FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1
Open accessAbstract
Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte–macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte–macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte–macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF–induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β–FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. Implications: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.
AI snapshot
Written from the paper’s full text.
TL;DR
The paper identifies PU.1 as a GSK3β substrate phosphorylated at Ser41 and Ser140, leading to FBW7-mediated ubiquitination and proteasomal degradation that suppresses monocyte-macrophage differentiation in acute myeloid leukemia. Inhibiting the GSK3β-FBW7 axis restores PU.1 and promotes myeloid differentiation, supporting GSK3β as a potential therapeutic target.
Key findings
- GSK3β phosphorylates PU.1 at Ser41 and Ser140, and this phosphorylation promotes FBW7 recognition and ubiquitin-mediated degradation of PU.1; the phospho-deficient PU.1-S41,S140A mutant neither bound FBW7 nor was degraded by it.
- FBW7 overexpression inhibited PMA-induced macrophage differentiation of U937 cells from 26% to 5.0%, while PU.1-S41,S140A overexpression promoted differentiation more than wild-type PU.1 even without PMA (23.8% vs. 18%). FBW7 overexpression only marginally inhibited PU.1-S41,S140A-induced differentiation (23%–17%) compared with wild-type PU.1 (18%–7.5%).
- FBW7 depletion promoted monocyte-macrophage differentiation of U937 and THP1 cells without inducers, and FBW7 overexpression inhibited M-CSF-induced macrophage differentiation of PBMCs from healthy volunteers.
- In PBMCs from patients with leukemia, FBW7 overexpression downregulated endogenous PU.1, and treatment with M-CSF plus GSK3 inhibitor SB216763 increased CD11b and CD14 expression compared with M-CSF alone.
Methods
- Cell culture and transfection: HEK293T, THP-1, U937, and PBMCs; transient transfections, siRNA/shRNA knockdowns, and inhibitors including SB216763, LiCl, MG132, PMA, oridonin, and M-CSF.
- Biochemical assays: Western blotting, co-immunoprecipitation, GST pull-down, His pull-down, in vivo ubiquitination, cycloheximide chase, in vitro kinase assay, EMSA, luciferase reporter assay, Giemsa staining, and flow cytometry.
- Patient samples: PBMCs from healthy volunteers and two patients with leukemia (P1 with AML and P2 with ALL; Table 1); differentiation markers CD11b and CD14 analyzed by FACS after M-CSF alone or with SB216763. Statistical comparisons used two-tailed Student t tests with P values reported as *P < 0.05, P < 0.001, *P < 0.0001.
Limitations
- The paper does not include a dedicated limitations section.
- Evident from the text: patient validation was limited to two patients (P1 and P2), and many mechanistic experiments relied on HEK293T cells or myeloid cell lines with overexpression/depletion systems.
- Evident from the text: no in vivo animal experiments are described in this paper, although prior mouse-model findings are cited in the Discussion.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
27 of 42 cited sources checked · 13 supported · 2 flagged · accuracy not yet measured
- Premisecitation
PMA is known to induce monocyte-macrophage differentiation in U937 cells through PU.1 upregulation.
“PMA is known induce monocyte-macrophage differentiation in U937 cells through PU.1 upregulation (43)”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [43] Carey, 1996, Phorbol ester-stimulated phosphorylation of PU.1: association with leukemic cell growth inhibition, Blood source
The section “TPA fails to increase the level of PU.1 mRNA transcripts” (page unavailable) reports that TPA did not increase PU.1 transcripts in U937 cells and that their levels slightly decreased after 72 hours. The paper associates TPA-induced maturation with PU.1 phosphorylation and enhanced PU.1-dependent activity, not PU.1 upregulation. (LLM, conf=0.91)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
- Premisecitation
Cellular levels of PU.1 direct different cell fates from hematopoietic progenitors, with highest amounts required for macrophage and lower concentrations for granulocyte or B-cell differentiation.
“cellular levels of PU.1 direct different cell fates from these hematopoietic progenitors where the highest amounts of PU.1 are required for macrophage and lower concentrations for granulocyte or B-cell differentiation (4,5).”
Quote in the paperSubstantial claim1 of 2 sources checked - Premisecitation
GSK3β phosphorylation consensus motif (S/TxxxS/T) very often coincides with FBW7 phosphodegron motifs.
“GSK3b phosphorylation consensus motif (S/TxxxS/T) very often coincides with FBW7 phosphodegron motifs (33).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [33] Doble, 2003, GSK-3: tricks of the trade for a multi-tasking kinase, J Cell Sci source
The Introduction (page not available) gives the GSK-3 substrate consensus as Ser/Thr–X–X–X–Ser/Thr-P, supporting the motif portion of the claim. Our copy does not say that this motif often coincides with FBW7 phosphodegron motifs; searches for FBW7 and phosphodegron did not find that connection. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Findingown data
Proteasome inhibition dramatically increases endogenous PU.1 protein levels in myeloid leukemia cells.
“proteasome inhibition dramatically increased endogenous PU.1 protein levels (Fig. 1C).”
Quote in the paperSubstantial claim - Findingown data
In vitro kinase assay shows GSK3β phosphorylates PU.1.
“in vitro kinase assay which showed that GSK3b did phosphorylate PU.1 which may augment its recognition and targeting by FBW7 (Fig. 2G).”
Quote in the paperSubstantial claim
- Findingown data
PMA inhibits GSK3β in myeloid cells, as measured by increased GSK3β-Ser9 and partially decreased GSK3β-Y216 phosphorylation.
“PMA, a known chemical inducer of PU.1, also inhibited GSK3b in myeloid cells as measured by increased GSK3b-Ser9 and partially decreased GSK3b-Y216 phosphorylation (Fig. 1B)”
Quote in the paperNot a substantial claim - Findingown data
GSK3β does not phosphorylate PU.1-S41,140A.
“GSK3b did not phosphorylate PU.1-S41,140AA suggesting that nonphosphorylated PU.1-S41,140AA could not be recognized by FBW7 (Fig. 5C).”
Quote in the paperSubstantial claim - Findingown data
FBW7 overexpression inhibits PMA-induced differentiation of U937 (from 26% to 5.0%).
“FBW7 overexpression in these cells potently inhibited PMAinduced differentiation (from 26% to 5.0%).”
Quote in the paperSubstantial claim - Findingown data
GSK3β inhibition increases PU.1 expression in myeloid leukemia cells (THP1 and U937).
“GSK3b inhibition indeed markedly increased PU.1 expression in THP1 and U937 cells (Fig. 1A).”
Quote in the paperSubstantial claim - Findingown data
FBW7 depletion enhances endogenous PU.1 in PBMCs.
“FBW7 depletion markedly enhanced endogenous PU.1 levels in these PBMCs (Fig. 4D).”
Quote in the paperSubstantial claim - Findingown data
PBMCs from leukemia patients treated with M-CSF + GSK3 inhibitor have higher CD11b and CD14 than M-CSF alone.
“cells treated with both M-CSF and GSK3 inhibitor together had higher levels of CD11b and CD14 (Fig. 6B, top and bottom graph, respectively”
Quote in the paperSubstantial claim - Findingown data
FBW7 interacts with PU.1 through its WD domain.
“FBW7 also interacted with PU.1 through its WD domain (Fig. 3D).”
Quote in the paperSubstantial claim - Findingown data
GSK3β co-precipitates with FBW7 and PU.1 in myeloid cells.
“GSK3b also co-precipitated together with these proteins in myeloid cells.”
Quote in the paperSubstantial claim - Findingown data
PU.1-S41,140A overexpression promotes myeloid differentiation more than wild-type PU.1 even without PMA (23.8% vs 18%).
“PU.1-S41, S140A overexpression in U937 cells promoted myeloid differentiation more than that of wild-type PU.1 even without PMA induction (23.8% vs. 18%).”
Quote in the paperSubstantial claim - Findingown data
GSK3β interacts with PU.1 mutants having both CPDs disrupted and various PU.1 deletion mutants.
