Research IndicatorsGraph generated 15 March 2017 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 15 March, 2017 using data from PubMed, MeSH and CancerIndex
Specific Cancers (4)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
TICdb, Universidad de Navarra
Search the database of Translocation breakpoints In Cancer for "MLLT3"
GEO Profiles, NCBI
Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: MLLT3 (cancer-related)
Wu J, Xiao L, Zhou H, et al.ZFX modulates the growth of human leukemic cells via B4GALT1.
Acta Biochim Biophys Sin (Shanghai). 2016; 48(12):1120-1127 [PubMed
] Related Publications
Zinc finger protein X-linked (ZFX) is a key regulator of both embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), which is required for both Notch intracellular domain (NotchIC)-induced acute T-cell leukemia and MLL-AF9-induced myeloid leukemia in mouse models. However, the role of ZFX and its underlying mechanism in human leukemic cells remain unclear yet, though accumulating data have demonstrated that ZFX is aberrantly expressed in various human tumors and plays an important role. Herein, we found that ZFX was aberrantly expressed in various human leukemic cell lines and primary cells from leukemia patients compared with control cells. The silence of ZFX led to the growth suppression through either the deregulated cell cycle or the induction of apoptosis in various cells including K562, Jurkat, Namalwa, and THP-1 cells. The gene expression analysis revealed that UDP-Gal:βGlcNAc β 1,4-galactosyltransferase, polypeptide 1 (B4GALT1) was significantly down-regulated upon ZFX silencing, which is implicated in the response of K562 cells to the treatment of imatinib mesylate (IM). In addition, lectin blot assay showed that the galactosylation of glycoproteins in K562 cells was suppressed upon ZFX silencing. Interestingly, overexpression of B4GALT1 restored the growth and conferred drug resistance to ZFX-silenced cells. Taken together, we have demonstrated that ZFX is aberrantly expressed in multiple human leukemic cells and it modulates the growth and drug response of leukemic cells partially via B4GALT1, which suggests that ZFX is a new regulator of leukemic cells and warrants intensive investigations on this 'stemness' regulator in these deadly diseases.
The currently available human tumor xenograft models permit modeling of human cancers in vivo, but in immunocompromised hosts. Here we report a humanized mouse (hu-mouse) model made by transplantation of human fetal thymic tissue plus hematopoietic stem cells transduced with a leukemia-associated fusion gene MLL-AF9. In addition to normal human lymphohematopoietic reconstitution as seen in non-leukemic hu-mice, these hu-mice showed spontaneous development of B-cell acute lymphoblastic leukemia (B-ALL), which was transplantable to secondary recipients with an autologous human immune system. Using this model, we show that lymphopenia markedly improves the antitumor efficacy of recipient leukocyte infusion (RLI), a GVHD-free immunotherapy that induces antitumor responses in association with rejection of donor chimerism in mixed allogeneic chimeras. Our data demonstrate the potential of this leukemic hu-mouse model in modeling leukemia immunotherapy, and suggest that RLI may offer a safe treatment option for leukemia patients with severe lymphopenia.
Sarashina T, Iwabuchi H, Miyagawa N, et al.Hematopoietic stem cell transplantation for pediatric mature B-cell acute lymphoblastic leukemia with non-L3 morphology and MLL-AF9 gene fusion: three case reports and review of the literature.
Int J Hematol. 2016; 104(1):139-43 [PubMed
] Related Publications
Mature B-cell acute lymphoblastic leukemia (B-ALL) is typically associated with French-American-British (FAB)-L3 morphology and MYC gene rearrangement. However, rare cases of mature B-ALL with non-L3 morphology and MLL-AF9 fusion have been reported, and such cases are characterized by a rapid and aggressive clinical course. We here report three such cases of pediatric mature B-ALL in female patients respectively aged 15 months, 4 years, and 4 months. Bone marrow smears at diagnosis showed FAB-L1 morphology in all patients. Immunophenotypically, they were positive for cluster of differentiation (CD)10, CD19, CD20 (or CD22), Human Leukocyte Antigen-DR, and surface immunoglobulin λ. No evidence of MYC rearrangement was detected in any of the cases by fluorescent in situ hybridization (FISH) analysis. However, MLL rearrangement was detected by FISH, and MLL-AF9 fusion was confirmed by reverse transcriptase-polymerase chain reaction. All patients achieved complete remission after conventional chemotherapy and subsequently underwent hematopoietic stem cell transplantation as high-risk ALL; patient 3 for infantile ALL with MLL rearrangement and the others for ALL with MLL rearrangement and hyperleukocytosis (white blood cell count at diagnosis >50 × 10(9)/L). At the latest follow-up for each case (12-98 months post-transplantation), complete remission was maintained. Moreover, we discuss the clinical, genetic, and immunophenotypic features of this rare disease.
