PU.1-Dependent Enhancer Inhibition Separates Tet2 -Deficient Hematopoiesis from Malignant Transformation

May combine two claims.

Loss of Tet2 increases hematopoietic stem cell self-renewal while progressively decreasing their ability to undergo differentiation commitment, yet is insufficient to drive leukemogenesis.

“For instance, loss of Tet2 increases hematopoietic stem cell (HSC) self-renewal while progressively decreasing their ability to undergo differentiation commitment, yet is insufficient to drive leukemogenesis (17).”

• cites [17] Moran-Crusio, 2011, Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation, Cancer Cell sourcePartly supported

  The Results sections on HSC self-renewal and progressive defects in hematopoiesis support increased self-renewal and impaired differentiation after Tet2 loss, though they do not establish a progressively declining ability to commit to differentiation specifically. The Results section on CMML-like disease reports that Tet2-deficient mice developed myeloproliferative disease, so the unqualified assertion that Tet2 loss is insufficient to drive leukemogenesis is not supported. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Combined loss of Tet2 and Dnmt3A or presence of AML-associated gene lesions impairing myeloid cell maturation triggers malignant transformation of Tet2-deficient mouse HSC.

“Only combined loss of Tet2 and Dnmt3A (18) or presence of AML-associated gene lesions impairing myeloid cell maturation triggers malignant transformation of Tet2-deficient mouse HSC (13).”

• cites [13] Shih, 2015, Mutational cooperativity linked to combinatorial epigenetic gain of function in acute myeloid leukemia, Cancer Cell sourcePartly supported

  The Results sections on Tet2 loss with Flt3ITD report AML in double-mutant mice, altered differentiation, and leukemia-initiating activity in LSK cells. The paper identifies the self-renewing leukemia-initiating population more specifically as multipotent progenitors, not long-term HSCs, and tests Flt3ITD rather than AML-associated lesions generally. The separate Tet2–Dnmt3A clause is attached to a different citation. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [18] Zhang, 2016, DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells, Nat Genet sourcePartly supported

  The Results section reports that combined Dnmt3a and Tet2 loss produced earlier hematologic disease and a synergistic increase in malignancies, supporting the combined-loss part of the claim. But the paper does not establish that combined loss is the only route to malignant transformation; its Introduction also describes malignancies associated with loss of either gene. The AML-associated-lesions clause is attached to a different citation and is not judged here. Page numbers were not supplied. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

PU.1 is an ETS transcription factor essential for hematopoietic multilineage commitment and differentiation.

“an E26-erythroblast transformation-specific (ETS) transcription factor essential for hematopoietic multilineage commitment and differentiation (23).”

• cites [23] Iwasaki, 2005, Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation, Blood sourceSupported

Classified as probably not a substantial claim. May combine two claims.

PU.1 acts in a highly dose- and context-specific manner as a master transcription factor governing hematopoiesis, but also contributes to leukemogenesis if perturbed.

“Past work has demonstrated that PU.1 acts in a highly dose- and context-specific manner as a master transcription factor governing hematopoiesis, but also contributes to leukemogenesis if perturbed.”

• cites [24] Scott, 1994, Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages, Science sourceNot checked — no full text of the source
• cites [25] Rosenbauer, 2004, Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1, Nat Genet sourceNot checked — no full text of the source
• cites [26] Staber, 2013, Sustained PU.1 levels balance cell-cycle regulators to prevent exhaustion of adult hematopoietic stem cells, Mol Cell sourceSupported

May combine two claims.

UREHET, Tet2HET and Tet2KO single mutant mice develop a late-onset myeloproliferative phenotype.

“both of the latter develop a late-onset myeloproliferative phenotype (Supplementary Fig. S2B–S2I; refs. 13, 17).”

• cites [13] Shih, 2015, Mutational cooperativity linked to combinatorial epigenetic gain of function in acute myeloid leukemia, Cancer Cell sourcePartly supported

  The Results section (page unavailable) reports that some Tet2 knockout mice died after more than a year from progressive myeloproliferative neoplasms, supporting a late-onset phenotype in that single-mutant group. The Introduction also says Tet2 mutant mice develop chronic myeloproliferative disease, but the copy does not establish this phenotype specifically for Tet2HET single mutants or address UREHET mice. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [17] Moran-Crusio, 2011, Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation, Cancer Cell sourceSupported

May combine two claims.

Donor cells from control UREHET BM showed low engraftment (1.5%-2.3%), whereas Tet2-deficient cells exhibited slightly higher donor cell chimerism (9.45%-53.5% and 6.52%-98.3% for Tet2HET and Tet2KO cells, respectively).

