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Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia

Nature Genetics volume 53, pages 672–682 (2021)Cite this article

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Abstract

Transposable elements or transposons are major players in genetic variability and genome evolution. Aberrant activation of long interspersed element-1 (LINE-1 or L1) retrotransposons is common in human cancers, yet their tumor-type-specific functions are poorly characterized. We identified MPHOSPH8/MPP8, a component of the human silencing hub (HUSH) complex, as an acute myeloid leukemia (AML)-selective dependency by epigenetic regulator-focused CRISPR screening. Although MPP8 is dispensable for steady-state hematopoiesis, MPP8 loss inhibits AML development by reactivating L1s to induce the DNA damage response and cell cycle exit. Activation of endogenous or ectopic L1s mimics the phenotype of MPP8 loss, whereas blocking retrotransposition abrogates MPP8-deficiency-induced phenotypes. Expression of AML oncogenic mutations promotes L1 suppression, and enhanced L1 silencing is associated with poor prognosis in human AML. Hence, while retrotransposons are commonly recognized for their cancer-promoting functions, we describe a tumor-suppressive role for L1 retrotransposons in myeloid leukemia.

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Fig. 1: CRISPR screens identify MPP8 as a vulnerability for myeloid leukemia.
Fig. 2: MPP8 is dispensable for hematopoiesis but required for AML development in vivo.
Fig. 3: MPP8 loss reactivates L1 retrotransposons in myeloid leukemia.
Fig. 4: Silencing of LINE-1 retrotransposons in myeloid leukemia.
Fig. 5: Reactivation of L1 retrotransposition impairs AML in vitro and in vivo.
Fig. 6: MPP8 suppresses L1s to safeguard genome stability in myeloid leukemia.

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Data availability

All raw and processed RNA-seq and ChIP–seq data are available at the Gene Expression Omnibus under accession number GSE150984. All other genomic datasets are listed in Supplementary Table 4. All key reagents and resources are listed in Supplementary Table 6. Source data are provided with this paper.

Code availability

RNA-seq analyses were conducted using STAR v.2.5.2b, DESeq2 v.1.14.1 and featureCounts v.1.6.2. ChIP–seq analyses were conducted using Bowtie v.2.1.0 and MACS v.2.1.2. Counting and normalization of sgRNAs were performed using MAGeCK v.0.5.5. The code for the analyses using other indicated software is available from the websites of the corresponding software.

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Acknowledgements

We thank S. J. Morrison, R. J. DeBerardinis, H. Zhu, M. Agathokleous and S. Chung at UTSW for discussion, G. G. Wang at the University of North Carolina at Chapel Hill for the OCI-AML3 cell line, J. Wysocka at Stanford University for assistance with the RNA-seq and ChIP–seq pipelines and other Xu laboratory members for technical support. Y.L. was supported by the Cancer Prevention and Research Institute of Texas (CPRIT) training grant no. RP160157. A.W. was supported by National Institutes of Health (NIH) Cancer Biology Training grant no. T32CA124334. W.A. was supported by NIH grant nos. R21OD017965 and R15GM131263 and the Markl Faculty Scholar Fund. J.X. is a Scholar of the Leukemia & Lymphoma Society and a Scholar of the American Society of Hematology. This work was supported by NIH grant nos. R01CA230631 and R01DK111430 (to J.X.), R01CA248736 (to C.C.Z.), R01GM115682 and R01CA222579 (to J.M.A.), by CPRIT grant nos. RR140025, RP180504, RP180826 and RP190417 (to J.X.) and RP170086 (to J.M.A.), by the Leukemia Texas Foundation research award and by the Welch Foundation grant no. I-1942 (to J.X.).

