AML1-ETO Research Articles

Rapid detection of AML1 associated fusion genes in patients with adult acute myeloid leukemia and its clinical significance.

This study was aimed to detect the expression of AML1 fusion genes in the patients with adult acute myeloid leukemia (AML) and further to investigate their association with the progression and prognosis of AML. Bone marrow samples were collected from 168 patients with de novo adult AML, and the expression of AML1 ETO, AML1-EVI1, AML1-MDS1, AML1-MTG16, AML1-PRDM16, AML1-LRP16, AML1-CLCA2 and AML1-PRDX4 was analyzed by a novel multiplex nested RT-PCR. Positive samples and minimal residual disease were further examined by real-time fluorescent quantitative PCR. The results showed that the AML1 fusion genes were found in 10.7% (18/168) patients. Among them, AML1-ETO in 12 (7.1%) cases were detected, AML1-EVI1 in 2 cases (1.2%), and AML1-MDS1, AML1-MTG16, AML1-PRDM16, and AML1-CLCA2 in 1 case (0.6%) each were detected. Among the patients with AML1-ETO, 10 patients (10/12, 83.33%) achieved complete remission (CR) after one cycle of chemotherapy, while 2 patients achieved CR after 2 cycles of chemotherapy. The 2 patients with AML1-EVI1 failed to achieve CR after one cycle of chemotherapy. Patients with AML1-MDS1, AML1-MTG16, AML1-PRDM16, or AML1-CACL2 did not achieve CR after one cycle of chemotherapy. It is concluded that AML1 fusion genes are more frequent and can provide the molecular markers for diagnostics and prognosis evaluation of AML and for monitoring MRD.

RUNX1–ETO induces a type I interferon response which negatively effects t(8;21)-induced increased self-renewal and leukemia development

The 8;21 translocation is the most common chromosomal aberration occurring in acute myeloid leukemia (AML). This translocation causes expression of the RUNX1–ETO (AML1–ETO) fusion protein, which cooperates with additional mutations in leukemia development. We report here that interferons (IFNs) and IFN-stimulated genes are a group of genes consistently up-regulated by RUNX1–ETO in both human and murine models. RUNX1–ETO-induced up-regulation of IFN-stimulated genes occurs primarily via type I IFN signaling with a requirement for the IFNAR complex. Addition of exogenous IFN in vitro significantly reduces the increase in self-renewal potential induced by both RUNX1–ETO and its leukemogenic splicing isoform RUNX1–ETO9a. Finally, loss of type I IFN signaling via knockout of Ifnar1 significantly accelerates leukemogenesis in a t(8;21) murine model. This demonstrates the role of increased IFN signaling as an important factor inhibiting t(8;21) fusion protein function and leukemia development and supports the use of type I IFNs in the treatment of AML.

Clinical significance of lymphoid enhancer-binding factor 1 expression in acute myeloid leukemia

Lymphoid enhancer-binding factor 1 (LEF1) is a downstream effector of the Wnt/β-catenin signaling pathway and its dysregulation is associated with a number of malignant diseases such as leukemia. We explored the expression profile of LEF1 in acute myeloid leukemia (AML) and determined its specific prognostic significance in this disease. The LEF1 mRNA level in patients with previously untreated AML was significantly higher than in normal controls. Patients with AML with relatively higher LEF1 expression were more likely to achieve a complete remission (CR) following induction therapy in comparison to those with a lower LEF1 level. Moreover, we provide the first evidence that primary AML samples with AML1–ETO or PML–RARα have a higher LEF1 level compared with those without each fusion gene. High LEF1 expression predicts a significantly better overall survival for patients with intermediate-risk cytogenetics. High LEF1 level was associated with a favorable relapse-free survival in patients with FLT3-ITD wild-type. Finally, a scoring system based on LEF1 level and mutation status of FLT3-ITD or NPM1 is reliable to predict the outcome for AML with intermediate-risk cytogenetics. Our results indicate that LEF1 contributes to the pathophysiology of AML and could serve as a novel predictor of better treatment response. LEF1 level may be incorporated into an improved risk classification system for certain specific subtypes of AML.