“GSK3b still interacted with the PU.1 mutants having both the CPDs disrupted (Fig. 3G) as well as various PU.1 deletion mutants (Fig. 3H)”
Quote in the paperNot a substantial claim - Findingown data
FBW7 depletion inhibits PU.1 ubiquitination.
“FBW7 depletion markedly inhibited PU.1 ubiquitination (Fig. 1J).”
Quote in the paperSubstantial claim - Findingown data
Depletion of GSK3β or FBW7 in THP1 shows similar differentiation results.
“Depletion of either GSK3b or FBW7 in another monocytic cell line THP1 showed similar results (Fig. 4B).”
Quote in the paperSubstantial claim - Findingown data
FBW7 overexpression only marginally inhibits PU.1-S41,140A induced differentiation (23%-17%) compared with wild-type PU.1 (18%-7.5%).
“while FBW7 overexpression potently mitigated PMA-induced macrophage differentiation of U937 cells as measured by CD14 levels through FACS (from 26% to 5.0%), it only marginally inhibited PU.1-S41, S140A induced differentiation (23%-17%) compared with wild-type PU.1 (18%-7.5%;Fig. 5G).”
Quote in the paperSubstantial claim - Findingown data
FBW7 downregulates PU.1 levels in a proteasome-dependent manner.
“increasing amounts of FBW7 concomitantly downregulated PU.1 levels while proteasome inhibition restored (Fig. 1E).”
Quote in the paperSubstantial claim - Findingown data
GSK3β depletion promotes endogenous PU.1 levels.
“GSK3b depletion promoted endogenous PU.1 levels (Fig. 2C).”
Quote in the paperSubstantial claim - Findingown data
PU.1 protein levels are regulated through ubiquitin-dependent proteasome pathway.
“In agreement with these data, we also demonstrate here that PU.1 protein levels are regulated through ubiquitin-dependent proteasome pathway where we identify FBW7 as the underlying E3 ubiquitin ligase.”
Quote in the paperSubstantial claim - Findingown data
Phosphorylation of PU.1 at Ser41 and Ser140, which coincide with two potential CPDs, is recognized by FBW7.
“Like other protein substrates, phosphorylation of PU.1 at these two serine residues which coincides with two potential CPDs within PU.1, is recognized by FBW7.”
Quote in the paperSubstantial claim - Findingown data
GSK3b cannot phosphorylate PU.1-S41,S140A mutant.
“Because GSK3b could not phosphorylate PU.1-S41, S140A and PU.1-S41, S140A neither interacted with FBW7 nor was”
Quote in the paperSubstantial claim - Findingown data
Overexpression of FBW7 in myeloid cell lines (U937, THP1) and primary PBMCs inhibits PMA- and cytokine (M-CSF)-induced macrophage differentiation.
“In line with essential cellular functions of PU.1, our data also demonstrate that overexpression of FBW7 in myeloid cell lines (U937 and THP1) and primary PBMCs potently inhibits even PMA as well as cytokine (M- CSF)-induced macrophage differentiation, respectively.”
Quote in the paperSubstantial claim - Findingown data
FBW7 is the E3 ubiquitin ligase underlying ubiquitin-dependent proteasomal regulation of PU.1.
“In agreement with these data, we also demonstrate here that PU.1 protein levels are regulated through ubiquitin-dependent proteasome pathway where we identify FBW7 as the underlying E3 ubiquitin ligase.”
Quote in the paperSubstantial claim - Findingown data
M-CSF treatment together with GSK3 inhibition enhances myeloid differentiation of PBMCs from AML patients.
“M-CSF treatment together with GSK3 inhibition enhances myeloid differentiation of PBMCs isolated from peripheral blood of patients with AML”
Quote in the paperSubstantial claim - Findingown data
Transient depletion of FBW7 in myeloid cell lines and primary PBMCs promotes macrophage differentiation.
“On the contrary, mere transient depletion of FBW7 in these cells promoted their macrophage differentiation.”
Quote in the paperSubstantial claim - Findingown data
Phosphorylation of PU.1 at Ser41 and Ser140 by GSK3 primes PU.1 for polyubiquitination and proteasomal degradation by FBW7.
“Consistently, our data also demonstrate that phosphorylation of PU.1 at these two serine residues by GSK3 primes PU.1 to be targeted for polyubiquitination and proteasomal degradation by FBW7.”
Quote in the paperSubstantial claim - Interpretationown data
GSK3β presumably interacts with the C-terminal region of PU.1.
“suggesting that GSK3b presumably interacted with C-terminal region of PU.1.”
Quote in the paperNot a substantial claim - Interpretationreasoning
GSK3β inhibition resolves PU.1 expression by rescuing it from FBW7-mediated ubiquitin-proteasome degradation leading to myeloid differentiation.
“Thus, GSK3b inhibition actually resolves PU.1 expression by rescuing it from FBW7-mediated ubiquitin-proteasome degradation leading to myeloid differentiation.”
Quote in the paperSubstantial claim - Interpretationown data
GSK3 inhibition may restore PU.1 along with other essential genomic/proteomic signatures required for induction of myeloid differentiation in patients with leukemia.
“which further suggests that GSK3 inhibition may restore PU.1 along with other essential genomic/proteomic signatures required for induction of myeloid differentiation in patients with leukemia.”
Quote in the paperSubstantial claim - Interpretationown data
Active GSK3β potentiates FBW7-mediated PU.1 degradation by phosphorylating it within two potential CPDs (starting at amino acids S41 and S140).
“In this study, we demonstrate that active GSK3b potentiates FBW7-mediated PU.1 degradation by phosphorylating it within two potential CPDs (starting at amino acids S41 and S140).”
Quote in the paperSubstantial claim - Interpretationreasoning
FBW7 is a potent negative regulator of macrophage differentiation.
“While earlier studies reported that FBW7 targeted some of the positive transcriptional regulators of PU.1 such as HSF1 (53), GSK3 by phosphorylating PU.1 within its potential CPDs may instigate its recognition by FBW7 for subsequent ubiquitinmediated degradation indicating that FBW7 is a potent negative regulator of macrophage differentiation.”
Quote in the paperSubstantial claim - Interpretationreasoning
GSK3 phosphorylation of PU.1 within its potential CPDs may instigate its recognition by FBW7 for subsequent ubiquitin-mediated degradation.
“While earlier studies reported that FBW7 targeted some of the positive transcriptional regulators of PU.1 such as HSF1 (53), GSK3 by phosphorylating PU.1 within its potential CPDs may instigate its recognition by FBW7 for subsequent ubiquitinmediated degradation indicating that FBW7 is a potent negative regulator of macrophage differentiation.”
Quote in the paperSubstantial claim - Interpretationown data
One plausible mechanism behind GSK3-mediated inhibition of myeloid differentiation, induction of hyperproliferation and leukemogenesis may involve negative regulation of PU.1 by GSK3-FBW7 axis.
“Thus, our data show that one of the plausible mechanisms behind GSK3-mediated inhibition of myeloid differentiation, induction of hyperproliferation and leukemogenesis may involve negative regulation of PU.1 by GSK3-FBW7 axis and therapeutic targeting of this axis may inhibit AML growth and overcome block in differentiation.”
Quote in the paperSubstantial claim- Rests on an assumption: Even a slight decrease in PU.1 levels can lead to leukemogenesis.
- Interpretationreasoning
Inhibition of PU.1 leading to reduced myeloid differentiation and maturation may be a critical step in leukemogenesis.
“This essentially indicates that inhibition of PU.1 leading to reduced myeloid differentiation and maturation may be a critical step in leukemogenesis.”
Quote in the paperSubstantial claim- Rests on an assumption: Even a slight decrease in PU.1 levels can lead to leukemogenesis.
- Premisecitation
PU.1 is a master regulator of monocyte to macrophage differentiation.
“PU.1 is a master regulator of monocyte to macrophage differentiation (4, 42)”
Quote in the paperSubstantial claim0 of 2 sources checked
- Classified as probably not a substantial claim. 29
- May combine two claims. 37
- The quote only partly states this claim. 2
Classified as probably not a substantial claim. May combine two claims.