Liu N, Wang C, Wang L, et al.Valproic acid enhances the antileukemic effect of cytarabine by triggering cell apoptosis.
Int J Mol Med. 2016; 37(6):1686-96 [PubMed
] Related Publications
Acute myeloid leukemia (AML) is an aggressive clonal malignancy of hematopoietic progenitor cells with a poor clinical outcome. The resistance of leukemia cells to contemporary chemotherapy is one of the most formidable obstacles to treating AML. Combining valproic acid (VPA) with other anti-leukemic agents has previously been noted as a useful and necessary strategy which can be used to specifically induce anticancer gene expression. In the present study, we demonstrated the synergistic antileukemic activities between VPA and cytarabine (Ara‑C) in a retrovirus-mediated murine model with MLL-AF9 leukemia, three leukemia cell lines (THP-1, K562 and HL-60) and seven primary human AML samples. Using RT-qPCR, we noted that the combination of VPA and Ara‑C significantly upregulated Bax expression and led to the arrest of leukemia cell proliferation, sub-G1 DNA accumulation and cell apoptosis, as demonstrated by flow cytometric analysis. Significantly, further experiments revealed that knockdown of Bax expression prevented VPA and Ara‑C‑induced cell apoptosis in THP-1 cells. The results of our present study demonstrated the synergistic antileukemic effect of combined VPA and Ara‑C treatment in AML, and thus we suggest that VPA be used an alternative treatment for AML.
BACKGROUND: The constitutive hyper-activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways has frequently been associated with acute myeloid leukemia (AML). While many inhibitors targeting these pathways have been developed, the anti-leukemic effect was not as robust as expected. As part of the molecular link between PI3K/Akt and mTOR kinase, the role of Rheb1 in AML remains unexplored. Our study aims to explore the role of Rheb1 in AML and estimate whether Rheb1 could be a potential target of AML treatment.
METHODS: The expressions of Rheb1 and other indicated genes were analyzed using real-time PCR. AML mouse model was established by retrovirus transduction. Leukemia cell properties and related signaling pathways were dissected by in vitro and in vivo studies. The transcriptional changes were analyzed via gene chip analysis. Molecular reagents including mTOR inhibitor and mTOR activator were used to evaluate the function of related signaling pathway in the mouse model.
RESULTS: We observed that Rheb1 is overexpressed in AML patients and the change of Rheb1 level in AML patients is associated with their median survival. Using a Rheb1-deficient MLL-AF9 murine AML model, we revealed that Rheb1 deletion prolonged the survival of AML mice by weakening LSC function. In addition, Rheb1 deletion arrested cell cycle progression and enhanced apoptosis of AML cells. Furthermore, while Rheb1 deletion reduced mTORC1 activity in AML cells, additional rapamycin treatment further decreased mTORC1 activity and increased the apoptosis of Rheb1 (Δ/Δ) AML cells. The mTOR activator 3BDO partially rescued mTORC1 signaling and inhibited apoptosis in Rheb1 (Δ/Δ) AML cells.
CONCLUSIONS: Our data suggest that Rheb1 promotes AML progression through mTORC1 signaling pathway and combinational drug treatments targeting Rheb1 and mTOR might have a better therapeutic effect on leukemia.