“Donor cells from control UREHET BM showed low engraftment (1.5%–2.3%), whereas Tet2-deficient cells exhibited slightly higher donor cell chimerism (9.45%–53.5% and 6.52%–98.3% for Tet2HET and Tet2KO cells, respectively; Supplementary Fig. S4A and S4B), consistent with previous reports (17).”

• cites [17] Moran-Crusio, 2011, Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation, Cancer Cell sourceSupported

May combine two claims.

Compound mutant cells exhibited greater serial colony-forming activity than Tet2KO cells, which showed extended serial replating activity.

“Compound mutant cells also exhibited greater serial colony-forming activity than Tet2KO cells, which showed extended serial replating activity, in line with past reports (17, 32).”

• cites [17] Moran-Crusio, 2011, Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation, Cancer Cell sourceSupported
• cites [32] Rasmussen, 2015, Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis, Genes Dev sourcePartly supported

  In Results, under “Loss of Tet2 and AML1-ETO (AE) expression collaborate to induce AML” (page unavailable), the paper reports that cells combining Tet2 loss with AE expression had dramatically and persistently more colonies than cells with Tet2 loss alone. It describes the Tet2-loss-only cells as having low basal colony-forming capacity, however, rather than establishing the claimed extended serial replating activity for those cells. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Many transcription factors, including several ETS family members, display reduced binding affinities to DNA motifs containing methylated cytosine residues.

“Although many TF, including several members of the ETS family, display reduced binding affinities to DNA motifs containing methylated cytosine residues (44)”

• cites [44] Yin, 2017, Impact of cytosine methylation on DNA binding specificities of human transcription factors, Science sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

ARCH-associated mutations leading to perturbation of key regulators of DNA methylation, such as TET2 and DNMT3A, alter DNA methylation in a highly locus-specific fashion.

“Past work has demonstrated that ARCH-associated mutations leading to perturbation of key regulators of DNA methylation, such as TET2 and DNMT3A, alter DNA methylation in a highly locus-specific fashion (18, 45).”

• cites [18] Zhang, 2016, DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells, Nat Genet sourcePartly supported

  The Introduction says DNMT3A and TET2 mutations are common in age-associated clonal hematopoiesis. The Results (‘DNMT3A and TET2 maintain DNA methylation in HSCs’; page unavailable) identify genotype-specific differentially methylated regions, supporting locus-specific effects. However, those methylation results come from knockout mouse HSCs, not from cells carrying ARCH-associated mutations. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [45] Kaasinen, 2019, Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans, Nat Commun sourcePartly supported

  The Results (Blood DNA methylation; page not provided) show that TET2 and DNMT3A mutations produce methylation changes enriched at particular regulatory regions, supporting the locus-specific point. But the studied carriers had germline mutations; these results do not directly establish the same pattern for acquired ARCH-associated mutations. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 mRNA levels moderately decline in aging HSCs.

“PU.1 mRNA levels moderately decline aging HSCs (27).”

• cites [27] Will, 2015, Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia, Nat Med sourceSupported

Classified as probably not a substantial claim.

PU.1 establishes gene-expression programs through binding and engagement of cell-type-specific enhancers.

“PU.1 establishes gene-expression programs, particularly through binding and engagement of cell-type–specific enhancers (35).”

• cites [35] Gupta, 2009, PU.1 and partners: regulation of haematopoietic stem cell fate in normal and malignant haematopoiesis, J Cell Mol Med sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 can physically interact with the myeloid master transcription factor.

“which can physically interact with the myeloid master transcription factor (TF; Supplementary Fig. S5K and S5L; ref. 34).”

• cites [34] Lio, 2016, Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility, eLife sourcePartly supported

  The Results section (‘Functional and physical interaction between TET proteins and E2A/PU.1’; page unavailable) reports a direct interaction between TET2 and PU.1. The Discussion identifies PU.1 as a key transcription factor in the myeloid lineage. The paper does not establish the claim as phrased—that PU.1 interacts with a separate myeloid master transcription factor. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Exceptions to the rule that PU.1 binding is largely unperturbed by DNA methylation have been reported.

“exceptions have been reported in the past (41).”

• cites [41] Minderjahn, 2020, Mechanisms governing the pioneering and redistribution capabilities of the non-classical pioneer PU.1, Nat Commun sourceSupported

Classified as probably not a substantial claim.

Sites with aberrant DNA methylation in TET2 or DNMT3A mutant cells are enriched for several key hematopoiesis-regulating transcription factors, including PU.1.

