The Role of MLL1 and MLL2 in MLL Fusion Oncoprotein-Initiated Leukemia

Abstract The MLL1 histone methyltransferase gene at 11q23 undergoes many distinct chromosomal translocations to yield poor prognosis leukemia. In these MLL-rearranged (MLL-r) leukemias, the remaining wild-type allele is typically expressed, and the fusion oncoprotein lacks the C terminal histone methyltransferase domain. The role of the wild-type, endogenous MLL1 allele in MLL-fusion protein mediated leukemogenesis is not fully understood, with some evidence suggesting an important functional role. On the other hand, the wild-type allele is lost in some patients and in the MLL-r ML2 cell line, suggesting that the wild-type allele is not absolutely required. Here we rigorously address the role of endogenous MLL1 in MLL-r leukemia. Using precise and independent animal models, we show that MLL1 is dispensable for MLL-AF9- or MLL-AF6-mediated acute myelogenous leukemia (AML). In parallel experiments, deletion of Menin, encoding a chromatin targeting cofactor for MLL-fusion proteins, has severe effects on the proliferation and survival of MLL-r leukemia, as previously published. Potential redundancy between MLL family methyltransferases was addressed by co-deleting the closest paralog, Mll2. Surprisingly, deletion of Mll2 by itself had a significant impact on survival of MLL-AF9 leukemia cells, and co-deletion of Mll1 and Mll2 more effectively killed these cells, suggesting a redundant or synthetic lethal relationship between the two genes. A comparison of gene expression and histone modification changes in Mll1, Mll2 and Mll1;Mll2 double knockout leukemia cells showed that 1) global defects in histone H3, lysine 4 methyation can be exclusively attributed to MLL2, 2) MLL1- versus MLL2-dependent genes are largely distinct, and 3) Mll1;Mll2 double knockout leukemia cells exhibit deficiencies in NFκB and integrin ß3 pathways, but exhibit little evidence of deregulation of either MLL1- or MLL-fusion protein target genes. These findings reshape our thinking about the role of endogenous MLL family proteins in MLL-fusion protein leukemogenesis. The data presented here, in conjunction with the minimal effect of Mll2 deletion on hematopoietic stem and progenitor cells make MLL2 an attractive candidate for targeted therapy in MLL-r leukemia. Disclosures Ernst: Amgen: Other: stocks. Yokoyama:Sumitomo Dainippon Pharma: Research Funding. Neff:Bristol Myers Squibb: Other: Travel; Janssen: Other: Travel; Epizyme: Patents & Royalties: patent filed.

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Short and sweet: foreleg abnormalities in Havanese and the role of the FGF4 retrogene

Canine Medicine and Genetics ◽

10.1186/s40575-020-00097-5 ◽

2020 ◽

Vol 7 (1) ◽

Author(s):

Kim K. L. Bellamy ◽

Frode Lingaas

Keyword(s):

Mutant Allele ◽

Large Degree ◽

Population Increase ◽

Wild Type Allele ◽

Wild Type ◽

Gradual Decline ◽

Type Allele ◽

Population Frequency ◽

Welfare Implications

Abstract Background Cases of foreleg deformities, characterized by varying degrees of shortened and bowed forelegs, have been reported in the Havanese breed. Because the health and welfare implications are severe in some of the affected dogs, further efforts should be made to investigate the genetic background of the trait. A FGF4-retrogene on CFA18 is known to cause chondrodystrophy in dogs. In most breeds, either the wild type allele or the mutant allele is fixed. However, the large degree of genetic diversity reported in Havanese, could entail that both the wild type and the mutant allele segregate in this breed. We hypothesize that the shortened and bowed forelegs seen in some Havanese could be a consequence of FGF4RG-associated chondrodystrophy. Here we study the population prevalence of the wild type and mutant allele, as well as effect on phenotype. We also investigate how the prevalence of the allele associated with chondrodystrophy have changed over time. We hypothesize that recent selection, may have led to a gradual decline in the population frequency of the lower-risk, wild type allele. Results We studied the FGF4-retrogene on CFA18 in 355 Havanese and found variation in the presence/absence of the retrogene. The prevalence of the non-chondrodystrophic wild type is low, with allele frequencies of 0.025 and 0.975 for the wild type and mutant allele, respectively (linked marker). We found that carriers of the beneficial wild type allele were significantly taller at the shoulder than mutant allele homozygotes, with average heights of 31.3 cm and 26.4 cm, respectively. We further found that wild type carriers were born on average 4.7 years earlier than mutant allele homozygotes and that there has been a gradual decline in the population frequency of the wild type allele during the past two decades. Conclusions Our results indicate that FGF4RG-associated chondrodystrophy may contribute to the shortened forelegs found in some Havanese and that both the wild type and mutant allele segregate in the breed. The population frequency of the wild type allele is low and appear to be decreasing. Efforts should be made to preserve the healthier wild type in the population, increase the prevalence of a more moderate phenotype and possibly reduce the risk of foreleg pathology.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽

10.1182/blood.v124.21.4344.4344 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4344-4344

Author(s):

Amanda Scholl ◽

Kentson Lam ◽

Alex Muselman ◽

Tingdong Tang ◽

Shinobu Matsuura ◽

...

Keyword(s):

Family Member ◽

Conflicts Of Interest ◽

Protein Family ◽

Dominant Negative ◽

Negative Regulator ◽

Wild Type ◽

Double Knockout ◽

Hematopoietic Stem ◽

Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

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Identification and Complementation of a Mutation Associated with Loss of Mycoplasma pneumoniae Virulence-Specific Proteins B and C

Journal of Bacteriology ◽

10.1128/jb.187.2.747-751.2005 ◽

2005 ◽

Vol 187 (2) ◽

pp. 747-751 ◽

Cited By ~ 16

Author(s):

Robert H. Waldo ◽

Jarrat L. Jordan ◽

Duncan C. Krause

Keyword(s):

Mycoplasma Pneumoniae ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Specific Proteins

ABSTRACT A mutation in gene MPN142 (orf6) was identified in the Mycoplasma pneumoniae cytadherence mutant III-4. MPN142 encodes virulence-specific proteins P90 and P40 (proteins B and C, respectively). Analysis of MPN142 in a cytadhering revertant and complementation using a recombinant wild-type allele confirmed the role of this mutation in the cytadherence defect.

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STAT5 signaling is required for the efficient induction and maintenance of CML in mice

Blood ◽

10.1182/blood-2005-10-4110 ◽

2006 ◽

Vol 107 (12) ◽

pp. 4917-4925 ◽

Cited By ~ 78

Author(s):

Dan Ye ◽

Nicholas Wolff ◽

Li Li ◽

Shumin Zhang ◽

Robert L. Ilaria

Keyword(s):

Cell Biology ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Dominant Negative ◽

Myelogenous Leukemia ◽

Wild Type ◽

Double Knockout ◽

Signal Transducers ◽

Efficient Induction

AbstractThe role of signal transducers and activators of transcription 5 (STAT5) in chronic myelogenous leukemia (CML) is controversial. To clarify the role of STAT5 signaling in P210BCR/ABL leukemogenesis, P210 was introduced into primary murine STAT5A-deficient (STAT5A–/–) bone marrow (BM) cells, which, unlike STAT5A/5B double knockout BM cells, have no major intrinsic hematopoietic defects. Interestingly, only 21% of mice reconstituted with P210-transduced STAT5A–/– BM cells developed classic CML, compared with 80% to 100% of P210/STAT5A+/+ and P210/STAT5A+/–-reconstituted animals. The remainder of P210/STAT5A–/– animals died from an acute B-cell lymphoblastic leukemia (ALL)–like disease (32%) or a CML/ALL mix (47%), reflecting impairment in the induction and maintenance of CML, which normally predominates in this mouse model. Of mice that ultimately developed CML, P210/STAT5A–/– animals had prolonged survival and increased myeloid immaturity. Importantly, reconstitution of wild-type mice with BM cells coexpressing P210 and dominant-negative STAT5 also profoundly reduced the incidence of CML, without impairing the induction of ALL. Altogether, these findings indicate that STAT5 and STAT5A play an important role in the pathogenesis of the CML-like disease in mice. A greater understanding of the STAT5 target genes involved in CML induction may lead to new therapeutic targets that influence CML progenitor cell biology.

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Hepatic Leukemia Factor Is Critical for Hematopoietic Stem Cells and Promotes MLL-AF9 Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-122661 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2466-2466

Author(s):

Aurelie Baudet ◽

Karolina Paulina Komorowska ◽

Hultmark Simon ◽

Marcus Järås ◽

Marion Chapellier ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Fusion Protein ◽

Fetal Liver ◽

Leukemia Cells ◽

Hematopoietic Stem ◽

Aged Mice ◽

Adult Mice ◽

Leukemia Development

The transcription factor Hepatic leukemia factor (HLF), initially identified as part of a leukemic fusion protein (E2A-HLF) causing B-lymphoid leukemia in children, is expressed in a wide range of tissues. In the mouse hematopoietic system, HLF is highly expressed in hematopoietic stem cells while being hardly detectable in more restricted progenitor populations. We have previously shown that in adult mice, HLF is essential for preserving the hematopoietic stem cell (HSC) pool during regeneration by maintaining their quiescence (Komorowska et al, Cell reports, 2017). Notably, despite this highly specific expression profile and a critical role in hematopoietic regeneration, mice with double knock-out of HLF alleles (KO) appeared to age normally (18-24 months survival > 90% for both WT and KO). In this study, we investigated the role of HLF in HSC during aging and leukemogenesis. We first confirmed that HLF is expressed all through ontogeny by performing quantitative PCR on HSC and progenitor populations from mouse fetal liver (E14.5 FL) and bone marrow (BM) of aged mice (18-month-old mice). We found that not only is HLF expressed in hematopoietic cells from embryonic to aged mice, but also that the expression profile was highly conserved. In fact, we found that HLF expression decreased during hematopoietic differentiation independently of ontogenic age (HLF/HPRT relative expression: 6 in HSC (LSK-CD150+CD48-), 3 in HSPC (LSK), and <0,2 in CMP (Lin-kit+sca1-CD34+CD16/32-), GMP (Lin-kit+sca1-CD34+CD16/32+), and MEP (Lin-kit+sca1-CD34-CD16/32-). Additionally, hematopoietic parameters as well as lineage distribution in peripheral blood and bone marrow of aged KO mice were within normal range (at the exception of a 50% reduction in platelet blood count, also reported in adult mice). While we found no significant difference in the frequency of immunophenotypic HSC in total BM (LSK-CD34-Flt3-; WT: 0,0006 %, KO: 0,0004 %), aged KO grafts (200 000 total BM cells in 1:1 ratio with competitor) demonstrated a defective capacity to reconstitute primary recipients (donor contribution, WT: 60%, KO: 45 %). In secondary recipients, the defect was further aggravated (donor contribution; WT: 60 %, KO: 20%). Because neither SLAM nor CD34 markers can be used in KO FL, we quantified the percentage of EPCR+ cells within the LSK compartment as a surrogate marker for HSC and found a 2-fold decrease compared to WT controls. In competitive transplantations, KO E14.5 fetal liver cells demonstrated an impaired regenerative capacity in primary (donor contribution; WT: 88.8% (SEM 6.2), KO 63% SEM (16.2); n>5) and secondary recipients (donor contribution: WT: 67% (SEM 2.7), KO: 8.4% (SEM 3.6), n=4). In addition to its function in healthy hematopoiesis, studies have suggested that HLF is a downstream mediator of the MLL/AF9 (MA9) leukemic fusion protein. To address the contribution of HLF to MA9 leukemogenesis, mice were transplanted with either KO- or WT-BM-derived c-Kit+ cells expressing the MA9 fusion protein. Preliminary results showed that HLF did not affect the latency of the leukemia in primary recipients with mice succumbing to a lethal disease at days 40-41 after transplant independently of the leukemic graft genetic background. However, KO leukemic cells had a significantly delayed leukemia development in secondary recipients (time to death; WT: 32-39 days, KO: 39-68 days). Moreover, semi-quantitative PCR showed that not only was HLF expressed in leukemia cells harvested from primary recipients but also that the expression level displayed a tendency to an increase in leukemia cells from secondary recipients. Altogether, our data suggest that while HLF may be dispensable for leukemic transformation, it promotes the development of MLL-AF9 leukemia in mice. We are currently in the process of confirming the role of HLF in MA9 leukemia development. Taken together, we here show that HLF is essential for HSCs from embryonic to aged mice. We further show that HLF is critical for serial propagation of AML cells, suggesting that HLF is needed to maintain leukemia stem cells. Disclosures No relevant conflicts of interest to declare.

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THE ROLE OF DOSAGE OF THE REGION 7D1–7D5-6 OF THE X CHROMOSOME IN THE PRODUCTION OF HOMEOTIC TRANSFORMATIONS IN DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/96.4.887 ◽

1980 ◽

Vol 96 (4) ◽

pp. 887-902 ◽

Cited By ~ 3

Author(s):

M Gans ◽

F Forquignon ◽

M Masson

Keyword(s):

X Chromosome ◽

Small Region ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Maternal Genotype ◽

External Conditions ◽

Ether Treatment ◽

Blastoderm Stage

ABSTRACT A high frequency of homeotic transformations appears in Df(3)red/+ progeny of Df(1)snC128 /+ females. Generally, the metathoracic appendages are partially transformed into mesothoracic ones. Df(1)snC128 includes a small region of the X chromosome: 7D1 to 7D5-6. Hypodosage of this region is mainly effective at the level of the maternal genotype, and the effect is probably due to hypodosage of the wild-type allele of the gene fs(1)h. Df(3)red has an effect that is mainly, if not exclusively, zygotic, probably due to hypodosage of the wild-type allele of Rg-bx. The frequencies of transformed flies resulting from the interaction between Df(1)snC128 and Df(3)red are not very sensitive to external conditions and genetic background. Studies of the interactions between Df(1)snC128 and other mutations or deficiencies of chromosome 3 [Rg-pbx, bx, pbx, Ubx1, Ubx130, Ubx80, Df(3)P9] reveal an analogy between the hypodosage effect of region 7D1–7D5-6 and the effects of ether treatment of blastoderm stage eggs. The role of the gene fs(1)h in the process of segment determination is discussed in the light of these results.

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CYP2C19 Polymorphisms in Indonesia: Comparison among Ethnicities and the Association with Clinical Outcomes

Biology ◽

10.3390/biology10040300 ◽

2021 ◽

Vol 10 (4) ◽

pp. 300

Author(s):

Muhammad Miftahussurur ◽

Dalla Doohan ◽

Ari Fahrial Syam ◽

Iswan Abbas Nusi ◽

Phawinee Subsomwong ◽

...

Keyword(s):

Proton Pump Inhibitor ◽

Clinical Outcomes ◽

Proton Pump ◽

Poor Metabolizers ◽

Wild Type Allele ◽

Wild Type ◽

Type Allele ◽

Sydney System ◽

Intermediate Metabolizers ◽

Cyp2c19 Polymorphisms

CYP2C19 polymorphisms are important factors for proton pump inhibitor-based therapy. We examined the CYP2C19 genotypes and analyzed the distribution among ethnicities and clinical outcomes in Indonesia. We employed the polymerase chain reaction-restriction fragment length polymorphism method to determine the CYP2C19 genotypes and evaluated inflammation severity with the updated Sydney system. For CYP2C19*2, 46.4% were the homozygous wild-type allele, 14.5% were the homozygous mutated allele, and 39.2% were the heterozygous allele. For CYP2C19*3, 88.6% were the homozygous wild-type allele, 2.4% were the homozygous mutated allele, and 9.0% were the heterozygous allele. Overall, the prevalence of rapid, intermediate, and poor metabolizers in Indonesia was 38.5, 41.6, and 19.9%, respectively. In the poor metabolizer group, the frequency of allele *2 (78.8%) was higher than the frequency of allele *3 (21.2%). The Papuan had a significantly higher likelihood of possessing poor metabolizers than the Balinese (OR 11.0; P = 0.002). The prevalence of poor metabolizers was lower compared with the rapid and intermediate metabolizers among patients with gastritis and gastroesophageal reflux disease. Intermediate metabolizers had the highest prevalence, followed by rapid metabolizers and poor metabolizers. Dosage adjustment should therefore be considered when administering proton pump inhibitor-based therapy in Indonesia.

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Monoallelic Expression of Pax5: A Paradigm for the Haploinsufficiency of Mammalian Pax Genes?

Biological Chemistry ◽

10.1515/bc.1999.077 ◽

1999 ◽

Vol 380 (6) ◽

Cited By ~ 31

Author(s):

S.L. Nutt ◽

M. Busslinger

Keyword(s):

Human Disease ◽

Mouse Genome ◽

Monoallelic Expression ◽

Wild Type Allele ◽

Loss Of Function ◽

Wild Type ◽

Type Allele ◽

Pax Genes ◽

Default State ◽

Mammalian Genes

AbstractIt is generally assumed that most mammalian genes are transcribed from both alleles. Hence, the diploid state of the genome offers the advantage that a loss-of-function mutation in one allele can be compensated for by the remaining wild-type allele of the same gene. Indeed, the vast majority of human disease syndromes and engineered mutations in the mouse genome are recessive, indicating that recessiveness is the ‘default’ state. However, a minority of genes are semi-dominant, as heterozygous loss-of-function mutation in these genes leads to phenotypic abnormalities. This condition, known as haploinsufficiency, has been described for five of the nine mammalian

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Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Blood ◽

10.1182/blood-2008-06-164889 ◽

2009 ◽

Vol 113 (1) ◽

pp. 66-74 ◽

Cited By ~ 52

Author(s):

Toshiki Ochi ◽

Hiroshi Fujiwara ◽

Koichiro Suemori ◽

Taichi Azuma ◽

Yoshihiro Yakushijin ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Cellular Immunotherapy ◽

Aurora A Kinase ◽

Aurora A ◽

Hematopoietic Stem ◽

Specific Ctls ◽

Leukemia Cell Lines

Abstract Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

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