The Warburg effect is probably the most prominent metabolic feature of cancer cells, although little is known about the underlying mechanisms and consequences. Here, we set out to study these features in detail in a number of leukemia backgrounds. The transcriptomes of human CB CD34+ cells transduced with various oncogenes, including BCR-ABL, MLL-AF9, FLT3-ITD, NUP98-HOXA9, STAT5A and KRASG12V were analyzed in detail. Our data indicate that in particular BCR-ABL, KRASG12V and STAT5 could impose hypoxic signaling under normoxic conditions. This coincided with an upregulation of glucose importers SLC2A1/3, hexokinases and HIF1 and 2. NMR-based metabolic profiling was performed in CB CD34+ cells transduced with BCR-ABL versus controls, both cultured under normoxia and hypoxia. Lactate and pyruvate levels were increased in BCR-ABL-expressing cells even under normoxia, coinciding with enhanced glutaminolysis which occurred in an HIF1/2-dependent manner. Expression of the glutamine importer SLC1A5 was increased in BCR-ABL+ cells, coinciding with an increased susceptibility to the glutaminase inhibitor BPTES. Oxygen consumption rates also decreased upon BPTES treatment, indicating a glutamine dependency for oxidative phosphorylation. The current study suggests that BCR-ABL-positive cancer cells make use of enhanced glutamine metabolism to maintain TCA cell cycle activity in glycolytic cells.
Guarnerio J, Bezzi M, Jeong JC, et al.Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations.
Cell. 2016; 165(2):289-302 [PubMed
] Related Publications
Chromosomal translocations encode oncogenic fusion proteins that have been proven to be causally involved in tumorigenesis. Our understanding of whether such genomic alterations also affect non-coding RNAs is limited, and their impact on circular RNAs (circRNAs) has not been explored. Here, we show that well-established cancer-associated chromosomal translocations give rise to fusion circRNAs (f-circRNA) that are produced from transcribed exons of distinct genes affected by the translocations. F-circRNAs contribute to cellular transformation, promote cell viability and resistance upon therapy, and have tumor-promoting properties in in vivo models. Our work expands the current knowledge regarding molecular mechanisms involved in cancer onset and progression, with potential diagnostic and therapeutic implications.
In this issue of Blood, Schulze et al use a tetracycline-inducible Dnmt3b knock-in mouse model to investigate how DNMT3B-mediated DNA methylation affects leukemogenesis. Increased DNMT3B expression prolonged survival in retrovirally induced Myc-Bcl2– or MLL-AF9–driven leukemia, and acute myeloid leukemia (AML) patients with high expression of DNMT3B target genes showed inferior overall survival.
To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen-specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54high or B2high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.
Fleischmann KK, Pagel P, von Frowein J, et al.The leukemogenic fusion gene MLL-AF9 alters microRNA expression pattern and inhibits monoblastic differentiation via miR-511 repression.
J Exp Clin Cancer Res. 2016; 35:9 [PubMed
] Free Access to Full Article Related Publications
BACKGROUND: In this study we explored the role of microRNAs (miRNAs) as mediators of leukemogenic effects of the fusion gene MLL-AF9, which results from a frequent chromosomal translocation in infant and monoblastic acute myeloid leukemia (AML).
METHODS: We performed a specific and efficient knockdown of endogenous MLL-AF9 in the human monoblastic AML cell line THP1.
RESULTS: The knockdown associated miRNA expression profile revealed 21 MLL-AF9 dependently expressed miRNAs. Gene ontology analyses of target genes suggested an impact of these miRNAs on downstream gene regulation via targeting of transcriptional modulators as well as involvement in many functions important for leukemia maintenance as e.g. myeloid differentiation, cell cycle and stem cell maintenance. Furthermore, we identified one of the most intensely repressed miRNAs, miR-511, to raise CCL2 expression (a chemokine ligand important for immunosurveillance), directly target cyclin D1, inhibit cell cycle progression, increase cellular migration and promote monoblastic differentiation. With these effects, miR-511 may have a therapeutic potential as a pro-differentiation agent as well as in leukemia vaccination approaches.
CONCLUSIONS: Our study provides new insights into the understanding of miRNAs as functional mediators of the leukemogenic fusion gene MLL-AF9 and opens new opportunities to further investigate specific therapeutic options for AML via the miRNA level.
