ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms. ASXL1 gene is often mutated in myeloid malignancies. Here, the authors show that mutant ASXL1 and BAP1 are in a positive feedback loop such that BAP1 induces monoubiquitination of mutant ASXL1, which in turn enhances BAP1 activity to potentiate myeloid transformation via HOXA clusters and IRF8. Mutations in the ASXL1 gene are frequently found in myeloid neoplasms, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML)8. These mutations are predominantly frameshift and nonsense mutations generating C-terminally truncated proteins, and are associated with worse prognosis8. ASXL1 mutations have also been implicated in clonal haematopoiesis of indeterminate potential, suggesting that it is among the earliest genetic events in the process of myeloid transformation11.Additional sex combs-like 1 (ASXL1) is a member of the ASXL family and is involved in epigenetic regulation12. It has been suggested that the PHD domain, which is lost in most ASXL1 mutations, binds histones with specific modifications and recruits chromatin modulators and transcriptional factors13. The ASXH domain mediates interaction with a partner protein BAP1. BAP1 is an essential component of the “polycomb repressive deubiquinase complex (PR-DUB),” in which it deubiquitinates monoubiquitinated histone H2A at lysine 119 (H2AK119ub), a modification that is catalyzed by the polycomb repressive complex 1 (PRC1)14. The mammalian PR-DUB complex contains ASXL family proteins, which are required for its deubiquinating activity15. In addition to BAP1, ASXL1 interacts directly with EZH2, EED, and SUZ12, catalytic and scaffold subunits of PRC2, which promotes trimethylation of H3 at lysine 27 (H3K27me3)17. Thus, ASXL1 may act as an epigenetic scaffold in the regulation of various histone modifications, including H2AK119ub and H3K27me3.Members of the ASXL family share a common domain architecture, which includes a highly conserved ASX homology (ASXH) domain at the N-terminal region and a plant homeodomain (PHD) finger at the C-terminal regionAsxl1 in haematopoietic cells promotes myeloid transformation18, indicating that mutations in ASXL1 produce loss of function. However, a growing body of evidence suggests that ASXL1 mutations in fact result in gain of function. Experiments using mouse bone marrow transplantation models have revealed that forced expression of a C-terminally truncated ASXL1 mutant in haematopoietic progenitor cells induces MDS-like diseases, and accelerates AML development in concert with Nras or SETBP1 mutations19. In patients with ASXL1 mutations, the mutations are typically heterozygous and occur near the 5′ end of exon 12, thus producing C-terminally truncated forms of ASXL1 probably escaping from nonsense-mediated decay (NMD) of mRNA, and indeed truncated ASXL1 proteins are expressed in MDS cells20. Thus, whether ASXL1 mutations promote myeloid transformation via a gain or loss of function remains an unresolved question.How ASXL1 mutations induce myeloid transformation is not fully understood. Previous studies have reported that ASXL1 knockdown and genetic deletion of Asxl1 deletion and mutant Asxl1 overexpression induce global reduction of H3K27me3 in haematopoietic cells18. These data suggest that loss of ASXL1 function in promoting H3K27me3 contributes to myeloid transformation. On the other hand, recent studies have shown that cancer-associated ASXL1 mutations aberrantly enhance BAP1 function in the deubiquination of H2AK119ub, raising the possibility that increased PR-DUB activity underlies the oncogenic effect of ASXL1 mutation21. However, the precise nature of the epigenetic dysregulation, which plays a major role in mutant ASXL1-induced leukaemogenesis, remains unknown.Mechanistically, it has been shown that both HOXA genes and IRF8 through inhibition of H2AK119ub, impairing multilineage differentiation of haematopoietic progenitors (except for that toward monocytes), and accelerates RUNX1-ETO-induced leukaemogenesis. Importantly, Bap1 depletion using CRISPR/Cas9 substantially inhibits the leukaemogenicity of myeloid leukemia cells expressing mutant ASXL1. BAP1 is also required for the growth of MLL-fusion leukemia cells through the upregulation of HOXA gene expression. These data indicate that BAP1, which has long been regarded as a beneficial tumor suppressor, also plays a tumor-promoting role in myeloid leukaemogenesis.In the present study, we report a mutually reinforcing effect between mutant ASXL1 and BAP1, which promotes myeloid leukaemogenesis. BAP1 induces stabilization and monoubiquitination of mutant ASXL1, and monoubiquitinated ASXL1-MT increases the catalytic function of BAP1. This hyperactive mutant ASXL1/BAP1 complex induces upregulation of posterior 17, which here we refer to as ASXL1-MT)] and BAP1 in 293T cells  in cells that were retrovirally transduced with ASXL1-MT without BAP1 overexpression  to promote ubiquitination of ASXL1-MT. To identify such ligases, we performed nano liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) using cell lysates from 293T cells transfected with ASXL1-MT alone or ASXL1-MT and BAP1 Fig. . This anNext, we compared the effect