PremisecitationGSK3β inhibition promotes myeloid differentiation and apoptosis in leukemia cells.
“GSK3b inhibition promotes myeloid differentiation and apoptosis in leukemia cells (27)(28)(29)(30); however, mechanisms underlying GSK3b inhibition leading to myeloid differentiation are poorly understood.”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [27] Banerji, 2012, The intersection of genetic and chemical genomic screens identifies GSK-3alpha as a target in human acute myeloid leukemia, J Clin Invest source
In “Loss of GSK-3α leads to differentiation of AML cells in vitro” (page unavailable), the paper reports that GSK-3β suppression was not enriched for monocyte-differentiation gene sets; it attributes the differentiation effect to GSK-3α suppression. In “Loss of GSK-3α decreases AML cell growth and proliferation” (page unavailable), it likewise reports apoptosis after GSK-3α suppression, not GSK-3β suppression. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured - cites [28] Gupta, 2012, GSK3 is a regulator of RAR-mediated differentiation, Leukemia source
The Results sections on AML differentiation and genetic evidence report that GSK3 inhibition promotes myeloid differentiation and that GSK3β knockdown increases differentiation. The Results section on AML growth reports growth inhibition, not apoptosis; the copy does not establish the apoptosis part of the claim. No page numbers are available in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [29] Zhou, 2011, GSK3beta inactivation induces apoptosis of leukemia cells by repressing the function of c-Myb, Mol Biol Cell source
The Results section reports that GSK3β inhibitor treatment increased apoptosis in leukemia cell lines. The paper does not report that inhibition promotes myeloid differentiation; its Introduction mentions differentiation only in connection with c-Myb, not GSK3β inhibition. No page numbers are available in our copy. (LLM, conf=0.90)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationATRA-mediated myeloid differentiation involves GSK3β inhibition.
“some of the studies suggested that ATRA-mediated myeloid differentiation involves GSK3b inhibition (32)”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [32] Brown, 2012, Retinoid differentiation therapy for common types of acute myeloid leukemia, Leuk Res Treatment source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationPU.1 is pivotal for normal hematopoiesis and directs differentiation of hematopoietic progenitors to macrophage, granulocyte, or B cell lineages.
“Ets family transcription factor PU.1 is pivotal for normal hematopoiesis as it directs differentiation of hematopoietic progenitors to different cell lineages such as macrophage, granulocyte or B cell (1).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [1] Dakic, 2005, PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis, J Exp Med source
The Abstract and Discussion (page not provided) support PU.1’s importance in hematopoiesis and its role in progenitor commitment, including pathways leading to B cells and macrophages. But the paper does not describe PU.1 as simply directing progenitors toward all three listed lineages: it reports that loss of PU.1 enhances granulocyte production in adults, while PU.1 helps restrict that production and supports later granulocyte maturation. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationFBW7 is the substrate recognition motif of multimeric E3 ligase SCF-FBW7 which binds to substrates by recognizing phosphorylated motifs via its WD40 repeat regions.
“F-box protein FBW7 is the substrate recognition motif of multimeric E3 ligase SCF-FBW7 (SKP-cullin-Fbox) which binds to substrates by recognizing phosphorylated motifs (phosphodegron motif) via its WD40 repeat regions (14,15).”
Quote in the paperSubstantial claim2 of 2 sources checkedClassified as probably not a substantial claim.
PremisecitationGSK3β drives protein destruction majorly through E3 ubiquitin ligase FBW7.
“GSK3b drives protein destruction majorly through E3 ubiquitin ligase FBW7 (also called FBXW7, hCDC4, and SEL-10; ref. 14).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [14] Davis, 2014, Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities, Cancer Cell source
The section “Substrate phosphorylation stimulates Fbw7 binding” (page not supplied) says GSK3 phosphorylates many Fbw7-recognized degrons, linking its activity to Fbw7-mediated degradation. It does not establish that GSK3β drives protein destruction *mainly* through FBW7; it notes that most substrates have multiple turnover pathways. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim.
PremisecitationPU.1 regulates vast number of myeloid gene promoters including GM-CSF receptor α, M-CSF receptor, CD163, and CD11b.
“PU.1 imparts its key functions by regulating vast number of myeloid gene promoters that include GM-CSF receptor a, M-CSF receptor (M-CSFR), scavenger receptor CD163, and CD11b, etc (6,7).”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [6] Hohaus, 1995, PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene, Mol Cell Biol source
The Discussion (page not supplied) says that most myeloid cell-specific promoters contain functional PU.1 sites, including the M-CSF receptor promoter, and identifies a functional PU.1 site in the GM-CSF receptor α promoter. The paper also names CD11b as a previously described myeloid PU.1 target. Our copy does not identify CD163 as a PU.1-regulated promoter, so the full list is not supported. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [7] Pahl, 1993, The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter, J Biol Chem source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationIn some AMLs, GSK3β acts as tumor promoter by targeting tumor suppressors such as p27.
“in some of the AMLs, GSK3b was shown to act as tumor promoter by targeting tumor suppressors such as p27 (13).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [13] Wang, 2008, Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy, Nature source
Classified as probably not a substantial claim.
PremisecitationATRA restores myeloid differentiation by resolving PU.1 expression in AML.
“all-trans retinoic acid (ATRA) restores myeloid differentiation by resolving PU.1 expression in AML (31)”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] Mueller, 2006, ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression, Blood source
The Introduction and Discussion report that ATRA restores PU.1 expression and induces myeloid differentiation in PML-RARA-positive, t(15;17) acute promyelocytic leukemia cells. The claim extends that finding to AML generally, which the paper does not establish. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLipopolysaccharide regulates PU.1 at protein levels in macrophages.
“In another study, it was shown that lipopolysaccharide also regulated PU.1 at protein levels in macrophages (50).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [50] Jung, 2011, The macrophage-specific transcription factor can be modified posttranslationally by ubiquitination in the lipopolysaccharide-treated macrophages, Tuberc Respir Dis source
Classified as probably not a substantial claim.
PremisecitationATRA restores PU.1 expression in AMLs bearing t(15;17).
“Moreover, because ATRA also restores PU.1 expression in AMLs bearing t(15;17) (31), we hypothesized that GSK3b negatively regulated PU.1 levels leading to AML”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [31] Mueller, 2006, ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression, Blood source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGSK3 regulates multiple cellular processes including cell growth, differentiation, and apoptosis in response to environmental stimuli.
“GSK3, originally identified as a key enzyme of glycolytic pathway, was subsequently shown to regulate multiple cellular processes, including cell growth, differentiation, and apoptosis in response to explicit environmental stimuli (11,33).”
Quote in the paperNot a substantial claim1 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationGSK3-mediated phosphorylation of substrates imparts differential actions ranging from protein localization, protein-protein interaction to protein stability.
“GSK3-mediated phosphorylation of substrates imparts differential actions to the substrate proteins that ranges from protein localization, protein-protein interaction to protein stability (33).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [33] Doble, 2003, GSK-3: tricks of the trade for a multi-tasking kinase, J Cell Sci source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationGSK3b negatively impacts expression and transcriptional activity of retinoic acid receptor by phosphorylating it at Ser445, so GSK3 inhibition promotes myeloid differentiation.
“Gupta and colleagues demonstrated that GSK3b negatively impacted expression and transcriptional activity of ATRA's receptor, the retinoic acid receptor by phosphorylating it at Ser445, hence GSK3 inhibition promoted myeloid differentiation (28).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [28] Gupta, 2012, GSK3 is a regulator of RAR-mediated differentiation, Leukemia source
The Results section (‘GSK3 modulates RAR expression and activity’; page unavailable) shows that GSK3β phosphorylates RARα at Ser445, that GSK3 inhibition increases RAR expression and transcriptional activity, and that a S443A/S445A mutant has increased RAR levels. Another Results section (‘GSK3 inhibition alone induces moderate AML differentiation’; page unavailable) reports differentiation after GSK3 inhibition. The paper does not establish that phosphorylation at Ser445 alone causes both the reduced expression and reduced transcriptional activity asserted in the sentence. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
PremisecitationSpecific cellular levels of PU.1 are critical for distinct cell fates; highest PU.1 amounts drive macrophage development and lower concentrations are required for granulocytic/B-cell fate adoption.