“Sites with aberrant DNA methylation in TET2 or DNMT3A mutant cells are enriched for several key hematopoiesis regulating TF, including PU.1 (28, 45)”

• cites [28] Tulstrup, 2021, TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis, Nat Commun sourcePartly supported

  The Results section (‘TET2-associated hypermethylation co-localizes with specific transcription factor motifs’; page unavailable) reports enrichment of hematopoietic transcription-factor motifs, including SPI1 (PU.1), near TET2-associated hypermethylation in CHIP and CCUS. It does not report the same enrichment for DNMT3A-mutant cells, so the DNMT3A portion is unsupported by this paper. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [45] Kaasinen, 2019, Impact of constitutional TET2 haploinsufficiency on molecular and clinical phenotype in humans, Nat Commun sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Adults and patients with myeloid malignancies typically lack the ETV6-RUNX1 oncogene.

“in line with clinical observations of adults and patients with myeloid malignancies typically lacking this oncogene (43).”

• cites [43] Cancer Genome Atlas Research N, 2013, Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia, N Engl J Med sourcePartly supported

  The Results > Gene Fusions section reports that 99 of 179 adult de novo AML samples had no detected gene fusion, so those samples lacked ETV6-RUNX1. The paper does not specifically discuss ETV6-RUNX1 or establish its prevalence across adults generally or across other myeloid malignancies. No page number is available in our copy. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Altered DNA methylation is prevalent in the elderly, as observed in ARCH, which often precedes AML.

“Altered DNA methylation is prevalent in the elderly, such as observed and well characterized in ARCH (28), which often precedes AML (5).”

• cites [28] Tulstrup, 2021, TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis, Nat Commun sourcePartly supported

  The Introduction and Results describe TET2-associated DNA hypermethylation in elderly people with CHIP, supporting the ARCH example. The source does not establish that altered DNA methylation is prevalent among elderly people generally. The separate claim that ARCH often precedes AML is attached to citation (5), not this source. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [5] Jan, 2012, Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia, Sci Transl Med sourcePartly supported

  The Results and Discussion (pages unavailable in our copy) identify pre-leukemic stem-cell clones in five of six selected patients with AML, supporting the idea that clonal changes can precede AML. The paper does not establish that ARCH often progresses to AML; it studies people who already have AML, not the subsequent AML risk of people with ARCH. The elderly methylation claim is attached to citation (28), not this source. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Despite moderately reduced PU.1 mRNA levels, its broader transcriptional network appears intact.

“Despite moderately reduced PU.1 mRNA levels, its broader transcriptional network appears intact, in line with the essentiality of the transcription factor in maintaining normal and malignant hematopoiesis (20, 22, 23, 46, 48).”

• cites [20] Mizuki, 2003, Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations, Blood sourceNot checked — no full text of the source
• cites [22] Mueller, 2006, ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression, Blood sourcePartly supported

  The Results report approximately half as much PU.1 RNA in t(15;17) AML samples as in AML-M2 and AML-M4 samples, although the difference was not statistically significant. The Discussion describes PU.1 as crucial to normal myeloid development and implicated in leukemia. The paper does not show that the broader PU.1 transcriptional network remains intact; its Results instead report loss of binding activity at a PU.1 target site in one APL cell line. Page numbers were not available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [23] Iwasaki, 2005, Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation, Blood sourcePartly supported

  The Discussion reports that PU.1 is necessary to maintain the normal HSC pool and links reduced PU.1 expression to AML development. It does not establish that PU.1 is essential for maintaining malignant hematopoiesis. The preceding observation about moderately reduced mRNA and an intact transcriptional network appears to be the citing authors’ own finding, not a finding attributed to this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In mice, loss of Tet2 triggers a late-onset myeloproliferative disease with penetrance inversely correlated with residual Tet2 gene dosage.

“In mice, loss of Tet2 triggers a late-onset myeloproliferative disease with penetrance inversely correlated with residual Tet2 gene dosage (17).”

• cites [17] Moran-Crusio, 2011, Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation, Cancer Cell sourcePartly supported

  The Results section, “Tet2 deficient animals develop CMML-like disease” (page unavailable), reports progressive myeloproliferation in mice, with approximately all Tet2−/− mice showing significant splenomegaly by week 20. The Results section on Tet2 haploinsufficiency (page unavailable) reports dose-dependent effects on hematopoiesis and myeloid transformation, but does not establish that disease penetrance is inversely correlated with residual Tet2 gene dosage. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 and RUNX1 jointly exclude transcriptional corepressors from multiprotein complexes driving expression of key myeloid genes.

