Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities.
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References
1.
Ben-NeriahY, DaleyGQ, Mes-MassonAM, et al.The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene. Science, 1986; 233:212–214. DOI: 10.1126/science.3460176.
2.
JanossyG, RobertsM, GreavesMF. Target cell in chronic myeloid leukaemia and its relationship to acute lymphoid leukaemia. Lancet, 1976; 2:1058–1061. DOI: 10.1016/s0140-6736(76)90970-3.
3.
ZhouH, XuR. Leukemia stem cells: the root of chronic myeloid leukemia. Protein Cell, 2015; 6:403–412. DOI: 10.1007/s13238-015-0143-7.
4.
TauchiT, BoswellHS, LeibowitzD, et al.Coupling between p210bcr-abl and Shc and Grb2 adaptor proteins in hematopoietic cells permits growth factor receptor-independent link to ras activation pathway. J Exp Med, 1994; 179:167–175. DOI: 10.1084/jem.179.1.167.
5.
KabarowskiJH, WitteON. Consequences of BCR-ABL expression within the hematopoietic stem cell in chronic myeloid leukemia. Stem Cells, 2000; 18:399–408. DOI: 10.1002/stem.180399.
6.
KonopkaJB, WatanabeSM, WitteON. An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell, 1984; 37:1035–1042. DOI: 10.1016/0092-8674(84)90438-0.
7.
KonopkaJB, WatanabeSM, SingerJW, et al.Cell lines and clinical isolates derived from Ph1-positive chronic myelogenous leukemia patients express c-abl proteins with a common structural alteration. Proc Natl Acad Sci U S A, 1985; 82:1810–1814. DOI: 10.1073/pnas.82.6.1810.
8.
McWhirterJR, GalassoDL, WangJY. A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins. Mol Cell Biol, 1993; 13:7587–7595. DOI: 10.1128/mcb.13.12.7587.
9.
MullerAJ, YoungJC, PendergastAM, et al.BCR first exon sequences specifically activate the BCR/ABL tyrosine kinase oncogene of Philadelphia chromosome-positive human leukemias. Mol Cell Biol, 1991; 11:1785–1792. DOI: 10.1128/mcb.11.4.1785.
10.
PendergastAM, GishizkyML, HavlikMH, et al.SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence. Mol Cell Biol, 1993; 13:1728–1736. DOI: 10.1128/mcb.13.3.1728.
11.
MaG, LuD, WuY, et al.Bcr phosphorylated on tyrosine 177 binds Grb2. Oncogene, 1997; 14:2367–2372. DOI: 10.1038/sj.onc.1201053.
12.
ReutherGW, FuH, CripeLD, et al.Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family. Science, 1994; 266:129–133. DOI: 10.1126/science.7939633.
13.
EganSE, GiddingsBW, BrooksMW, et al.Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature, 1993; 363:45–51. DOI: 10.1038/363045a0.
14.
ShuaiK, HalpernJ, ten HoeveJ, et al.Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene, 1996; 13:247–254.
15.
IlariaRLJ, Van EttenRA. P210 and P190(BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members. J Biol Chem, 1996; 271:31704–31710. DOI: 10.1074/jbc.271.49.31704.
16.
CarlessoN, FrankDA, GriffinJD. Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. J Exp Med, 1996; 183:811–820. DOI: 10.1084/jem.183.3.811.
17.
FrankDA, VarticovskiL. BCR/abl leads to the constitutive activation of Stat proteins, and shares an epitope with tyrosine phosphorylated Stats. Leukemia, 1996; 10:1724–1730.
18.
WongS, WitteON. The BCR-ABL story: bench to bedside and back. Annu Rev Immunol, 2004; 22:247–306. DOI: 10.1146/annurev.immunol.22.012703.104753.
19.
CheredaB, Melo JV. Natural course and biology of CML. Ann Hematol, 2015; 94Suppl 2:S107-21. DOI: 10.1007/s00277-015-2325-z.
20.
BowerH, BjörkholmM, DickmanPW, et al.Life expectancy of patients with chronic myeloid leukemia approaches the life expectancy of the general population. J Clin Oncol, 2016; 34:2851–2857. DOI: 10.1200/JCO.2015.66.2866.