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Author notes

  1. These authors contributed equally: Zhimin Gu, Yuxuan Liu.

Authors and Affiliations

  1. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhimin Gu, Yuxuan Liu, Yuannyu Zhang, Hui Cao, Junhua Lyu, Xun Wang, Michael Lee Jr., Giovanni A. Botten, Kathryn E. Dickerson & Jian Xu

  2. Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhimin Gu, Yuxuan Liu, Yuannyu Zhang, Hui Cao, Junhua Lyu, Michael Lee Jr., Giovanni A. Botten, Kathryn E. Dickerson & Jian Xu

  3. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Annika Wylie, Amanda E. Jones & John M. Abrams

  4. Department of Pharmaceutical Sciences, South Dakota State University, Brookings, SD, USA

    Simon J. Newkirk & Wenfeng An

  5. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Mi Deng & Cheng Cheng Zhang

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Contributions

Z.G., Y.L. and J.X. conceptualized the study. Z.G., Y.L., Y.Z., H.C., J.L., X.W., A.W., S.J.N., A.E.J., M.L., G.A.B., M.D., K.E.D. and J.X. devised the methodology. Z.G., H.C., J.L., X.W., S.J.N. and J.X. carried out the investigation. Z.G., Y.L. and J.X. wrote the original manuscript draft. J.X. reviewed and edited the draft. C.C.Z., W.A., J.M.A. and J.X. acquired the funding. C.C.Z., W.A., J.M.A. and J.X. supervised the study.

Corresponding author

Correspondence to Jian Xu.

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The authors declare no competing interests.

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Peer review information Nature Genetics thanks Ross Levine, Haig Kazazian and the other, anonymous, reviewer for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Design and validation of epigenetic regulator-focused CRISPR screens.

a, Schematic of the CRISPR screen system containing a TET-on-3G trans-activator, a tetracycline (TRE3G)-inducible Cas9 with an mCherry reporter, and the sgRNA. b, Schematic of the experiments to validate the inducible CRISPR screen system by targeting the stably expressed GFP reporter gene in MOLM-13 cells. c, Dox-induced expression of Cas9 and GFP-targeting sgRNAs in mCherry+ cells significantly decreased GFP expression compared to Cas9-negative (mCherry−) cells after 4 days of doxycycline treatment. d, Schematic of the customized sgRNA library containing 3,248 sgRNAs targeting 498 conserved chromatin-interacting domains in 266 annotated human epigenetic regulators and 1,800 sgRNAs targeting the 5’ exon of the other epigenetic regulators, together with 239 positive and 390 negative control sgRNAs. e, Comparisons of replicate experiments of CRISPR screens in MOLM-13 cells. The x- and y-axis of each graph represent the normalized sgRNA counts at day0 and day14. Spearman correlation value is shown for each comparison. f, MPP8 expression was determined by Western blot in MOLM-13 cells before xenograft (control), one week after Dox-induced MPP8 KO in xenografted NSG mice, and in the moribund mice (6-week post-xenograft) by independent MPP8-targeting sgRNAs (sg1 and sg2). g, Depletion of MPP8 by RNAi-mediated knockdown using shRNAs against the coding or 3’UTR sequences. shRNA against the luciferase gene (shLuc) was used as a negative control. Schematic of the MPP8 gene and the positions of shRNAs (sh1 to sh4) are shown. Results are mean ± SD (N = 3 independent experiments) and analyzed by a one-way ANOVA with Dunnett’s test. h, Growth curves of MOLM-13 AML cells after doxycycline-induced expression of MPP8−targeting shRNAs. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Tukey’s test.

Source data

Extended Data Fig. 2 HUSH components and other L1 regulators are required for myeloid leukemia cells.