A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression1. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia (AML), is a transcription factor implicated in both gene repression and activation2. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis3–6, making it important to identify coregulatory factors that “read” the NHR2 oligomerization and contribute to leukaemogenesis4. We now show that, in leukaemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO–induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO and a potential therapeutic target in t(8;21)+ AML.

AML1-ETO mediates hematopoietic self-renewal and leukemogenesis through a COX/β-catenin signaling pathway

AbstractDeveloping novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs), leading to the development of leukemia stem cells. Previously, using a zebrafish model of AE and a whole-organism chemical suppressor screen, we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin–dependent pathway. Here, we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE+ mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE+ mouse HSPCs and human leukemia cells. In addition, treatment with nimesulide, a COX-2 selective inhibitor, dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary, our data indicate an important role of a COX/β-catenin–dependent signaling pathway in tumor initiation, growth, and self-renewal, and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments.

Holding back

A recent paper published in Blood indicates that the interaction between AML1–ETO and the transcriptional co-repressor NCOR1 is partly responsible for limiting the leukaemogenic capacity of this fusion gene.

A novel recurrent AML1–ETO fusion: tight in vivo association with BCR–ABL1

A novel recurrent AML1–ETO fusion: tight in vivo association with BCR–ABL1

ATRA and the specific RARα agonist, NRX195183, have opposing effects on the clonogenicity of pre-leukemic murine AML1-ETO bone marrow cells

All-trans retinoic acid (ATRA) is used successfully in the treatment of acute promyelocytic leukemia (APL). ATRA enhances hematopoietic stem cell self-renewal through retinoic acid receptor (RAR)γ activation while promoting differentiation of committed myeloid progenitors through RARα activation. Its lack of success in the treatment of non-APL acute myeloid leukemia (AML) may be related to ATRA's non-selectivity for the RARα and RARγ isotypes, and specific RARα activation may be more beneficial in promoting myeloid differentiation. To investigate this hypothesis, the effects of ATRA and the specific RARα agonist NRX195183 was assessed in AML1-ETO (AE)-expressing murine bone marrow (BM) progenitors. ATRA potentiated the in vitro clonogenicity of these cells while NRX195183 had the opposite effect. Morphological and flow cytometric analysis confirmed a predominantly immature myeloid population in the ATRA-treated AE cells while the NRX195183-treated cells demonstrated an increase in the mature myeloid population. Similarly, NRX195183 treatment promoted myeloid differentiation in an AE9a in vivo murine model. In the ATRA-treated AE cells, gene expression analyses revealed functional networks involving SERPINE1 and bone morphogenetic protein 2; AKT phosphorylation was upregulated. Collectively, these findings confirm the contrasting roles of specific RARα and RARγ activation in the clonogenicity and differentiation of AE cells with potential significant implications in the treatment of non-APL AML using a specific RARα agonist.

The Activation of STAT3 and 5 in t(6;9)(DEK/CAN)-Positive Acute Myeloid Leukemia Models Is Inhibited by Exposure to Arsenic Trioxide