“consistent with the known function of PU.1 and RUNX1 to jointly exclude transcriptional corepressors from multiprotein complexes driving the expression of key myeloid genes (58).”

• cites [58] Gu, 2014, Runx1 regulation of Pu.1 corepressor/coactivator exchange identifies specific molecular targets for leukemia differentiation therapy, J Biol Chem sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Upon DNA recruitment, Tet2 provides a hub for subsequent lineage-instructing transcription factor binding at cell type-specific enhancers residing in open chromatin.

“Upon DNA recruitment, Tet2 also provides a hub for subsequent lineage-instructing transcription factor binding at cell type–specific enhancers residing in open chromatin (52).”

• cites [52] Rasmussen, 2019, TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells, Genome Res sourcePartly supported

  The Results sections on TET2 binding show that it occupies open, cell-type-specific regulatory regions with enhancer features and motifs for master transcription factors. The Discussion supports a model in which TET2 shapes chromatin to permit transcription-factor binding, but it does not establish that TET2 acts as a physical hub or demonstrate the claimed sequence of subsequent binding. Page numbers were not available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Perturbed PU.1 gene regulation and impaired expression drives malignant transformation of mismatch-repair-deficient HSCs.

“We previously uncovered that perturbed PU.1 gene regulation and impaired expression drives malignant transformation of mismatch-repair–deficient HSCs (27)”

• cites [27] Will, 2015, Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia, Nat Med sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 is inhibited in most patients with AML through various molecular pathways.

“PU.1 is also inhibited in most patients with AML through various molecular pathways (19, 20, 22, 46, 47).”

• cites [19] Vangala, 2003, The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia, Blood sourceNot checked — no full text of the source
• cites [20] Mizuki, 2003, Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations, Blood sourceNot checked — no full text of the source
• cites [22] Mueller, 2006, ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression, Blood sourcePartly supported

  The Discussion reports PU.1 suppression in acute promyelocytic leukemia (APL) and mentions PU.1 mutations in patients with other AML subtypes. It does not establish that PU.1 is inhibited in most patients with AML; the Results found a nonsignificant difference in PU.1 RNA between the t(15;17) group and two other AML groups. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 has a known tumor-suppressive function in myeloid malignancies.

“consistent with the known tumor-suppressive function of PU.1 in myeloid malignancies (25, 27).”

• cites [25] Rosenbauer, 2004, Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1, Nat Genet sourceNot checked — no full text of the source
• cites [27] Will, 2015, Minimal PU.1 reduction induces a preleukemic state and promotes development of acute myeloid leukemia, Nat Med sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

TET2-mutant immature and myeloid cells present with hypermethylation of active enhancers and perturbed TF activity.

“Consistently, TET2-mutant immature and myeloid cells present with hypermethylation of active enhancers and perturbed TF activity (28, 32, 53).”

• cites [28] Tulstrup, 2021, TET2 mutations are associated with hypermethylation at key regulatory enhancers in normal and malignant hematopoiesis, Nat Commun sourcePartly supported

  The Results sections on chromatin states and AML report TET2-associated hypermethylation at monocyte and hematopoietic stem-cell enhancers. The transcription-factor analysis finds enriched binding motifs and suggests a link to transcription-factor recruitment, but does not establish that transcription-factor activity is perturbed. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [32] Rasmussen, 2015, Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis, Genes Dev sourcePartly supported

  The Results section on GMP and AML cells reports enhancer hypermethylation after TET2 disruption, including in human TET2-mutant AML. The Results section on enhancer activity also reports altered nearby gene expression and enrichment of transcription-factor binding motifs, but it does not establish perturbed transcription-factor activity itself. Page numbers are unavailable in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [53] Figueroa, 2010, Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation, Cancer Cell sourcePartly supported

  The Results section on TET2-mutant AMLs reports promoter hypermethylation, supporting the general hypermethylation finding, but it does not identify active enhancers as the affected sites. The Results discussion of transcription-factor binding sites suggests perturbed transcription-factor function in IDH1/2-mutant AMLs, not specifically in TET2-mutant cells. Page numbers are not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

Moribund UREHETTet2HET and UREHETTet2KO compound mutant mice presented with splenomegaly, accompanied by elevated white blood cell counts and reduced red blood cells, compared with age-matched WT and single-gene mutant controls.