of BAP1 and BAP1-C91S on the ubiquitination of ASXL1-MT. Immunoprecipitation assay revealed that expression of BAP1-C91S efficiently induced both mono- and polyubiquitination of ASXL1-MT, while BAP1 induced only its monoubiquitination Fig. . Given t23, we assessed the effect of ASXL1-MT on the subcellular localization of BAP1 using immunofluorescence analyses. BAP1 normally showed a diffuse localization pattern in both nucleus and cytoplasm. Overexpression of ASXL1-MT promoted nuclear import of BAP1, which was observed only weakly when we used the ubiquitination-deficient ASXL1-MT-K351R  CD34+ cells were transduced with ASXL1-MT or ASXL1-MT-K351R together with BAP1, and the cells were cultured with cytokines to induce myeloid or erythroid differentiation. ASXL1-MT expression decreased the ratio of CD33+CD66b+ granulocytes in the cultures designed to promote myeloid differentiation, and this effect was enhanced with BAP1 coexpression  in promoting myeloid leukaemogenesis. RUNX1-ETO is a leukemia fusion protein and has been shown to increase self-renewal of HSPCsWe then assessed the effect of coexpression of BAP1 and ASXL1-MT on the leukaemogenicity of RUNX1-ETO leukemia. After transducing fetal liver cells with RUNX1-ETO9a along with multiple combinations of ASXL1-MT, ASXL1-MT-K351R, and BAP1, we transplanted the cells into recipient mice. Coexpression of ASXL1-MT and BAP1 promoted RUNX1-ETO9a-induced leukemia progression with higher efficiencies than did ASXL1-MT alone. In contrast, ASXL1-MT-K351R showed no effect, indicating that BAP1-induced monoubiquitination of ASXL1-MT plays an important role in enhancing leukaemogenicity Fig. .+ cells, and recapitulate many features of human RUNX1-ETO leukemia34. We transduced a control vector, ASXL1-MT, or ASXL1-MT-K531R (coexpressing GFP) into RUNX1-ETO-expressing CB cells and monitored changes of GFP frequency in culture. Again, ASXL1-MT, but not ASXL1-MT-K351R, substantially promoted the growth of RUNX1-ETO cells  were also upregulated in cells expressing both ASXL1-MT and BAP139  targeting Bap1. Both gRNAs induced nearly complete depletion of Bap1 in 32Dcl3 cells Fig. . We trancombined expression of SETBP1 and ASXL1 Mutations). SETBP1-D868N is an oncogenic mutation of SETBP1, which collaborates with ASXL1-MT in the induction of myeloid transformation19. We transduced Cas9 and Bap1-targeting gRNAs into cSAM cells, and cultured the cells in semisolid medium or directly transplanted them into sublethally irradiated recipient mice  into the MLL-AF9 cells, and examined the effect of BAP1 depletion in MLL-AF9 leukemia cells. Similar to our results using cSAM cells, Bap1 depletion profoundly inhibited the colonogenicity and leukaemogenicity of MLL-AF9 leukemia cells, and this effect was rescued by expression of exogeneous BAP1  and RUNX1-ETO fusion. MEG-01 and TS9;22 are other myeloid leukemia cell lines with ASXL1 mutations . MOLM-13 and THP-1 are cell lines harboring MLL-AF9. We transduced Cas9 and gRNAs targeting human BAP1 (sgBAP1 #3 and #4) into these cells. BAP1 depletion resulted in substantial growth inhibition in all of these myeloid leukemia cell lines. We also confirmed downregulation of posterior ASXL1 mutations are frequently identified in a variety of myeloid neoplasms and are associated with poor prognosis, making the development of molecular therapies targeting these mutations an important goal. Our data indicate an obligate role for BAP1 in mutant ASXL1-induced myeloid leukaemogenesis, and suggest this enzyme as a promising therapeutic target. Research to date has primarily focused on the tumor-suppressing role of BAP1. Clinical data have shown that BAP1 gene is frequently mutated in various tumors, including mesothelioma and melanoma45. Experimentally, it has been shown that Bap1 deletion in adult mice causes MDS-like diseases46. Although these studies have clearly shown a tumor suppressor role for BAP1, several lines of evidence also raise the possibility that BAP1 activity drives the growth of myeloid leukemia cells with ASXL1 mutations. First, BAP1 is rarely mutated in patients with myeloid neoplasms, and BAP1 and ASXL1 genes are mutated in distinct cancer types47. Second, BAP1 and its Drosophila homolog Calypso have been shown to maintain posterior HOXA genes and it has also been shown that ASXL1 deletion/mutations lead to increased expression of posterior HOXA genes17. Third, one recent study showed that BAP1 loss results in transformation, independent of ASXL1 activity48. In addition to these reports implicating BAP1 in myeloid transformation, the present results identify the underlying molecular mechanisms by which ASXL1-MT/BAP1 complex induce myeloid transformation; BAP1 plays a necessary role in maintaining aberrant posterior HOXA expression in ASXL1-mutant leukemia and in sustaining their leukemic proliferation. Maintenance of the leukemic phenotype in MLL-fusion leukemia, a well-known AML subtype with posterior HOXA dysregulation50, also depends on the BAP1 activity. Inhibition of BAP1 activity, rather than enhancing it, may thus represent a promising therapeutic strategy for myeloid neoplasms involving posterior HOXA dysregulation.52, it appears that