AbstractAbstract 1384▪▪This icon denotes a clinically relevant abstractIn acute myeloid leukemia (AML) chimeric genes derived from specific chromosomal translocations, such as t(15;17), t(8;21) or t(6;9) encode aberrant fusion proteins (FPs). These Fps, i.e. PML/RARα (PR), AML-1/ETO (AE) or DEK/CAN (DC), recapitulate the leukemic phenotype in the mouse. In about 50% af AMLs constitutive activation of STAT3 and/or 5 is observed and it is postulated that the aberrant stem cell capacity in leukemia is correlated to the activation of STATs. Furthermore it has been shown in models of the t(15;17)(PR)-positive AML that there is a relationship between STAT-activation and response to arsenic trioxide (ATO). t(6;9)(DC)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The mechanisms by which DC induces leukemia are nearly completely unknown.In order to investigate whether the STAT-activation plays a role for the pathogenesis of t(6;9)-positive AML we investigated the activation of STAT3 and 5 in models of DEK/CAN positive leukemia.As models of DC-induced AML we used either primary Sca1+/lin- murine hematopoietic stem and progenitor cells (mHSPC) retrovirally transduced with DC or as controls with PR. Samples of DC-positive pre-leukemia as well as primary leukemia and 2° leukemia from established DC-induced AML were used for the in vivo studies. Expression and activation of STAT3 and 5 was assessed by either intracellular FACS or Western blotting using antibodies against the phosphorylated (activated) and total STAT3 or 5. The effect of constitutively activated STAT3 (STAT3*) on the stem cell capacity was studied by the retroviral expression of STAT3* in mHSPC which then were subjected to serial replating and colony-forming unit spleen day 12 (CFU-S12) assays. ATO treatment was performed on sublethally irradiated recipients (8 mice/group), from day 5 after inoculation of DC-positive primary leukemic spleen cells for the induction of a 2° leukemia for 14 days. Leukemia development and response to ATO was assessed 30 days post-transplantation by spleen size and determination of the proportion of leukemic cells in the peripheral blood (PB), spleen and bone marrow (BM) in comparison to solvent treated controls.Here we show that i.) both DC and PR strongly activated both STAT3 and 5 in primary blasts from mouse leukemia as as well as in retrovirally transduced primary HSCPs; ii) the expression of constitutively activated STAT3 (STAT*) in HSC led to an increased replating efficiency in the presence of mIL-3, mIL-6 and SCF; iii.) STAT* increased CFU-S12 as a read out for short-term stem cell and early progenitor potential to a similar extent as PR. iv.) the pre-leukemic state of DC-positive AML was characterized by the only activation of STAT3 but not of STAT5; v.) STAT5 activation appeared only in the full blown leukemia; v.) exposure to ATO inhibited phosphorylation of STAT3 and STAT5 in vivo in the PB of the leukemic mice; vi.) the treatment with ATO significantly decreased spleen size and the proportion of leukemic cells in the BM and spleen as compared to solvent treated controls.In summary, our results strongly suggest a direct relationship between the expression of the leukemogenic oncogenes DC or PR and the activation of STATs, which seems to play an important role for the maintenance of the leukemic stem cell. Furthermore ATO seems to represent a novel therapeutic option for the t(6;9)-positive AML. Disclosures:No relevant conflicts of interest to declare.

Oncogene Dosage Is a Major Determinant of Leukemogenic Transformation by AE9a

Abstract 1358Chromosomal translocation (8;21) results in the formation of the AML1-ETO (AE) leukemia-associated fusion protein. Roughly 70% of AE+ patient samples also harbor an alternatively spliced variant of AE, termed AE9a. AE9a promotes the leukemic transformation of mouse hematopoietic stem/progenitor cells without the need for secondary external cooperating oncogenes. By comparison, leukemia development in response to expression of full length AE requires one or more additional events. We extended these findings into a human cell system to determine whether AE9a is able to promote the transformation of human CD34+ cord blood (CB) cells to acute myeloid leukemia (AML). Transduction of CD34+ CB cells with AE9a promotes long-term culture potential, enhanced replating capacity, and expansion of CD34+ progenitor cells similar to full length AE. While we have not yet been able to generate AE9a leukemia in xenograft assays (despite several attempts), subcutaneous injection of AE9a cells promotes formation of a robust granulocytic sarcoma which is not seen using cells expressing full length AE. Several of our analyses have highlighted the importance of increased AE9a fusion protein expression in its enhanced function relative to full length AE, a concept that was also observed using the MLL-AF9 leukemia oncogene (Chen et. al, Cancer Cell, 2008). Indeed, flow cytometric sorting of freshly transduced AE or AE9a cultures based on high and low fusion protein expression revealed the importance of expression level for both AE and AE9a. High AE expression completely blocked colony formation and decreased cell expansion. These effects correlated with a dramatic upregulation of the cell cycle inhibitor p21. Conversely, high levels of AE9a did not upregulate p21 to the same degree, and colony formation and cell expansion were not as dramatically affected. In contrast, flow sort for cells expressing low levels of AE or AE9a had significantly less remarkable effects on cell function and p21 expression. Unexpectedly, AE9a-low cells eventually came to resemble the AE9a-high in terms of AE9a protein levels. These effects were not seen for full length AE sorted cultures. To determine whether similar effects were occurring in the murine AML model, we examined the effects of low and high expression levels of AE9a in mouse fetal liver cells. Corroborating our findings with the human cell model, AE9a-low samples promoted significantly less AML as compared to AE9a-high cells. This was not due to lack of engraftment of these cells or to loss of AE9a expression. Similar effects were found in vitro, where AE9a-low cells were unable to replate in methylcellulose assays while AE9a-high cells as well as AE-high and AE-low cells showed enhanced replating ability. Strikingly, the AML that did develop in mice transplanted with AE9a-low cells reverted to a very high level of AE9a protein expression, implying that increased expression of AE9a is essential for function. To better understand the mechanistic link between increased AE9a expression and AML development, we performed global gene expression analysis of murine progenitor cells expressing AE or AE9a (after three weeks of in vivo expansion). Analysis revealed a subset of genes that were highly repressed in AE9a expressing cells as compared to full length AE. Given that AE9a lacks the carboxy-terminal NHR3 and NHR4 domains of AE that interact with the NCoR and SMRT corepressors, the finding of significantly more repressed genes in AE9a cells is quite surprising. Network analysis has uncovered potential signaling pathways that may be involved in AE9a mediated transformation. Uncovering the mechanism of AE9a function could lead to a better understanding of t(8;21)-associated function in human AML. Disclosures:No relevant conflicts of interest to declare.