Overexpression of HOXA/MEIS1/PBX3 homeobox genes is the hallmark of mixed lineage leukemia (MLL)-rearranged acute myeloid leukemia (AML). HOXA9 and MEIS1 are considered to be the most critical targets of MLL fusions and their coexpression rapidly induces AML. MEIS1 and PBX3 are not individually able to transform cells and were therefore hypothesized to function as cofactors of HOXA9. However, in this study, we demonstrate that coexpression of PBX3 and MEIS1 (PBX3/MEIS1), without ectopic expression of a HOX gene, is sufficient for transformation of normal mouse hematopoietic stem/progenitor cells in vitro. Moreover, PBX3/MEIS1 overexpression also caused AML in vivo, with a leukemic latency similar to that caused by forced expression of MLL-AF9, the most common form of MLL fusions. Furthermore, gene expression profiling of hematopoietic cells demonstrated that PBX3/MEIS1 overexpression, but not HOXA9/MEIS1, HOXA9/PBX3, or HOXA9 overexpression, recapitulated the MLL-fusion-mediated core transcriptome, particularly upregulation of the endogenous Hoxa genes. Disruption of the binding between MEIS1 and PBX3 diminished PBX3/MEIS1-mediated cell transformation and HOX gene upregulation. Collectively, our studies strongly implicate the PBX3/MEIS1 interaction as a driver of cell transformation and leukemogenesis, and suggest that this axis may play a critical role in the regulation of the core transcriptional programs activated in MLL-rearranged and HOX-overexpressing AML. Therefore, targeting the MEIS1/PBX3 interaction may represent a promising therapeutic strategy to treat these AML subtypes.
Schulze I, Rohde C, Scheller-Wendorff M, et al.Increased DNA methylation of Dnmt3b targets impairs leukemogenesis.
Blood. 2016; 127(12):1575-86 [PubMed
] Related Publications
The de novo DNA methyltransferases Dnmt3a and Dnmt3b are of crucial importance in hematopoietic stem cells. Dnmt3b has recently been shown to play a role in genic methylation. To investigate how Dnmt3b-mediated DNA methylation affects leukemogenesis, we analyzed leukemia development under conditions of high and physiological methylation levels in a tetracycline-inducible knock-in mouse model. High expression of Dnmt3b slowed leukemia development in serial transplantations and impaired leukemia stem cell (LSC) function. Forced Dnmt3b expression induced widespread DNA hypermethylation inMyc-Bcl2-induced leukemias, preferentially at gene bodies.MLL-AF9-induced leukemogenesis showed much less pronounced DNA hypermethylation upon Dnmt3b expression. Nonetheless, leukemogenesis was delayed in both models with a shared core set of DNA hypermethylated regions and suppression of stem cell-related genes. Acute myeloid leukemia patients with high expression of Dnmt3b target genes showed inferior survival. Together, these findings indicate a critical role for Dnmt3b-mediated DNA methylation in leukemia development and maintenance of LSC function.
Leukemogenesis occurs under hypoxic conditions within the bone marrow (BM). Knockdown of key mediators of cellular responses to hypoxia with shRNA, namely hypoxia-inducible factor-1α (HIF-1α) or HIF-2α, in human acute myeloid leukemia (AML) samples results in their apoptosis and inability to engraft, implicating HIF-1α or HIF-2α as therapeutic targets. However, genetic deletion of Hif-1α has no effect on mouse AML maintenance and may accelerate disease development. Here, we report the impact of conditional genetic deletion of Hif-2α or both Hif-1α and Hif-2α at different stages of leukemogenesis in mice. Deletion of Hif-2α accelerates development of leukemic stem cells (LSCs) and shortens AML latency initiated by Mll-AF9 and its downstream effectors Meis1 and Hoxa9. Notably, the accelerated initiation of AML caused by Hif-2α deletion is further potentiated by Hif-1α codeletion. However, established LSCs lacking Hif-2α or both Hif-1α and Hif-2α propagate AML with the same latency as wild-type LSCs. Furthermore, pharmacological inhibition of the HIF pathway or HIF-2α knockout using the lentiviral CRISPR-Cas9 system in human established leukemic cells with MLL-AF9 translocation have no impact on their functions. We therefore conclude that although Hif-1α and Hif-2α synergize to suppress the development of AML, they are not required for LSC maintenance.
Baker A, Gregory GP, Verbrugge I, et al.The CDK9 Inhibitor Dinaciclib Exerts Potent Apoptotic and Antitumor Effects in Preclinical Models of MLL-Rearranged Acute Myeloid Leukemia.
Cancer Res. 2016; 76(5):1158-69 [PubMed
] Related Publications
Translocations of the mixed lineage leukemia (MLL) gene occur in 60% to 80% of all infant acute leukemias and are markers of poor prognosis. MLL-AF9 and other MLL fusion proteins aberrantly recruit epigenetic regulatory proteins, including histone deacetylases (HDAC), histone methyltransferases, bromodomain-containing proteins, and transcription elongation factors to mediate chromatin remodeling and regulate tumorigenic gene expression programs. We conducted a small-molecule inhibitor screen to test the ability of candidate pharmacologic agents targeting epigenetic and transcriptional regulatory proteins to induce apoptosis in leukemic cells derived from genetically engineered mouse models of MLL-AF9-driven acute myeloid leukemia (AML). We found that the CDK inhibitor dinaciclib and HDAC inhibitor panobinostat were the most potent inducers of apoptosis in short-term in vitro assays. Treatment of MLL-rearranged leukemic cells with dinaciclib resulted in rapidly decreased expression of the prosurvival protein Mcl-1, and accordingly, overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. Administration of dinaciclib to mice bearing MLL-AF9-driven human and mouse leukemias elicited potent antitumor responses and significantly prolonged survival. Collectively, these studies highlight a new therapeutic approach to potentially overcome the resistance of MLL-rearranged AML to conventional chemotherapies and prompt further clinical evaluation of CDK inhibitors in AML patients harboring MLL fusion proteins.
Wang Y, Chen C, Dong F, et al.NK cells play a significant role in immunosurveillance at the early stage of MLL-AF9 acute myeloid leukemia via CD226/CD155 interactions.
Sci China Life Sci. 2015; 58(12):1288-98 [PubMed
] Related Publications
Acute myeloid leukemia (AML) is an aggressive hematological malignancy, and the mechanism underlying immune system involvement in leukemia development is unclear. In the present study, we utilized a myeloid/lymphoid or mixed-lineage leukemia; translocated to, 3 (MLLT3/MLL-AF9)-induced AML mouse model with or without exposure to irradiation. We found that the leukemia cells could survive and expand in hosts with intact immune systems, whereas leukemia progression was accelerated in mice with impaired immune systems. Moreover, the leukemia cells escaped from host immunosurveillance via editing their immunogenicity, including the up-regulation of an inhibitory antigen (i.e., CD47) and the down-regulation of active antigens (i.e., CD86, CD54, retinoic acid early transcript (RAE), histocompatibility 2, D region locus b (H2-Db) and H2-Dd). Natural killer (NK) cells were activated in the early phase of AML progression, whereas T cells were stimulated in the late phase. Furthermore, NK cell depletion showed that NK cells were necessary for the elimination of leukemia cells in our AML mouse model. Notably, CD155/CD226 primarily mediated the interaction between NK cells and leukemia cells and contributed to the antitumor effects of NK cells during the early phase of AML. Clinical data from patients with diverse hematological malignancies showed that CD155 expression was decreased in hematological malignancies. Taken together, our results demonstrate that NK cells play a pivotal role in immunosurveillance against leukemia cells during the early stage of AML primarily through the CD226/CD155 interaction; however, NK cells are not sufficient to eliminate leukemia cells.
Nowek K, Sun SM, Dijkstra MK, et al.Expression of a passenger miR-9* predicts favorable outcome in adults with acute myeloid leukemia less than 60 years of age.