a, Schematic of the HUSH complex in epigenetic silencing of retrotransposons. b, Depletion of HUSH proteins (PPHLN1 and TASOR) impaired MOLM-13 cell growth by the negative-selection competition assays. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. c, Depletion of other L1 regulators (MORC2, ATPIP7 and SETDB1) impaired MOLM-13 cell growth. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. d, Schematic of the xenotransplantation assay. e, Depletion of HUSH proteins (TASOR or PPHLN1) impaired MOLM-13 cell growth in NSG mice. Bioluminescence intensity is shown at 4 hours and 3 weeks post-transplantation. f, Quantification of bioluminescent imaging. Results are mean ± SD (N = 5 mice per group) and analyzed by a two-way ANOVA with Dunnett’s test. g, Representative image is shown for spleens of the xenografted NSG mice 3 weeks post-transplantation. h, Quantification of spleen weight 3 weeks post-transplantation. Results are mean ± SD (N = 3 mice per genotype) and analyzed by a one-way ANOVA with Dunnett’s test. i, Quantification of leukemia burden in PB, BM, spleen and liver of the xenografted NSG mice 3 weeks post-transplantation. Results are mean ± SD (N = 3 mice per group) and analyzed by a two-way ANOVA with Dunnett’s test. j, Kaplan-Meier survival curves of NSG mice xenografted with MOLM-13 cells transduced with control (sgNT) or sgRNAs against TASOR or PPHLN1. N = 5 mice per group. P values by a log-rank Mantel-Cox test.

Extended Data Fig. 3 Cellular phenotypes of MPP8-deficient myeloid leukemia cells.

a, Validation of shRNA-mediated MPP8 depletion by qRT-PCR in human normal CD34+ HSPCs (control) and four independent primary AML samples (AML1 to AML4). Cells transduced with shLuc were analyzed as controls. Results are mean ± SD (N = 4 independent experiments) and analyzed by a two-way ANOVA with Tukey’s test. b, MPP8 depletion impaired the colony-forming activity of AML cells but not normal CD34+ HSPCs in serial plating assays. Results are mean ± SD (N = 5 experiments) and analyzed by a two-way ANOVA with Tukey’s test. c, MPP8 depletion significantly increased CD11b expression in MOLM-13 and OCI-AML3 cells. Cells were analyzed after 10 days of Dox-induced MPP8 KO. Results are mean ± SD (N = 3 experiments) and analyzed by a two-way ANOVA with Dunnett’s test. d, MPP8 depletion increased apoptosis (Annexin V+) in MOLM-13 and OCI-AML3 cells after 10 days of Dox-induced MPP8 KO. Results are mean ± SD (N = 3 experiments) and analyzed by a two-way ANOVA with Dunnett’s test. e, MPP8 depletion led to a G0/G1 cell cycle arrest after 10 days of Dox-induced MPP8 KO. Results are mean ± SD (N = 3 experiments) and analyzed by a two-way ANOVA with Dunnett’s test. f, Expression of myeloid differentiation genes were upregulated in MPP8-deficient MOLM-13 cells after 10 days of Dox-induced MPP8 KO. Results are mean ± SD (N = 4 experiments) and analyzed by a one-way ANOVA with Dunnett’s test.

Extended Data Fig. 4 Generation of MPP8 constitutive and conditional KO mouse models.

a, Schematic of the design and genotyping strategies of the MPP8 constitutive and conditional KO mouse models. The locations and sizes of the genotyping primers and PCR products are shown, respectively. b, Representative genotyping results are shown for the MPP8 constitutive heterozygous and homozygous KO mice. c, Representative genotyping results are shown for the MPP8 conditional heterozygous and homozygous KO mice (by Mx1-Cre) 2 weeks after pIpC-induced MPP8 deletion. Genomic DNA from lineage-negative bone marrow cells were analyzed. d, Validation of MPP8 KO by Western blot analysis of bone marrow and spleen cells in MPP8 WT and homozygous constitutive KO mice. The non-specific signal (*) was also observed by the MPP8 antibody. e, Validation of pIpC-induced MPP8 KO by Western blot of bone marrow and spleen cells in MPP8 conditional WT and homozygous KO mice (by Mx1-Cre) 2 weeks after pIpC-induced MPP8 deletion. f, Birth ratios are shown for MPP8 WT, heterozygous and homozygous constitutive KO mice from the breeding of MPP8 heterozygous KO mice. g, Representative images are shown for male and female mice with the indicated genotypes at 6-weeks old. h, Growth curves (body weight) of male or female mice of the indicated genotypes up to 20 weeks. Results are mean ± SD (N = 8 independent male or female mice per genotype) and analyzed by a two-way ANOVA with Dunnett’s test.

