To further rule out the potential off-target effects of sgSsty1 and sgSsty2, we introduced them into HSPCs from female Cas9 mice. Red, H2AX; blue, DAPI-labeled DNA. Transcriptome analyses showed that the expression levels of chromosome Yspecific genes, Uty, Eif2s3y, and Kdm5d, were significantly reduced in sgSsty1-sgCas9 and sgSsty2-sgCas9 HSPCs compared with those with sgScr-sgCas9 (Figure 1C). 
Zhao, L. Flow cytometry. HSPCs were transfected by spinoculation. Reference information: JCI Insight.
sgRNAs were cloned into the pLentiCRISPR-mCherry vector(U6-sgCas9-U6-sgRNA-EFS-mCherry).
These sgSsty1-sgCas9 and sgSsty2-sgCas9 HSPCs were analyzed by fluorescence in situ hybridization (FISH) with FITC-labeled wholechromosome Y probes to visualize chromosome Y loss with Texas Redlabeled chromosome XqA7.3 probes as staining control, with sgScr-sgCas9 cells as control (27). Herens C, et al. Among the 29 common downregulated genes was that encoding lysine demethylase 5D (KDM5D), a chromosome Yspecific H3K4 demethylase (Figure 4, AC). Leukemic blasts were observed in the peripheral blood of all recipient mice (Figure 2C). It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. in: LZ performed bioinformatics analysis and wrote the manuscript. *FDR q < 0.05, **FDR q < 0.01 (Kruskal-Wallis test). Human and mouse mLOY AML also shared common pathways important for leukemogenesis, such as the HALLMARK_MYC_TARGETS_V2 pathway (human: NES = 2.12, P = 0.00; mouse: NES = 1.39, P = 0.03) (Figure 2H). Data shown as mean SD (n = 6). Krauth MT, et al. The sample images were captured using a ZEISS LSM880 confocal microscope.
(B and C) The expression levels of KDM5D in mLOY AML patients with AML1-ETO (B, n = 5; C, n = 3) and other AML1-ETO+ patients (B, n = 4; C, n = 3) in 2 AML cohorts (TARGET AML and BEAT AML), from analysis of The Cancer Genome Atlas data.
Generating mLOY in murine HSPCs. mLOY is strongly associated with a high risk of leukemia (6, 12, 30). Mosaic loss of chromosome Y in peripheral blood is associated with shorter survival and higher risk of cancer. Copyright: 2022, Zhang et al. Loss of the Y chromosome in bone marrow cells: results on 1907 consecutive cases of leukaemia and preleukaemia. Further, all of the p53-intact AML patients with mLOY had significant downregulation of the HALLMARK_p53 pathway compared with those without mLOY (TCGA LAML, NES = 1.82, P = 0.00; BEAT AML, NES = 1.33, P = 0.01) (Supplemental Figure 3, C and D). | Green, FITC-labeled whole-chromosome probe for Y chromosome; red, Texas redlabeled X chromosome probe for XqA7.3; blue, DAPI-labeled DNA. Kdm5d was disrupted in HSPCs with CRISPR/Cas9, and H2AX staining and the comet assay were performed to measure their DNA damage. Kdm5d cDNA was cloned by PCR from the central nervous system cDNA library of an E14.5 male mouse embryo. in: Longitudinal changes in the frequency of mosaic chromosome Y loss in peripheral blood cells of aging men varies profoundly between individuals. All recipients were monitored by complete blood count (CBC) assay and blood smear for leukemogenesis.
*P < 0.05 (log-rank test). mLOY gives rise to clonal hematopoiesis in mice. Lastly, we explored the molecular consequences of Kdm5d loss in HSPCs by RNA-seq analyses (Supplemental Figure 6A). *FDR q < 0.05, **FDR q <0.01 (Kruskal-Wallis test). PubMed Because there are more than 300 copies of Ssty1 and Ssty2 repeats specifically located on chromosome Y, multiple cuts by sgRNAs on the same chromosome would result in the deletion of the whole chromosome, at least in embryos (27, 29). YY and SL performed DNA damage assays. Electronic publication. RNA was extracted from AML tumor cells (bone marrow cells or splenocytes) or HSPCs with the RNeasy Mini Kit (Qiagen, catalog 74104) following the manufacturers instructions.
(E) The frequencies of mLOY in HSPCs before injection and blast cells in AML mouse tumor cells; tumor cells were harvested from bone marrow at each of the endpoints when recipient mice developed full-blown AML (BM).