“Volumes of evidences indicate that specific cellular levels of PU.1 are critical for distinct cell fates, where highest PU.1 amounts drive macrophage development and lower concentrations are required for granulocytic/B-cell fate adoption (4,5).”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [4] DeKoter, 2000, Regulation of B lymphocyte and macrophage development by graded expression of PU.1, Science source
- cites [5] Kueh, 2013, Positive feedback between PU.1 and the cell cycle controls myeloid differentiation, Science source
The supplied passage labeled Abstract (page not provided) reports that macrophages up-regulated PU.1, B cells down-regulated it, and developing granulocytes maintained progenitor-like levels. The paper also reports that increased PU.1 promotes macrophage differentiation and inhibits B-cell development. It does not establish that a lower PU.1 concentration is required for granulocyte fate. (LLM, conf=0.84)
Experimental — accuracy not yet measured
May combine two claims.
PremisecitationActive GSK3b regulates pathways that influence leukemogenesis, leukemia cell survival, myeloid differentiation, and resistance to chemotherapy.
“Plethora of studies in last decade showed that active GSK3b is involved in the regulation of number of pathways that can influence leukemogenesis, leukemia cell survival (13,46), myeloid differentiation, and resistance to chemotherapy.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [13] Wang, 2008, Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy, Nature source
The Discussion and the section ‘GSK3 dependence is a general feature of MLL transformed cells’ link GSK3 activity to MLL leukemogenesis and continued proliferation, and report myeloid differentiation after GSK3 inhibition. The paper does not establish resistance to chemotherapy. It also finds that GSK3α and GSK3β cooperate, so these findings cannot be assigned solely to active GSK3β. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [46] Holmes, 2008, Glycogen synthase kinase-3beta inhibition preserves hematopoietic stem cell activity and inhibits leukemic cell growth, Stem Cells source
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationK63-ubiquitination of PU.1 promotes its degradation in a p62-dependent selective autophagy manner in Th9 cells, leading to reduced protein turnover and antitumor immunity.
“Rivera Vargas and colleagues, recently demonstrated K63-ubuqtination of PU.1 promoted its degradation in a p62-depenent selective autophagy manner in Th 9 cells leading to reduced protein turnover and antitumor immunity (49).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [49] Rivera Vargas, 2017, Selective degradation of PU.1 during autophagy represses the differentiation and antitumour activity of TH9 cells, Nat Commun source
The Discussion (page unavailable) states that K63-ubiquitinated PU.1 is recruited by p62 for selective autophagic degradation in TH9 cells, and that blocking autophagy enhances their anticancer function. The claim's 'reduced protein turnover' does not follow: degradation increases PU.1 turnover; blocking that degradation stabilizes PU.1. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Classified as probably not a substantial claim. May combine two claims.
PremisecitationGSK3b inhibition sensitizes AML cells to ATRA and to 1,25 vitamin D-mediated differentiation.
“While GSK3b inhibition was independently shown to sensitize AML cells to ATRA (48) as well 1,25 vitamin D-mediated differentiation (30), the underlying molecular mechanism remained unknown.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [48] Si, 2011, GSK3 inhibitors enhance retinoic acid receptor activity and induce the differentiation of retinoic acid-sensitive myeloid leukemia cells, Leukemia source
- cites [30] Gupta, 2016, GSK-3 inhibition sensitizes acute myeloid leukemia cells to 1,25D-mediated differentiation, Cancer Res source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationGSK3b inhibition overcomes differentiation block and induces apoptosis in leukemia.
“Owing to its oncogenic role in leukemia, GSK3b inhibition was shown to overcome differentiation block and induce apoptosis (29).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [29] Zhou, 2011, GSK3beta inactivation induces apoptosis of leukemia cells by repressing the function of c-Myb, Mol Biol Cell source
The Results section reports that GSK3β inhibitors increased apoptosis in Jurkat and K562 leukemia cells. The paper does not report that inhibition overcame a differentiation block; its discussion focuses on apoptosis and reduced expression of survival factors. Page numbers are unavailable in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationUSP22 positively regulates PU.1 protein levels and its target genes by deubiquitinating PU.1, leading to myeloid differentiation; USP22 loss causes leukemogenesis upon K-RAS activation in a PU.1-dependent mechanism.
“In addition, a recent study demonstrated that USP22 positively regulated PU.1 protein levels and its target genes by deubiquitinating PU.1 leading to myeloid differentiation while USP22 loss caused leukemogenesis upon K-RAS activation in a PU.1-dependent mechanism (51).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites [51] Melo-Cardenas, 2018, USP22 deficiency leads to myeloid leukemia upon oncogenic Kras activation through a PU.1-dependent mechanism, Blood source
Classified as probably not a substantial claim. May combine two claims.
PremisecitationAn aberrant pool of nuclear GSK3b is present in some AMLs and drives AML growth and drug resistance.
“Previous studies also showed that an aberrant pool of nuclear GSK3b is seen in some of AMLs which drives AML growth and drug resistance.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [47] Ignatz-Hoover, 2018, Aberrant GSK3beta nuclear localization promotes AML growth and drug resistance, Blood Adv source
May combine two claims.
PremisecitationNuclear GSK3b enhances AML colony formation and AML growth in mouse models.
“This nuclear, but not cytoplasmic, GSK3b was shown to enhance AML colony formation and AML growth in mouse models (47).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [47] Ignatz-Hoover, 2018, Aberrant GSK3beta nuclear localization promotes AML growth and drug resistance, Blood Adv source
May combine two claims.
Findingown dataFBW7 overexpression inhibits endogenous PU.1 in PBMCs; MG132 restores.
“FBW7 drastically inhibited endogenous PU.1 levels while MG132 treatment restored (Fig. 4C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 ubiquitinates and degrades PU.1 in a proteasome-dependent manner; FBW7ΔF-box does not.
“FBW7 indeed ubiquitinated and degraded PU.1 in a proteasome-dependent manner, FBW7DF-box did not (Fig. 1I).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 and PU.1 interact in vitro and in vivo.
“FBW7 and PU.1 indeed interacted with each other both in vitro (GST-pull down; Fig. 3A) and in vivo (co-IP) in heterologous HEK293T (Fig. 3B) and myeloid cells (Fig. 3C)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPU.1 and PU.1-S41,140A enhance M-CSFR promoter luciferase activity; FBW7 inhibits transactivation of PU.1 but not PU.1-S41,140A.
“while both PU.1 and PU.1-S41,140A enhanced luciferase promoter activity, contransfection of FBW7 significantly inhibited transactivation potential of PU.1 but not PU.1-S41,140A (Fig. 5D).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPU.1 single-point mutants S41A and S140A are still downregulated by GSK3β and FBW7; double mutant S41,140A or del71-160,140A are resistant to degradation by FBW7.
“both GSK3b and FBW7 still downregulated these single-point mutants of PU.1; however, the PU.1 mutants having both CPDs (PU.1-S41,140A) or all the three putative CPDs (PU.1-del71-160,140A) disrupted were resistant to degradation by FBW7 (Fig. 2I).”
Quote in the paperSubstantial claimMay combine two claims. The quote only partly states this claim.
Findingown dataPU.1-S41,140A has enhanced half-life and is resistant to ubiquitination by FBW7.
“PU.1-S41,140AA having both CPDs disrupted showed enhanced half-life (Fig. 5A) and was resistant to ubiquitination by FBW7 (Fig. 5B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 interacts with PU.1 mutants having one CPD disrupted, but almost no interaction with PU.1 mutants having all CPDs disrupted.