C-CBL Inactivation Promotes Cytokine-Driven Growth of Human CD34+ Cells and AML1-ETO Expressing Cells

Abstract 2464C-CBL is an E3 ubiquitin ligase and negatively regulates activated receptor tyrosine kinases. C-CBL mutations have been frequently found in a variety of myeloid neoplasms, most frequently in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The mutations occur almost exclusively in exons 8–9 that encode linker/RING finger domains, which result in loss of E3 ligase activity. These mutants are considered to have dominant-negative function over the wild-type C-CBL. In acute myeloid leukemia (AML), C-CBL mutations are associated with core binding factor (CBF) leukemia. Although the physiologic roles of C-CBL and its mutations have been elucidated with several mouse models, the precise roles of C-CBL in primary human hematopoietic cells remain unclear.We have developed a unique culture system to model myeloid leukemogenesis using human CD34+ cord blood (CB) cells cultured in cytokines (SCF, TPO, Flt3 ligand, IL-3, IL-6), and have shown that CBF fusion oncoproteins, AML1-ETO (AE) and CBFB-MYH11 (CM), enable long-term proliferation in vitro by increasing self-renewal of CD34+ cells. Using this system, we assessed functions of C-CBL and its disease-related mutants in primary human CD34+ CB cells. All 3 C-CBL mutants (Gln367Pro, Tyr371Ser, DExon8/9), but not wild-type C-CBL, promoted the growth of CD34+ CB cells. However, unlike cells expressing AE or CM, C-CBL mutant-expressing cells stopped growing at about the same time as normal CB cells (3–4 weeks), suggesting that the mutants do not increase the self-renewal of CD34+ cells. Similar to the results obtained for normal CB cells, all 3 C-CBL mutants accelerated the cytokine-driven expansion of AE cells.Next, to test functions of endogenous C-CBL, we knocked down C-CBL expression in AE-expressing cells using 5 different shRNAs. To our surprise, one shRNA promoted, while the other 4 inhibited the growth of AE cells. The disparate effects of shRNAs were not due to different knockdown efficiencies of each shRNA, because the two best shRNAs exhibited nearly complete C-CBL depletion but showed either growth-promoting (shCBL-1) or growth-inhibitory (shCBL-2) effects. To determine which effect of shRNA is actually due to C-CBL downregulation, we cotransduced shRNA-resistant C-CBL together with shCBL-1 or shCBL-2 in AE cells, and compared the growth of shRNA-transduced cells and shRNA/C-CBL cotransduced cells. C-CBL reintroduction suppressed the enhanced AE cell growth by shCBL-1, while it did not rescue the defective growth of shCBL-2-transduced AE cells. These results indicate that the growth-promoting effect of shCBL-1 is actually due to C-CBL knockdown, while the growth inhibitory effect of shCBL-2 (and possibly the other 3 shRNAs) is caused by off-target and/or nonspecific toxicity of shRNAs. Transduction of the shCBL-1 also promoted the proliferation of normal CB cells. Interestingly, neither expression of C-CBL mutants nor C-CBL depletion by shCBL-1 showed growth-promoting effects on a cytokine-independent AE cell line (Kasumi-1), indicating that C-CBL inactivation promotes cell growth by enhancing the response to cytokines.To characterize the increased cell growth induced by C-CBL mutations/depletion, we assessed expression of a stem cell marker CD34 in the mutant expressing or C-CBL depleted CB and AE cells. In contrast to effects seen with CBF fusion genes, C-CBL inactivation results in decreased expression of CD34. We also performed cell cycle and apoptosis analyses, and found a significant increase in the proportion of S/G2/M phase cells upon C-CBL inactivation. These results indicate that C-CBL inactivation promotes cell growth mainly through increased cell cycle progression.In summary, we found that C-CBL mutant expression and C-CBL depletion promote cytokine-dependent growth, but do not increase self-renewal of human CD34+ CB cells (Fig.1). C-CBL mutations/depletion also showed growth-promoting effects on AE-expressing cells (Fig.1), which support the notion that C-CBL inactivation collaborates with CBF-rearranged genes (AE and CM) to promote AML. Functional cooperation between C-CBL mutations and AE in vivo is currently being tested using a xenotransplantation assay. These unique human cell models for myeloid neoplasms with C-CBL mutations/deletion will be useful to reveal their molecular pathogenesis and to find novel therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pro-Survival Role of RUNX1 in Acute Myeloid Leukemia with Common Fusion Proteins