Leukemia. 2016; 30(2):303-9 [PubMed
] Related Publications
In double-stranded miRNA/miRNA* duplexes, one of the strands represents an active miRNA, whereas another, known as a passenger strand (miRNA*), is typically degraded. MiR-9* is not detectable in normal myeloid cells. Here we show that miR-9* is expressed in 59% of acute myeloid leukemia (AML) cases and we investigate its clinical impact in 567 adults with de novo AML (age⩽60 years). AML cases with detectable miR-9* included a lower percentage of cases with favorable risk (P<0.001) as compared with those with no detectable miR-9*. High levels of miR-9* expression independently predicted for higher complete remission (odds ratio=1.28, P=0.013) and better event-free survival (EFS) (hazard ratio (HR)=0.86, P=0.001), relapse-free survival (RFS) (HR=0.84, P=0.008) and overall survival (OS) (HR=0.86, P=0.002). Among the subgroup of adverse risk patients, high miR-9* expressers had strikingly longer median survival than low miR-9* expressers (EFS: 16 vs 5 months, P=0.020; RFS: 12 vs 4, P=0.060; OS: 23 vs 8, P=0.021). Comparative transcriptome analysis suggests that miR-9* regulates genes involved in leukemogenesis, for example, MN1 and MLLT3. This is the first report showing that an miRNA* has prognostic value in AML.
Fong CY, Gilan O, Lam EY, et al.BET inhibitor resistance emerges from leukaemia stem cells.
Nature. 2015; 525(7570):538-42 [PubMed
] Related Publications
Bromodomain and extra terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic opportunity by directly targeting bromodomain proteins that bind acetylated chromatin marks. Early clinical trials have shown promise, especially in acute myeloid leukaemia, and therefore the evaluation of resistance mechanisms is crucial to optimize the clinical efficacy of these drugs. Here we use primary mouse haematopoietic stem and progenitor cells immortalized with the fusion protein MLL-AF9 to generate several single-cell clones that demonstrate resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism, but is shown to emerge from leukaemia stem cells both ex vivo and in vivo. Chromatin-bound BRD4 is globally reduced in resistant cells, whereas the expression of key target genes such as Myc remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors, in human and mouse leukaemia cells, is in part a consequence of increased Wnt/β-catenin signalling, and negative regulation of this pathway results in restoration of sensitivity to I-BET in vitro and in vivo. Together, these findings provide new insights into the biology of acute myeloid leukaemia, highlight potential therapeutic limitations of BET inhibitors, and identify strategies that may enhance the clinical utility of these unique targeted therapies.
One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.
Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.
Comprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non-Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): -7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk-7p abnormalities; poor risk-normal karyotypes, -7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, -13/13q-, and -15; and intermediate risk-others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.
Cytopenias resulting from the impaired generation of normal blood cells from hematopoietic precursors are important contributors to morbidity and mortality in patients with leukemia. However, the process by which normal hematopoietic cells are overtaken by emerging leukemia cells and how different subsets of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are distinctly influenced during leukemic cell infiltration is poorly understood. To investigate these important questions, we used a robust nonirradiated mouse model of human MLL-AF9 leukemia to examine the suppression of HSCs and HPCs during leukemia cell expansion in vivo. Among all the hematopoietic subsets, long-term repopulating HSCs were the least reduced, whereas megakaryocytic-erythroid progenitors were the most significantly suppressed. Notably, nearly all of the HSCs were forced into a noncycling state in leukemic marrow at late stages, but their reconstitution potential appeared to be intact upon transplantation into nonleukemic hosts. Gene expression profiling and further functional validation revealed that Egr3 was a strong limiting factor for the proliferative potential of HSCs. Therefore, this study provides not only a molecular basis for the more tightened quiescence of HSCs in leukemia, but also a novel approach for defining functional regulators of HSCs in disease.
Ney Garcia DR, Liehr T, Emerenciano M, et al.Molecular studies reveal a MLL-MLLT3 gene fusion displaced in a case of childhood acute lymphoblastic leukemia with complex karyotype.
Cancer Genet. 2015; 208(4):143-7 [PubMed
] Related Publications
Rearrangement of the mixed lineage-leukemia gene (MLL-r) is common in hematological diseases and is generally associated with poor prognosis. The mixed-lineage leukemia gene translocated to, 3 (MLLT3) gene (9p22) is a frequent MLL-r partner (∼18% of leukemias with MLL rearrangement) and is characterized by the translocation t(9;11) (p22;q23), forming an MLL-MLLT3 gene fusion. MLL-r are usually simple reciprocal translocations between two different chromosomes, although karyotypes with complex MLL-r have been observed. We present a rare case of a child with acute lymphoblastic leukemia with a complex karyotype in which the classical t(9;11) (p22;q23) was cryptically relocated into a third chromosome in a balanced three-way translocation. At the genome level, however, the MLL-MLLT3 three-way translocation still displayed both reciprocal fusion transcripts. This argues in favor for a model where a simple two-way t(9;11) (p22;q23) was likely the first step that then evolved in to a more complex karyotype. Multicolor banding techniques can be used to greatly refine complex karyotypes and its chromosomal breakpoints. Also in the presence of putative new rearrangements, Long distance inverse-PCR is an important tool to identify which gene fusion is involved.
Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a low-oxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment.
Chromosomal rearrangements of the MLL gene are associated with high-risk infant, pediatric, adult, and therapy-induced acute leukemias. So far, about 80 different direct MLL fusions and about 120 reciprocal MLL fusions have been characterized at the molecular level. The common theme in these leukemia-associated genetic rearrangements is the genetic disruption of the MLL gene. This leads to MLL-X fusion proteins that still bind to nuclear factors (e.g., MEN1, LEDGF), which in turn allow them to target promoters and cause ectopic gene transcription. In addition, the most frequent MLL fusions (MLL-AF4, MLL-AF9, MLL-AF10, and MLL-ENL) are all recruiting the wild-type AF4 multiprotein complex that contains the target proteins P-TEFb, BRD4, and DOT1L. Vice versa, reciprocal X-MLL fusions exhibit a PHD domain (H3K4me3 reader domain), sequester the histone acetyltransferases CREBBP and MOF1 and bear a histone methyltransferase domain at their very C-terminus (SET domain). Except for AF4-MLL, the functional consequences deriving from reciprocal fusion proteins are not very well understood. However, based on our knowledge about the above-mentioned MLL fusions, it is reasonable to inhibit their oncogenic activity in a targeted fashion. Recent efforts in developing such inhibitors and their mode of action will be critically discussed.
PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 data set. We examined the role of mutated Shp2 in leukemias harboring MLL translocations, which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9-mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. Although the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K, suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small-molecule-mediated Mcl1 inhibition, suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2.
UNLABELLED: Protein arginine methyltransferase 5 (PRMT5) has been implicated as a key modulator of lymphomagenesis. Whether PRMT5 has overt oncogenic function in the context of leukemia/lymphoma and whether it represents a therapeutic target remains to be established. We demonstrate that inactivation of PRMT5 inhibits colony-forming activity by multiple oncogenic drivers, including cyclin D1, c-MYC, NOTCH1, and MLL-AF9. Furthermore, we demonstrate that PRMT5 overexpression specifically cooperates with cyclin D1 to drive lymphomagenesis in a mouse model, revealing inherent neoplastic activity. Molecular analysis of lymphomas revealed that arginine methylation of p53 selectively suppresses expression of crucial proapoptotic and antiproliferative target genes, thereby sustaining tumor cell self-renewal and proliferation and bypassing the need for the acquisition of inactivating p53 mutations. Critically, analysis of human tumor specimens reveals a strong correlation between cyclin D1 overexpression and p53 methylation, supporting the biomedical relevance of this pathway.
SIGNIFICANCE: We have identified and functionally validated a crucial role for PRMT5 for the inhibition of p53-dependent tumor suppression in response to oncogenic insults. The requisite role for PRMT5 in the context of multiple lymphoma/leukemia oncogenic drivers suggests a molecular rationale for therapeutic development.
Translocation of the mixed-lineage leukemia (MLL) gene with AF4, AF9, or ENL results in acute leukemia with both lymphoid and myeloid involvement. We characterized leukemia-initiating cells (LICs) in primary infant MLL-rearranged leukemia using a xenotransplantation model. In MLL-AF4 patients, CD34(+)CD38(+)CD19(+) and CD34(-)CD19(+) cells initiated leukemia, and in MLL-AF9 patients, CD34(-)CD19(+) cells were LICs. In MLL-ENL patients, either CD34(+) or CD34(-) cells were LICs, depending on the pattern of CD34 expression. In contrast, in patients with these MLL translocations, CD34(+)CD38(-)CD19(-)CD33(-) cells were enriched for normal hematopoietic stem cells (HSCs) with in vivo long-term multilineage hematopoietic repopulation capacity. Although LICs developed leukemic cells with clonal immunoglobulin heavy-chain (IGH) rearrangement in vivo, CD34(+)CD38(-)CD19(-)CD33(-) cells repopulated recipient bone marrow and spleen with B cells, showing broad polyclonal IGH rearrangement and recipient thymus with CD4(+) single positive (SP), CD8(+) SP, and CD4(+)CD8(+) double-positive (DP) T cells. Global gene expression profiling revealed that CD9, CD32, and CD24 were over-represented in MLL-AF4, MLL-AF9, and MLL-ENL LICs compared with normal HSCs. In patient samples, these molecules were expressed in CD34(+)CD38(+) and CD34(-) LICs but not in CD34(+)CD38(-)CD19(-)CD33(-) HSCs. Identification of LICs and LIC-specific molecules in primary human MLL-rearranged acute lymphoblastic leukemia may lead to improved therapeutic strategies for MLL-rearranged leukemia.
Zhang Y, Peng L, Hu T, et al.La-related protein 4B maintains murine MLL-AF9 leukemia stem cell self-renewal by regulating cell cycle progression.
Exp Hematol. 2015; 43(4):309-18.e2 [PubMed
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Our recent study identified a nonsense mutation of La-related protein 4B (LARP4B) from whole genome sequencing of a 3-year-old female monozygotic twin pair discordant for MLL-associated acute myeloid leukemia (AML). To study the role of LARP4B in AML, we established a LARP4B-knockdown MLL-AF9 AML mouse model. Using this mouse model, we found that LARP4B knockdown significantly decreased leukemia cells in the peripheral blood, spleen, and bone marrow and prolonged the survival of AML recipient mice. Additional studies showed that LARP4B knockdown reduced leukemia stem cells (LSCs) and impaired the self-renew capacity of LSCs. Cell cycle analysis revealed that LARP4B knockdown arrested more LSCs in the G0 phase. The transcription of the cell cycle inhibitors p16, p19, and p21 and of the lineage-specific transcription factor CCAAT-enhancer-binding protein α was increased in the LARP4B-knockdown LSCs. Thus, our results demonstrate that LARP4B plays an important role in the maintenance of LSCs and suggest that LARP4B may regulate the cell cycle of LSCs via suppressing the expression of the cell cycle inhibitors p16, p19, and p21 and the myeloid specific transcription factor CCAAT-enhancer-binding protein α.
BACKGROUND: A fundamental challenge for cancer therapy is that each tumor contains a highly heterogeneous cell population whose structure and mechanistic underpinnings remain incompletely understood. Recent advances in single-cell gene expression profiling have created new possibilities to characterize this heterogeneity and to dissect the potential intra-cancer cellular hierarchy.
RESULTS: Here, we apply single-cell analysis to systematically characterize the heterogeneity within leukemic cells using the MLL-AF9 driven mouse model of acute myeloid leukemia. We start with fluorescence-activated cell sorting analysis with seven surface markers, and extend by using a multiplexing quantitative polymerase chain reaction approach to assay the transcriptional profile of a panel of 175 carefully selected genes in leukemic cells at the single-cell level. By employing a set of computational tools we find striking heterogeneity within leukemic cells. Mapping to the normal hematopoietic cellular hierarchy identifies two distinct subtypes of leukemic cells; one similar to granulocyte/monocyte progenitors and the other to macrophage and dendritic cells. Further functional experiments suggest that these subtypes differ in proliferation rates and clonal phenotypes. Finally, co-expression network analysis reveals similarities as well as organizational differences between leukemia and normal granulocyte/monocyte progenitor networks.
CONCLUSIONS: Overall, our single-cell analysis pinpoints previously uncharacterized heterogeneity within leukemic cells and provides new insights into the molecular signatures of acute myeloid leukemia.
Sánchez-Aguilera A, Arranz L, Martín-Pérez D, et al.Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.
Cell Stem Cell. 2014; 15(6):791-804 [PubMed
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Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs.