IL1beta and soluble crosstalk modulate NF-κB activity in living Multiple Myeloma and stromal cells
Ontology highlight
ABSTRACT: Multiple myeloma (MM) progression is linked to chronic NF-κB activation in myeloma cells. However, the identity and source of autocrine/paracrine signals driving NF-κB activation and the role of the 3D microenvironment have been scarcely investigated both in vivo and in vitro. To investigate them, we knocked-in the Venus (YFP) ORF in the NF-κB p65 gene in both MM and stromal cells. Surprisingly, a large fraction of p65-YFP MM cells engrafted in mouse bone marrow showed overall low levels of NF-κB activation whereas a small fraction was highly activated. To understand these in vivo data, we investigated NF-κB dynamics in MM and stromal cells, both alone and in co-culture. In vitro experiments exploiting microfluidics, bioreactor and microchip cell cultures highlighted crosstalk between the myeloma and stromal components that leads to mild basal activation. In contrast, we found that high-density cultures within 3D scaffolds dampen NF-κB activation in MM and stromal cells, both in basal and inflammatory conditions. It has been recently hypothesized that IL1β, and the inflammatory ME, shape the overall activity of ME components and promote the transition of Mesenchimal Stromal Cells (MSCs) toward an inflammatory NF-κB driven transcriptional phenotype (iMSCs). We tested this hypothesis in our system and found that IL1β strongly activates NF-κB in stromal but not in myeloma cells. In addition, secreted molecules from IL1β-stimulated MSCs strongly activate NF-κB only in a small fraction of MM cells. We propose that the balance between activating stimuli from iMSCs and dampening feedbacks from the 3D ME, maintains a mild NF-κB activation in myeloma cells in the patients’ BM to avoid exceedingly harmful responses.
Project description:By generating a paired single cell RNA-sequencing database of the tumor niche from 10 newly diagnosed MM patients, we created a unique dataset allowing the in-depth analyses of stromal-immune interactions within the tumor microenvironment. Using this database, we identified the presence of inflammatory stromal fibroblasts in the bone marrow of Myeloma patients.The stromal inflammation was associated with NF-κB signaling, and sources of IL-1β or TNFα were specific immune subsets previously shown to be altered in MM, suggesting the presence of an immune cell-mediated feed-forward loop of bone marrow inflammation in MM. By tracking inflammatory signatures over time in individual patients undergoing first-line treatment using bulk RNA sequencing, we show that bone marrow inflammation is not reverted by successful anti-tumor therapy (see related accession number), suggesting a role for stromal fibroblasts and bone marrow inflammation in disease persistence or relapse.
Project description:By generating a paired single cell RNA-sequencing database of the tumor niche from 10 newly diagnosed MM patients, we created a unique dataset allowing the in-depth analyses of stromal-immune interactions within the tumor microenvironment (see related accession number). Using this database, we identified the presence of inflammatory stromal fibroblasts in the bone marrow of Myeloma patients.The stromal inflammation was associated with NF-κB signaling, and sources of IL-1β or TNFα were specific immune subsets previously shown to be altered in MM, suggesting the presence of an immune cell-mediated feed-forward loop of bone marrow inflammation in MM. By tracking inflammatory signatures over time in individual patients undergoing first-line treatment using bulk RNA sequencing, we show that bone marrow inflammation is not reverted by successful anti-tumor therapy (this dataset), suggesting a role for stromal fibroblasts and bone marrow inflammation in disease persistence or relapse. Raw sequencing data files will be deposited to EGA.
Project description:Recent studies have delineated cancer type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM); and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. TNFα or bone marrow stromal cell culture supernatants induce KDM6B, which is blocked by IKKβ inhibitor MLN120B, suggesting KDM6B is regulated by NF-κB signaling in MM cells. RNA-sequencing and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS, and upregulates these genes expression without affecting H3K27 methylation level. Overexpression of catalytically-inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.
Project description:In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.
Project description:In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.
Project description:In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.
Project description:In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.
Project description:In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a novel p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.
Project description:Pro-inflammatory cytokines were shown to promote growth and survival of cancerous cells. TNF induced RelA:p50 NF-κB dimer via the canonical pathway is thought to link inflammation with cancer. Integrating biochemical and computational studies we identify that deficiency of non-canonical signal transducer p100 triggers a positive autoregulatory loop, which instead perpetuates an alternate RelB:p50 containing NF-κB activity upon TNF treatment. TNF stimulated RelB:p50 dimer is sufficient for mediating NF-κB target gene-expressions and suppressing apoptotic cellular death independent of principal NF-κB subunit RelA. We further demonstrate that activating mutations in non-canonical NF-κB module deplete multiple myeloma cells of p100, thereby, provoking autoregulatory RelB:p50 activation. Finally, autoregulatory control reinforces protracted pro-survival NF-κB response, albeit comprising of RelB:p50, upon TNF priming that protects myeloma cells with dysfunctional p100 from subsequent apoptotic insults. In sum, we present evidence for positive autoregulation mediated through the NF-κB system and its potential involvement in human neoplasm.
Project description:Immunomodulatory drugs (IMiDs) have markedly improved patient outcome in multiple myeloma (MM); however, resistance to IMiDs commonly underlies relapse of disease. Here we identify and validate that TNF receptor associated factor protein family TRAF2 KD/KO in MM cells mediates IMiDs resistance via activation of non-canonical NF-kB and MEK-ERK signaling. Within MM bone marrow (BM) stromal cell supernatants, TNF-a induces proteasomal degradation of TRAF2, non-canonical NF-kB, and downstream ERK signaling in MM cells, whereas IL-6 directly triggers ERK activation. RNA sequencing of MM patient samples shows nearly universal ERK pathway activation at relapse on lenalidomide maintenance therapy, confirming its clinical relevance. Combination MEK inhibitor treatment restores IMiDs sensitivity of TRAF2 KO cells both in vitro and in vivo using an inducible TRAF2 KD MM xenograft model. Our studies provide the framework for clinical trials of MEK inhibitors to overcome IMiDs resistance in the BM microenvironment and improve patient outcome in MM.