“Specifically, moribund UREHETTet2HET and UREHETTet2KO compound mutant mice presented with splenomegaly (Fig. 2B), accompanied by elevated white blood cell counts (WBC) and reduced red blood cells (RBCs), in comparison with age-matched WT and single-gene mutant (UREHET, Tet2HET, and Tet2KO) control animals (Fig. 2C and D).”

May combine two claims.

Compound mutant mice exhibited an accumulation of differentiation-impaired blast cells in the peripheral blood, bone marrow, and spleen, staining positive for myeloperoxidase, and disrupting the tissue architecture of these hematopoietic organs, in contrast to control mice.

“Compound mutant mice exhibited an accumulation of differentiation-impaired blast cells in the peripheral blood, bone marrow, and spleen, staining positive for the myeloid marker myeloperoxidase (MPO), and disrupting the tissue architecture of these hematopoietic organs, in contrast to control mice (Fig. 2E and F; Supplementary Fig. S2B–S2D).”

May combine two claims.

Transplantation of total bone marrow cells and purified HSPC-enriched cKit+ cells from compound mutant donors was followed by moribund states of the recipients within 4 to 13.5 weeks (TBM: 4-13.5 weeks, cKit+ cells: 4-9 weeks).

“In contrast, transplantation of total bone marrow (TBM) cells and purified HSPC-enriched cKit+ cells from compound mutant donors was followed by moribund states of the recipients within 4 to 13.5 weeks (TBM: 4–13.5 weeks, cKit+ cells: 4–9 weeks; Fig. 3F; Supplementary Fig. S4C).”

May combine two claims.

UREHETTet2KO showed a minor decrease in the abundance of phenotypical immature CD4− CD8a− B220− cKit+ cells in the bone marrow and a concomitant 3-fold (P < 0.001) expansion in the spleen, compared with WT controls.

“Albeit UREHETTet2KO showed a minor decrease in the abundance of phenotypical immature CD4− CD8a− B220− cKit+ cells in the bone marrow (Supplementary Fig. S1M–S1O) and a concomitant 3-fold (P < 0.001) expansion in the spleen (Supplementary Fig. S1M and S1N and S1P), compared with WT controls.”

May combine two claims.

Approximately half (42.9% for UREHETTet2HET and 50% for UREHETTet2KO) of the mice harbored leukemic blasts hallmarked by immature cell morphology and a dominant cKit-expressing cell population with high Gr-1 antigen presentation in the bone marrow and spleen (immature AML); the remainder presented with blast cells showing some degree of myeloid differentiation (metamyelocyte-like cell morphology,

“Approximately half (42.9% for UREHETTet2HET and 50% for UREHETTet2KO) of the mice harbored leukemic blasts hallmarked by immature cell morphology and a dominant cKit-expressing cell population with high Gr-1 antigen presentation in the bone marrow and spleen (classified further as immature AML); the remainder presented with blast cells showing some degree of myeloid differentiation (metamyelocyte”

May combine two claims.

RNA-sequencing of HSPCs from leukemic mice mirrored this morphologic heterogeneity; PCA showed a similar heterogeneity of the compound mutant cells, with HSPCs from mice with a mature AML phenotype more closely related with WT control-derived cells of the same immunophenotype.

“RNA-sequencing of HSPCs from leukemic mice (Fig. 4C and D) mirrored this morphologic heterogeneity; compared with phenotypical control cells from control animals, principal component analysis (PCA) showed a similar heterogeneity of the compound mutant cells, with HSPCs from mice with a mature AML phenotype more closely related with WT control-derived cells of the same immunophenotype (Fig. 4E).”

May combine two claims.

Some leukemic compound mutant mice showed PU.1 mRNA expression similar to parental control mice (PU.1High), while others exhibited a decrease in expression of the transcription factor (PU.1Low) in comparison with control-derived cells of the same immunophenotype.

“Although some of the leukemic compound mutant mice showed PU.1 mRNA expression similar to parental control mice (PU.1High), others exhibited a decrease in expression of the transcription factor (PU.1Low) in comparison with control-derived cells of the same immunophenotype (Supplementary Fig. S5C).”

May combine two claims.

Compound mutant mice presented with blast cell phenotypes of varying morphology regardless of the underlying genotype which was reminiscent of human AML, and distinct from the previously reported UREKO myeloid malignancy which presents with early-onset AML homogenously hallmarked by the expansion of myeloblast-like cells.

“Compound mutant mice presented with blast cell phenotypes of varying morphology regardless of the underlying genotype which was reminiscent of human AML, and distinct from the previously reported UREKO myeloid malignancy which presents with early-onset AML homogenously hallmarked by the expansion of myeloblast-like cells (25).”