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Extended Data Fig. 5 MPP8 loss has no detectable effect on steady-state hematopoiesis.

a, Complete blood counts (CBC) of PB red blood cells (RBC), hemoglobin (Hgb), white blood cells (WBC) and platelets in mice at 10-weeks old. N = 5 mice. b, Frequencies of BM erythroid (Ter119+), B-lymphoid (B220+), T-lymphoid (CD3+) and myeloid (Mac1+Gr1+) cells in mice at 8-weeks old. N = 5 mice. c, Cellularity and frequencies of spleen cell populations at 10-weeks old. N = 5 mice. d, Frequencies of donor-derived CD45.2+, myeloid (Mac1+), B (B220+) and T (CD3+) cells at 4 to 24 weeks after BMT. N = 5 mice. e, Schematic of experimental approach. f, CBC of PB RBC, Hgb, WBC and platelets. N = 5 mice. g, Cellularity and frequencies of BM cell populations. N = 5 mice. h, Cellularity and frequencies of spleen cell populations. N = 5 mice. i, Homing of AE9a or MLL-AF9-transformed cells in recipient BM. Results are shown for the % of GFP+ leukemia cells 16 hours after tail vein injection. N = 5 mice. j, MPP8 KO by Mx1-Cre had no effect on leukemia engraftment before pIpC administration in PB 4 weeks after tail vein injection. N = 8 control and 9 Mx1-Cre+;MPP8loxP/loxP mice (for AE9a), or N = 7 control and 9 Mx1-Cre+;MPP8loxP/loxP (for MLL-AF9). k, Expression of known AE9a and MLL-AF9 gene targets in AE9a or MLL-AF9-transformed BM cells, respectively. N = 4 experiments. l, Expression of MPP8 protein in MLL-AF9-transformed cells 2 to 8 weeks after pIpC-induced MPP8 deletion in recipients or the moribund mouse (12 weeks post-pIpC). Cells before transplantation were analyzed as the control. For a, b, c, f, g, h, results are mean ± SD and analyzed by a one-way ANOVA with Tukey’s test. For d, i, j, k, l, results are mean ± SD and analyzed by a two-way ANOVA with Bonferroni’s test.

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Extended Data Fig. 6 Expression and regulation of L1 retrotransposons in myeloid leukemia.

a, Expression of young L1s was significantly upregulated upon depletion of other HUSH components or SETDB1. Results are mean ± SD (N = 4 experiments) and analyzed by a one-way ANOVA with Dunnett’s test. b, Heatmap depicting the genome-wide co-localization between MPP8 and H3K9me3, but not H3K4me3, ChIP-seq signals in MOLM-13 cells. MPP8 occupied genome regions (N = 6,292 peaks) were ranked according to the normalized MPP8 ChIP-seq intensities, and the regions upstream (-5kb) and downstream (+5 kb) of the ChIP-seq peak summit are shown. Independent ChIP-seq experiments were merged for the heatmap illustration. c, Browser view of representative loci showing enrichment of MPP8 and H3K9me3 ChIP-seq signals at L1s. d, Validation of MPP8 chromatin occupancy at L1Hs in MOLM-13 cells and four independent primary AML samples (AML1 to AML4). Primers for the β-actin locus was analyzed as the negative control. Results are mean ± SD (N = 4 experiments) and analyzed by a two-way ANOVA with Bonferroni’s test. e, Depletion of MPP8 reactivated L1s in MOLM-13 cells, human normal CD34+ HSPCs and primary AML samples. Fold changes of L1 expression were calculated in MPP8-depleted versus shLuc-transduced cells. Results are mean ± SD (N = 4 experiments) and analyzed by a two-way ANOVA with Dunnett’s test. f, MPP8 KO in AML cells increased L1 retrotransposition, which were inhibited by 10 µM of 3TC. Representative flow cytometry graphs are shown for control (sgNT) or MPP8 KO (MPP8-sg1 and MPP8-sg2) cells harboring the LRE3-EGFP retrotransposition reporter or the retrotransposition-deficient JM111 control, respectively. g, Lower L1Hs expression is associated with poor survival in AML patients. Univariate and multivariate Cox regression analyses were performed using the AML samples from the TCGA cohort (N = 134 samples). L1Hs-high (top 50%) and L1Hs-low (bottom 50%) samples were compared. P values by the two-sided Wald test.

Extended Data Fig. 7 Reactivation of L1 retrotransposition impairs myeloid leukemia in vitro and in vivo.

a, Schematic of 10 sgRNAs against the 5’UTR promoter sequences of the full-length L1Hs. b, CRISPRa-mediated activation of endogenous L1s in MOLM-13 cells using L1-promoter-targeting sgRNAs individually. Relative L1 expression was determined by qRT-PCR in non-transduced cells (control) and cells expressing non-targeting (sgNT) or L1-promoter-targeting sgRNAs (L1-sg1 to L1-sg10). Results are mean ± SD (N = 4 independent experiments) and analyzed by a one-way ANOVA with Dunnett’s test. c, CRISPRa of endogenous L1s in MOLM-13 cells using combinations of multiple L1-promoter-targeting sgRNAs. Results are mean ± SD (N = 4 independent experiments) and analyzed by a one-way ANOVA with Dunnett’s test. d, CRISPRa-mediated activation of endogenous L1s increased the expression of L1-encoded ORF1p in MOLM-13 and OCI-AML3 cells by Western blot. GAPDH was analyzed as a loading control. e, CRISPRa of endogenous L1s increased L1 retrotransposition in MOLM-13 and OCI-AML3 cells. Representative flow cytometry graphs are shown for control (sgNT) or L1 activated (L1-sg278 and L1-sg457) cells harboring the LRE3-EGFP or JM111 control, respectively. f, Activation of L1 retrotransposition impaired MOLM-13 cell growth by the negative-selection competition assay. Treatment with 3TC (10 µM) abrogated L1 retrotransposition-induced cell growth defects. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. g, Activation of L1 retrotransposition impaired OCI-AML3 cell growth, whereas 3TC treatment (10 µM) abrogated L1 retrotransposition-induced defects. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. h, 3TC treatment had no significant effect on AML cell viability as determined by treating MOLM-13 and OCI-AML3 cells with escalating doses (0 to 640 µM) of 3TC for 5 days. Results are mean ± SD (N = 3 independent experiments) and analyzed by a one-way ANOVA with Dunnett’s test.

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Extended Data Fig. 8 p21 KO abrogates MPP8 KO or L1 reactivation-induced phenotypes in AML cells in vitro.

a, Schematic of CRISPR-mediated KO of p21 (CDKN1A) gene in human leukemia cells. The positions of sgRNAs and genotyping primers (F1, F2, R1 and R2) for WT or KO alleles are indicated. b, Representative genotyping results are shown for p21 KO in MOLM-13 and OCI-AML3 cells with MPP8 KO or CRISPRa (for L1 reactivation). c, p21 KO abrogated MPP8-deficiency-induced cell growth defects by the negative-selection competition assays in MOLM-13 and OCI-AML3 cells. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. d, p21 KO abrogated L1-reactivation-induced cell growth defects by the negative-selection competition assays in AML cells. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test.

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Extended Data Fig. 9 p21 KO abrogates MPP8 KO or L1 reactivation-induced phenotypes in AML cells in vivo.

