in late B cell development and it is required for optimal T cell-dependent humoral immune responses. In the plasma cell (PC) cancer multiple myeloma, interaction of constitutively expressed ZBP1 with TBK1 and IRF3 results in IRF3 phosphorylation. Notably, rather than IFN type I response genes, IRF3 directly activates, in part through co-operation with the PC lineage-defining TF IRF4, cell cycle genes thus promoting myeloma cell proliferation. This generates a novel, potentially therapeutically targetable and relatively selective myeloma cell addiction to the ZBP1-IRF3 axis. These data expand our knowledge of the role of cellular immune sensors in cancer biology.

cancer biology↗

bioRxiv2020

Signaling input from divergent pathways subverts malignant B-cell transformation

Malignant transformation typically involves multiple genetic lesions whose combined activity gives rise to cancer1. Our analysis of 1,148 patient-derived B-cell leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway characteristic for the differentiation status of these transformed B cells. Specifically, we show here the JAK/STAT5 signaling pathway supports the developmental stage-specific expansion of pro-B ALL whereas the ERK-pathway that of pre-B ALL. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (through activation of STAT5)2-7 or the pre-B cell receptor (through activation of ERK)8-13. STAT5- (372 cases) and ERK- (386 cases) activating lesions were frequently found but only co-occurred in [~]3% (37) of cases (P=2.2E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation while convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways14-17. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways.

cancer biology↗

bioRxiv2020

Lgr5-mediated restraint of β-catenin is essential for B-lymphopoiesis and leukemia-initiation

Upon productive immunoglobulin gene rearrangement, expression of a functional pre-B cell receptor (pre-BCR) initiates positive selection of pre-B cells, clonal expansion and self-renewal1-2. Studying mechanisms driving this first wave of B-lymphopoiesis, we identified the G-protein coupled receptor Lgr5 as an essential initiator of positive selection. Lgr5 was extensively studied as determinant of stem cell populations in multiple tissues3-6, but not in B-cells. While undetectable throughout the hematopoietic system, positively selected pre-B cells were marked with a sharp peak of Lgr5 expression. Conditional deletion of Lgr5 preceding the pre-BCR checkpoint induced negative selection and complete abortion of B-cell development. Proteomic studies of Lgr5-ablation revealed massive (>250-fold) accumulation of {beta}-catenin and suppression of MYC. Lgr5-deficient pre-B cells fully recovered by concurrent {beta}-catenin-deletion, demonstrating a central role of Lgr5-mediated restraint of {beta}-catenin at the pre-BCR checkpoint. In other cell types, {beta}-catenin/TCF4 complexes drive transcriptional activation of MYC7-9. Instead of TCF4, proximity-based interactome studies in pre-B cells identified the B-lymphoid transcription factors IKZF1 and IKZF310-11 as {beta}-catenin-binding partners, which had the opposite effect and caused transcriptional repression of MYC. On positively selected pre-B cells, Lgr5 prevented accumulation of {beta}-catenin and formation of complexes with IKZF1 and IKZF3, which relieved transcriptional repression of MYC. Activating {beta}-catenin-mutations are common throughout all main types of cancer7-8, but were conspicuously absent in pre-B leukemia (B-ALL). Like pre-B cells, B-ALL cells were uniquely sensitive to genetic and pharmacological {beta}-catenin hyperactivation, which recapitulated the effects of Lgr5-deletion and compromised colony formation and leukemia-initiation. A new LGR5 antibody-drug conjugate targeted leukemia-initiating cells in patient-derived B-ALL and achieved long-term disease-control. Likewise, small molecule hyperactivation of {beta}-catenin selectively killed B-ALL but not other cell types. Hence, Lgr5-mediated restraint of {beta}-catenin activation is essential for B-lymphopoiesis and revealed an unexpected vulnerability that can be leveraged for the treatment of drug-resistant B-ALL.

cancer biology↗