Abstract 870RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Blocking RUNX1 function has been implicated in development of core-binding factor (CBF) leukemia and MLL-rearranged leukemia. In addition, inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in CBF- and MLL-rearranged leukemias, raising the possibility that a certain level of RUNX1 activity is required for efficient propagation of these leukemia cells.To determine the precise role of RUNX1 in specific types of myeloid neoplasms, we assessed RUNX1 functions in primary human CD34+ cord blood cells and those transduced with CBF related fusion oncoproteins [AML1-ETO (AE) or CBFB-MYH11 (CM)] or a MLL fusion oncoprotein, MLL-AF9 (MA9). RUNX1 was abundantly expressed and phosphorylated in AE-, CM-, and MA9-expressing long-term cultured cells. RUNX1 overexpression induced myeloid differentiation in normal CD34+ cells and prevented their long-term proliferation. Leukemogenic RUNX1 mutants lost the ability to induce differentiation, and a C-terminal truncated RUNX1 mutant conferred long-term (over 3 months) proliferative ability to CD34+ cells. RUNX1 overexpression also induced differentiation in CBF leukemia cells (AE- or CM-expressing cells). Interestingly, block of proper RUNX1 function, either by shRNA driven knockdown or forced expression of dominant-negative type mutants, showed growth inhibitory effects on CBF leukemia cells, suggesting that a certain level of RUNX1 activity is required for CBF leukemogenesis.Strikingly, block of RUNX1 function, but not RUNX1 overexpression, resulted in substantial growth inhibition of MA9 cells through enhanced apoptosis and cell cycle arrest. A xenotransplantation assay further demonstrated that RUNX1 knockdown inhibited human AML development by MA9 in vivo. The growth inhibitory effect of shRNA-mediated RUNX1 knockdown on MA9 cells was rescued by reintroduction of RUNX1, and partially restored by another RUNX transcription factor RUNX2. Thus, RUNX proteins have a growth-promoting role during MA9-driven leukemogenesis. These results contrast with those obtained using a mouse transplantation model that showed loss of Runx1 accelerates the development of MLL-ENL driven leukemia. The cause of this discrepancy is unclear, but it could be explained by species differences (human vs mouse), different experimental assays (homologous transplantation vs xenotransplantation), or the compensatory mechanism of Runx1 deletion with other Runx proteins (Runx2 and Runx3) in Runx1 knockout mice. Further studies will be needed to determine the precise roles of RUNX1 in human MLL leukemias.Finally, we assessed molecular changes in RUNX1-depleted MA9 cells and found CDKN1A upregulation and BCL2 downregulation. We also confirmed that CDKN1A depletion and BCL2 overexpression have growth-promoting effects on MA9 cells. Therefore, it appears that these molecular changes contribute to the attenuated growth of RUNX1-depleted MA9 cells. However, MA9 cells with CDKN1A depletion or BCL2 overexpression were not fully rescued from the effects of RUNX1 depletion, indicating the importance of other RUNX1 targets to support cell survival and proliferation.In conclusion, our human cell system confirmed a tumor suppressor role of RUNX1 in normal CD34+ cells, and unveiled an unexpected growth-promoting role of RUNX1 in MLL-rearranged human leukemia cells. In CBF leukemia cells, precise control of RUNX1 level appears to be important for optimal cell growth (Fig.1). Taken together, these findings suggest that partial reduction of RUNX1 activity expands myeloid progenitors by blocking differentiation, while further reduction of RUNX1 results in cell cycle arrest and increased cell death in human cells. Therefore, inhibiting RUNX1 activity rather than enhancing it will be a promising therapeutic strategy for certain types of leukemia, particularly for leukemias with common fusion proteins. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

