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 USP39 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 USP39 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
USP39 binds with USP4 via its UBL domain and correspondingly enhances the stability of USP4 in T cells [XREF_BIBR, XREF_BIBR], implying that USP39 might be deubiquitylating USP4.
USP39 promotes malignant proliferation and angiogenesis of renal cell carcinoma by inhibiting VEGF-A165b alternative splicing via regulating SRSF1 and SRPK1.
The inducible shRNA mediated downregulation of USP39 expression markedly reduced the proliferation and colony forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation.
Moreover, recent studies indicated that knocking down USP39 promoted cell proliferation in different types of cancers [XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR].
USP39 overexpression deprived miR-133a inhibitor mediated suppression of cell proliferation, suggesting that USP39 is involved in miR-133a-mediated biological role in gastric cancer cell HGC-27 (P < 0.0001) (XREF_FIG).
Furthermore, knockdown of USP39 inhibited VSMC cell proliferation and the expression of cyclin D1 and cyclin-dependent kinase 4, as analyzed via cell counting, MTT assay and western blotting.
In addition, it has been reported that overexpression of MGC-803 cells and knockdown of USP39 may inhibit MGC-803 cell proliferation and induce cell cycle arrest.
Knockdown of USP39 suppressed the proliferation and cell cycle progression, and induced apoptosis, accompanied by the reduction of EGFR in both mRNA and protein levels in PC-3 and DU145 cells.
Meanwhile, knockdown of USP39 could arrest cell cycle at G2/M via cyclin dependent pathway and promoted apoptosis through PARP cleavage [XREF_BIBR - XREF_BIBR].
Collectively, our data suggest that knocking down USP39 induces cell cycle arrest at S phase and G2/M phase, and results in apoptosis, and that the mechanism of apoptosis induced by USP39 knockdown may be related to DNA damage.
In order to further demonstrate the mechanisms of apoptosis induced by USP39 knockdown, the levels of cleaved cas3 and cleaved cas9 were increased in response to USP39 knockdown (XREF_FIG E, F).
Furthermore, we demonstrate that USP39 depletion promotes apoptosis induced by cisplatin, which is related with the induction of oxidative stress and DNA damage response.
These results suggested that knockdown of USP39 in SMMC-7721 cells could induce cell apoptosis via altering the expression of Bax, Caspase 9, Caspase 3 and PARP.
Knockdown of USP39 suppressed the proliferation and cell cycle progression, and induced apoptosis, accompanied by the reduction of EGFR in both mRNA and protein levels in PC-3 and DU145 cells.
The group of silencing USP39 increased the apoptotic cells (early apoptosis and late apoptosis) by 13-fold, as compared to the group of shCon (Additional file 1 : Figure S1A and B).
Second, we found that knocking down USP39 induces apoptosis of A549 and HCC827 cells, upregulates cleaved cas3, cleaved cas9 and DNA damage makers (53BP1 and gammaH2AX) [XREF_BIBR].
In addition, flow cytometry analysis in the present study identified that the knockdown of USP39 induced cell cycle arrest in the G 2 / M phases, which may have induced the inhibition of proliferation.
Our loss-of-function experiments demonstrated that knockdown of the expression of USP39 repressed the proliferation of leukemia cells, induced cell cycle arrest, and cell apoptosis.
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
To evaluate the role of cell cycle-regulatory molecules in knockdown of USP39 induced G2/M cell cycle arrest, we examined the effect of USP39 knockdown on cell cycle-regulatory molecules, including p21, CDK1 and cyclin A2.
Meanwhile, knockdown of USP39 could arrest cell cycle at G2/M via cyclin dependent pathway and promoted apoptosis through PARP cleavage [XREF_BIBR - XREF_BIBR].
Knockdown of endogenous USP39 expression could suppress the oncogenic properties of osteosarcoma cells and induce cell cycle arrest at G2/M phase, promote apoptosis through PARP cleavage.
Taken together, our findings implicate that USP39 promotes the development of human leukemia by regulating cell cycle, survival, and proliferation of the cells.
Subsequent functional experiments revealed that USP39 promoted the proliferation and invasion of ovarian cancer cells in vitro and tumor growth in vivo.
Moreover, USP39 knockdown significantly inhibited migration and invasion of A549 and HCC827 cells, also via activation of the p53 pathway, and downregulation of MMP2 and MMP9.
Third , we demonstrated that USP39 knockdown inhibits cell migration and invasion by upregulating p53 and the downstream proteins MMP2 and MMP9 [ 31 ] .