ASXL1 mutations do not confer the capacity for self-renewal to HSPCs. The ASXL1-MT/BAP1 complex is not a simple growth-accelerator, but rather inhibits terminal differentiation of HSPCs toward granulocytes, macrophages, mast cells, and erythrocytes. Interestingly, ASXL1-MT/BAP1 promotes monocyte differentiation, while it inhibits terminal differentiation to macrophage, which may account for the frequent (40–50%) detection of ASXL1 mutations in CMML patients. We also identified IRF8, a master regulator of monocyte differentiation, as a novel target gene of the ASXL1-MT/BAP1 complex. Consistent with this finding, one previous study showed substantial downregulation of IRF8 in Bap1-deleted murine HSPCs46.The ASXL1-MT/BAP1 complex enhances transient myeloid proliferation, but does not increase the long-term proliferation potential of murine and human HSPCs. Thus, unlike other epigenetic genes related to clonal haematopoiesis  whose disruption leads to increased HSPC function23, activation of UBE2O may represent an alternative approach to suppress the hyperactivity of the ASXL-MT/BAP1 complex. However, UBE2O is dispensable for BAP1-induced monoubiquitination of ASXL1-MT at lysine 351. We also examined the roles of additional 19 candidate ubiquitination-related proteins in 293T cells that coexpress ASXL1-MT, BAP1, and Cas9, but depletion of any individual ubiquitination-related protein did not reduce the monoubiquitination of ASXL1-MT  8- to 12-week-old male mice  transduced with the combinations of ASXL1-MT, ASXL1-MT-K351R, and BAP1 were plated in M3234 (STEMCELL Technologies) methylcellulose containing 20 ng/ml SCF, 10 ng/ml IL-3, and IL-6. A total of 1 × 104 cells were plated for each round of plating. Colony counting and replating were performed every 7 days. To generate RUNX1-ETO9a leukemia in vivo, E14.5 c-kit+ fetal liver cells derived from C57BL/6 (Ly5.2) mice were transduced with RUNX1-ETO9a together with the combinations of ASXL1-MT, ASXL1-MT-K351R and BAP1, and were transplanted into sublethally irradiated 8- to 12-week-old female C57/B6J (Ly5.1) mice 53. cSAM cells and MLL-AF9 cells were generated by transducing ASXL1-MT and SETBP1-D868N or MLL-AF9 into murine bone marrow progenitors, respectively54. In some experiments, these cells were transduced with Cas9 and Bap1-targeting sgRNAs, and were transplanted into sublethally irradiated 8- to 12-week-old female C57/B6J (Ly5.1) mice. We also used c-Kit+ cells derived from 8- to 12-week-old Rosa26-LSL-Cas9 knockin male mice40 (Jackson Laboratory) to establish RUNX1-ETO9a leukemia cells that coexpress ASXL1-MT, BAP1, and Cas9. Randomization and blinding were not performed in this study.All animal studies were approved by the Animal Care Committee of the Institute of Medical Science at the University of Tokyo , and were conducted in accordance with the Regulation on Animal Experimentation at University of Tokyo based on International Guiding Principles for Biomedical Research Involving Animals. Mouse bone marrow c-Kit55, MOLM-13 , THP-1 , Jurkat , and Raji  were cultured in RPMI-1640 medium supplemented with 10% FBS. TF-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 2 ng/ml GM-CSF. For erythroid differentiation assay, TF-1 cells were washed with PBS three times and were incubated with RPMI-1640 supplemented with 10% FBS and 2 U/ml erythropoietin (EPO)56. All cell lines used in this study were authenticated by short-tandem repeat analyses and tested for mycoplasma contamination in our laboratory. Mouse bone marrow c-Kit+ cells were separated using CD117 MicroBead Kit (Miltenyi Biotec) and were cultured in Iscove’s modified Dulbecco’s media (IMDM) containing 20% FBS and 50 ng/ml murine SCF, TPO, and FLT-3L for 16 h, and then were induced differentiation. For the mast cell maturation assay, we incubated cells with IMDM containing 20% FBS, 50 ng/ml SCF, 10 ng/ml IL-3 and IL-621. For the monocyte/macrophage differentiation assay, cells were incubated with DMEM containing 10% FBS and 100 ng/ml macrophage colony-stimulating factor (M-CSF)25. Human CB cells were obtained from Riken BRC or the Japanese Red Cross Kanto-Koshinetsu Cord Blood Bank  following an institutional review board-approved protocol. Informed consent was obtained in accordance with the Declaration of Helsinki. CD34+ cells were separated using CD34 MicroBead Kit (Miltenyi Biotec). For myeloid differentiation assay, cells were cultured in StemSpan  containing 10 ng/ml human SCF, megakaryocyte growth and development factor (TPO), FLT3 ligand (FLT-3L), IL-3, and IL-6 34. For erythroid differentiation assay, cells were cultured in StemSpan containing 100 ng/ml SCF, 2 U/ml erythropoietin (EPO), insulin, transferrin, selenium solution (ITS-G) (Thermo Fisher scientific), and insulin growth factor-1 (IGF-1) , and 10 μM dexamethasone 57. In stem cell culture condition, cells were cultured in StemSpan containing 50 ng/ml human SCF, TPO, FLT-3L, and 750 nM stemreginin-158.HEK293T  and Hela  were cultured in DMEM containing 10% fetal bovine serum (FBS). 