The Dependency Map (DepMap) is a genome-wide pooled
CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many
different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene
knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of
CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell
lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can
include activity in the same or parallel pathways or membership in the same protein complex or the
same pathway.
We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment
analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein
interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The
“Evidence” column contains the PPIDs in which the interaction appears as well as whether there is
support for the association by an INDRA statement. As another approach to identify potential
interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for
proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB;
it has previously been observed that proteins in the same complex are frequently significantly
co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided.
And, we determined whether co-dependent genes yield similar transcriptomic signatures
in the Broad Institute's Connectivity
Map (CMap). A CMap score greater than 90 is considered significantly similar.
Using the biological processes and other Gene Ontology terms from well characterized DUBs as a
positive control, several gene set enrichment analyses were considered. Threshold-less methods
like GSEA had relatively poor results.
Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map
correlations yielded the best results and is reported below.
GO Identifier
GO Name
GO Type
p-value
p-value (adj.)
q-value
Transcriptomics
The following table shows the significantly differentially expressed genes after knocking
out USP24 using CRISPR-Cas9.
There were too few differentially expressed genes to run a meaningful GSEA.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to USP24 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
These data suggest that USP24 normally negatively regulates ubiquitination and stability of ULK1, therefore interrupting the positive feed-back amplifying induction of autophagy.
Indeed, we observed that USP24 interacts with p300, and knockdown of USP24 increased p300 ubiquitination, and this effect of USP24 knockdown was abolished after treatment with the proteasome inhibitor MG132.
USP24 deubiquitinates and stabilizes the histone acetyltransferase p300, thereby facilitating histone H3 acetylation of the IL-6 promoter and thus IL-6 transcription.
To further clarify whether USP24 directly targets beta-TrCP for deubiquitination, an in vitro deubiquitination assay was performed, and purified human USP24 significantly decreased the beta-TrCP ubiquitination signal.
In agreement with previous reports indicating that USP24 may de-ubiquitinate the pro apoptotic BH3 protein BAX [31], we also observed a decrease in BAX protein levels in USP24 knockdown cells).
In agreement with previous reports indicating that USP24 may de-ubiquitinate the pro apoptotic BH3 protein BAX [31], we also observed a decrease in BAX protein levels in USP24 knockdown cells).
These results indicate that USP24 may upregulate IL-6 expression through directly stabilizing p300 and indirectly induce NF-kappaB expression through p300 in M2 macrophages.
In this study, another interesting point is the discovery of distinct molecular mechanisms regulating IL-6 expression in M2 macrophages and lung cancer cells in response to USP24.
In this study, we found that USP24 increased IL-6 expression by decreasing the stability of DNMT1 by stabilizing beta-TrCP in lung cancer cells and increasing the stability of p300 in both lung cancer cells and M2 macrophages, thereby resulting in the acetylation of histone H3 and demethylation of the IL-6 promoter, respectively.
Finally, we found that knockdown of DNMT1 in USP24-knockdown A549 cells can rescue IL-6 level, suggesting that USP24 mediated DNMT1 degradation is really involved in IL-6 expression.
In addition to the p300 stabilization induced by USP24 in cancer cells to regulate IL-6 expression through increased histone H3 acetylation, USP24 also reduces DNA methylation in the promoter region of IL-6 in lung cancer cells, but not in M2 macrophages, to increase IL-6 transcription by decreasing DNMT1 protein stability.
The IL-6 levels secreted from M2 macrophages and A549 cells decreased after USP24 knockdown, suggesting that USP24 upregulation in the tumor associated microenvironment and cancer cells during tumorigenesis increased IL-6 expression.
While studying the role of USP24 upregulation in M2 macrophages, we found that USP24 expression in M2 macrophages enhanced cancer metastasis by inducing IL-6 expression.
USP24 induces IL-6 in TME through deubiquitinating p300 and beta-TrCP, and whether WP1130 inhibiting USP24 affects IL-6 in TME needs more investigation [XREF_BIBR].
Adding the physiological dose of IL-6 to the media of USP24-knockdown cells can rescue the effect of USP24 knockdown, suggesting that the USP24 mediated IL-6 in M2 macrophages and in lung cancer is an important factor that affects USP24 induced lung cancer malignancy.
To examine whether USP24 mediated IL-6 was indeed functioning in lung cancer metastasis and angiogenesis, IL-6 was added to conditioned medium derived from USP24-knockdown M2 macrophages to address malignant lung cancer cell activity.