a, Survival curves of NSG mice xenografted with WT or p21 KO MOLM-13 cells transduced with control (sgNT) or MPP8-targeting sgRNAs. P values by a log-rank Mantel-Cox test. b, KO of p21 abrogated MPP8-deficiency-induced cell growth defects of MOLM-13 cells in NSG mice. Bioluminescence intensity is shown at 4 hours and 3 weeks post-transplantation. c, Quantification of bioluminescent imaging. The numbers of independent mice are indicated. Results are mean ± SD and analyzed by mixed-effects analysis with Dunnett’s test. d, Survival curves of NSG mice xenografted with WT or p21 KO MOLM-13 cells transduced with control or L1-promoter-targeting sgRNAs for CRISPRa-mediated L1 reactivation. P values by a log-rank Mantel-Cox test. e, KO of p21 abrogated L1-reactivation-induced cell growth defects of MOLM-13 cells in xenografted NSG mice. f, Quantification of bioluminescent imaging. The numbers of independent mice are indicated. Results are mean ± SD and analyzed by mixed-effects analysis with Dunnett’s test. g, Schematic of the leukemia initiation experiments. h, Schematic of the leukemia maintenance experiments. i, Survival curves of recipient mice engrafted with WT (N = 7 mice), MPP8−/− (N = 10), or MPP8−/−p21−/− (N = 7) BM lineage-negative cells transduced with MLL-AF9. P values by a log-rank Mantel-Cox test. Quantification of leukemia burden by % of GFP+ leukemia cells at day 38 post-transplantation is shown. Results are mean ± SEM and analyzed by a two-way ANOVA with Tukey’s test. j, Survival curves of recipient mice engrafted with cells of the indicated genotypes transduced with MLL-AF9 for the leukemia maintenance experiments. N = 7, 8, and 8 WT, Mx1-Cre+;MPP8loxP/loxP, and Mx1-Cre+;MPP8loxP/loxP;p21−/− mice, respectively. P values by a log-rank Mantel-Cox test. Quantification of leukemia burden at day 60 post-transplantation is shown. Results are mean ± SEM and analyzed by a two-way ANOVA with Tukey’s test.

Extended Data Fig. 10 p53 is required for MPP8 KO or L1 reactivation-induced phenotypes in AML cells.

a, MPP8 loss increased p53 expression in AML cells. b, L1 reactivation increased p53 expression in AML cells. c, Validation of p53 KO by Western blot. d, p53 KO blunted MPP8-deficiency-induced cell growth defects. Results are mean ± SD (N = 3 experiments) and analyzed by a two-way ANOVA with Dunnett’s test. e, p53 KO blunted L1-reactivation-induced cell growth defects. Results are mean ± SD (N = 3 independent experiments) and analyzed by a two-way ANOVA with Dunnett’s test. f, Validation of p53 depletion in AE9a or MLL-AF9-transformed BM cells. Results are mean ± SD (N = 4 experiments) and analyzed by a two-way ANOVA with Bonferroni’s test. g, Schematic of the leukemia initiation experiments. h, Survival curves of recipients engrafted with AE9a-transformed WT or MPP8−/− cells with control (shLuc) or p53 depletion. N = 7 mice. P values by a log-rank Mantel-Cox test. Quantification of leukemia burden at day 50 post-transplantation (N = 5 mice) is shown. i, Survival curves of recipients engrafted with MLL-AF9-transformed WT or MPP8−/− cells with or without p53 depletion. N = 7, 7, and 6 mice for WT, MPP8−/−;shLuc, and MPP8−/−;shp53, respectively. Quantification of leukemia burden at day 37 (N = 5 mice) is shown. j, Schematic of the leukemia maintenance experiments. k, Survival curves of recipients engrafted with AE9a-transformed cells. N = 7, 8, and 6 mice for WT, Mx1-Cre+;MPP8loxP/loxP;shLuc, and Mx1-Cre+;MPP8loxP/loxP;shp53, respectively. Quantification of leukemia burden at day 62 (N = 5 mice) is shown. l, Survival curves of recipients engrafted with MLL-AF9-transformed cells. N = 7, 7, and 8 mice for WT, Mx1-Cre+;MPP8loxP/loxP;shLuc, and Mx1-Cre+;MPP8loxP/loxP;shp53, respectively. Quantification of leukemia burden at day 47 (N = 5 mice) is shown. For h, i, k and l, results are mean ± SD and analyzed by a two-way ANOVA with Tukey’s test.

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Gu, Z., Liu, Y., Zhang, Y. et al. Silencing of LINE-1 retrotransposons is a selective dependency of myeloid leukemia. Nat Genet 53, 672–682 (2021). https://doi.org/10.1038/s41588-021-00829-8

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