PIG7, transactivated by AML1, promotes apoptosis and differentiation of leukemia cells with AML1–ETO fusion gene

AML1, the potent transcription factor in hematopoiesis, is antagonized by AML1-ETO in t(8;21) leukemia. Our previous study showed that the differentiation and apoptosis of Kasumi-1 induced by sodium phenylbutyrate (PB), were accompanied by significant upregulation of PIG7 and AML1b (one of the AML1 isoforms). Here, we further investigated the relationship between AML1b and PIG7, also the effects of PIG7 on leukemia cells. The results demonstrated that exogenous AML1b could upregulate PIG7 expression in HEK-293 and CV-1 cells in sequence-specific and dosage-dependent manners, and this effect was antagonized by AML1-ETO. The specific AML1-binding site required for p53-induced gene 7 (PIG7) transactivation was located between nucleotides -1511 and -1503 in the PIG7 promoter. Overexpression of PIG7 could induce the apoptosis and differentiation of Kasumi-1 and SKNO-1 cells, but showed less effect on NB4 cells directly. Moreover, ectopic expression of PIG7 could sensitize these cell lines to PB or all-trans retinoic acid, respectively, which could then be abrogated by downregulation of PIG7 expression. Furthermore, the primary acute myeloid leukemia cells showed similar response to the ectopic expression of PIG7. In conclusion, PIG7 could be transactivated by AML1, which subsequently induces differentiation and apoptosis of leukemia cells, especially those with AML1-ETO fusion gene.

Dynamics of epigenetic modifications in leukemia

Chromatin modifications at both histones and DNA are critical for regulating gene expression. Mis-regulation of such epigenetic marks can lead to pathological states; indeed, cancer affecting the hematopoietic system is frequently linked to epigenetic abnormalities. Here, we discuss the different types of modifications and their general impact on transcription, as well as the polycomb group of proteins, which effect transcriptional repression and are often mis-regulated. Further, we discuss how chromosomal translocations leading to fusion proteins can aberrantly regulate gene transcription through chromatin modifications within the hematopoietic system. PML-RARa, AML1-ETO and MLL-fusions are examples of fusion proteins that mis-regulate epigenetic modifications (either directly or indirectly), which can lead to acute myeloblastic leukemia (AML). An in-depth understanding of the mechanisms behind the mis-regulation of epigenetic modifications that lead to the development and progression of AMLs could be critical for designing effective treatments.

The t(8;21) Fusion Protein AML1/ETO Promotes Susceptibility to Mutation In Acute Myeloid Leukemia.

AbstractAbstract 3368Translocation (8;21)(q22;q22) is the most common cytogenetic abnormality found in acute myeloid leukemia (AML), presenting in around 15% of all cases and is also reported in lymphoid leukemia. This subtype of AML has a favourable prognosis in contrast to other AML subtypes, however the mechanisms underlying this good response to treatment are not well understood. The translocation between AML1 (RUNX1) and the almost full-length ETO (RUNX1T1, MTG8) gene produces a 752 amino acid fusion protein and changes a transcriptional modulator essential for hematopoiesis into a constitutive repressor. When AML1/ETO (AE) is expressed in animal models, further mutations are required for the development of leukemia. AE has also been detected at a considerable frequency in the Guthrie cards of individuals who developed t(8;21) AML up to twelve years later, suggesting that AE+ hematopoietic precursor cells can remain latent for many years before transformation. AE+ cells have an activated p53 pathway, increased levels of p53 response genes and display an increased susceptibility to DNA damaging agents. AE also decreases the levels of DNA repair genes, including those involved in base-excision repair. This evidence predicts the existence of a pre-leukemic, AE-expressing hematopoietic precursor, which easily acquires additional mutations required for transformation, but that also responds well to treatment. There are multiple alternative forms of AE, the best described being AML1/ETO9a (AE9a), which codes for a truncated version of the protein lacking a significant proportion of ETO. AE9a has been found to be more leukemogenic than AE in mouse models by causing leukemia without the need for additional mutations.Our investigation aims to test the effect of cellular expression of AE or AE9a on mutation, cell death and transformation following exposure to leukemogens and to identify co-operating mutations involved in t(8;21) AML. TK6 lymphoblastoid cells are heterozygous for the TK gene and when exposed to mutagens, can lose their one functional copy and become TK-/-. These mutants can then be selected for and quantified. TK6 were lentivirally transduced with vectors containing EGFP and either AE or AE9a. AE was cloned into the pHR-SINcPPT-SIEW lentiviral transfer vector and a similar AE9a construct acquired. Virus was produced utilising the 293T cell line and TK6 were transduced by a spinfection technique. Expression was confirmed by flow cytometry, western blotting and qRT-PCR. Single clonal populations were generated and fusion gene expression levels of 28% (AE) and 40% (AE9a) relative to AE expression in Kasumi-1 (a t(8;21) AML patient-derived cell line) were achieved.We used these transduced cell line models to determine whether the presence of AE or AE9a affected cell viability and susceptibility to the acquisition of mutations. Spontaneous TK-/- mutants were first purged from the population by treatment with CHAT media. Two weeks post CHAT treatment, AE+ TK6 were shown to acquire a greater number of mutations than controls, both spontaneously and when exposed to doxorubicin (Figure 1). AE+ TK6 had a 2.2 fold higher background mutation frequency than controls, suggesting that AE promotes spontaneous acquisition of mutations. When treated with low-dose doxorubicin, AE+ TK6 had a 2–3 fold higher mutation frequency (3.5 × 10−5) in comparison to controls; wild-type TK6 and backbone transduced TK6 (1.4 × 10−5 and 1.1 × 10−5 respectively). High density SNP array analysis of AE-expressing TK6 has recently identified deletions at 10q25.1 and 3p24.3. The deletion at 3p24.3 potentially affects expression of SGOL1, coding for a protein involved in sister chromatid segregation and PCAF, an acetyl transferase which has been implicated in the regulation of p53. The effect of the deletion on these genes is currently being evaluated. [Display omitted] Our data show that TK6 expressing AE or AE9a have an enhanced sensitivity to the cytotoxic effects of the anthracycline doxorubicin. This contributes to the understanding of the relatively good prognosis of t(8;21) AML. We have shown that expression of AE in a cell system promotes the acquisition of more mutations than that of non-expressing cells. This provides further understanding as to how the pre-leukemic AE+ hematopoetic clone transforms and results in leukemia. Disclosures:No relevant conflicts of interest to declare.

ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

AbstractAbstract 999All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures:No relevant conflicts of interest to declare.

Hoxa9 Suppresses Cellular Senescence and Sustains the Development of Leukemic Stem Cells Induced by Fusion Proteins In the Absence of Bmi1.

AbstractAbstract 1578While self renewal is an essential feature for the maintenance of both normal hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs), very little is known about the underlying molecular pathways. Here we report a critical functional interplay between Bmi1 and Hox in establishment of HSCs and LSCs. Using Bmi1-/- bone marrow cells, we observe that leukemia-associated fusion proteins have distinctive Bmi1 requirements. AML1-ETO (AE) and PLZF-RARα (PR) fail to transform Bmi1-/- primary hematopoietic cells, and induce expression of p16/Arf leading to oncogene-induced senescence (OIS). In contrast, MLL-AF9 driving expression of multiple Hox genes can bypass oncogene-induced senescence and exhibits modest Bmi1-dependence for establishment of LSCs, which can induce leukemia upon serial transplants. Since members of Hox genes with proclaimed self-renewal property are specifically up-regulated by MLL fusions in patient samples and our murine models, we asked the question if these Hox genes may partly compensate the functions associated with the loss of Bmi1. To this end, we generated compound Bmi1-/-Hoxa9-/- mice, which have even more compromised hematopoietic stem cell/progenitor compartments than those of Bmi1-/- or Hoxa9-/- mice. Bmi1-/-Hoxa9-/- mice have a greater than eight-fold reduction in the absolute number of Lin-Sca+kit+ (LSK) in the bone marrow as compared to Bmi1-/- mice and a very significant forty-fold reduction for long term hematopoietic stem cells (LT-HSC). More importantly, while MAF9 is able to transform wild type, Bmi1-/- and Hoxa9-/-, it fails to transform Bmi1-/-Hoxa9-/- cells for establishment of LSCs, which can however be resurrected by re-expression of either Bmi1 or Hoxa9, indicating a critical functional interplay between these protein in development of MLL LSCs. Consistent with the known function of Bmi1 in suppressing cellular senescence and the expression of p16/Arf loci, we showed that Hoxa9 alone can also inhibit replicative senescence and Ras-induced senescence in primary human fibroblast. Forced expression of Hoxa9 can suppress p16/Arf expression, as well as cellular senescence induced by AE and PR in Bmi1-/- cells. Together, these results reveal a previously unrecognized functional interplay between Hox and Bmi1 in regulating cell senescence and development of LSCs induced by fusion proteins, which also suggests that synergistic targeting of both molecules may be required for certain LSCs. Disclosures:No relevant conflicts of interest to declare.

Proteomics of AML1/ETO Target Proteins: AML1–ETO Targets a C/EBP–NM23 Pathway

Abstract Introduction The rational design of targeted therapies for acute myeloid leukemia (AML) requires the discovery of novel protein pathways in the systems biology of a specific AML subtype. We have shown that in the AML subtype with translocation t(8;21), the leukemic fusion protein AML1–ETO inhibits the function of transcription factors PU.1 and C/EBPα via direct protein–protein interaction. In addition, recently using proteomics, we have also shown that the AML subtypes differ in their proteome, interactome, and post-translational modifications. Methods We, therefore, hypothesized that the systematic identification of target proteins of AML1–ETO on a global proteome-wide level will lead to novel insights into the systems biology of t(8;21) AML on a post-genomic functional level. Thus, 6 h after inducible expression of AML1–ETO, protein expression changes were identified by two-dimensional gel electrophoresis and subsequent mass spectrometry analysis. Results Twenty-eight target proteins of AML1–ETO including prohibitin, NM23, HSP27, and Annexin1 were identified by MALDI-TOF mass spectrometry. AML1–ETO upregulated the differentiation inhibitory factor NM23 protein expression after 6 h, and the NM23 mRNA expression was also elevated in t(8;21) AML patient samples in comparison with normal bone marrow. AML1–ETO inhibited the ability of C/EBP transcription factors to downregulate the NM23 promoter. These data suggest a model in which AML1–ETO inhibits the C/EBP-induced downregulation of the NM23 promoter and thereby increases the protein level of differentiation inhibitory factor NM23. Conclusions Proteomic pathway discovery can identify novel functional pathways in AML, such as the AML1–ETO–C/EBP–NM23 pathway, as the main step towards a systems biology and therapy of AML.

RUNX1 repression‐independent mechanisms of leukemogenesis by fusion genes CBFB–MYH11 and AML1–ETO (RUNX1–RUNX1T1)

The core binding factor (CBF) acute myeloid leukemias (AMLs) are a prognostically distinct subgroup that includes patients with the inv(16) and t(8:21) chromosomal rearrangements. Both of these rearrangements result in the formation of fusion proteins, CBFB-MYH11 and AML1-ETO, respectively, that involve members of the CBF family of transcription factors. It has been proposed that both of these fusion proteins function primarily by dominantly repressing normal CBF transcription. However, recent reports have indicted that additional, CBF-repression independent activities may be equally important during leukemogenesis. This article will focus on these recent advances.

Enrichment of N-Cadherin and Tie2-bearing CD34 +/CD38 −/CD123 + leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1–ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34 +/CD38 −/CD123 + compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34 +/CD38 −/CD123 + LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34 +/CD38 −/CD123 + LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34 +/CD38 −/CD123 + LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.