USP24 induces IL-6 in TME through deubiquitinating p300 and beta-TrCP , and whether WP1130 inhibiting USP24 affects IL-6 in TME needs more investigation [ 100 ] .
After analyzing the five CpG sites upstream of the transcription start site, we found that the methylation status of a CpG site located at -123 was increased in A549 cells but not in M2 macrophages after USP24 knockdown, indicating that a decrease in IL-6 methylation induced by USP24 in A549 cells, but not in M2 macrophages, increases IL-6 mRNA levels.
In cancer cells, USP24 increased p300 and beta-TrCP, thus increasing the acetylation of histone H3 and the degradation of DNMT1 and IkappaB, resulting in the recruitment of histone H3 acetylation to the promoter region of IL-6, the reduction of DNA methylation, and the promotion of NF-kappaB nuclear translocation, thereby facilitating IL-6 expression.
Thus, GFP-USP24 increased p300 protein stability, whereas knockdown USP24 decreased its stability (XREF_FIG; XREF_SUPPLEMENTARY) and USP24 interacted with p300 (XREF_FIG).
Because our previous study confirmed that p300 is a USP24 substrate in lung cancer cells 31, we wanted to confirm whether p300 is also directly regulated by USP24 in M2 macrophages.
Knockdown of USP24 decreased the Bax and caspase-3 levels; conversely, these protein levels were increased in GFP-USP24-expressing cells, although no alterations were observed in the Bax mRNA level (XREF_FIG; XREF_SUPPLEMENTARY).
The localization of Bax in cells expressing GFP-USP24 indicated that USP24 expression increased Bax 's mitochondrial localization, implying that USP24 induced cell apoptosis (XREF_FIG).
MG132 treatment rescued the Bax level, indicating that USP24 increased the Bax level by enhancing protein stability, thereby contributing to cell apoptosis (XREF_SUPPLEMENTARY).
Knockdown of USP24 resulted in a decreased level of DDB2, suggesting that USP24 stabilizes DDB2 by removing the ubiquitin moiety from modified DDB2, thereby preventing DDB2 degradation.
To examine if USP24 can target ubiquitinated DDB2 in vitro, we treated HeLa cells expressing Hig tagged DDB2 with MG132, a proteosome inhibitor, XREF_BIBR for 1 h and exposed HeLa cells to UVC (10 J/m 2) to stimulate DDB2 uniquitination for another hour, Ni-NTA beads were then used to pulldown modified His DDB2.
Our group previously reported that ubiquitinated DDB2 can be targeted by USP24, and in this study, we demonstrate that USP24 is a p53 deubiquitinase, required for p53 stabilization in unstressed cells, as well as for p53 stabilization and PUMA activation after DNA damage.
Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24 mediated DDB2 deubiquitination prevents DDB2 degradation.
Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24 mediated DDB2 deubiquitination prevents DDB2 degradation.
Furthermore, we observed elevated levels of USP24 in the substantia nigra of a subpopulation of idiopathic PD patients, suggesting that USP24 may negatively regulate autophagy in PD.
Furthermore, our data demonstrate elevated levels of USP24 in some cases of idiopathic PD, suggesting that USP24 may negatively regulate autophagy in PD.
Our data indicate that USP24 mRNA and protein levels are also elevated in the substantia nigra of a subset of idiopathic PD patients, suggesting that suppression of autophagy by USP24 could also occur in at least some cases of non familial PD.
Consistently, in the original screen [25] USP24 knockdown failed to upregulate autophagy in cells overexpressing BCL-2, which binds and inhibits activity of the class III PtdIns3K subunit BECN1 [34]).
USP24 is another modulator of autophagy which may also influence PD progression as it can regulate dopaminergic neurite outgrowth, but the potential as a disease modulator is not evaluated yet.
Blocking lysosomal function with bafilomycin led to significant increase in accumulation of GFP-LC3 positive autophagosomes in USP24 knockdown cells, indicating that USP24 is negatively regulating autophagy flux without affecting lysosomal degradation).
Knockdown of USP24 in cell lines and in human induced-pluripotent stem cells (iPSC) differentiated into dopaminergic neurons resulted in elevated ULK1 protein levels and increased autophagy flux in a manner independent of MTORC1 but dependent on the class III phosphatidylinositol 3-kinase (PtdIns3K) activity.
Knockdown of USP24 also led to increase in levels of the autophagosome associated lipidated form of LC3 (LC3-II) [30]), confirming increase in autophagy.