(A) Schematic of mLOY AML mouse model. Right: Plot of H2AX foci per cell, shown as the mean SD. | The mouse RNA-seq reads were aligned to the reference genome GRCm38 by STAR (42). Dumanski JP, et al. Zhang, Q. (C) Representative images of blood smear at 7 weeks from recipient mice. Although it has been proposed that mLOY might be a consequence of genomic instability, it is also possible that mLOY might be a cause of genomic instability. NS, not significant, *P < 0.05, ***P < 0.001 (2-way ANOVA). Data shown as mean SD. And more importantly, it provides a mechanism for the different disease spectrum of patients with Turner syndrome and those with mLOY (24, 25). The company carried out preliminary quality control of the raw data, removing the adapter, poly-N, and low-quality reads to obtain clean data. And conversely, 35.9% (14/39) of AML1-ETO+ AML were also mLOY (31). YL and CC conceived the project and wrote the manuscript. (B) RBC counts of recipient mice 9 weeks after transplantation with sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs.
Tyner JW, et al. PubMed The platelet numbers in their peripheral blood were similar (Supplemental Figure 4A).
*P < 0.05, **P < 0.01 (2-tailed t test). RNA quality was analyzed using Agilent picochips. To further exclude the possibility of continuous cutting by CRISPR/Cas9 and thus potential off-target effects, we cointroduced a suicide sgRNA against Cas9, which was linked in tandem with sgSsty1 or sgSsty2 (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.153768DS1). The distinct mechanisms of DNA damage responses in embryonic and somatic cells (22, 23) might explain the distinct phenotypes of XO mice previously reported (21) and mLOY mice generated in the current study.
(C) Heatmap showing the relative expression levels of the chromosome Yspecific genes in sgSsty1-sgCas9 and sgSsty2-sgCas9 HSPCs compared with sgScr-sgCas9 HSPCs, measured by RNA-seq 8 days after infection (n = 3 for each group). Role of H3K4 demethylases in complex neurodevelopmental diseases.
It would be interesting to identify the downstream targets of KDM5D in HSPCs and AML. The slides were air dried for 13 minutes and fixed with 4% paraformaldehyde solution for 30 minutes. GSEA used statistical approaches to identify significant similarities and differences between 2 given clusters by identifying a prioridefined gene sets (44). Pilie PG, et al.
JCI (D) Representative photomicrographs of FISH of sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs before injection (top) and bone marrow cells from recipient mice that developed full-blown AML (BM, bottom). By immunofluorescence staining, we found that sgCas9 completely depleted Cas9 in HSPCs (Supplemental Figure 1B). All statistics are indicated for each figure and analyses were conducted using GraphPad Prism 8. RNA-seq analysis. Scale bars: 10 m. Bone marrow cells were enriched from 8-week-old Trp53/; Cas9 male mice or mCD45.1; Cas9 male mice. Google Scholar, Find articles by
JCI ***FDR q < 0.001, ****FDR q < 0.0001 (Kruskal-Wallis test). Forsberg LA, et al.
Although mLOY mice were harvested earlier than control mice, their liver and spleen weights were similar to those of the control animals (Supplemental Figure 4, B and C).
in: Grimwade D, et al. | Love MI, et al. FISH analyses showed that 50% of sgSsty1-sgCas9; AML1-ETO and 65% of sgSsty2-sgCas9; AML1-ETO leukemic cells had lost their Y chromosome, more than a 4-fold increase from their initial mLOY ratio before transplantation (Figure 2E and Supplemental Figure 4F). The comet assay showed that Kdm5d significantly reduced the tail moment of both sgSsty1 and sgSsty2 HSPCs (Figure 4, G and H).
Google Scholar, Find articles by |. White arrows indicate XO cells. Matsuura S, et al. Flow cytometric analysis was performed using FlowJo (RRID: SCR_008520), and flow cytometry was performed on an LSRFortessa (BD Biosciences). The results showed that there were significantly more H2AX foci in the sgKdm5d cells than the control sgScr HSPCs (Figure 4E).
mLOY leads to increased DNA damage in HSPCs. (E) Left: Representative images of comet assay of sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs.
The expansion of these mLOY cells in the recipients and associated anemia resembled CH in elderly males. NS, not significant, *P < 0.05, **P < 0.01 (2-way ANOVA). JCI PubMed All mouse experiments were approved by the Institutional Animal Care and Use Committee of of West China Hospital of Sichuan University (approval number 2021822A). Phone: 86.18980606532; Email: chongchen@scu.edu.cn. Study approval.