21.
KantarjianH, O'BrienS, JabbourE, et al.Improved survival in chronic myeloid leukemia since the introduction of imatinib therapy: a single-institution historical experience. Blood, 2012; 119:1981–1987. DOI: 10.1182/blood-2011-08-358135.
22.
DeiningerMW, GoldmanJM, MeloJV. The molecular biology of chronic myeloid leukemia. Blood, 2000; 96:3343–3356. DOI: 10.1182/blood.V96.10.3343.
23.
MilojkovicD, ApperleyJ. Mechanisms of resistance to imatinib and second-generation tyrosine inhibitors in chronic myeloid leukemia. Clin Cancer Res, 2009; 15:7519–7527. DOI: 10.1158/1078-0432.CCR-09-1068.
24.
GrahamSM, JorgensenHG, AllanE, et al.Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood, 2002; 99:319–325. DOI: 10.1182/blood.v99.1.319.
25.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337:816–821. DOI: 10.1126/science.1225829.
26.
MojicaFJM, MontoliuL. On the origin of CRISPR-Cas technology: from prokaryotes to mammals. Trends Microbiol, 2016; 24:811–820. DOI: 10.1016/j.tim.2016.06.005.
27.
WassefM, LuscanA, BattistellaA, et al.Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines. Methods, 2017; 121–122:45–54. DOI: 10.1016/j.ymeth.2017.05.003.
28.
Garcia-TuñonI, Hernandez-SanchezM, OrdonezJL, et al.The CRISPR/Cas9 system efficiently reverts the tumorigenic ability of BCR/ABL in vitro and in a xenograft model of chronic myeloid leukemia. Oncotarget, 2017; 8. DOI: 10.18632/oncotarget.15215.
29.
Garcia-TuñonI, Alonso-PerezV, VueltaE, et al.Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency. PLoS One, 2019; 14:e0216674. DOI: 10.1371/journal.pone.0216674.
30.
LugoTG, PendergastAM, MullerAJ, et al.Tyrosine kinase activity and transformation potency of bcr-abl oncogene products. Science, 1990; 247:1079–1082. DOI: 10.1126/science.2408149.
31.
LuoZ, GaoM, HuangN, et al.Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells. J Exp Clin Cancer Res, 2019; 38:224. DOI: 10.1186/s13046-019-1229-5.
32.
HuangN, HuangZ, GaoM, et al.Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases. J Exp Clin Cancer Res, 2018; 37:62. DOI: 10.1186/s13046-018-0732-4.
33.
ChenS-H, HsiehY-Y, TzengH-E, et al.ABL genomic editing sufficiently abolishes oncogenesis of human chronic myeloid leukemia cells in vitro and in vivo. Cancers (Basel), 2020; 12:1399. DOI: 10.3390/cancers12061399.
34.
GroffenJ, StephensonJR, HeisterkampN, et al.Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell, 1984; 36:93–99. DOI: 10.1016/0092-8674(84)90077-1.
35.
HeisterkampN, StamK, GroffenJ, et al.Structural organization of the bcr gene and its role in the Ph′ translocation. Nature, 1985; 315:758–761. DOI: 10.1038/315758a0.
36.
ShtivelmanE, LifshitzB, GaleRP, et al.Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature, 1985; 315:550–554. DOI: 10.1038/315550a0.
37.
StamK, HeisterkampN, GrosveldG, et al.Evidence of a new chimeric bcr/c-abl mRNA in patients with chronic myelocytic leukemia and the Philadelphia chromosome. N Engl J Med, 1985; 313:1429–1433. DOI: 10.1056/NEJM198512053132301.
38.
HondaH, OdaH, SuzukiT, et al.Development of acute lymphoblastic leukemia and myeloproliferative disorder in transgenic mice expressing p210bcr/abl: a novel transgenic model for human Ph1-positive leukemias. Blood, 1998; 91:2067–2075. DOI: 10.1182/blood.V91.6.2067.
39.
PalaciosR, SteinmetzM. Il-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo. Cell, 1985; 41:727–734. DOI: 10.1016/s0092-8674(85)80053-2.
40.