This third, independent assay format thus supports a direct interaction between USP24, p53 and orf10.Novel modulators of p53 signaling encoded by unknown genes of emerging viruses Next, we deleted USP24 gene in U2OS cells using CRISPR and Cas9, which generated three independent US2OS-DeltaUSP24 cell populations upon long-term selection.
Knockdown of USP24 failed to prevent stabilization of p53 following etoposide treatment of H4 cells), suggesting that in this cell type USP24 may be dispensable for DNA damage responses or that the very low residual USP24 protein may be sufficient.
Using this deactivated CRISP-Cas9-SAM system we successfully reactivated USP24 and demonstrate that endogenously induced USP24 could partially rescue the apoptosis induced by WP1130 in Jurkat cells.
These results indicate that USP24 promotes UV induced apoptosis in HCT116 cells, consistent with the notion that p53 directed apoptosis is attenuated in USP24 depleted cells which lack robust p53 stabilization after UV treatment.
Knockdown of USP24 decreased the E2F4, p130 and TFDP1 levels, which form a complex to regulate the G1-S transition, but did not alter the E2F4 and TDFP1 mRNA levels (XREF_FIG; XREF_SUPPLEMENTARY).
The molecular mechanism involves a decrease in the USP24 level, which reduces the expression of E2F4 and its partner TFDP1, and thus increases the G1/S transition.
Taken together, our results demonstrated that USP24 knockdown decreased E2F4, p130 and TFDP1, resulting in an increase in the E2F1 levels and a subsequent increase in the G1-S transition, leading to tumorigenesis.
Taken together, our results suggested that USP24 was decreased by APC/C cdc20 to decrease the securin level, which was beneficial for the metaphase-anaphase transition and enhanced cell cycle progression.
Finally, we found that knockdown of DNMT1 in USP24-knockdown A549 cells can rescue IL-6 level, suggesting that USP24 mediated DNMT1 degradation is really involved in IL-6 expression.
beta-TrCP overexpression in USP24-knockdown cells prevented the upregulation of DNMT1and IkappaB induced by USP24 knockdown and demonstrated that beta-TrCP is critical in the USP24 induced increase of DNMT1 and IkappaB expression.
However, in this study, USP24-activated NF-κB did not increase CCL2, implying that other factors regulated by USP24 might negatively regulate CCL2 expression.
In this study, we discovered the underlying mechanisms by which USP24 not only promotes IkappaB degradation by stabilizing beta-transduction repeat containing E3 ubiquitin protein ligase (beta-TrCP) in lung cancer cells but also induces the upregulation of NF-kappaB in M2 macrophages, resulting in an increase in IL-6 expression.
beta-TrCP overexpression in USP24-knockdown cells prevented the upregulation of DNMT1and IkappaB induced by USP24 knockdown and demonstrated that beta-TrCP is critical in the USP24 induced increase of DNMT1 and IkappaB expression.
In this study, we discovered the underlying mechanisms by which USP24 not only promotes IkappaB degradation by stabilizing beta-transduction repeat containing E3 ubiquitin protein ligase (beta-TrCP) in lung cancer cells but also induces the upregulation of NF-kappaB in M2 macrophages, resulting in an increase in IL-6 expression.
beta-TrCP overexpression in USP24-knockdown cells prevented the upregulation of DNMT1and IkappaB induced by USP24 knockdown and demonstrated that beta-TrCP is critical in the USP24 induced increase of DNMT1 and IkappaB expression.
Taken together, these data revealed that Kras G12D - or EGFR L858R -mediated pathways negatively regulated USP24 expression, which might trigger lung cancer formation.
XREF_BIBR, XREF_BIBR Although their levels were increased in some specimens from late-stage lung cancer patients, inhibition of Kras G12D - or EGFR L858R -mediated activities in all cell lines at different stages increased the USP24 level.
Based on the data in this study and our previous study, we propose that EGFR L858R - and Kras G12D -mediated signaling pathways inhibit USP24 expression, which is beneficial for cancer formation during the early stage.
XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR Our recent study showed that USP24 variants from SNPs and RNA editing products increased the levels of USP24 and MDM2, which regulates Suv39h1 in lung cancer cells, subsequently resulting in an increase in metastatic activities during lung cancer progression.
Here we found that the phosphorylation of several USP24 residues was regulated by EGF treatment and mitotic CDK1 to enhance USP24 degradation in a polyubiquitination dependent manner.
Mutations at these residues increased the USP24 half-life during mitosis, suggesting that USP24 phosphorylation during mitosis decreased USP24 expression (XREF_FIG).
Our results demonstrated that USP24 rs487230 T allele and CT/TT genotype notably decreased the risk of developing PD in the AAO> = 60 year subgroup of patients.
In cancer cells, USP24 increased p300 and beta-TrCP, thus increasing the acetylation of histone H3 and the degradation of DNMT1 and IkappaB, resulting in the recruitment of histone H3 acetylation to the promoter region of IL-6, the reduction of DNA methylation, and the promotion of NF-kappaB nuclear translocation, thereby facilitating IL-6 expression.
To further clarify whether USP24 directly targets beta-TrCP for deubiquitination, an in vitro deubiquitination assay was performed, and purified human USP24 significantly decreased the beta-TrCP ubiquitination signal.
Moreover, UV induced USP24 accumulation appears to be ATM dependent; inhibition of ATM by either KU-55933 or a specific siRNA prevented USP24 accumulation after UV (XREF_FIG).
Taken together, these data suggest that the ATM kinase mediated phosphorylation of USP24 is involved in USP24 stabilization/up regulation following UV irradiation.
Knockdown of USP24 suppressed the cell death during pUL38 deficient HCMV infection, suggesting that pUL38 achieved its function by antagonizing the function of USP24.
In p53 +/+ cells, cleaved PARP started to appear as early as three hours after UV irradiation and depletion of USP24 resulted in slow and reduced PARP cleavage (XREF_FIG, right panel).
To examine whether USP24 regulates the apoptotic response to UV through the p53 pathway, we compared the effects of USP24 depletion on UV induced PARP cleavage in two isogenic cell lines : HCT116 p53 +/+ and HCT116 p53-/-.
The molecular mechanism involves a decrease in the USP24 level, which reduces the expression of E2F4 and its partner TFDP1, and thus increases the G1/S transition.
Knockdown of USP24 decreased the E2F4, p130 and TFDP1 levels, which form a complex to regulate the G1-S transition, but did not alter the E2F4 and TDFP1 mRNA levels (XREF_FIG; XREF_SUPPLEMENTARY).
Taken together, our results demonstrated that USP24 knockdown decreased E2F4, p130 and TFDP1, resulting in an increase in the E2F1 levels and a subsequent increase in the G1-S transition, leading to tumorigenesis.
Taken together, these data revealed that Kras G12D - or EGFR L858R -mediated pathways negatively regulated USP24 expression, which might trigger lung cancer formation.
Therefore, during the initiation of tumor formation, EGFR mutations decrease USP24 to increase the degradation of p300 and Bax, and thus repress cell apoptosis.
After IL-6 was added to the M2 macrophages conditioned medium, this effect was rescued, indicating that the USP24 induced metastasis activity is due to upregulated IL-6, which can be expressed by M2 macrophages or cancer cells.
Overexpression of E2F4 in USP24 silenced cells rescued only the E2F1 mRNA level and partially abolished the effect of the USP24 knockdown on the G1-S transition and colony formation (XREF_FIG; XREF_SUPPLEMENTARY), implying that E2F4 might be involved in USP24 mediated cell cycle progression.
Overexpression of GFP-USP24 increased the cyclin B1 level, suggesting that USP24 was beneficial for cells that remained in mitosis (XREF_SUPPLEMENTARY).
Knockdown of USP24 increased the CDK1 and cyclin B1 levels, and increased the ratio of cells in the G2/M stage, indicating that USP24 reduction was beneficial for cell cycle progression (XREF_SUPPLEMENTARY).
Overexpression of GFP-USP24 increased the cyclin B1 level, suggesting that USP24 was beneficial for cells that remained in mitosis (XREF_SUPPLEMENTARY).
Knockdown of USP24 increased the CDK1 and cyclin B1 levels, and increased the ratio of cells in the G2/M stage, indicating that USP24 reduction was beneficial for cell cycle progression (XREF_SUPPLEMENTARY).
Knockdown of USP24 decreased the E2F4, p130 and TFDP1 levels, which form a complex to regulate the G1-S transition, but did not alter the E2F4 and TDFP1 mRNA levels (XREF_FIG; XREF_SUPPLEMENTARY).
Taken together, our results demonstrated that USP24 knockdown decreased E2F4, p130 and TFDP1, resulting in an increase in the E2F1 levels and a subsequent increase in the G1-S transition, leading to tumorigenesis.
These results indicate that USP24 promotes UV induced apoptosis in HCT116 cells, consistent with the notion that p53 directed apoptosis is attenuated in USP24 depleted cells which lack robust p53 stabilization after UV treatment.
Gefitinib and FTI-276, which inhibited the EGFR and Kras activities 21 in the A431 and A549 cell lines, respectively, increased USP24 expression (XREF_FIG).
Given that USP2-4 is strikingly induced by aldosterone [ 17 ] , the induction of USP2-4 may therefore function as a node of the negative feedback loop for MR signaling .
The localization of Bax in cells expressing GFP-USP24 indicated that USP24 expression increased Bax 's mitochondrial localization, implying that USP24 induced cell apoptosis (XREF_FIG).
The data indicated that the knockdown of USP24 in M2 macrophages decreased Angiopoirtin-2, CD40L, IL-6, tumor necrosis factor alpha (TNF-alpha), Thrombospondin-1 and vascular endothelial growth factor (VEGF) levels, indicating that USP24 is involved in angiogenesis.
USP24 was also found to target histones by controlling the levels of the histone-lysine N-methyltransferase Suv39h1, resulting in a modulation of the H3K9me levels.
However, unlike HAUSP, USP24 was unable to interact with RUNX2 and reduce ubiquitination levels of RUNX2, suggesting that HAUSP, not USP24, functions as a bona-fide RUNX2-DUB.
In addition, we found that USP24 phosphorylation at Ser2604 was declined by U0126, which inhibited the Erk1/2 activities in A549 cells (XREF_FIG); however, USP24 phosphorylation was not decreased by LY294002, which inhibited the PI3K activities (XREF_SUPPLEMENTARY).
XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR Our recent study showed that USP24 variants from SNPs and RNA editing products increased the levels of USP24 and MDM2, which regulates Suv39h1 in lung cancer cells, subsequently resulting in an increase in metastatic activities during lung cancer progression.
12 In this study, we found that the modification of USP24 by phosphorylation decreased its protein stability in Kras G12D - and EDFR L858R -induced lung cancer mouse models, lung cancer cell lines and mitotic cells.
Herein, we found that USP24 not only repressed DNA-damage repair (DDR) activity by decreasing Rad51 expression to cause the tumor genomic instability and cancer stemness, but also increased the levels of the ATP binding cassette (ABC) transporters P-gp, ABCG2, and ezrin to enhance the pumping out of Taxol from cancer cells, thus resulted in drug resistance during cancer therapy.
Knockdown of USP24 increased the CCNA2 and E2F1 mRNA levels (XREF_FIG); the same results were obtained in the cDNA array performed under USP24 knockdown conditions (XREF_SUPPLEMENTARY).
Herein, we found that USP24 not only repressed DNA-damage repair (DDR) activity by decreasing Rad51 expression to cause the tumor genomic instability and cancer stemness, but also increased the levels of the ATP binding cassette (ABC) transporters P-gp, ABCG2, and ezrin to enhance the pumping out of Taxol from cancer cells, thus resulted in drug resistance during cancer therapy.
Finally, USP24 knockdown increased the cell numbers and cell viability in A549 and lung primary cells, suggesting that USP24 negatively regulated lung cancer formation (XREF_FIG; XREF_SUPPLEMENTARY).
USP24 phosphorylation increased the recruitment of the E3 ligase cdc20, which is the subunit of the APC/C complex that provides the E3 ligase activity.
However, in this study, USP24 activated NF-kappaB did not increase CCL2, implying that other factors regulated by USP24 might negatively regulate CCL2 expression.
Knockdown of USP24 decreased the Bax and caspase-3 levels; conversely, these protein levels were increased in GFP-USP24-expressing cells, although no alterations were observed in the Bax mRNA level (XREF_FIG; XREF_SUPPLEMENTARY).
Herein, we found that USP24 not only repressed DNA-damage repair (DDR) activity by decreasing Rad51 expression to cause the tumor genomic instability and cancer stemness, but also increased the levels of the ATP binding cassette (ABC) transporters P-gp, ABCG2, and ezrin to enhance the pumping out of Taxol from cancer cells, thus resulted in drug resistance during cancer therapy.
Overexpression of E2F4 in USP24 silenced cells rescued only the E2F1 mRNA level and partially abolished the effect of the USP24 knockdown on the G1-S transition and colony formation (XREF_FIG; XREF_SUPPLEMENTARY), implying that E2F4 might be involved in USP24 mediated cell cycle progression.
Overexpression of E2F4 in USP24 silenced cells rescued only the E2F1 mRNA level and partially abolished the effect of the USP24 knockdown on the G1-S transition and colony formation (XREF_FIG; XREF_SUPPLEMENTARY), implying that E2F4 might be involved in USP24 mediated cell cycle progression.