“although FBW7 still interacted with PU.1 mutants having one of the CPDs disrupted, almost no interaction was observed with PU.1 mutants having all the CPDs disrupted.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPU.1-S41,S140A neither binds to FBW7 nor is degraded by it.
“a phospho-deficient PU.1 mutant (PU.1-S41,S140A) neither bound to FBW7 nor was degraded by it.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 overexpression downregulates endogenous PU.1 in myeloid cells (THP1, U937); FBW7 depletion enhances endogenous PU.1.
“FBW7 overexpression downregulated endogenous PU.1 protein levels in myeloid cells (Fig. 1K), its depletion markedly enhanced endogenous PU.1 levels (Fig. 1L).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGSK3β overexpression inhibits PU.1; simultaneous FBW7 depletion restores PU.1, indicating interdependence of FBW7 and GSK3β in targeting PU.1 for degradation.
“GSK3b potently inhibited, simultaneous FBW7 depletion in GSK3b overexpressed condition restored endogenous PU.1 levels (Fig. 2D) indicating for an interdependence of FBW7 and GSK3b in targeting PU.1 for degradation.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIndividual depletion of FBW7 and GSK3β promotes monocyte-macrophage differentiation of U937; simultaneous PU.1 depletion substantially mitigates.
“while individual depletion of FBW7 and GSK3b promoted monocytemacrophage differentiation of U937 cells, simultaneous PU.1 depletion substantially mitigated suggesting that RNAi-mediated FBW7 and GSK3b inhibition indeed promotes macrophage differentiation by targeting PU.1 (Fig. 4A: bottom)”
Quote in the paperSubstantial claimMay combine two claims. The quote only partly states this claim.
Findingown dataFBW7 depletion induces monocyte-macrophage differentiation in U937 without PMA; GSK3β overexpression did not.
“depletion of FBW7 either alone or even in GSK3b overexpressed condition markedly induced monocyte-macrophage differentiation in promonocytic U937 cells without induction with any known differentiation inducers such as PMA (Fig. 4A; top).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 overexpression inhibits M-CSF-induced macrophage differentiation of PBMCs; FBW7 depletion induces differentiation.
“FBW7 overexpression inhibited even cytokine (M-CSF) induced macrophage differentiation of the PBMCs while mere depletion of FBW7 induced them to differentiate toward macrophages (Fig. 4E).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGSK3β overexpression inhibits ectopic and endogenous PU.1 in HEK293T and THP1; MG132 restores PU.1 levels.
“GSK3b overexpression also inhibited both ectopically expressed as well as endogenous PU.1 in heterologous HEK293T (Fig. 2A) and physiologically relevant THP1 cells (Fig. 2B), respectively, while MG312 restored PU.1 levels.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 ligase activity is required to downregulate PU.1; ligase-dead FBW7 mutants (F-box deleted or WD domain only) have almost no inhibitory effect on PU.1 protein levels.
“ligase activity of FBW7 was required to downregulate PU.1 because liagse-dead FBW7 expression plasmids (F-box deleted or only WD domain containing FBW7 mutants) had almost no inhibitory effect on PU.1 protein levels (Fig. 1H).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFBW7 inhibits PU.1 binding to M-CSFR promoter but not PU.1-S41,140A DNA-binding.
“while FBW7 cotransfection had no effect on PU.1-S41, S140A DNA-binding ability, it markedly inhibited PU.1 binding on M-CSFR promoter (Fig. 5E).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPMA-treated leukemia cells show increased PU.1 coupled with reduced GSK3β activity (enhanced Ser9 phosphorylation) and partial reduction in FBW7 protein levels.
“copiously increased PU.1 levels in PMA-treated leukemia cells was not only coupled with reduced GSK3b activity (enhanced GSK3b phosphorylation at Ser9; Fig. 1B) but also with partial reduction in FBW7 protein levels (Fig. 1M)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataOverexpression of degradation-resistant phospho-deficient mutant of PU.1 enhances transactivation and promotes macrophage differentiation even without external inducer such as PMA.
“Not only FBW7 depletion, overexpression of degradation-resistant phospho-deficient mutant of PU.1 also led to enhanced transactivation and promoted macrophage differentiation even without any external inducer such as PMA.”
Quote in the paperSubstantial claimMay combine two claims.
FindingreasoningPU.1-S41,S140A neither interacts with FBW7 nor is degraded by it.
“Because GSK3b could not phosphorylate PU.1-S41, S140A and PU.1-S41, S140A neither interacted with FBW7 nor was degraded by it, It appears that like in case of other GSK3 substrates (17), PU.1 phosphorylation by GSK apparently creates highaffinity binding site for FBW7 which eventually targets PU.1 but not the interaction-resistant PU.1-S41, S141A for polyubiquitination and proteasomal degradation.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGSK physically interacts with and apparently phosphorylates PU.1 at Ser41 and Ser140.
“In accordance with protein stability regulation of these substrates, our data also show that GSK physically interacts and apparently phosphorylates PU.1 at Ser41 and Ser140.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationGSK3β is constitutively active and acts as tumor suppressor in solid tumors by promoting proteolysis of oncogenic proteins such as β-catenin, c-myc, MCL1.
“GSK3b is constitutively active and has been shown to act as tumor suppressor by promoting proteolysis of key oncogenic proteins such as b-catenin, c-myc, MCL1, etc. in solid tumors (11).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11] Ruvolo, 2017, GSK-3 as a novel prognostic indicator in leukemia, Adv Biol Regul source
May combine two claims.
PremisecitationPU.1-/- mice lack mature myeloid cells and PU.1-deficient hematopoietic stem cells do not develop into earliest myeloid and lymphoid progenitors.
“PU.1 À/À mice lacks mature myeloid cells and PU.1-difiecient hematopoietic stem cells also do not develop into earliest myeloid and lymphoid progenitors which results in a block in the development of both lineages (2,3).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMutations in PU.1 gene are reported in patients with acute myeloid leukemia (AML).
“mutations in PU.1 gene are reported in patients with acute myeloid leukemia (AML; ref. 9).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9] Mueller, 2002, Heterozygous PU.1 mutations are associated with acute myeloid leukemia, Blood source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationFBW7 targets positive transcriptional regulators of PU.1 such as HSF1.
“While earlier studies reported that FBW7 targeted some of the positive transcriptional regulators of PU.1 such as HSF1 (53), GSK3 by phosphorylating PU.1 within its potential CPDs may instigate its recognition by FBW7 for subsequent ubiquitinmediated degradation indicating that FBW7 is a potent negative regulator of macrophage differentiation.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [53] Jego, 2014, Dual regulation of SPI1/PU.1 transcription factor by heat shock factor 1 (HSF1) during macrophage differentiation of monocytes, Leukemia source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenInformed and written consent was obtained from participating individuals.
“after obtaining informed and written consent from participating individuals.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductnone givenThe study was approved by institutional research board of KGMU and adherent to Declaration of Helsinki.
“The study was approved by institutional research board (Ref No. 83rd ECM II-A/P21) of KGMU and was adherent to the regulations of the declaration of Helsinki.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataData are representative of three independent experiments.
“Data are representative of three independent experiments.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningTherapeutic targeting of GSK3-FBW7 axis may inhibit AML growth and overcome block in differentiation.
“Thus, our data show that one of the plausible mechanisms behind GSK3-mediated inhibition of myeloid differentiation, induction of hyperproliferation and leukemogenesis may involve negative regulation of PU.1 by GSK3-FBW7 axis and therapeutic targeting of this axis may inhibit AML growth and overcome block in differentiation.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataData shown are representative of three independent experiments.
“Data are representative of three independent experiments.”
- Also stated in: § Authors' Disclosures · plain
“Data are representative of three independent experiments.”
- Also stated in: § Authors' Disclosures · plain
“Data are representative of three independent experiments.”
Quote in the paperNot a substantial claim- Also stated in: § Authors' Disclosures · plain
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataMycoplasma negativity in cell lines was confirmed by PCR.
“Mycoplasma negativity in these cells was confirmed by PCR using Universal Mycoplasma Detection Kit (ATCC, catalog no.30-1012K).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationATRA inhibits GSK3b in AML cells in a time- and concentration-dependent manner by phosphorylating its N-terminal serine, as an early step to induce myeloid differentiation.
“However, ATRA itself was also shown to inhibit GSK3b in AML cells in a time-and concentration-dependent manner by phosphorylating its N-terminal serine (48) as an early step to induce myeloid differentiation highlighting the importance of GSK-3b inhibition in myeloid differentiation.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [48] Si, 2011, GSK3 inhibitors enhance retinoic acid receptor activity and induce the differentiation of retinoic acid-sensitive myeloid leukemia cells, Leukemia source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataCell lines were authenticated by short tandem repeat profiling.
“their authentication was confirmed by short tandem repeat profiling on payment basis through DNA forensics laboratory private limited.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
InterpretationreasoningRapid changes in PU.1 protein turnover are needed for cells to react to external cues/signals.
“In view of the fact that precise levels of PU.1 are required for distinct cell fates, rapid changes in PU.1 protein turnover is needed for cells to allow them to react to respective external cues/signals.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningSubstantial inhibition of macrophage differentiation by FBW7 overexpression may be due to reduced stability of PU.1, reflected by diminished transactivation and DNA-binding ability of PU.1 on its target gene.
“Substantial inhibition of macrophage differentiation in these cells may likely be due to reduced stability of PU.1 which is also reflected by diminished transactivation and DNA-binding ability of PU1 on its target gene.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningPU.1 phosphorylation by GSK creates a high-affinity binding site for FBW7, which targets PU.1 but not interaction-resistant PU.1-S41,S140A for polyubiquitination and proteasomal degradation.
“It appears that like in case of other GSK3 substrates (17), PU.1 phosphorylation by GSK apparently creates highaffinity binding site for FBW7 which eventually targets PU.1 but not the interaction-resistant PU.1-S41, S141A for polyubiquitination and proteasomal degradation.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [17] Wei, 2005, The v-Jun point mutation allows c-Jun to escape GSK3-dependent recognition and destruction by the Fbw7 ubiquitin ligase, Cancer Cell source
The paper relies on this without support.
PU.1 reduced expression leads to leukemia in mice.
“Moreover, PU.1 reduced expression also leads to leukemia in mice.”
The paper relies on this without support.
PMA is a known chemical inducer of PU.1.
“PMA, a known chemical inducer of PU.1”
The paper relies on this without support.
The molecular mechanism by which GSK3b inhibition promotes myeloid differentiation was unknown.
“While GSK3b inhibition was independently shown to sensitize AML cells to ATRA (48) as well 1,25 vitamin D-mediated differentiation (30), the underlying molecular mechanism remained unknown.”
The paper relies on this without support.
Even a slight decrease in PU.1 levels can lead to leukemogenesis.
“In wake of perturbation, even a slight decrease in PU.1 levels can lead to leukemogenesis.”
The paper relies on this without support.
How PU.1 protein turnover is regulated was largely unknown.
“However, unlike extensive data available about PU.1 gene regulation itself, how the PU.1 protein turnover is regulated remains wide open.”
The paper relies on this without support.
β-TRCP and FBW7 are potential F-box E3 ligases that regulate stability of GSK3-phosphorylated substrates.
“b-TRCP and FBW7 are the two potential F-box E3 ligases that have been shown to regulate stability of protein substrates phosphorylated by GSK3.”
The paper relies on this without support.
PU.1 is the master regulator of monocyte-macrophage differentiation and its expression/activity is often inhibited in some AMLs.
“PU.1 is the master regulator of monocyte-macrophage differentiation as it regulates key regulators (CD11b, GM-CSF receptor a, M-CSFR, etc.) of monocyte-macrophage differentiation and maturation, therefore, expression and activity of PU.1 is often inhibited in some cases of myelogenous leukemia (AML).”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Three loci in PU.1 match FBXW7 phosphodegron motifs.
“Specifically, we analyzed the amino acid sequence of PU.1 and found three loci in accordance with the FBXW7 phosphodegron motifs (S/TxxxS/T), which was consistent with a previous study29 (Fig. 4f).”
From Targeting Msx2 as a brake in the fusion fate of osteoclasts and an anabolic therapy in pre-clinical models of osteoporosis (2025) · cites this paper as [29]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
AML patient group includes a patient listed as having ALL
MinorThe section describes the two patients studied in Figure 6 as patients with AML, but Table 1 identifies P2 as having ALL. The stated AML-patient conclusion therefore includes a patient the paper classifies differently.
“M-CSF treatment together with GSK3 inhibition enhances myeloid differentiation of PBMCs isolated from peripheral blood of patients with AML”
“| P2 | 21/F | Peripheral blood | ALL | CD10, CD38, cCD79a, cCD22, CD99 | Normal karyotype |”
Final mutant designation changes Ser140 to Ser141
MinorThe concluding description names an S41,141A mutant, while the degradation-resistant mutant described and used elsewhere is S41,140A.
“a PU.1 mutant (PU.1-S41,141A) with disrupted CPDs is resistant to FBW7 degradation”
“PU.1-S41, S140A is resistant to degradation by FBW7”
SB216763 dose differs between methods and Figure 1
MinorThe methods state that 20 mmol/L SB216763 was used, whereas the Figure 1 caption says those cells received 30 mmol/L.
“A total of 20 mmol/L SB216763 (Sigma) and 10 mmol/L MG132 (Abcam) were used as GSK3b and proteasome inhibitors, respectively.”
“WCEs of THP-1 and U937 cells treated with 30 mmol/L SB216763 and 20 mmol/L LiCl”
Deleted PU.1 mutant has conflicting point-mutation labels
MinorThe results identify the deletion mutant as carrying a 140A substitution, whereas the Figure 2I description identifies it as carrying S41A. These are different mutant constructs.
“PU.1-del71-160,140A”
“FLAG-PU.1D71-160,S41A-His”
Figure 5 differentiation mutant changes from 140A to 140S
MinorThe description of the Figure 5 differentiation result names PU.1-S41,140S, but the Figure 5 caption identifies the transfected mutant as PU.1-S41,140A.