The samples were resolved by electrophoresis at 25 V and 300 mA for 50 minutes at 4C, after which the gels were neutralized in neutralization buffer for 30 minutes at 4C. The gray dotted line shows the growth trend. sgSsty1-sgCas9 and sgSsty2-sgCas9 target repeat sequences located on chromosome Y, which causes chromosome Y elimination. With aging, the frequency of mLOY in the population and the levels of mLOY are dramatically increased.
Notably, the genes significantly upregulated in the sgKdm5d HSPCs were positively enriched in both AML patients and mice with mLOY, while those significantly downregulated in the sgKdm5d HSPCs were negatively enriched in both mouse and human cells with mLOY (Supplemental Figure 6, D and E). Statistics. The solution was removed, and the slides were washed 3 times with PBS, permeabilized with 0.3% Triton X-100 for 15 minutes, and then washed 3 times with PBS. **P < 0.01 (log-rank test). FISH. 1 The tail moment is shown as the mean SD. Mice. Whether it also plays a critical role in other mLOY-associated syndromes requires further study.
The RNA-seq data were sequenced using an Illumina NovaSeq 6000, and 150-bp paired-end reads were obtained.
Taking advantage of recent advances in genome editing (27), we designed a strategy to generate mLOY by introducing 2 independent single guide RNAs (sgRNAs) targeting Ssty1 (spermiogenesis-specific transcript on Y 1) or Ssty2 (spermiogenesis-specific transcript on Y 2) in c-Kit+ HSPCs from Rosa-Cas9 mice (Figure 1A). Insights into clonal haematopoiesis from 8,342 mosaic chromosomal alterations. (D) Kaplan-Meier tumor-free survival curves of recipient mice (n = 6).
Loh PR, et al.
Then, we analyzed the genetics and cytogenetics of the resulting AML cells. ****P < 0.0001 (2-tailed Mann-Whitney test).
Statistical analysis of all survival data was accomplished with the log-rank test in GraphPad Prism (RRID: SCR_002798). Data availability. (D) Left: Representative immunofluorescence images of H2AX foci in sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs. The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions.
After washing the slides 3 times with PBS for 5 minutes each, they were incubated for 1 hour with goat anti-rabbit Alexa Fluor 633 diluted 1:1000 in PBS containing 1% BSA, followed by washing 3 times with PBS for 5 minutes each. To further investigate the mLOY spectrum in AML, we analyzed 3 independent AML cohorts with a total of 620 patients.
RNA-seq data of AML patients were downloaded from TARGET AML, TCGA LAML, and BEAT AML (32), transcripts were normalized by DESeq2 (43), and differential expression analysis was performed. Consistently with the increased level of DNA damage, the DNA damage checkpoint gene signature was significantly positively enriched in Kdm5d-deficient cells. Similarly, we also observed a strong association in the BEAT AML and The Cancer Genome Atlas (TCGA) LAML cohorts (32, 33) (P = 1.5 1011 and P = 1.1 1015, respectively) (Supplemental Figure 2, B and C). State-of-the-art strategies for targeting the DNA damage response in cancer. Thompson DJ, et al. The tail moment is shown as the mean SD. Further, through bioinformatics and functional studies, we showed that KDM5D loss partially mediated mLOY in AML, CH, and DNA damage. Previous whole-genome sequencing showed that these sgRNAs had minimal off-target effects and could give rise to healthy animals without chromosome Y (27). mLOY collaborates with AML1-ETO to promote leukemogenesis. mLOY is associated with CH in elderly males (3, 30). 17 Peoples South Road, Chengdu 610041, China.
Li, S. Consistently with the preleukemic HSPCs in vitro, sgCas9 completely depleted the expression of Cas9 in the resulting sgSsty1-sgCas9; AML1-ETO and sgSsty2-sgCas9; AML1-ETO AML cells (Supplemental Figure 4D). In the TARGET AML cohort, 16 out of the total 250 male patients showed mLOY and 14 of them (87.5%) were also AML1-ETO+. Taken together, our results show that mLOY itself led to DNA damage in HSPCs. In contrast, there were approximately 30% mLOY cells in the sgSsty1-sgCas9 and sgSsty2-sgCas9 mice at 12 weeks after transplantation, and more than 50% mLOY cells at 21 weeks, respectively, compared with approximately 15% mLOY in preinjected cells (Figure 3, C and D). The truncated Kdm5d (gene ID:20592) gene produces a 659amino acid protein product (aa 1695, including the JmjN, ARID, PHD-type 1, and JmjC domains). Immunofluorescence. Consistent with mLOY HSPCs, mLOY AML cells directly harvested from sick mice displayed significantly higher levels of ongoing DNA damage in leukemic cells, as measured by the comet assay (Figure 2F). Most of the leukemic cells from either group expressed high levels of c-Kit and myeloid markers Gr-1 and/or Mac-1, but not lymphoid marker CD3 or B220 (Supplemental Figure 4E). QZ performed experiments, analyzed data, and wrote the manuscript.