• cites [25] Rosenbauer, 2004, Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1, Nat Genet sourceNot checked — no full text of the source

May combine two claims.

Compound mutant mice presented with normal blood cell counts in the periphery and showed no overt pathology in primary or secondary sites of hematopoiesis.

“Compound mutant mice presented with normal blood cell counts in the periphery (Supplementary Fig. S1G and S1H) and showed no overt pathology in primary or secondary sites of hematopoiesis (Supplementary Fig. S1I–S1L).”

May combine two claims.

Bone marrow cells from compound mutant mice harbored a comparable number of colony-initiating progenitors in primary colony-forming assays, albeit lacking multipotent and erythroid potential.

“Compared with single-gene mutant cells, bone marrow cells from compound mutant mice harbored a comparable number of colony-initiating progenitors in primary colony-forming assays, albeit lacking multipotent and erythroid potential (Fig. 3A; Supplementary Fig. S3A).”

May combine two claims.

Only a minor fraction of the compound mutant cells gave rise to colonies lacking morphologic features of myeloid cell maturation in the first plating; these blastlike colonies gradually became the dominant type upon serial replating.

“Although only a minor fraction of the compound mutant cells gave rise to colonies lacking morphologic features of myeloid cell maturation in the first plating, these blastlike colonies gradually became the dominant type upon serial replating (Fig. 3A and B; Supplementary Fig. S3B).”

May combine two claims.

Network perturbations affected pathways involved in myeloid cell function, macrophage-mediated inflammatory response, innate immune system function, cell fate commitment and maturation, and leukemia.

“these network perturbations affected pathways involved in myeloid cell function, macrophage-mediated inflammatory response, innate immune system function, cell fate commitment and maturation, and leukemia”

May combine two claims.

Some leukemic UREHETTet2KO LSK cells had further reduced mRNA levels (AML; 84% ± 2% reduction of Young), which was inconsistent with PU.1 protein abundance, found not markedly changed in healthy, aged compound mutant mice.

“we detected further reduced mRNA levels in some leukemic UREHETTet2KO LSK cells (AML; 84% ± 2% reduction, of Young; Fig. 4I), which was inconsistent with PU.1 protein abundance, found not markedly changed in healthy, aged compound mutant mice (Supplementary Fig. S5E).”

The quote only partly states this claim.

Nonleukemic mice shared most regulatory element patterns defining the epigenome of immature cells and progenitors and mature cells of most hematopoietic lineages, with only about one-third overlap with erythroid and mature CD8a+, CD4+ T cells and natural killer cells.

“Hierarchical clustering of all differential chromatin peaks that correspond to differentially expressed eRNAs (cCRE) further showed that nonleukemic mice shared most of the regulatory element patterns defining the epigenome of immature cells (HPC-7 and LSK cells) and progenitors and mature cells of most hematopoietic lineages”

May combine two claims.

HOMER motif analysis revealed several variations of an ETS motif composed of a core PU box (NN GGAAGT) along with cytosine compositions (CA and CC) at the NN position as top ranking and strongly enriched DNA-binding motifs within ETV6-RUNX1 gene set-regulating cCRE, as well as all closed chromatin cCRE compared with WT controls.

“HOMER motif analysis of cCRE sequences in chromatin with reduced accessibility in compound mutant leukemic HSPC revealed several variations of an ETS motif composed of a core PU box (NN GGAAGT) along with cytosine compositions (CA and CC) at the NN position as top ranking and strongly enriched DNA-binding motifs”

May combine two claims.

Close to 90% of cCREs localized in closed chromatin in compound mutant leukemic HSPCs overlapped with PU.1 and/or Tet2 occupancy, whereas less than half of cCREs within loci with increased accessibility are bound by either regulator.

“Intersection with PU.1 and Tet2 ChIP-seq data uncovered that close to 90% of the cCREs localized in the closed chromatin section in compound mutant leukemic HSPCs overlapped with PU.1 and/or Tet2 occupancy, whereas less than half of cCREs within loci with increased accessibility are bound by either regulator”

May combine two claims.

Comparative pathway analyses of human healthy ARCH HSCs and leukemic stem cells showed significant alterations in this gene set exclusively in TET2mut leukemic HSPCs and dissimilar alterations in canonical pathways and upstream regulators between compound mutant mice and human healthy ARCH HSCs, including in the ETV6-RUNX1 signature.