32Dcl3  were cultured in RPMI-1640 medium supplemented with 10% FBS and 1 ng/ml IL-3. K562 , Kasumi-1 , MEG-01 , TS9;2222 or pcDNA3.1 vector. We also used 3xFLAG-tagged, and Myc-tagged ASXL1-MT, FLAG-tagged and Myc-tagged ASXL1-MT-K351R, FLAG-tagged ASXL1-Y591X and Myc-tagged ASXL1-WT in the pMYs-IRES-GFP vector and HA-tagged ASXL1-MT in the pMYs-IRES-Blastcidin vector. For BAP1 expression, we used pEF-4xHA-BAP1 WT, pMMP-puro-BAP1 WT, and pMMP-puro-BAP1 C91S [gifts from Dr. Machida59], and HA-tagged BAP1 WT/C91S in pMYs-IRES-nerve growth factor receptor (NGFR) vector and pMYs-IRES-tdTomato vector. For UBE2O expression, we used pLV-Myc-UBE2O WT and pLV-Myc-UBE2O C885S [gifts from Dr. Dijke60]. For RUNX1-ETO expression, we used HA-tagged RUNX1-ETO or RUNX1-ETO9a in a pMSCV-IRES-Thy1.1 retroviral vector54. For MLL-AF9 expression, we used pMSCV-MLL-AF9-pgk-EGFP retroviral vector54. For HOXA7 and HOXA9 expression, we used Flag-tagged HOXA7 and HOXA9 in pMSCV-neo retroviral vector. The full-length cDNAs of HOXA7 and HOXA9 were purchased from Promega, and we cloned them into the pMSCV-neo vector (Clontech).We used ASXL1-WT, ASXL1-MT, ASXL1-G646WfsX12, and ASXL1-MT-K351R in the pMYs-IRES-GFP (pMYs-IG) vector61. Retroviruses for human cells and lentiviruses were produced by transient transfection of 293T cells with viral plasmids along with gag-, pol-, and env-expressing plasmids using the calcium-phosphate method34. Retrovirus transduction to the cells was performed using Retronectin .Retroviruses for mouse cells were produced by transient transfection of Plat-E packaging cells with retroviral constructs using the calcium-phosphate method62, which was obtained from Addgene (#52963). Cas9-expressing vector (lentiCas9-Blast #52962); and lentiviral packaging vectors [(pMD2.G #12259) and (psPAX2 #12260)] were also purchased from Addgene. Lentiviruses were generated by transient transfection of 293T cells with these lentiviral constructs. Cells were infected with the virus for 24 h, and were selected for stable expression of Cas9 using blasticidin (10 μg/ml) and for stable expression of sgRNAs using puromycin (1 μg/ml). The cells were then harvested to examine the depletion of targeted gene by immunoblotting. Sequence of the sgRNAs targeting for mouse Bap1, human BAP1, human UBE2O, mouse Hoxa5, mouse Hoxa7, mouse Hoxa9, and mouse Irf8 from 5′ to 3′ are provided as follows: CCTGATCGTAGGTGTCAAAG (sgBap1#1), AGTGGACAGATAAAGCTCGA (sgBap1#2), GAACCGTCAGACAGTACTAG (sgBAP1#3), TCTACCCCATTGACCATGGT (sgBAP1#4), GCCCGACGTAGAGCGCAAGG (sgUBE2O#1), GACTTCGTGGTAGATAAGCG (sgUBE2O#2), CCCCACTCGAACCCCCTACG (sgUBE2O#3), AATGGCATGGATCTCAGCGT (sgHoxa5#1), GTCCCTGAATTGTTCGCTCA (sgHoxa5#2), GCCTGCGACAAGGCGGACGA (sgHoxa7#1), CCTGGATGCGCAGTTCAGGT (sgHoxa7#2), CGCGTGCACTGGGTTCCACG (sgHoxa9#1), ACCGGGCCATTAATAGCGTG (sgHoxa9#2), AGTTTACCGAATTGTCCCCG (sgIrf8#1), and TCGACAGCAGCATGTACCCG (sgIrf8#2).To generate short guide RNA (sgRNA) constructs, annnealed oligos were inserted into pLentiguide-puro vector293T cells transfected with FLAG-tagged vector, ASXL1 (MT or MT-K351R) and HA-tagged BAP1 (WT or C91S) were fixed with 2% paraformaldehyde, permealized with 0.2% Triton-X, blocked with 2% BSA and 5% goat serum, and were then incubated with anti-FLAG rabbit monoclonal antibody  and anti-HA mouse monoclonal antibody , followed by labeling with Alexa Fluor 568-conjugated anti-rabbit  and Alexa Fluor 488-conjugated anti-mouse antibody . Nuclei were visualized with DAPI . Fluorescent images were analyzed on a confocal microscope (Nikon A1).Cells were stained by fluoro-conjugated antibodies for 30 min at 4 °C. After staining, cells were washed with cold PBS for several times, and were resuspended with PBS containing 2% FBS. Cells were analyzed on a FACSCalibur or a FACS Verse, and were sorted with a FACSAria . Cell cycle analysis  and apoptosis analysis  were performed according to the manufacturer’s recommendations. Analyses were performed using Flowjo software (Tree Star Inc.). The following fluoro-conjugated antibodies were used in this study at indicated dilutions: anti-mouse B220 , anti-mouse CD11b , anti-mouse CD3 , anti-mouse CD117 , anti-mouse F4/80 , anti-mouse Gr-1 , anti-mouse Fce1Rα , anti-mouse CD115 , anti-mouse CD11b , anti-mouse CD45.2 , anti-mouse CD90.1 (Thy-1.1) , anti-human CD33 , anti-human CD66b , anti-human CD235ab , anti-human CD71 , anti-human CD34 , anti-human CD271 (NGFR) .63. Uncropped blots are provided in Supplementary Figs. 293T cells were transiently transfected with plasmids using polyethylenimine (PEI). Forty-eight hours after transfection, cells were lysed with cell lysis buffer . For immunoprecipitation, cell lysates were incubated with anti-FLAG  or anti-HA  antibody for 30 min at 4 °C. The samples were then incubated with protein-G-Sepharose  for 30 min at 4 °C. The precipitates were washed stringently three times with lysis buffer  containing 1 mM phenylmethanesulfonyl fluoride, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were analyzed by western blotting. Signals were detected with SuperSignalWest Pico , and the immunoreactive bands were visualized by LAS-4000 Luminescent Image Analyser (FUJIFILM). The intensity of band was measured using LabWorks Version 4.5 software . The following antibodies were used in this study at indicated dilutions: anti-DYKDDDDK tag (Wako (Japan), catalog #012-22384, clone 1E6, 1:2000), anti-HA , anti-MYC , anti-GAPDH , anti-α-tubulin , anti-ubiquitin , anti-ASXL1 , anti-UBE2O polyclonal antibody , anti-ubiquityl-histone H2A (Lys119) , anti-BAP1 antibody , anti-HOXA5 , anti-HOXA7 polyclonal antibody , anti-HOXA9 polyclonal antibody , anti-IRF8 . Anti-histone H3 and anti-H3K27me3 were kind gifts from Dr. KimuraHoxa5 Forward), CGCAAGCTGCACATTAGTCA (mouse Hoxa5 Reverse), AGGTAGCGGTTGAAATGGAA (mouse Hoxa7 Forward), GAAGCCAGTTTCCGCATCTA (mouse Hoxa7 Reverse), GTTCCAGCGTCTGGTGTTTT (mouse Hoxa9 Forward), ACAATGCCGAGAATGAGAGC (mouse Hoxa9 Reverse), CGTGGAAGACGAGGTTACGCTG (mouse Irf8 Forward), GCTGAATGGTGTGTGTCATAGGC (mouse Irf8 Reverse), AATTCCCAGTCAGAGGCGCTATGT (mouse Pten Forward), GATTGCAAGTTCCGCCACTGAACA (mouse Pten Reverse), GTGGGCCTTGATTCCACAGG (mouse Sf3b1 Forward), GGCTTCTTCTGACCGAGCAA (mouse Sf3b1 Reverse), TTGATGGCAACAATCTCCAC (mouse Gapdh Forward), CGTCCCGTAGACAAAATGGT (mouse Gapdh Reverse), TGGAACTCCTTCTCCAGCT (human HOXA5 Forward), AGATCTACCCCTGGATGCG (human HOXA5 Reverse), CGTCAGGTAGCGGTTGAAGT (human HOXA7 Forward), AATTTCCGCATCTACCCCTG (human HOXA7 Reverse), CAGTTCCAGGGTCTGGTGTT (human HOXA9 Forward), AATGCTGAGAATGAGAGCGG (human HOXA9 Reverse), TTCCCTTTAAAAACTGCCCA (human IRF8 Forward), CCAGGACTGATTTGGGAGAA (human IRF8 Reverse), TGGATTCGACTTAGACTTGACCT (human PTEN Forward), GCGGTGTCATAATGTCTCTCAG (human PTEN Reverse), GTGGGCCTCGATTCTACAGG (human SF3B1 Forward), GATGTCACGTATCCAGCAAATCT (human SF3B1 Reverse), CTCAGGGGTGAATTCTTTGC (human HBG1 Forward), GTGACAAGCTGCATGTGGAT (human HBG1 Reverse), GCTGGTCCTCAGACTTCACG (human KLF1 Forward), CACACAGGATGACTTCCTCAA (human KLF1 Reverse), TTGAGGTCAATGAAGGGGTC (human GAPDH Forward), GAAGGTGAAGGTCGGAGTCA (human GAPDH Reverse).Total RNA was extracted using the RNeasy Mini kit (QIAGEN). RNA was reverse transcribed using Omniscript RT kit (Qiagen). Complementary DNA (cDNA) was then subjected to quantitative RT-PCR using a SYBR Select Master Mix (Applied Biosystems). Sequences of the primers used for qRT-PCR in this study, from 5′ to 3′ are as follows: AGGTAGCGGTTGAAGTGGAA (mouse Hoxa5 Forward), GGGGTCGAATTGAGGTTACA (mouse Hoxa5 Reverse), CTGTGAGGGCTGCTGAGATT (mouse Hoxa7 Forward), GCAGCTTTCAGTGTCGGTTT (mouse Hoxa7 Reverse), AGTACAGAGGCAAGGCCAGA (mouse Hoxa9 Forward), CCGGTGTTTTGCAGTCATAA (mouse Hoxa9 Reverse), AACAGCCTTTCGGTTTTCCT (mouse Irf8 Forward), ACTGCCGGAAGATCAGAGGT (mouse Irf8 Reverse), TGCGAGGATTATCCGTCTTC (mouse Pten Forward), GCTGGATGGTTGCAGAGACT (mouse Pten Reverse), GAGAGCGGCCTCTGTTTCT (mouse Sf3b1 Forward), ATCTTCGCCATTTTGTCCAC (mousee Sf3b1 Reverse).ChIP was performed using Simple chip kit  with antibodies against H2AK119  and HA , following the manufacturer’s recommendations. Purified DNA was then subjected to quantitative RT-PCR using a SYBR Select Master Mix (Applied Biosystems). Sequences of the primers used for ChIP-qPCR in this study, from 5′ to 3′, are as follows: CTCCCCCGAATCCTCTGTAT (mouse https://ccb.jhu.edu/software/tophat/index.shtml). The expression level for each gene was calculated from mapped read counts using HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) and normalized with the DESeq2 pipeline (http://bioconductor.org/packages/release/bioc/html/DESeq2.html). For clustering analysis, normalized read counts were further transformed by the variance-stabilizing transformation method in DESeq2. They were then subjected to hierarchical clustering analysis with Ward’s method.Total RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA integrity was examined with the TapeStation (Agilent Technologies). cDNA was prepared from polyA-selected RNA by using an NEBNext Ultra Directional RNA Library Prep kit (New England BioLabs) and was subjected to next-generation sequencing from both ends with the HiSeq 2500 platform (Illumina). For expression profiling with RNA-seq data, paired-end reads were aligned to the mm10 mouse genome assembly using the TopHat2 computational pipeline . A 10% (vol/vol) volume of the eluted proteins that co-precipitated with ASXL1-MT was subjected to SDS/PAGE, followed by silver staining. The remaining 90% (vol/vol) volume of eluted proteins was trypsinized and subjected to nano liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) analysis to identify co-immunoprecipitated host proteins. For this analysis, we used LTQ-Orbitrap Velos (Thermo Fisher Scientific) coupled with Dina-2A (KYA Technologies). The MS/MS signals were processed against the RefSeq  human protein database  using the Mascot algorithm . Carbamidomethylation of cysteine was set as a fixed modification, whereas oxidation (Met), protein N-terminal acetylation, pyroglutamination (Gln), phosphorylation , and diglycine (Lys) were set as variable modifications. Trypsin was defined as a proteolytic enzyme and a maximum of two missed cleavages were allowed. The mass tolerance was set to three parts per million (ppm) for peptide masses and 0.8 Da for MS/MS peaks. In the process of peptide identification, we conducted decoy database searching by Mascot and applied a filter to satisfy a false-positive rate lower than 1%.t test or by analysis of variance (ANOVA) with Dunnett’s or Turkey’s post hoc test after testing for normal distribution and equal variance. The survival distributions were compared by the log-rank test. GraphPad Prism 7 was used for these statistical analyses. Samples sizes were estimated by preliminary experiments. No specific statistical methods were used to predetermine the sample size.Statistical analyses were performed by the unpaired and two-tailed Student’s All animal experiments were approved by the Animal Care Committee at the Institute of Medical Science, the University of Tokyo . All experiments using human cord blood cells were approved by the Ethics Committee at the Institute of Medical Science, the University of Tokyo .GSE114861[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE114861]. The data that support the findings of this study are available from the authors upon request.RNA-seq data from this study have been deposited into the NCBI Gene Expression Omnibus (GEO) under accession number Supplementary Informationpeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


ER-positive mutation carriers also had a 3.4-times higher risk of death due to BC compared with ER-positive non-carriers . Moreover, prognosis in ER-negative BRCA1/BRCA2 mutation carriers was comparable with that in ER-positive non-carriers. Our study demonstrates that ER-positivity worsens BC prognosis in BRCA1/BRCA2 mutation carriers, while prognosis for carriers with ER-negative tumors (including early-onset) is significantly better and comparable with that in ER-positive, older BC non-carriers. These observations indicate that BRCA1/BRCA2 mutation carriers with ER-positive BC represent high-risk patients.Breast cancer (BC) prognosis in BRCA1 and BRCA2 mutation carriers has been reported contradictorily, and the significance of variables influencing prognosis in sporadic BC is not established in BC patients with hereditary BRCA1/BRCA2 mutations. In this retrospective cohort study, we analyzed the effect of clinicopathological characteristics on BC prognosis  in hereditary BRCA1/BRCA2 mutation carriers. We enrolled 234 BRCA1/BRCA2 mutation carriers and 899 non-carriers, of whom 191 carriers and 680 non-carriers, with complete data, were available for survival analyses. We found that patients with ER-positive tumors developed disease recurrence 2.3-times more likely when they carried a BRCA1/BRCA2 mutation (23/60; 38.3% ER-positive carriers vs. 74/445; 16.6% ER-positive non-carriers;  BRCA1 or BRCA2 (BRCA1/BRCA2) account for most hereditary BC cases [BRCA1 vs. BRCA2 mutations is population-specific, with BRCA1 mutations being dominant among Czech patients [BRCA1/BRCA2 mutations have a 70–80% risk of BC development by age 80 [Approximately 5–10% of all breast cancers (BC) have a hereditary background . MutatioBC cases . The propatients . Women cy age 80 . BesidesBRCA1-associated BC cases have typical histopathological features including high-grade and triple-negative tumors [BRCA2 mutation carriers are less indicative , resembling sporadic tumors [Breast tumors are classified into distinct molecular subtypes with different prognoses and require specific therapeutic approaches ,9. Most e tumors ,11,12,13e tumors ,15. Pathc tumors .BRCA1/BRCA2 mutation carriers has been contradictorily reported to be worse [BRCA1/BRCA2 mutations and non-carriers or unselected BC patients revealed that current evidence does not suggest worsened BC survival in mutation carriers [BRCA1/BRCA2 mutation carriers [BRCA1/BRCA2 mutation carriers had better OS than non-carriers at two years , but comparable at subsequent time points.BC prognosis in be worse ,17,18 orbe worse ,20,21 ascarriers . Indeed,BRCA1/BRCA2 mutations undoubtedly increase BC risk, it is unclear whether the overall prognosis and clinicopathological prognostic factors differ between mutation carriers and non-carriers. This question is of considerable clinical importance because the age at BC onset is over a decade lower among mutation carriers than in non-carriers [While germline carriers ,12,22.BRCA1/BRCA2 mutation carriers and 899 non-carriers.To determine clinicopathological characteristics influencing BC prognosis , we analyzed 1133 Czech BC patients, including 234 BRCA1 (N = 183) or BRCA2 (N = 51) mutations . Median follow-up of 1133 patients eligible for subsequent analyses was 9.8 years. A comparison of the clinicopathological characteristics of BRCA1/BRCA2 mutation carriers and non-carriers is summarized in BRCA1/BRCA2 mutation carriers were diagnosed with BC at an earlier age, with more advanced disease, different BC morphology, higher grade, and more frequent TNBC. These differences were driven mainly by BRCA1 mutation carriers, as BRCA2 mutation carriers differed from non-carriers only in terms of higher tumor stage and lower HER2-positivity. BRCA1/BRCA2 mutation carriers were more often treated by chemotherapy; however, surgical treatment and radiotherapy were comparable in both groups. BRCA1/BRCA2 mutation carriers also developed distant metastases and second BC more often than non-carriers, but the frequency of loco-regional recurrences was similar. Differences in age at diagnosis between mutation carriers and non-carriers are displayed in Among 1133 enrolled BC patients, 234 (19.5%) were carriers of pathogenic utations . The remBRCA1 and 40 BRCA2 mutation carriers) and 680 non-carriers , already apparent at 5 years. The non-significant difference in DFS (p = 0.241) for BRCA1/BRCA2 mutation carriers and non-carriers, respectively. DFS was affected mainly by a higher relapse rate in carriers of mutations in BRCA2 , including patients ≥35 years (HR 1.81 95% CI 1.10–2.99), patients ≥45 years (HR 3.98 95% CI 1.62–9.81), and postmenopausal patients (HR 3.72 95% CI 1.16–11.90), when compared with age-matched non-carriers. Mutation carriers with ER-positive tumors also had significantly worse DFS  than non-carriers with ER-positive tumors, with similar significant differences also detected for DSS . The opposite prognostic effect in BRCA1/BRCA2 mutation carriers and non-carriers was not limited to ER status only as we have observed the same trend with age at diagnosis and menopausal status. Subsequent analyses within each group confirmed the observed differences in DFS with similar trends for DSS  and menopausal status only slightly modified the risk . In non-carriers, 10-year DFS and DSS were significantly worsened in younger patients’ subgroups  with ER-negative patients relapsing substantially earlier, as expected .BRCA1/BRCA2 mutation carriers, but patients with ER-positive tumors had worsened DFS (with a similar non-significant trend for DSS), compared with patients with ER-negative BC (BRCA1/BRCA2 mutations diagnosed with ER-positive tumors at ≥35 years showed a significantly increased risk of recurrence (HR 2.53 95% CI 1.15–5.57), compared with ER-negative mutation carriers of the same age. Similarly, increased risk of recurrence was shown for ER-positive patients ≥45 years (HR 4.03 95% CI 1.36–12.00), compared with ER-negative patients of the same age.In contrast, 10-year DFS was not significantly influenced by age or menopausal status in ative BC . PatientBRCA1/BRCA2 mutation carriers and non-carriers is shown in BRCA1/BRCA2 mutation carriers with ER-positive tumors diagnosed at ≥35 years had worse DFS (HR 4.56 95% CI 2.00–10.37) and DSS (HR 8.24 95% CI 2.37–28.72) than ER-positive non-carriers of the same age. Similarly, BRCA1/BRCA2 mutation carriers with ER-positive tumors diagnosed at ≥45 years or with post-menopausal BC also faced increased risk of recurrence.A direct comparison of the combined effects of ER status with age at disease onset  on survival in p = 0.039) than in BRCA1/BRCA2 mutation carriers, with the same trend observed for DSS. The non-significant trend was also observed in patients diagnosed at <45 years, but not in premenopausal patients.The recurrence risk in young (<35 years) ER-negative patients was higher in non-carriers . A similar observation was recently reported in a meta-analysis by Baretta and colleagues, showing decreased BC-specific survival (HR = 1.42 95% CI 1.05–1.92) but non-significantly changed DFS for BRCA1/BRCA2 mutation carriers [BRCA1/BRCA2 mutation carriers [BRCA1/BRCA2 mutation carriers [BRCA1/BRCA2 mutation carriers have only a slight prognostic disadvantage over non-carriers. However, this conclusion is clinically contra-intuitive because we and others have noted that BRCA1 mutation carriers (predominating among our mutation-positive patients) are mostly young, TNBC patients who otherwise represent a subpopulation of BC patients with poor prognoses [BRCA1/BRCA2 mutation carriers and non-carriers.Our initial analysis revealed slightly worsened DFS and DSS in carriers . A meta-carriers . A recencarriers . All therognoses ,24,25. TBRCA1/BRCA2 mutation carriers who developed disease recurrence 2.3-times more likely  than ER-positive non-carriers . ER-positive BRCA1/BRCA2 mutation carriers also had 3.4-times higher risk of death due to BC compared with ER-positive non-carriers . We must emphasize that the prognosis for ER-negative BRCA1/BRCA2 mutation carriers was comparable with that for ER-positive non-carriers. Inferior survival associated with ER-positive tumors was also reported in a prospective POSH study in BRCA1 mutation carriers (HR = 1.96 95% CI 1.41–2.71) and BRCA2 mutation carriers (HR = 2.24 95% CI 1.56–3.22) at 10 years [BRCA2 ER-positive patients in our study contributed to a worsened prognosis more than BRCA1 ER-positive patients  [BRCA2 mutations (HR = 2.04 95% CI 1.22–3.39) but not BRCA1 mutations [BRCA2 mutation carriers  for ER-positive and ER-negative patients, respectively) [BRCA1/BRCA2 mutation carriers and non-carriers.Indeed, we observed an inverse correlation between ER status and age in these two groups in initial univariate analyses . As expe10 years . Interespatients . Jonassoctively) . All theBRCA1 and BRCA2 mutation carriers share similar specific genomic profiles of DNA somatic copy number alterations, different from those in ER-positive sporadic tumors and in ER-negative tumors in BRCA1 mutation carriers [BRCA1 genomic locus in ER-positive tumors from BRCA1 mutation carriers found in this study (83%) and in a study by Tung and colleagues (81%) indicates that BRCA1 impairment directly contributes to the formation of ER-positive tumors [BRCA1/BRCA2 mutation carriers is unknown; however, preclinical data