| Clonal hematopoiesis (CH) is a common aging-related hematopoietic abnormality in which a single hematopoietic stem or progenitor cell gives rise to a substantial proportion of peripheral blood cells or bone marrow cells (hematopoietic stem and progenitor cells, HSPCs) (11).
Generating mLOY in mouse HSPCs. In AML, low KDM5D expression was associated with AML1-ETO translocation, similar to mLOY (Supplemental Table 1). PubMed Reikvam H, et al. Kuchenbauer F, et al. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. Plasmid constructs. Comet assay.
Cancer incidence in women with Turner syndrome in Great Britain: a national cohort study. To test the function of KDM5D in AML, we transplanted Trp53/; AML1-ETO; Cas9 leukemic cells with sgScr or sgKdm5d into sublethally irradiated recipient mice. Slides were then submerged in cold working alkaline lysis buffer overnight at 4C, followed by alkaline unwinding for 40 minutes in cold alkaline electrophoresis buffer. (F) Comet assay of AML tumor cells (3 mice per group). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Whether mLOY is a common feature among different species remains an open question. However, given the complexity of the cell populations involved in AML and CH, further studies are needed to dissect the effects of mLOY in different HSC, lineage-committed progenitors, and other populations in more details. Scale bars: 50 m. Chen, C. Thus, Kdm5d loss partially resembled mLOY in HSPCs.
Zuo E, et al. van Zeventer IA, et al. Consistently, accumulating evidence indicates that Turner syndrome patients have a reduced risk of cancer, including hematopoietic malignancies, which is in sharp contrast to the strong association of mLOY with multiple cancers in aging males (24, 25). Previous meta-analyses showed that approximately half of AML1-ETO+ AML had mLOY (12, 17, 20). Given that many other human diseases, such as Alzheimers disease, cardiovascular events, and various solid cancers have also been associated with mLOY in patients and mLOY has also been detected in nonhematopoietic cells (37), it would be interesting to test whether mLOY also plays roles in other associated conditions.
To test the latter hypothesis, we infected Cas9 HSPCs with sgScr-sgCas9, sgSsty1-sgCas9, or sgSsty2-sgCas9 and then analyzed the ratio of mLOY cells to non-mLOY cells by FISH and DNA damage by the comet assay and H2AX staining over time. The target sequence of Cas9 is GATCGGCGACCAGTACGCC; the target sequences of Ssty2 are Ssty2-A ATCACTCAAGAAGAAGAGT and Ssty2-B GGAGCTCCACAGCGATGAG; the target sequences of Ssty1 are Ssty1-A ATCCCTCATGAAGAAGAGG and Ssty1-B GGAGCTCTACAGTGATGAC; and the target sequences of Kdm5d are Kdm5d-A ATGGTACCTACAGAAGTTG and Kdm5d-B GACTTATCTCCTGAAGAAA. mLOY was first observed more than half a century ago (1). Slides were rinsed for 2 minutes in 0.3% NP-40/2 SSC at 72C and 1 minute in 0.1% NP-40/2 SSC at room temperature. HSPCs from male mCD45.1; Cas9 mice were infected with mCherry-linked Y-chromosome-targeting sgRNA (sgSsty1 and sgSsty2)-sgCas9 and then transplanted into sublethally irradiated (7 Gy) mCD45.2 female recipient mice, with sgScr-sgCas9 as a negative control. Each group consisted of 3 replicates. Chromosome-specific accumulation of aneuploidy in the aging mouse brain. https://doi.org/10.1172/jci.insight.153768, https://doi.org/10.1172/jci.insight.153768DS1, https://www.rdocumentation.org/packages/pheatmap/versions/0.2/topics/pheatmap, American Society for Clinical Investigation. Scale bars: 10 m. Samples with an RNA integrity number (RIN) of 7.5 or greater were analyzed by RNA-seq. in:
Zhao, L. Flow cytometry. HSPCs were transfected by spinoculation. Reference information: JCI Insight.
sgRNAs were cloned into the pLentiCRISPR-mCherry vector(U6-sgCas9-U6-sgRNA-EFS-mCherry).