HerreroAB, San MiguelJ, GutierrezNC. Deregulation of DNA double-strand break repair in multiple myeloma: implications for genome stability. PLoS One, 2015; 10:e0121581. DOI: 10.1371/journal.pone.0121581.
41.
VouillotL, ThélieA, PolletN. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda), 2015; 5:407–15. DOI: 10.1534/g3.114.015834.
42.
PatroR, DuggalG, LoveMI, et al.Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods, 2017; 14:417–419. DOI: 10.1038/nmeth.4197.
43.
LoveMI, HuberW, AndersS. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014; 15:550. DOI: 10.1186/s13059-014-0550-8.
44.
YoungMD, WakefieldMJ, SmythGK, et al.Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol, 2010; 11:R14. DOI: 10.1186/gb-2010-11-2-r14.
45.
PrietoC, BarriosD. RaNA-Seq: Interactive RNA-Seq analysis from FASTQ files to functional analysis. Bioinformatics, 2020; 36:1955–1956. DOI: 10.1093/bioinformatics/btz854.
46.
BraunTP, EideCA, DrukerBJ. Response and resistance to BCR-ABL1-targeted therapies. Cancer Cell, 2020; 37:530–542. DOI: 10.1016/j.ccell.2020.03.006.
AnkathilR, AzlanH, DzarrAA, et al.Pharmacogenetics and the treatment of chronic myeloid leukemia: how relevant clinically? An update. Pharmacogenomics, 2018; 19:393–475. DOI: 10.2217/pgs-2017-0193.
49.
JacquelA, HerrantM, LegrosL, et al.Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl-positive K562 cell line and its differentiation toward the erythroid lineage. FASEB J, 2003; 17:2160–2162. DOI: 10.1096/fj.03-0322.
50.
NagarB, BornmannWG, PellicenaP, et al.Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res, 2002; 62:4236–4243.
51.
SchwartzbergPL, StallAM, HardinJD, et al.Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations. Cell, 1991; 65:1165–1175. DOI: 10.1016/0092-8674(91)90012-n.
52.
TybulewiczVL, CrawfordCE, JacksonPK, et al.Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell, 1991; 65:1153–1163. DOI: 10.1016/0092-8674(91)90011-m.
53.
Martinez-LageM, Torres-RuizR, Puig-SerraP, et al.In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells. Nat Commun, 2020; 11:5060. DOI: 10.1038/s41467-020-18875-x.
54.
TanY-T, YeL, XieF, et al.CRISPR/Cas9-mediated gene deletion efficiently retards the progression of Philadelphia-positive acute lymphoblastic leukemia in a p210 BCR-ABL1(T315I) mutation mouse model. Haematologica, 2020; 105:e232–e236. DOI: 10.3324/haematol.2019.229013.
55.
BauerDE, CanverMC, OrkinSH. Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9. J Vis Exp, 2015;e52118. DOI: 10.3791/52118.
56.
MandalPK, FerreiraLMR, CollinsR, et al.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell, 2014; 15:643–652. DOI: 10.1016/j.stem.2014.10.004.
57.
ShultzLD, LyonsBL, BurzenskiLM, et al.Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol, 2005; 174:6477–6489. DOI: 10.4049/jimmunol.174.10.6477.
58.
PearWS, MillerJP, XuL, et al.Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood, 1998; 92:3780–3792. DOI: 10.1182/blood.V92.10.3780.
59.
DaleyGQ, Van EttenRA, BaltimoreD. Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome. Science, 1990; 247:824–830. DOI: 10.1126/science.2406902.
60.
KelliherMA, McLaughlinJ, WitteON, et al.Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL. Proc Natl Acad Sci U S A, 1990; 87:6649–6653. DOI: 10.1073/pnas.87.17.6649.
61.
ItoM, HiramatsuH, KobayashiK, et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood, 2002; 100:3175–3182. DOI: 10.1182/blood-2001-12-0207.
62.
McDermottSP, EppertK, LechmanER, et al.Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood, 2010; 116:193–200. DOI: 10.1182/blood-2010-02-271841.
63.
EistererW, JiangX, ChristO, et al.Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease. Leukemia, 2005; 19:435–441. DOI: 10.1038/sj.leu.2403649.
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