Third, we demonstrated that USP39 knockdown inhibits cell migration and invasion by upregulating p53 and the downstream proteins MMP2 and MMP9 [XREF_BIBR].
The results demonstrated that the level of the 3 '-end of EGFR decreased (P = 0.0015) more sharply than that of the 5 '-end (P = 0.0069), and the ratio ofthe 3 '-end to 5 '-end levels was significant decreased (P = 0.0297), implying that knockdown of USP39 might inhibit the transcription elongation of EGFR, and might produce unstable EGFR mRNA fragments lacking the 3 '-UTR.
To verify whether the regulatory effect of USP39 was EGFR specific, ectopic expression of USP39 were performed in PC-3 and DU145 cells, which showed that overexpressed USP39 upregulated EGFR mRNA and protein levels, indicating that EGFR is a downstream target of USP39.
Silencing of USP39 inhibited the expression of EGFR 3 '-end, and presented a remarkable block to the maturation of EGFR mRNA, suggesting that silencing of USP39 decreased the transcriptional elongation and maturation of EGFR mRNA.
Recently, scientists reported aberrant USP39 expression could inhibit breast cancer cell growth in vitro [XREF_BIBR], however, little is known about how USP39 functions in human osteosarcoma and whether it can be used as an potential therapeutic target.
Further studies demonstrated that depletion of USP39 results in an upregulation of p53 through prolonging its half-life and activating its transcriptional activation activity .
on A549 cells to confirm the key role of p53, the WB result demonstrated that exposure to PFT-alpha (30 or 40 mum/48 h) clearly inhibited the p53 pathway activation, which activated by USP39 knockdown in A549 cells.
Altogether, these results suggest that USP39 knockdown causes a significant accumulation of p53 via regulating both transcriptional levels and post-translational modifications of p53.
In addition, by employing a double luciferase reporter system assay, we found that depletion of USP39 enhances p53 responsive transcriptional reporter activity.
The effect of c-MYC on USP39 expression was further investigated by performing a luciferase assay, and the result showed that forced expression of c-MYC increased luciferase activity of the USP39 promoter reporter containing the wild-type but not the mutant c-MYC binding site.
It showed that the down-regulation of USP39 gene can cause rb1 mRNA splicing abnormalities, which then leaded to downstream target genes e2f4 up-regulated in zebrafish.
We then performed an rb1 mRNA overexpression experiment in usp39 mutants and observed partial rescue of the adenohypophysis phenotype (XREF_FIG, mutant N = 34, ~ 50% showed rescue), validating the importance of usp39 mediated rb1 mRNA splicing in controlling pituitary lineage expansion during development.
Our studies reveal a novel role of usp39 mediated mRNA splicing of rb1 in pituitary cell growth control, which is critical for maintaining embryonic pituitary homeostasis.
Silencing of USP39 could specifically reduce the Aurora B mRNA expression [XREF_BIBR], and mutation of zebrafish USP39 induces rb1 splicing defects and pituitary lineage expansion [XREF_BIBR].
USP39 promotes tumor progression by increasing HMGA2 levels in ovarian cancer cells We next investigated the functional significance of the USP39-HMGA2 axis .
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
Third, we demonstrated that USP39 knockdown inhibits cell migration and invasion by upregulating p53 and the downstream proteins MMP2 and MMP9 [XREF_BIBR].
Third , we demonstrated that USP39 knockdown inhibits cell migration and invasion by upregulating p53 and the downstream proteins MMP2 and MMP9 [ 31 ] .
As shown in XREF_FIG A-F, USP39 knockdown reduced the cell migration and invasion of both USP39KD cells in comparison to the control cells (* p < 0.05, ** p < 0.01, **** p < 0.0001).
Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK and STAT downstream of type I signaling by enhancing IFN stimulated response elements promoter activity and expression of IFN stimulated genes.
Intriguingly, USP39 promotes IFN-mediated antiviral responses by decreasing K6-linked but not canonical K48-linked polyubiquitination of STAT1 for degradation (118), eventhough K6-linked ubiquitin chains are often related to DNA damage instead of protein degradation (142).
Unlike USP2A, the deubiquitinating enzymes BRCC36, USP13, and USP39 positively regulate IFN activities by attenuating the polyubiquitination level of STAT1, and this process is independent of IFN treatment, which suggests divergent functional roles of these DUBs under differential contexts.Additionally, ATXN3 does not affect IFN-I production during viral infection but positively regulates IFNAR1-mediated downstream signaling by targeting HDAC3 (108).
Knockdown of USP39 in VSMCs inhibited cyclinD1 and CDK4 protein expression compared with transfection with control siRNA (XREF_FIG), which are essential proteins for G1/S phase transformation.
Furthermore, knockdown of USP39 inhibited VSMC cell proliferation and the expression of cyclin D1 and cyclin dependent kinase 4, as analyzed via cell counting, MTT assay and western blotting.
In addition, we performed quantitative RT-PCR analysis on the rb1 mRNA injected usp39 embryos and observed a 30% reduction of e2f4 expression compared to control uninjected usp39 mutants (XREF_SUPPLEMENTARY), indicating that e2f4 upregulation in usp39 mutant is secondary to Rb1 loss of function.
Consequently, e2f4 upregulation in usp39 mutants may contribute to increased proliferation of terminally differentiated pituitary cells leading to lineage expansion as seen in our BrdU studies (XREF_SUPPLEMENTARY).
The results suggest that knockdown of USP39 inhibits the growth of HCC in vitro and in vivo, potentially through the induction of G2/M arrest by regulating the pre-mRNA splicing of FoxM1.
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
To verify whether the regulatory effect of USP39 was EGFR specific, ectopic expression of USP39 were performed in PC-3 and DU145 cells, which showed that overexpressed USP39 upregulated EGFR mRNA and protein levels, indicating that EGFR is a downstream target of USP39.
A previous study indicated that USP39 silencing induced defective chromosome segregation and cytokinesis in U2OS cells, indicating USP39 is critical in the regulation of mitosis.
As a confirmed spliceosome factor, USP39 is critical to maintain the spindle checkpoint and promote successful cytokinesis through the regulation of Aurora B mRNA splicing in mammalian cells.
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
Altogether, our data shows that USP39 induces mRNA maturation and elevates the expression of ADAM9 in glioma cells and may thus be considered as potential target for treating patients with glioma.
The results demonstrated that the level of the 3 '-end of EGFR decreased (P = 0.0015) more sharply than that of the 5 '-end (P = 0.0069), and the ratio ofthe 3 '-end to 5 '-end levels was significant decreased (P = 0.0297), implying that knockdown of USP39 might inhibit the transcription elongation of EGFR, and might produce unstable EGFR mRNA fragments lacking the 3 '-UTR.
In yeast, the ABH1 homolog CBP80 and the SAD1 homolog Lsm5 are active components of the spliceosome and mediate various steps of RNA metabolism [47], but these two proteins do not seem to interact dir[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Altogether , our data shows that USP39 induces mRNA maturation and elevates the expression of ADAM9 in glioma cells and may thus be considered as potential target for treating patients with glioma .
A previous study indicated that USP39 silencing induced defective chromosome segregation and cytokinesis in U2OS cells, indicating USP39 is critical in the regulation of mitosis.
USP39 promotes malignant proliferation and angiogenesis of renal cell carcinoma by inhibiting VEGF-A165b alternative splicing via regulating SRSF1 and SRPK1.
USP39 knockdown upregulated the expression of VEGF-A 165b , and USP39 overexpression downregulated the expression of VEGF-A 165b significantly (both P < 0.05).
To test whether USP39 modulate spliceosome activity via SF3B1 , we measured phosphorylated SF3B1 in ovarian cancer cells with USP39 overexpression or knockdown .
Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK and STAT downstream of type I signaling by enhancing IFN stimulated response elements promoter activity and expression of IFN stimulated genes.
Mechanistically, USP39 does not affect the production of type I IFN but significantly promotes JAK and STAT downstream of type I signaling by enhancing IFN stimulated response elements promoter activity and expression of IFN stimulated genes.
The depletion of USP39 has been found to cause activation of the p53 signaling pathway, an important regulatory mechanism in cell cycle and DNA replication, by stabilizing the p53 target p21 in multiple kinds of cancer cell lines [23, 26, 27].
USP39 promotes malignant proliferation and angiogenesis of renal cell carcinoma by inhibiting VEGF-A165b alternative splicing via regulating SRSF1 and SRPK1.
Knockdown of USP39 in VSMCs inhibited cyclinD1 and CDK4 protein expression compared with transfection with control siRNA (XREF_FIG), which are essential proteins for G1/S phase transformation.
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
However, treated with PFT-alpha reduced the up-regulation of p21 and BAX induced by USP39 knockdown, but had no effect on the down-regulation of cell cycle related proteins (CDK1 and CyclinB1 and CDK2 and CyclinA2) induced by USP39 knockdown.
Furthermore, injection of mRNA encoding wild-type usp39 rescued the mutant phenotype (XREF_TABLE), indicating that pituitary pomc upregulation in usp39 mutants results from the nonsense mutation in zebrafish usp39.