These sgSsty1-sgCas9 and sgSsty2-sgCas9 HSPCs were analyzed by fluorescence in situ hybridization (FISH) with FITC-labeled wholechromosome Y probes to visualize chromosome Y loss with Texas Redlabeled chromosome XqA7.3 probes as staining control, with sgScr-sgCas9 cells as control (27). Herens C, et al. Among the 29 common downregulated genes was that encoding lysine demethylase 5D (KDM5D), a chromosome Yspecific H3K4 demethylase (Figure 4, AC). Leukemic blasts were observed in the peripheral blood of all recipient mice (Figure 2C). It is strongly associated with clonal hematopoiesis, hematopoietic malignancies, and other hematopoietic and nonhematopoietic diseases. in: LZ performed bioinformatics analysis and wrote the manuscript. *FDR q < 0.05, **FDR q < 0.01 (Kruskal-Wallis test). Human and mouse mLOY AML also shared common pathways important for leukemogenesis, such as the HALLMARK_MYC_TARGETS_V2 pathway (human: NES = 2.12, P = 0.00; mouse: NES = 1.39, P = 0.03) (Figure 2H). Data shown as mean SD (n = 6). Krauth MT, et al. The sample images were captured using a ZEISS LSM880 confocal microscope.
(B and C) The expression levels of KDM5D in mLOY AML patients with AML1-ETO (B, n = 5; C, n = 3) and other AML1-ETO+ patients (B, n = 4; C, n = 3) in 2 AML cohorts (TARGET AML and BEAT AML), from analysis of The Cancer Genome Atlas data.
Generating mLOY in murine HSPCs. mLOY is strongly associated with a high risk of leukemia (6, 12, 30). Mosaic loss of chromosome Y in peripheral blood is associated with shorter survival and higher risk of cancer. Copyright: 2022, Zhang et al. Loss of the Y chromosome in bone marrow cells: results on 1907 consecutive cases of leukaemia and preleukaemia. Further, all of the p53-intact AML patients with mLOY had significant downregulation of the HALLMARK_p53 pathway compared with those without mLOY (TCGA LAML, NES = 1.82, P = 0.00; BEAT AML, NES = 1.33, P = 0.01) (Supplemental Figure 3, C and D). | Green, FITC-labeled whole-chromosome probe for Y chromosome; red, Texas redlabeled X chromosome probe for XqA7.3; blue, DAPI-labeled DNA. Kdm5d was disrupted in HSPCs with CRISPR/Cas9, and H2AX staining and the comet assay were performed to measure their DNA damage. Kdm5d cDNA was cloned by PCR from the central nervous system cDNA library of an E14.5 male mouse embryo. in: Longitudinal changes in the frequency of mosaic chromosome Y loss in peripheral blood cells of aging men varies profoundly between individuals. All recipients were monitored by complete blood count (CBC) assay and blood smear for leukemogenesis.
*P < 0.05 (log-rank test). mLOY gives rise to clonal hematopoiesis in mice. Lastly, we explored the molecular consequences of Kdm5d loss in HSPCs by RNA-seq analyses (Supplemental Figure 6A). *FDR q < 0.05, **FDR q <0.01 (Kruskal-Wallis test). PubMed Because there are more than 300 copies of Ssty1 and Ssty2 repeats specifically located on chromosome Y, multiple cuts by sgRNAs on the same chromosome would result in the deletion of the whole chromosome, at least in embryos (27, 29). YY and SL performed DNA damage assays. Electronic publication. RNA was extracted from AML tumor cells (bone marrow cells or splenocytes) or HSPCs with the RNeasy Mini Kit (Qiagen, catalog 74104) following the manufacturers instructions.
(E) The frequencies of mLOY in HSPCs before injection and blast cells in AML mouse tumor cells; tumor cells were harvested from bone marrow at each of the endpoints when recipient mice developed full-blown AML (BM).
(A) Schematic of mLOY AML mouse model. Right: Plot of H2AX foci per cell, shown as the mean SD. | The mouse RNA-seq reads were aligned to the reference genome GRCm38 by STAR (42). Dumanski JP, et al. Zhang, Q. (C) Representative images of blood smear at 7 weeks from recipient mice. Although it has been proposed that mLOY might be a consequence of genomic instability, it is also possible that mLOY might be a cause of genomic instability. NS, not significant, *P < 0.05, ***P < 0.001 (2-way ANOVA). Data shown as mean SD. And more importantly, it provides a mechanism for the different disease spectrum of patients with Turner syndrome and those with mLOY (24, 25). The company carried out preliminary quality control of the raw data, removing the adapter, poly-N, and low-quality reads to obtain clean data. And conversely, 35.9% (14/39) of AML1-ETO+ AML were also mLOY (31). YL and CC conceived the project and wrote the manuscript. (B) RBC counts of recipient mice 9 weeks after transplantation with sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs.
Tyner JW, et al. PubMed The platelet numbers in their peripheral blood were similar (Supplemental Figure 4A).
*P < 0.05, **P < 0.01 (2-tailed t test). RNA quality was analyzed using Agilent picochips. To further exclude the possibility of continuous cutting by CRISPR/Cas9 and thus potential off-target effects, we cointroduced a suicide sgRNA against Cas9, which was linked in tandem with sgSsty1 or sgSsty2 (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.153768DS1). The distinct mechanisms of DNA damage responses in embryonic and somatic cells (22, 23) might explain the distinct phenotypes of XO mice previously reported (21) and mLOY mice generated in the current study. (C) Heatmap showing the relative expression levels of the chromosome Yspecific genes in sgSsty1-sgCas9 and sgSsty2-sgCas9 HSPCs compared with sgScr-sgCas9 HSPCs, measured by RNA-seq 8 days after infection (n = 3 for each group). Role of H3K4 demethylases in complex neurodevelopmental diseases.
It would be interesting to identify the downstream targets of KDM5D in HSPCs and AML. The slides were air dried for 13 minutes and fixed with 4% paraformaldehyde solution for 30 minutes. GSEA used statistical approaches to identify significant similarities and differences between 2 given clusters by identifying a prioridefined gene sets (44). Pilie PG, et al.
JCI (D) Representative photomicrographs of FISH of sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs before injection (top) and bone marrow cells from recipient mice that developed full-blown AML (BM, bottom). By immunofluorescence staining, we found that sgCas9 completely depleted Cas9 in HSPCs (Supplemental Figure 1B). All statistics are indicated for each figure and analyses were conducted using GraphPad Prism 8. RNA-seq analysis. Scale bars: 10 m. Bone marrow cells were enriched from 8-week-old Trp53/; Cas9 male mice or mCD45.1; Cas9 male mice. Google Scholar, Find articles by
JCI ***FDR q < 0.001, ****FDR q < 0.0001 (Kruskal-Wallis test). Forsberg LA, et al.
Although mLOY mice were harvested earlier than control mice, their liver and spleen weights were similar to those of the control animals (Supplemental Figure 4, B and C).
in: Grimwade D, et al. | Love MI, et al. FISH analyses showed that 50% of sgSsty1-sgCas9; AML1-ETO and 65% of sgSsty2-sgCas9; AML1-ETO leukemic cells had lost their Y chromosome, more than a 4-fold increase from their initial mLOY ratio before transplantation (Figure 2E and Supplemental Figure 4F). The comet assay showed that Kdm5d significantly reduced the tail moment of both sgSsty1 and sgSsty2 HSPCs (Figure 4, G and H).
Google Scholar, Find articles by |. White arrows indicate XO cells. Matsuura S, et al. Flow cytometric analysis was performed using FlowJo (RRID: SCR_008520), and flow cytometry was performed on an LSRFortessa (BD Biosciences). The results showed that there were significantly more H2AX foci in the sgKdm5d cells than the control sgScr HSPCs (Figure 4E).
mLOY leads to increased DNA damage in HSPCs. (E) Left: Representative images of comet assay of sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs.
The expansion of these mLOY cells in the recipients and associated anemia resembled CH in elderly males. NS, not significant, *P < 0.05, **P < 0.01 (2-way ANOVA). JCI PubMed All mouse experiments were approved by the Institutional Animal Care and Use Committee of of West China Hospital of Sichuan University (approval number 2021822A). Phone: 86.18980606532; Email: chongchen@scu.edu.cn. Study approval.
The samples were resolved by electrophoresis at 25 V and 300 mA for 50 minutes at 4C, after which the gels were neutralized in neutralization buffer for 30 minutes at 4C. The gray dotted line shows the growth trend. sgSsty1-sgCas9 and sgSsty2-sgCas9 target repeat sequences located on chromosome Y, which causes chromosome Y elimination. With aging, the frequency of mLOY in the population and the levels of mLOY are dramatically increased.
Notably, the genes significantly upregulated in the sgKdm5d HSPCs were positively enriched in both AML patients and mice with mLOY, while those significantly downregulated in the sgKdm5d HSPCs were negatively enriched in both mouse and human cells with mLOY (Supplemental Figure 6, D and E). Statistics. The solution was removed, and the slides were washed 3 times with PBS, permeabilized with 0.3% Triton X-100 for 15 minutes, and then washed 3 times with PBS. **P < 0.01 (log-rank test). FISH. 1 The tail moment is shown as the mean SD. Mice. Whether it also plays a critical role in other mLOY-associated syndromes requires further study.
The RNA-seq data were sequenced using an Illumina NovaSeq 6000, and 150-bp paired-end reads were obtained.
Taking advantage of recent advances in genome editing (27), we designed a strategy to generate mLOY by introducing 2 independent single guide RNAs (sgRNAs) targeting Ssty1 (spermiogenesis-specific transcript on Y 1) or Ssty2 (spermiogenesis-specific transcript on Y 2) in c-Kit+ HSPCs from Rosa-Cas9 mice (Figure 1A). Insights into clonal haematopoiesis from 8,342 mosaic chromosomal alterations. (D) Kaplan-Meier tumor-free survival curves of recipient mice (n = 6).
Loh PR, et al.
Then, we analyzed the genetics and cytogenetics of the resulting AML cells. ****P < 0.0001 (2-tailed Mann-Whitney test).
Statistical analysis of all survival data was accomplished with the log-rank test in GraphPad Prism (RRID: SCR_002798). Data availability. (D) Left: Representative immunofluorescence images of H2AX foci in sgScr-sgCas9, sgSsty1-sgCas9, and sgSsty2-sgCas9 HSPCs. The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions.
After washing the slides 3 times with PBS for 5 minutes each, they were incubated for 1 hour with goat anti-rabbit Alexa Fluor 633 diluted 1:1000 in PBS containing 1% BSA, followed by washing 3 times with PBS for 5 minutes each. To further investigate the mLOY spectrum in AML, we analyzed 3 independent AML cohorts with a total of 620 patients.
RNA-seq data of AML patients were downloaded from TARGET AML, TCGA LAML, and BEAT AML (32), transcripts were normalized by DESeq2 (43), and differential expression analysis was performed. Consistently with the increased level of DNA damage, the DNA damage checkpoint gene signature was significantly positively enriched in Kdm5d-deficient cells. Similarly, we also observed a strong association in the BEAT AML and The Cancer Genome Atlas (TCGA) LAML cohorts (32, 33) (P = 1.5 1011 and P = 1.1 1015, respectively) (Supplemental Figure 2, B and C). State-of-the-art strategies for targeting the DNA damage response in cancer. Thompson DJ, et al. The tail moment is shown as the mean SD. Further, through bioinformatics and functional studies, we showed that KDM5D loss partially mediated mLOY in AML, CH, and DNA damage. Previous whole-genome sequencing showed that these sgRNAs had minimal off-target effects and could give rise to healthy animals without chromosome Y (27). mLOY collaborates with AML1-ETO to promote leukemogenesis. mLOY is associated with CH in elderly males (3, 30). 17 Peoples South Road, Chengdu 610041, China.
Li, S. Consistently with the preleukemic HSPCs in vitro, sgCas9 completely depleted the expression of Cas9 in the resulting sgSsty1-sgCas9; AML1-ETO and sgSsty2-sgCas9; AML1-ETO AML cells (Supplemental Figure 4D). In the TARGET AML cohort, 16 out of the total 250 male patients showed mLOY and 14 of them (87.5%) were also AML1-ETO+. Taken together, our results show that mLOY itself led to DNA damage in HSPCs. In contrast, there were approximately 30% mLOY cells in the sgSsty1-sgCas9 and sgSsty2-sgCas9 mice at 12 weeks after transplantation, and more than 50% mLOY cells at 21 weeks, respectively, compared with approximately 15% mLOY in preinjected cells (Figure 3, C and D). The truncated Kdm5d (gene ID:20592) gene produces a 659amino acid protein product (aa 1695, including the JmjN, ARID, PHD-type 1, and JmjC domains). Immunofluorescence. Consistent with mLOY HSPCs, mLOY AML cells directly harvested from sick mice displayed significantly higher levels of ongoing DNA damage in leukemic cells, as measured by the comet assay (Figure 2F). Most of the leukemic cells from either group expressed high levels of c-Kit and myeloid markers Gr-1 and/or Mac-1, but not lymphoid marker CD3 or B220 (Supplemental Figure 4E). QZ performed experiments, analyzed data, and wrote the manuscript.