“PU.1-S41,140S promoted macrophage differentiation even in FBW7 cotransfection conditions”
“U937 cells transfected either with Flag-PU.1-S41,140A-His and FBW7 alone or both together”
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BackgroundPositive Feedback Between PU.1 and the Cell Cycle Controls Myeloid Differentiation
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Self-citationGSK3 is a regulator of RAR-mediated differentiation
Gupta, K., Gulen, F., Sun, L., Aguilera, R., Chakrabarti, A., Kiselar, J., Agarwal, M. K., & Wald, D. N. (2012) · Leukemia · doi:10.1038/leu.2012.2 · Cited by 50 · see citing works
GSK-3 inhibition sensitizes acute myeloid leukemia cells to 1,25D-mediated differentiation
Gupta, K., Stefan, T., Ignatz-Hoover, J., Moreton, S., Parizher, G., Saunthararajah, Y., & Wald, D. N. (2016) · Cancer Research · doi:10.1158/0008-5472.can-15-2290 · Cited by 44 · see citing works
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Retinoid Differentiation Therapy for Common Types of Acute Myeloid Leukemia
Brown, G., & Hughes, P. (2012) · Leukemia Research and Treatment · doi:10.1155/2012/939021 · Cited by 39 · see citing works
GSK-3 as a novel prognostic indicator in leukemia
Ruvolo, P. P. (2017) · Advances in Biological Regulation · doi:10.1016/j.jbior.2017.05.001 · Cited by 39 · see citing works
Hu, S., Ueda, M., Stetson, L. C., Ignatz-Hoover, J. J., Moreton, S. A., Chakrabarti, A., Xia, Z., Karan, G., Lima, M. J. G. D., Agrawal, M., et al. (2016) · Molecular Cancer Therapeutics · doi:10.1158/1535-7163.mct-15-0566 · Cited by 37 · see citing works
Lochab, S., Pal, P., Kapoor, I., Kanaujiya, J. K., Sanyal, S., Behre, G., & Trivedi, A. K. (2013) · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research · doi:10.1016/j.bbamcr.2013.06.018 · Cited by 35 · see citing works
Self-citationHeterozygous PU.1 mutations are associated with acute myeloid leukemia
Mueller, B. U., Pabst, T., Osato, M., Asou, N., Johansen, L. M., Minden, M. D., Behre, G., Hiddemann, W., Ito, Y., & Tenen, D. G. (2003) · Blood · doi:10.1182/blood-2002-12-3903 · Cited by 35 · see citing works
Pal, P., Lochab, S., Kanaujiya, J. K., Kapoor, I., Sanyal, S., Behre, G., & Trivedi, A. K. (2013) · Cell Death and Disease · doi:10.1038/cddis.2013.120 · Cited by 34 · see citing works
Self-citationPU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding
Zhang, P., Zhang, X., Iwama, A., et al. (2000) · Blood · doi:10.1182/blood.v96.8.2641.h8002641_2641_2648 · Cited by 34 · see citing works
Si, J., Mueller, L., & Collins, S. J. (2011) · Leukemia · doi:10.1038/leu.2011.171 · Cited by 30 · see citing works
Lentiviral PU.1 overexpression restores differentiation in myeloid leukemic blasts
Durual, S., Rideau, A., Ruault-Jungblut, S., Cossali, D., Beris, P., Piguet, V, & Matthes, T. (2007) · Leukemia · doi:10.1038/sj.leu.2404645 · Cited by 30 · see citing works
Aberrant GSK3β nuclear localization promotes AML growth and drug resistance
Ignatz-Hoover, J. J., Wang, V., Mackowski, N. M., Roe, A. J., Ghansah, I. K., Ueda, M., Lazarus, H. M., de Lima, M., Paietta, E., Fernandez, H., et al. (2018) · Blood Advances · doi:10.1182/bloodadvances.2018016006 · Cited by 27 · see citing works
Durgan, J., & Parker, P. J. (2010) · Biochemical Journal · doi:10.1042/bj20100799 · Cited by 26 · see citing works
GSK3β/Axin-1/β-Catenin Complex Is Involved in Semaphorin3A Signaling
Hida, T., Yamashita, N., Usui, H., Nakamura, F., Sasaki, Y., Kikuchi, A., & Goshima, Y. (2012) · Journal of Neuroscience · doi:10.1523/jneurosci.6139-11.2012 · Cited by 25 · see citing works
Ito, T., Nishiyama, C., Nishiyama, M., Matsuda, H., Maeda, K., Akizawa, Y., Tsuboi, R., Okumura, K., & Ogawa, H. (2005) · Journal of Immunology · doi:10.4049/jimmunol.174.1.376 · Cited by 25 · see citing works
Proteomic identification of E 6 AP as a molecular target of tamoxifen in MCF 7 cells
Lochab, S., Pal, P., Kanaujiya, J. K., Tripathi, S. B., Kapoor, I., Bhatt, M. L. B., Sanyal, S., Behre, G., & Trivedi, A. K. (2012) · PROTEOMICS · doi:10.1002/pmic.201100572 · Cited by 23 · see citing works
Self-citationA Nucleolar Isoform of the Fbw7 Ubiquitin Ligase Regulates c-Myc and Cell Size
Welcker, M., Orian, A., Grim, J. E., Eisenman, R. N., & Clurman, B. E. (2005) · Current Biology · doi:10.1016/j.cub.2005.12.001 · Cited by 23 · see citing works
Kapoor, I., Kanaujiya, J. K., Kumar, Y., Thota, J. R., Bhatt, M. L. B., Chattopadhyay, N., Sanyal, S., & Trivedi, A. K. (2015) · Oncotarget · doi:10.18632/oncotarget.6156 · Cited by 19 · see citing works
Self-citationThacker, G., Mishra, M., Sharma, A., et al. (2019) · Journal of Cellular Biochemistry · doi:10.1002/jcb.29516 · Cited by 17 · see citing works
Self-citationKumar, Y., Shukla, N., Thacker, G., Kapoor, I., Lochab, S., Bhatt, M. L. B., Chattopadhyay, N., Sanyal, S., & Trivedi, A. K. (2016) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-16-0138 · Cited by 14 · see citing works
Self-citationOptimization of a cAMP response element signal pathway reporter system
Shan, Q., & Storm, D. R. (2010) · Journal of Neuroscience Methods · doi:10.1016/j.jneumeth.2010.06.003 · Cited by 12 · see citing works
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Axin-1/β-catenin/GSK3β complex is involved in Semaphorin3A signaling
Hida, T., Yamashita, N., Usui, H., et al. (2011) · Neuroscience Research · doi:10.1016/j.neures.2011.07.571
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GSK-3: a multifaceted player in acute leukemias
Martelli, A. M., Evangelisti, C., Paganelli, F., Chiarini, F., & McCubrey, J. A. (2021) · Leukemia · doi:10.1038/s41375-021-01243-z · Cited by 34 · see citing works
“1 transcription factor at Ser41 and Ser140, thereby leading to its recognition by FBXW7 and subsequent degradation via the proteasome [65].”
BackgroundTumor Suppressor FBXW7 and Its Regulation of DNA Damage Response and Repair
Lan, H., & Sun, Y. (2021) · Frontiers in Cell and Developmental Biology · doi:10.3389/fcell.2021.751574 · Cited by 22 · see citing works
“…FBXW7 phosphorylation at Ser227 by either serum and glucocorticoidregulated kinase 1 (SGK1) or phosphoinositide 3-kinase (PI3K), respectively, switched the catalytic activity of FBXW7 toward its substrates instead of targeting itself for self-ubiquitylation (Mo et al., 2011; Schulein et al., 2011).”
BackgroundMishra, M., Singh, A., Thacker, G., et al. (2023) · Cell Biology International · doi:10.1002/cbin.12017 · Cited by 17 · see citing works
“Western blot analysis was performed as previously described (Mishra et al., 2021; Thacker et al., 2021).”
MethodInfluentialSelf-citationUbiquitin-Mediated Control of ETS Transcription Factors: Roles in Cancer and Development
Ducker, C. E., & Shaw, P. E. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22105119 · Cited by 17 · see citing works
“1 contains two phosphodegrons that can be phosphorylated by GSK-3β and subsequently bound by FBXW7, leading to its ubiquitination and degradation (Figure 1a) [63].”
BackgroundSharma, A., Thacker, G., Mishra, M., et al. (2022) · Life Sciences · doi:10.1016/j.lfs.2022.120682 · Cited by 16 · see citing works
Self-citationMa, Q., Wang, S., Xue, H., Ni, L., Yuan, P., Shen, Y., Zheng, B., Wang, Q., Zhang, J., Wang, H., et al. (2025) · Nature Communications · doi:10.1038/s41467-025-61938-0 · Cited by 15 · see citing works
Acute Myeloid Leukemia-Related Proteins Modified by Ubiquitin and Ubiquitin-like Proteins
Park, S. S., & Baek, K. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23010514 · Cited by 13 · see citing works
Role of the ubiquitin–proteasome system on macrophages in the tumor microenvironment
Xiao, Y., Liu, R., Li, N., Li, Y., & Huang, X. (2024) · Journal of Cellular Physiology · doi:10.1002/jcp.31180 · Cited by 12 · see citing works
“Regulation of PU.1 levels by FBXW7 and USP22 may be a novel way to regulate macrophage differentiation in the future.”
BackgroundInfluentialChowdhury, S., Singh, A., Srivastava, S., Upadhyay, V., Sethi, A., Siddiqui, S. I., & Trivedi, A. K. (2023) · Journal of Cellular Biochemistry · doi:10.1002/jcb.30421 · Cited by 10 · see citing works
“Western blot analysis was performed as previously described.(23) Briefly, cells were harvested after 36 h using RIPA lysis buffer (1% NP40, 0.”