“Comparative pathway analyses of human healthy ARCH HSCs and leukemic stem cells further showed significant alterations in this gene set exclusively in TET2mut leukemic HSPCs (Fig. 1D) and dissimilar alterations in canonical pathways and upstream regulators between our compound mutant mice and human healthy ARCH HSCs”

May combine two claims.

Mutational analysis of transplantable leukemic HSPCs did not reveal any prevalent AML driver mutations, but uncovered highly recurrent and defined nonsense insertion mutations in Cux1 at subclonal levels.

“Mutational analysis of transplantable leukemic HSPCs did not reveal any prevalent AML driver mutations, but uncovered highly recurrent and defined nonsense insertion mutations in Cux1 at subclonal levels.”

May combine two claims.

Compound mutant mice lacking one copy of the PU.1 URE, along with either heterozygous or homozygous deletion of Tet2, developed aggressive AML hallmarked by splenomegaly, anemia, leukocytosis, and accumulation of differentiation-impaired blast cell populations with varying phenotypes.

“Compound mutant mice lacking one copy of the PU.1 URE, along with either heterozygous or homozygous deletion of Tet2, developed aggressive AML hallmarked by splenomegaly, anemia, leukocytosis, and the accumulation of differentiation-impaired blast cell populations with varying phenotypes.”

May combine two claims.

Expression levels of PU.1 are comparable in aged HSCs with and without ARCH mutations; they appear to drop moderately in LSCs.

“Using published data sets, we found that expression levels of the hematopoietic master regulator are comparable in aged HSCs with and without ARCH mutations; yet they appear to drop moderately in LSCs”

May combine two claims.

Only a fraction of PU.1-controlled enhancers had aberrant gain of accessibility and activity, in contrast to remarkable enrichment of PU.1-associated enhancers in aberrantly closed chromatin.

“In contrast to the remarkable enrichment of PU.1-associated enhancers in aberrantly closed chromatin, we found only a fraction of PU.1-controlled enhancers with an aberrant gain of accessibility and activity.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A lack of PU.1 is incompatible with multilineage hematopoiesis.

“Although a lack of PU.1 is incompatible with multilineage hematopoiesis (24)”

• cites [24] Scott, 1994, Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages, Science sourceNot checked — no full text of the source

May combine two claims.

Homozygous deletion of the −14 kb URE of PU.1 (resulting in 80% reduced PU.1 mRNA expression) in mouse hematopoietic stem and progenitor cells allows for multilineage differentiation, but leads to an early-onset fully penetrant leukemia or lymphoma phenotype.

“homozygous deletion of a critical upstream regulatory element (−14 kb UREΔ/Δ) of PU.1 (resulting in 80% reduced PU.1 mRNA expression) in mouse hematopoietic stem and progenitor cells allows for multilineage differentiation, but leads to an early-onset fully penetrant leukemia or lymphoma phenotype (25).”

• cites [25] Rosenbauer, 2004, Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1, Nat Genet sourceNot checked — no full text of the source

May combine two claims.

Compound mutant phenotypic HSPCs harbor cell-autonomous defects resulting in enhanced self-renewal, impaired myeloid differentiation, and leukemia-initiating capacity.

“These data demonstrate that compound mutant phenotypic HSPCs harbor cell-autonomous defects resulting in enhanced self-renewal, impaired myeloid differentiation, and leukemia-initiating capacity.”

• AssumptionRests on an assumption: The compound mutant mouse model (URE deletion plus Tet2 deficiency) recapitulates human Tet2-deficient ARCH/AML and PU.1 deregulation.

May combine two claims.

PU.1 mRNA levels may decline moderately upon leukemic transformation, but its global network activity is preserved at the stem cell level.

“These observations suggested that albeit PU.1 mRNA levels may decline moderately upon leukemic transformation, its global network activity is preserved at the stem cell level.”

May combine two claims.

The observed alterations in PU.1 mRNA and protein levels in compound mutant mice are to a certain extent reflective of the underlying cellular differentiation state, and not a sole feature of their malignant state.

“This strongly suggests that the observed alterations in PU.1 mRNA and protein levels in compound mutant mice are to a certain extent reflective of the underlying cellular differentiation state (also observed at the global gene-expression level; Supplementary Tables S4 and S5), and not a sole feature of their malignant state.”

• AssumptionRests on an assumption: PU.1 mRNA and protein abundance reflect functional PU.1 activity in HSPCs.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The ETV6-RUNX1 translocation fuses the N terminus of ETV6 to nearly full-length RUNX1, retaining the RUNX1 DNA-binding domain and turning the oncogenic TF into a repressor via ETV6-mediated recruitment of corepressors and epigenetic modifiers.

“The translocation fuses the N terminus of the ETS family member ETV6 (TEL) to nearly full-length RUNX1 (AML1), retaining the DNA-binding domain of RUNX1 (Runt) and turning the oncogenic TF into a repressor via ETV6-mediated recruitment of transcriptional corepressors (NCOR or SIN3A) and epigenetic modifiers (HDAC; ref. 42).”

• cites [42] Hiebert, 1996, The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription, Mol Cell Biol sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Lower PU.1 protein abundance results in enhanced granulocytic lineage differentiation.

“which is consistent with lower PU.1 protein abundance resulting in enhanced granulocytic lineage differentiation (33).”

• cites [33] Dahl, 2003, Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor, Nat Immunol sourceNot checked — no full text of the source

May combine two claims.

Reduced PU.1 mRNA expression can occur in aging compound mutant mice in the absence of alterations at the protein level, and is compatible with a nonleukemic state.

“Together, this strongly suggests that reduced PU.1 mRNA expression can occur in aging compound mutant mice in the absence of alterations at the protein level, and is compatible with a nonleukemic state.”

• AssumptionRests on an assumption: PU.1 mRNA and protein abundance reflect functional PU.1 activity in HSPCs.
• AssumptionRests on an assumption: Aged compound mutant mice that do not develop AML represent a nonleukemic/healthy clonal hematopoiesis state.

Classified as probably not a substantial claim.

Regions with significant differential methylation across comparison groups were determined via the Fisher exact test and corrected for multiple comparisons with the Benjamini-Hochberg procedure.

“Regions with significant differential methylation across comparison groups were determined via the Fisher exact test on the numbers of methylated/unmethylated bases and corrected for multiple comparisons with the Benjamini–Hochberg procedure.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

RNA quantity and quality were assessed using a NanoDrop 1000 full-spectrum spectrophotometer and a 2100 Bioanalyzer device.

“RNA quantity and quality were assessed using a NanoDrop 1000 full-spectrum spectrophotometer (Thermo Fisher) and 2100 Bioanalyzer (Agilent) device, respectively.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Post-sort enrichment of bead-sorted BM cells was quantified by FACS analysis.

“post-sort enrichment was quantified by FACS analysis (using the anti-CD117-PE antibody, Miltenyi #130-091-730).”

May combine two claims.

Moderate perturbation of PU.1 gene regulation through deletion of an URE critical for high expression of PU.1 in HSCs and differentiating myeloid cells causes malignant transformation of Tet2-deficient HSPC, whereas it is not sufficient to induce malignancy in Tet2 WT cells.

“Here, we uncovered that moderate perturbation of PU.1 gene regulation through deletion of an URE critical for high expression of PU.1 in HSCs and differentiating myeloid cells (54) causes malignant transformation of Tet2-deficient HSPC, whereas not sufficient to induce malignancy in Tet2 WT cells.”

• cites [54] Li, 2001, Regulation of the PU.1 gene by distal elements, Blood sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

For real-time PCR, triplicate samples and six serial dilutions of standards were prepared for each target gene.

“Triplicate samples and six serial dilutions of standards were prepared for each target gene.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Sample sizes were not based on formal power calculations to detect prespecified effect sizes.

“Sample sizes chosen are indicated in the individual figure legends and were not based on formal power calculations to detect prespecified effect sizes.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

CUX1-inactivating mutations are frequently found in human myeloid malignancies and associated with poor survival and trigger an MDS/MPN phenotype in mice.

“CUX1-inactivating mutations are frequently found in human myeloid malignancies and associated with poor survival and trigger an MDS/MPN phenotype in mice (56).”

• cites [56] An, 2018, Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS, Blood sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

FACS-purified cKit+ cells with high viability (>90%) were used for Omni-ATAC-seq.

“FACS-purified, high viability (>90%) cKit+ cells were used.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Duplicate reads were marked before methylation calls in bisulfite-seq analysis.

“Duplicate reads were marked (picard-tools v1.92) before methylation calls.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Investigators were not blinded to genotype group allocations.

“Investigators were not blinded to genotype group allocations.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Multiple-testing-adjusted P values are reported.

“Multiple-testing–adjusted P values are reported.”

Probably not a substantial claim: may not pass S3 (load-bearing).

High-quality RNA (RNA integrity number ≥8) was prepared and sent for library construction and sequencing.

“high-quality RNA (RNA integrity number ≥8) was prepared and sent for library construction and 100 bp, paired-end, Illumina-HiSeq 2500/4000 sequencing to the Beijing Genomics Institute (BGI) Group.”