demonstrated estrogen-dependent progression of mammary tumorigenesis in BRCA1-defficient cells [BRCA1/BRCA2 mutation carriers with ER-positive tumors and found a high proportion of patients with a high recurrence score who may benefit from adjuvant chemotherapy [Interestingly, Lips and colleagues have shown that ER-positive tumors in both carriers . A high e tumors . The mecnt cells ,30. Shahotherapy .BRCA1/BRCA2 mutation carriers  in BRCA1/BRCA2 carriers and 0.66 (95% CI 0.45–0.98) in non-carriers with a similar trend for survival  is currently considered a cancer with a favorable prognosis, allowing the omission of adjuvant chemotherapy, shorter course of adjuvant hormonal treatment (no longer than five years), and other “de-escalation” approaches. In contrast, our data suggest that ER-positive BRCA1/BRCA2 mutation carriers exert an extremely dismal prognosis. We suppose that this patient group should be considered a high-risk group for BC recurrence and BC-related death.Hormone receptor-positive BC  were excluded due to incomplete data  [BRCA1/BRCA2 mutation carriers’ prognoses by Baretta and colleagues identified 60 studies and revealed the median number of BRCA1/BRCA2 mutation carriers as 39.5 (range 5–326). The size limitation affected especially BRCA2 mutation carriers . Further limitations resulted from the study design and a retrospective character of data. We performed a two-round, independent review of all clinicopathological data (irrespectively to the BRCA1/BRCA2 mutation status) obtained from the medical records. However, we cannot exclude a potential selection bias as 23.2% of all enrolled patients were excluded due to incomplete data from the univariate and multivariable analyses. On the other hand, no patient was excluded due to the loss of follow-up from the survival analyses. Retrospective study design is also sensitive to changes made in diagnostics and treatment procedures during the 19-year study period (1997–2015). Histopathological assessments for ER, PR, and HER-2 positivity have not been identical for all patients throughout the study period. Potential bias represent changes in BC treatment guidelines  during the study period, including the introduction of taxanes and trastuzumab, routinely available in the last 15 years. We are also aware that a further extension of the median follow-up (currently 9.8 years) will be necessary to better evaluate the effect of ER-positivity on DSS. Future studies screening BRCA1/BRCA2 mutations in an unselected BC population prospectively should be conducted to further examine differences in clinicopathological characteristics in BRCA1/BRCA2-positive and BRCA1/BRCA2-negative patients in the general BC population.Our study has several strengths. All genotyping was done in a single center using systematic counseling and testing criteria. We analyzed a homogenous set of BC patients excluding patients with breast and ovarian/pancreatic cancer duplicity and patients carrying mutations in non-H trial) . We usedH trial) . A subseBRCA1/BRCA2 mutation carriers and 899 non-carriers) who were tested for the presence of mutations in BRCA1/BRCA2 and other cancer-susceptibility genes at the Laboratory of Oncogenetics, First Faculty of Medicine, Charles University, in 1997–2015. All patients met national criteria for genetic/familial high-risk assessment in BC/OC , as described previously [An analysis of mutations in BC-susceptibility genes was initially performed using a protein truncation test or direct sequencing, the presence of large genomic eviously ,33,34. Aeviously . The bioeviously ,36.BRCA1/BRCA2 mutation carriers and non-carriers using χ2 or Fisher exact tests, where appropriate. Continuous variables (age at diagnosis and follow-up period) were tested by the Mann–Whitney test.Categorical variables  were compared between N = 46) or primarily metastatic BC (N = 25) were excluded from survival analyses. DFS was defined as the interval between BC diagnosis and the first loco-regional or distant recurrence or the last follow-up. The development of a second tumor was not considered a DFS event. DSS was defined as the interval between BC diagnosis and death from BC or the last follow-up.The Kaplan–Meier product-limit method was used for survival analyses and differences were tested using the log-rank and Mantel–Haenszel tests. BC patients with carcinoma in situ  and multivariate analyses  were performed using Cox proportional hazard regression.p values < 0.05 and 95% confidence intervals (CI) excluding 1 were considered statistically significant.All analyses were performed using the GraphPad Prism v8.0.1  and Statistica v12  programs. Two-sided BRCA1/BRCA2 mutation carriers with ER-positive tumors have a poor prognosis with increased BC recurrence and BC-related death rate; therefore, their specific treatment  should be considered. The BC prognosis for these patients is worse than that for young ER-negative BC non-carriers. In contrast, the prognosis for BRCA1/BRCA2 mutation carriers with ER-negative tumors, even with early BC onset, is comparable with ER-positive, older BC non-carriers, who are generally considered lower-risk patients.The present study indicates that