MethodSelf-citationSrivastava, S., Siddiqui, S. I., Singh, S., et al. (2023) · Steroids · doi:10.1016/j.steroids.2023.109269 · Cited by 10 · see citing works
Self-citationUpadhyay, V., Sharma, S., Sethi, A., et al. (2024) · Journal of Cellular Physiology · doi:10.1002/jcp.31388 · Cited by 9 · see citing works
“Total RNA was extracted and reverse ‐ transcribed into complementary DNA (cDNA) as previously described (Mishra et al., 2021; Thacker et al., 2016; Upadhyay et al., 2024).”
Self-citationThe roles of ubiquitination in AML
Wei, Z., Su, L., & Gao, S. (2023) · Annals of Hematology · doi:10.1007/s00277-023-05415-y · Cited by 7 · see citing works
“It is worth noting that phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it, resulting in differentiation of monocyte–macrophage, which confirms that FBW7 degrades PU.1 in a GSK3β-dependent manner [65].”
Origin, production and molecular determinants of macrophages for their therapeutic targeting
Chowdhury, S., & Trivedi, A. K. (2022) · Cell Biology International · doi:10.1002/cbin.11914 · Cited by 7 · see citing works
“…in macrophage polarization in the tumor microenvironment (TME) but recent research shows that myeloid cell‐specific E3 ubiquitin ligase FBXW7 (a critical tumor suppressor gene) can inhibit M2‐like TAM polarization in the TME by mediating c‐Myc degradation (Mishra et al., 2021; Zhong et al., 2020).”
BackgroundSelf-citationSrivastava, S., Siddiqui, S. I., Chowdhury, S., & Trivedi, A. K. (2023) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2023.01.079 · Cited by 6 · see citing works
Self-citationNemo‐like kinase blocks myeloid differentiation by targeting tumor suppressor C/ EBPα in AML
Singh, A. K., Thacker, G., Upadhyay, V., Mishra, M., Sharma, A., Sethi, A., Chowdhury, S., Siddiqui, S. I., Verma, S. P., Pandey, A., et al. (2024) · FEBS Journal · doi:10.1111/febs.17245 · Cited by 5 · see citing works
“The luciferase promoter assay was performed as described previously [39].”
InfluentialSelf-citationKaranjin counteracts OVX-induced bone loss by dual regulating bone remodeling
Ma, Q., Li, C., Lin, G., Shi, J., Wang, C., Wang, H., Cai, J., Zhang, Y., Li, J., Chang, M., et al. (2025) · Phytomedicine · doi:10.1016/j.phymed.2025.157099 · Cited by 5 · see citing works
Upadhyay, V., Singh, A., Sharma, S., Sethi, A., Srivastava, S., Chowdhury, S., Siddiqui, S. I., Chattopadhyay, N., & Trivedi, A. K. (2024) · Journal of Cellular Physiology · doi:10.1002/jcp.31217 · Cited by 5 · see citing works
“Western blot analysis was performed as previously described (Mishra et al., 2021; A. Sharma, Thacker, et al., 2022; A. Sharma, Upadhyay, et al., 2022; S. Sharma, Porwal, et al., 2022).”
MethodSelf-citationE3 ubiquitin ligases in the acute leukemic signaling pathways
Zhan, Q., Zhang, H., Wu, B., Zhang, N., & Zhang, L. (2022) · Frontiers in Physiology · doi:10.3389/fphys.2022.1004330 · Cited by 4 · see citing works
Sharma, A., Thacker, G., Mishra, M., et al. (2022) · SSRN Electronic Journal · doi:10.2139/ssrn.4062543 · Cited by 4 · see citing works
Self-citationFu, Q., Wang, X., Zhao, S., Wang, C., Sun, R., Wang, H., Du, P., Wang, J., Wang, X., & Jiang, G. (2022) · Oncology Letters · doi:10.3892/ol.2022.13393 · Cited by 3 · see citing works
Singh, A., Upadhyay, V., Sethi, A., et al. (2024) · Biochemical Journal · doi:10.1042/bcj20240027 · Cited by 3 · see citing works
Self-citationBarabino, S. M. L., Citterio, E., & Ronchi, A. E. (2021) · Cancers · doi:10.3390/cancers13153753 · Cited by 4 · see citing works
“Interestingly, the reduction in PU1 and CEBPa levels caused by aberrant ubiquitination signals have been recently described in AML, further demonstrating the importance of a proper TF protein dosage [125,126].”
BackgroundChowdhury, S., Sethi, A., Srivastava, S., et al. (2025) · Oncogene · doi:10.1038/s41388-025-03610-x · Cited by 2 · see citing works
“Current study builds on our previous fi nding where we demonstrated that FBXW7 inhibits monocyte-to-macrophage differentiation by suppressing PU.1, a master regulator of this developmental process [8].”
Self-citationE3 Ligases and COVID-19: Insights into Viral Control and Therapeutic Potential
Mishra, M., Islam, S., & Khan, D. (2025) · Biocell · doi:10.32604/biocell.2025.058038 · Cited by 2 · see citing works
“Cullin-1 forms complexes with S-phase kinase-associated protein 1 (SKP1) and various substrate receptor F-box proteins, such as Fbxw1, Fbxw7, and Fbx8 [22,23].”
Self-citationSrivastava, S., Chowdhury, S., Upadhyay, V., et al. (2026) · FEBS Journal · doi:10.1111/febs.70422 · Cited by 1 · see citing works
“In vitro kinase reaction was performed as previously described [29].”
Self-citationKozub, A., Szarek, R., Szczęsny, M., et al. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27020933
“Concerning the leukaemic cells, several studies also confirmed that PMA induced AML and T-ALL cells to produce and/or activate transcription factors and other important proteins related to differentiation [49–54].”
Saw, S., Parihar, P. S., Sahasrabuddhe, A. A., et al. (2026) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2026.150395
Self-citationGSK3β-FBXW7 axis regulates MAFB protein stability through ubiquitin-mediated proteasome degradation
Chowdhury, S., Sahoo, P., Shome, A., Srivastava, S., Upadhyay, V., Mishra, S., & Trivedi, A. K. (2026) · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research · doi:10.1016/j.bbamcr.2026.120215
Self-citationMishra, M., Thacker, G., Sharma, A., et al. (2026) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-26-0616
Self-citationRamani, M., Nguyen, N., Solis, J., Tenen, D. G., & Trinh, B. Q. (2026) · Biology · doi:10.3390/biology15181657
“Ubiquitination regulates protein stability and turnover, allowing rapid adjustment of intracellular PU.1 levels during lineage commitment or immune activation [45].”
Influential
- Authors
- Mishra, M., Thacker, G., Sharma, A., Singh, A. K., Upadhyay, V., Sanyal, S., Verma, S. P., Tripathi, A. K., Bhatt, M. L. B., & Trivedi, A. K.
- Affiliations
- Central Drug Research Institute (IN)
- King George's Medical University (IN)
- Corresponding author
- Trivedi, Arun Kumar
- ORCID
- Venue
- Molecular Cancer Research
- Published
- 2020-11-13
- Volume
- 19
- Issue
- 2
- Pages
- 261-273
- ISSN
- 1541-7786 (ISSN-L)
- 1557-3125
- OpenAlex
- W3098191540
- Cited by
- 30
- References
- 58
- Citations by year
- 2026: 6 · 2025: 4 · 2024: 5 · 2023: 5 · 2022: 6 · 2021: 4
- FWCI
- 2.03
- Type
- journal article
- Language
- en
- Update notices
- correction (2026-09-02)
- Open access
- hybrid
- doi.org
- Licence
- cc-by
- Locations
- Molecular Cancer Research (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Lady Tata Memorial Trust (GAP0239)
- Topics
- Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
- Keywords
- FBXW7, GSK3β, PU.1, acute myeloid leukemia, myeloid differentiation, ubiquitination, protein degradation, monocyte-to-macrophage differentiation
- MeSH terms
- F-Box-WD Repeat-Containing Protein 7, Animals, Cell Differentiation, Humans, Phosphorylation, Transfection, Leukemia, Myeloid, Acute
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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