| Clonal hematopoiesis (CH) is a common aging-related hematopoietic abnormality in which a single hematopoietic stem or progenitor cell gives rise to a substantial proportion of peripheral blood cells or bone marrow cells (hematopoietic stem and progenitor cells, HSPCs) (11).
Generating mLOY in mouse HSPCs. In AML, low KDM5D expression was associated with AML1-ETO translocation, similar to mLOY (Supplemental Table 1). PubMed Reikvam H, et al. Kuchenbauer F, et al. Here, we generated mLOY in murine hematopoietic stem and progenitor cells (HSPCs) with CRISPR/Cas9 genome editing. Plasmid constructs. Comet assay.
Cancer incidence in women with Turner syndrome in Great Britain: a national cohort study. To test the function of KDM5D in AML, we transplanted Trp53/; AML1-ETO; Cas9 leukemic cells with sgScr or sgKdm5d into sublethally irradiated recipient mice. Slides were then submerged in cold working alkaline lysis buffer overnight at 4C, followed by alkaline unwinding for 40 minutes in cold alkaline electrophoresis buffer. (F) Comet assay of AML tumor cells (3 mice per group). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Whether mLOY is a common feature among different species remains an open question. However, given the complexity of the cell populations involved in AML and CH, further studies are needed to dissect the effects of mLOY in different HSC, lineage-committed progenitors, and other populations in more details. Scale bars: 50 m. Chen, C. Thus, Kdm5d loss partially resembled mLOY in HSPCs.
Zuo E, et al. van Zeventer IA, et al. Consistently, accumulating evidence indicates that Turner syndrome patients have a reduced risk of cancer, including hematopoietic malignancies, which is in sharp contrast to the strong association of mLOY with multiple cancers in aging males (24, 25). Previous meta-analyses showed that approximately half of AML1-ETO+ AML had mLOY (12, 17, 20). Given that many other human diseases, such as Alzheimers disease, cardiovascular events, and various solid cancers have also been associated with mLOY in patients and mLOY has also been detected in nonhematopoietic cells (37), it would be interesting to test whether mLOY also plays roles in other associated conditions.
To test the latter hypothesis, we infected Cas9 HSPCs with sgScr-sgCas9, sgSsty1-sgCas9, or sgSsty2-sgCas9 and then analyzed the ratio of mLOY cells to non-mLOY cells by FISH and DNA damage by the comet assay and H2AX staining over time. The target sequence of Cas9 is GATCGGCGACCAGTACGCC; the target sequences of Ssty2 are Ssty2-A ATCACTCAAGAAGAAGAGT and Ssty2-B GGAGCTCCACAGCGATGAG; the target sequences of Ssty1 are Ssty1-A ATCCCTCATGAAGAAGAGG and Ssty1-B GGAGCTCTACAGTGATGAC; and the target sequences of Kdm5d are Kdm5d-A ATGGTACCTACAGAAGTTG and Kdm5d-B GACTTATCTCCTGAAGAAA. mLOY was first observed more than half a century ago (1). Slides were rinsed for 2 minutes in 0.3% NP-40/2 SSC at 72C and 1 minute in 0.1% NP-40/2 SSC at room temperature. HSPCs from male mCD45.1; Cas9 mice were infected with mCherry-linked Y-chromosome-targeting sgRNA (sgSsty1 and sgSsty2)-sgCas9 and then transplanted into sublethally irradiated (7 Gy) mCD45.2 female recipient mice, with sgScr-sgCas9 as a negative control. Each group consisted of 3 replicates. Chromosome-specific accumulation of aneuploidy in the aging mouse brain. https://doi.org/10.1172/jci.insight.153768, https://doi.org/10.1172/jci.insight.153768DS1, https://www.rdocumentation.org/packages/pheatmap/versions/0.2/topics/pheatmap, American Society for Clinical Investigation. Scale bars: 10 m. Samples with an RNA integrity number (RIN) of 7.5 or greater were analyzed by RNA-seq. in: