Downregulation of MiR-449a Is Essential for the Survival of EVI1 Positive Leukemic Cells through Modulation of NOTCH1 and BCL2.

Abstract Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p<0.05) were shown to be related to the EVI1 expression status. Amongst these, miR-449a was selected for further study based on its homology to the known cancer associated miRNA miR-34a. Downregulation of miR-449a by EVI1 was further confirmed in the leukemic cell line U937 with tetracycline controllabel (tet-off) EVI1 overexpression. Next, direct transcriptional regulation of miR-449a expression by EVI1 was demonstrated by chromatin immunoprecipitation (ChIP). To test the functional consequences of downregulation of miR-449a in AML cells, reconstitution of the expression of miR-449a in the Kasumi-3 and UCSD-AML1 cell lines was performed, which resulted in significantly decreased cell viability, increased apoptosis and differentiation towards the megakaryocytic and monocytic lineages. Interestingly, siRNA mediated knockdown of EVI1 expression in Kasumi-3 or UCSD-AML1 almost completely abrogated the miR-449a induced reduction in cell viability, while electroporation of both cell lines with EVI1 siRNAs alone had essentially no effect on cell viability. These data strongly suggest that repression of miR-449a expression is essential for the survival and growth of EVI1 overexpressing cells and that this requirement is specifically imposed by EVI1 itself. We next demonstrated that the predicted miR-449a targets NOTCH1 and BCL2 were bona fide miR-449a targets using promoter reporter assays. To asses the contribution of these target genes to the observed phenotype upon miR-449a upregulation, knockdown of NOTCH1 and BCL2 was performed, revealing similar effects on cell viability and apoptosis. These results indicated that the effects seen upon treatment of cells with a precursor miR-449a are at least partly mediated through NOTCH1 and BCL2. In conclusion, we provided for the first time evidence that EVI1 mediated downregulation of miR-449a leads to NOTCH1 and BCL2 upregulation and is required for sustained proliferation and survival of EVI1 overexpressing cells. These data also open new perspectives for therapeutic intervention through modulation of miR-449a and/or its target genes. Disclosures: No relevant conflicts of interest to declare.

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A Novel Peptide Derived from Ginger Induces Apoptosis through the Modulation of p53, BAX, and BCL2 Expression in Leukemic Cell Lines

Planta Medica ◽

10.1055/a-1408-5629 ◽

2021 ◽

Author(s):

Chawalit Chatupheeraphat ◽

Sittiruk Roytrakul ◽

Narumon Phaonakrop ◽

Kamolchanok Deesrisak ◽

Sucheewin Krobthong ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Mononuclear Cells ◽

Raji Cell ◽

Leukemic Cell ◽

B Cell Lymphoma ◽

Leukemic Cells ◽

Peripheral Blood Mononuclear ◽

Normal Peripheral Blood ◽

Peptide Fraction

AbstractDespite the efficacy of chemotherapy, the adverse effects of chemotherapeutic drugs are considered a limitation of leukemia treatment. Therefore, a chemotherapy drug with minimal side effects is currently needed. One interesting molecule for this purpose is a bioactive peptide isolated from plants since it has less toxicity to normal cells. In this study, we extracted protein from the Zingiber officinale rhizome and performed purification to acquire the peptide fraction with the highest cytotoxicity using ultrafiltration, reverse-phase chromatography, and off-gel fractionation to get the peptide fraction that contained the highest cytotoxicity. Finally, a novel antileukemic peptide, P2 (sequence: RALGWSCL), was identified from the highest cytotoxicity fraction. The P2 peptide reduced the cell viability of NB4, MOLT4, and Raji cell lines without an effect on the normal peripheral blood mononuclear cells. The combination of P2 and daunorubicin significantly decreased leukemic cell viability when compared to treatment with either P2 or daunorubicin alone. In addition, leukemic cells treated with P2 demonstrated increased apoptosis and upregulation of caspase 3, 8, and 9 gene expression. Moreover, we also examined the effects of P2 on p53, which is the key regulator of apoptosis. Our results showed that treatment of leukemic cells with P2 led to the upregulation of p53 and Bcl-2-associated X protein, and the downregulation of B-cell lymphoma 2, indicating that p53 is involved in apoptosis induction by P2. The results of this study are anticipated to be useful for the development of P2 as an alternative drug for the treatment of leukemia.

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Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽

10.1182/blood-2020-138622 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hsiangyu Hu ◽

Nirmalya Saha ◽

Yuting Yang ◽

Sierrah Marie Grigsby ◽

Rolf Marschalek ◽

...

Keyword(s):

Cell Growth ◽

Acute Leukemia ◽

Transcriptional Activation ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Leukemic Cells ◽

Fusion Partner ◽

Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

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All-Trans-Retinoic Acid (ATRA) Causes Extensive Differentiation In the NPM Mutant, Non-APL Leukemic Cell Line OCI-AML3

Blood ◽

10.1182/blood.v116.21.3305.3305 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3305-3305 ◽

Cited By ~ 1

Author(s):

Matthew A. Kutny ◽

Steven J. Collins ◽

Keith Loeb ◽

Roland B. Walter ◽

Soheil Meshinchi

Keyword(s):

Cell Viability ◽

Cell Count ◽

Cell Line ◽

Cell Lines ◽

Time Course ◽

Conflicts Of Interest ◽

Live Cell ◽

Leukemic Cells ◽

Viable Cell Number ◽

Nuclear Segmentation

Abstract Abstract 3305 The differentiating agent ATRA has been used successfully in the treatment of acute promyelocytic leukemia (APL). By comparison, non-APL AML has not shown similar sensitivity to ATRA induced differentiation. Recent data has suggested that a subset of de novo AML patients with nucleophosmin (NPM1) mutations may benefit from addition of ATRA to conventional therapy. The NPM1 gene has several functions affecting cell cycle proliferation including regulation of ribosome biogenesis and centrosome duplication and it acts as a histone chaperone. Mutation of the NPM1 gene leads to differentiation arrest contributing to AML pathogenesis. We hypothesized that leukemia cells with NPM1 mutations could be induced to undergo differentiation. We tested this hypothesis with the NPM1 mutant AML cell line OCI-AML3 and compared the results to identical assays using the AML cell line HL-60 which has been previously well documented to differentiate in response to ATRA therapy. OCI-AML3 and HL-60 cell lines were treated for 5 days with control media and four ATRA doses including 0.2 μM, 1 μM, 5 μM, and 25 μM. Cell viability was assessed by flow cytometry. Compared to the control condition, OCI-AML3 cells treated with the lowest dose of ATRA (0.2 μM) had a live cell count 21.6% of the control. HL-60 cells treated at even the highest ATRA dose (25 uM) had a live cell count 79.3% of the control. Due to the sensitivity of OCI-AML3 cells to the toxic effects of ATRA, the experiment was repeated with lower doses of ATRA including 0.001 μM, 0.01 μM and 0.1 μM. At the lowest dose of ATRA (0.001 μM), OCI-AML3 cells demonstrated a cell viability of 49% with further decrease to 26% at 0.1 μM dose of ATRA. At similar ATRA doses, cell viability for HL-60 cells was 91% and 85%, respectively (see table 1). Table 1: Cell viability as a percent of control cells after 5 days of treatment at three different doses of ATRA in OCI-AML3 and HL-60 cell lines. Cell Line: ATRA 0.001 μM ATRA 0.01 μM ATRA 0.1 μM OCI-AML3 49% 33% 26% HL-60 91% 91% 85% We subsequently determined the time course of changes in cell growth and the extent of differentiation at each point was determined by morphologic assessment. Both cell lines were treated with ATRA at doses of 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM for a total of 4 days. Each day viable cell number was determined. In contrast to the HL-60 cells which had continued growth in lower ATRA doses, OCI-AML3 cells demonstrated exquisite sensitivity to growth arrest at the lowest doses of ATRA. Cell morphology was assessed daily with modified Wright-Giemsa staining of cells. Cells were examined for signs of myeloid differentiation including decrease in nuclear to cytoplasmic (N/C) ratio, nuclear segmentation, and cytoplasmic granules and vacuoles. At the lowest dose of ATRA (0.001 μM), after 4 days of exposure, significant number of OCI-AML3 cells demonstrated morphologic evidence of differentiation. At this ATRA dose and exposure interval, HL-60 cells showed no evidence of differentiation. At an ATRA dose of 1 μM (considered a standard dose used for differentiation of HL-60 cells), the OCI-AML3 cells showed differentiation changes as early as day 2 with nuclear segmentation and decreased N/C ratio while HL-60 cells did not show any change at this time point. After 4 days of ATRA exposure, most OCI-AML3 cells showed segmented nuclei and vacuolated cytoplasm, whereas HL-60 cells showed less distinct signs of differentiation with some cytoplasm granules and cup shaped nuclei. This data suggests that leukemic cells with NPM mutations may be susceptible to the pro-differentiating properties of ATRA. Further substantiation of this data with primary human specimens may ultimately provide the rationale for a novel therapeutic option using ATRA-based differentiation therapy for subsets of non-APL AML. Disclosures: No relevant conflicts of interest to declare.

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Mine the Stability of the G2/M Checkpoint to Break Down Acute Lymphoblastic Leukemia Defenses Against Antineoplastic Drugs

Blood ◽

10.1182/blood.v128.22.2808.2808 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2808-2808

Author(s):

Andrea Ghelli Luserna di Rorà ◽

Ilaria Iacobucci ◽

Neil Beeharry ◽

Maria Vittoria Falzacappa ◽

Chiara Ronchini ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Leukemic Cell ◽

Leukemic Cells ◽

Antineoplastic Drugs ◽

Primary Cells ◽

Dna Damages ◽

Leukemic Cell Lines

Abstract Although impressive developments have been made in the treatment of Acute Lymphoblastic Leukemia (ALL) patients, the overall survival is still very poor. With the exception of novel therapeutic strategies based on monoclonal antibodies (Bi-specific T-cell engagers, BiTEs) or immunogenic cells (CART cells), the therapeutic approaches for adult ALL patients are still base on non-selective chemotherapy or on tyrosine kinase inhibitors (TKIs) for the patients harboring the BCR-ABL1 fusion transcript. In addition a large percentage of initial successfully treated patients frequently develop relapses. Thus there is a need to improve the efficacy of conventional therapies, in particular those related to TKIs and to DNA damaging agents, in order to reduce the off-target toxicity and avoid relapses. In the present study we evaluated the in vitro, ex vivo and in vivo efficacy of MK-1775, a specific Wee1 inhibitor, in single agent and in combination with different therapeutic agents normally used for the treatment of B-/T-ALL. We firstly started by evaluated the efficacy of the compound in single agent on a panel of human B and T ALL cell lines (n=8) and on primary cells isolated from the bone marrow of adult B-ALL patients (n=8). The inhibition of Wee1 deeply reduced the cell viability and the proliferation rate, induced the apoptosis and increased the DNA damages of both leukemic cell lines and primary cells. Further cell-cycle analysis showed that in leukemic cell lines the treatment increased the number of cell in late S and G2/M phase. Light microscopy analyses, looking for nuclei morphology, confirmed that MK-1775 increased the number of mitotic cells but it interfered with normal mitotic division (induction of aberrant mitosis as showed by the increment of DNA bridges and micro-nuclei). The effects of the compound on the cell cycle profile and on the G2/M checkpoint were confirmed also in immunoblotting analyses, by the increment of phospho-HH3(ser10) and of Myt1 (mitotic isoform), and by gene expression analysis looking to specific genes involved in the G2/M checkpoints (PrimePcr DNA damage assay, Biorad). In particular genes like GADD45A and CCNB1/CCNB2 were significantly up-regulated between treated and untreated samples. Finally using a T-ALL mouse model we evaluated the effect of MK-1775 in single agent. Although no significative differences were seen between treated and un-treated samples, due to a very aggressive phenotype of the disease (all animal died after only 18 days from the engraftment), molecular analyses confirmed that the treatment induced DNA damages (increase of H2A.X and p-Chk1 ser317) and inhibited Wee1 functionality (reduction of pCDC2) on leukemic blasts isolated from both spleens and bone marrows. To evaluate if the inhibition of the G2/M checkpoint could sensitize leukemic cells to the toxicity of antineoplastic drugs, Philadelphia-negative ALL cell lines and primary leukemic cells (n=9) where treated with increasing concentration of MK-1775 and increasing concentration of the nucleotide analogue, clofarabine. Statistical analyses (Combination index value) confirmed the synergy of the combination in the reduction of the cell viability, in the inhibition of the proliferation and in the induction of the apoptosis. Similar results were seen on Philadelphia-positive ALL cell lines and primary cells (n=3) combining the MK-1775 with the TKI, bosutinib. The simultaneously inhibition of the Wee1 and the BCR-ABL downstream pathway resulted in a synergic inhibition of the cell viability, reduction of the proliferation and induction of apoptosis. In our opinion the pre-clinical results of this study are the basis for a future clinical evaluation of MK-1775 for the treatment of ALL patients. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Speakers Bureau; BMS: Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Genentech: Consultancy; Celgene: Consultancy, Speakers Bureau.

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A New Protocol for Efficient and Non-Toxic Transfection of siRNA Into Leukemic Cell Lines and Primary Cells – a Direct Comparison Between Nucleofection and Accell Delivery Using Stem Cell Related Antigens As Validation Tools

Blood ◽

10.1182/blood.v118.21.4802.4802 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4802-4802

Author(s):

Hanne Østergaard Larsen ◽

Anne Stidsholt Roug ◽

Claus Svane Sondergaard ◽

Peter Hokland

Keyword(s):

Cell Lines ◽

Target Genes ◽

Conflicts Of Interest ◽

Transfection Efficiency ◽

Leukemic Cell ◽

Housekeeping Gene ◽

Target Cells ◽

Primary Cells ◽

Cyclophilin B

Abstract Abstract 4802 BACKGROUND: Transient downregulation of genes in vitro by use of siRNA is a time-honored approach to study gene function. This can readily be applied in haematological malignancies, where many lesions derive from altered RNA activity arising e.g. from balanced translocations. PROBLEM FORMULATION: A crucial requirement to obtain this downregulation is an efficient and non-toxic delivery of the siRNA into the target cells, which has proven difficult to accomplish with some protocols; in particular, cells in suspension, such as leukemic blasts. HYPOTHESIS: Directly comparing the range of new transfection methodologies to unravel protocols suitable for cells in suspension. RESULTS: From pilot data, where a range of other methods were excluded due to either low transfection efficiencies or low viability of transfected cells, we compared the gold standard Nucleofection methodology to the novel non-viral based delivery method Accell in both suspension cell lines as well as primary patient AML and CML cells. As control the housekeeping gene Cyclophilin B was used, while hMICL and CD96, recently suggested to be associated with the putative leukemic stem cells, were chosen as target genes. Comparison of the two transfection methods yielded superior results using Accell methodology, as this technique not only yielded higher transfection rates but also retained superior cell viabilities for both cell lines and primary cells. Three AML and two CML samples were included in this study for the validation of transfection efficiency in primary cell employing the programs T-20 and U-15 for Nucleofection. Here, there was a large span in viabilities pre-experimentation among the five patient samples included in this study, probably reflecting the conditions of the patients. However, following transfection there was a significantly lower viability after Nucleofection (mean 37%, range 23%-65%) compared to Accell (mean 82%, range 57%-95%), in the two groups (p<0.001). Corroborating the cell line data transfection efficiencies were clearly superior after Accell transfection of primary patient cells (mean 85%, range 71%-97%) compared to Nucleofection transfection (mean 38%, range 23%-65%), p<0.01. Transfection efficiencies in primary cells after Accell delivery was 90–97% compared to 25–30% using Nucleofection for siRNA delivery. As a functional correlate to these data we performed CFU-GM assays on selected hMICL+ AML patients exposed to anti-hMICL siRNAs. While the AML blast growth of some patients were unaffected by the siRNA treatment others showed a clear inhibition of blast colony formation, suggesting a differential role of hMICL between AML patients, which needs to be further elucidated. CONCLUSION: Accell delivery is thus the method of choice for non-viral transfection of cells in suspension including primary AML and CML cells. These data should provide a platform for further studies of genes involved in early leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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The Three E Proteins Define a Heterogeneity of the AML1-ETO-Containing Transcription Factor Complex (AETFC) and Differentially Regulate t(8;21) Leukemogenesis

Blood ◽

10.1182/blood-2018-99-112372 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5247-5247

Author(s):

Na Liu ◽

Junhong Song ◽

Yangyang Xie ◽

Xiao-Lin Wang ◽

Bowen Rong ◽

...

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Binding Capacity ◽

Ectopic Expression ◽

Cellular Heterogeneity ◽

Leukemic Cells ◽

Molecular Heterogeneity ◽

E Proteins

Abstract The leukemogenic AML1-ETO fusion protein is produced by the t(8;21) translocation, which is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). In leukemic cells, AML1-ETO resides in and functions through a stable protein complex, AETFC, that contains multiple transcription factors and cofactors. Among these AETFC components, E2A (also known as TCF3) and HEB (also known as TCF12), two members of the ubiquitously expressed E proteins, directly interact with AML1-ETO, confer new DNA (E-box) binding capacity to AETFC, and are functionally essential for leukemogenesis. However, we find that the third E protein, E2-2 (also known as TCF4), is specifically silenced in AML1-ETO-expressing leukemic cells, suggesting E2-2 as a negative factor of leukemogenesis. Indeed, ectopic expression of E2-2 selectively inhibits the growth of AML1-ETO-expressing leukemic cells, and this inhibition requires the basic helix-loop-helix (bHLH) DNA-binding domain of E2-2. Gene expression profiling and ChIP-seq analysis reveal that, despite some overlap, the three E proteins differentially regulate many target genes. In particular, consistent with the fact that E2-2 is a critical transcription factor in dendritic cell (DC) development, our studies show that E2-2 both redistributes AETFC to, and activates, some genes associated with DC differentiation, and that restoration of E2-2 triggers a partial differentiation of the AML1-ETO-expressing leukemic cells into the DC lineage. Meanwhile, E2-2, but not E2A or HEB, represses MYC target genes, which may also contribute to leukemic cell differentiation and apoptosis. In AML patients, the expression of E2-2 is relatively lower in the t(8;21) subtype, and an E2-2 target gene, THPO, is identified as a potential predictor of relapse. In a mouse model of human t(8;21) leukemia, E2-2 suppression accelerates the development of leukemia. Taken together, these results reveal that, in contrast to HEB and E2A, which facilitate AML1-ETO-mediated leukemogenesis, E2-2 compromises the function of AETFC and negatively regulates leukemogenesis. The three E proteins thus define a molecular heterogeneity of AETFC, which merits further study in different t(8;21) AML patients, as well as in its potential regulation of cellular heterogeneity of AML. These studies should improve our understanding of the precise mechanism of leukemogenesis and assist development of diagnostic and therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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Exogenous Expression of WNT7A in Leukemia-Derived Cell Lines Induces Resistance to Chemotherapeutic Agents

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200521114100 ◽

2020 ◽

Vol 20 (12) ◽

pp. 1504-1514

Author(s):

Monserrat Alvarez-Zavala ◽

Christian Barreto-Vargas ◽

Luis A. Torres-Reyes ◽

Roberto F. De la Peña-Castro ◽

Adriana Aguilar-Lemarroy ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Cell Lines ◽

Acquired Resistance ◽

Ectopic Expression ◽

Leukemic Cell ◽

Chemotherapeutic Agents ◽

Canonical Pathway ◽

Leukemic Cells ◽

Cell Counting

Background: Dysregulations of the WNT pathway are implicated in the malignant transformation of different types of neoplasia. WNT7A is expressed in normal peripheral lymphocytes, but is decreased in the tumoral counterpart. Furthermore, the treatment of leukemic cells with recombinant WNT7A decreases proliferation, suggesting its possible use as a therapeutic biomolecule. This study aimed to evaluate the concomitant action of WNT7A and different chemotherapeutic agents over proliferation and cell death of leukemia/ lymphoma derived cell lines. Methods: Ectopic expression of WNT7A was induced in CEM and BJAB cell lines by using a lentiviral system. RNA expression was analyzed by microarrays and qPCR, and protein expression was determined by Western Blot. Cell proliferation was measured by cell counting, metabolic activity by WST-1 assay, cell death and DNA content by flow cytometry. Results: WNT7A ectopic expression was shown to decrease cell proliferation, but the apoptosis rate of leukemic cells was not altered. Moreover, these cells acquired resistance to doxorubicin, vincristine and MG-132. Cell cycle analysis reveals a decrease in G1 and an increase in S and G2 phases with a further upregulation of senescence- associated genes. Microarray analysis reveals that most gene expression changes were related to cancer and metabolic associated pathways. All those changes appear to be independent of the WNT canonical pathway regulation. Conclusion: WNT7A negatively regulates cell proliferation in leukemic cell lines and promotes resistance to chemotherapeutic agents by inducing a senescence-like phenotype independently of the WNT canonical pathway.

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MicroRNA Profiling of EVI1 Deregulated Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.4146.4146 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4146-4146

Author(s):

An De Weer ◽

Bruce Poppe ◽

Pieter Mestdagh ◽

Nadine Van Roy ◽

Anne De Paepe ◽

...

Keyword(s):

Transcriptional Activation ◽

Myeloid Leukemia ◽

Target Genes ◽

Chromosomal Rearrangements ◽

Ectopic Expression ◽

Stem Loop ◽

Functional Studies ◽

Mirna Genes ◽

Cancer Initiation ◽

Overexpressing Cell

Abstract Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression of, or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) patients, and is a diagnostic marker which predicts a poor outcome. Recently, microRNA deregulation was identified as a major contributor to cancer initiation and progression. Moreover, miRNA genes were shown to be directly regulated by activated proto-oncogenes. In this study, we investigated which miRNA genes are implicated in the transcriptional pathways governed by the EVI1 oncogene. A total of 384 miRNAs were profiled through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR (Applied Biosystems). In a first step, differential miRNA expression was determined in 5 EVI1 rearranged/EVI1 overexpressing samples, 5 non-rearranged/EVI1 overexpressing samples and 5 non-rearranged/non-EVI1 overexpressing bone marrow samples. Next, up and down regulated miRNAs were analyzed in the leukemic EVI1 overexpressing cell lines Kasumi-3 and UCSD-AML1 following transient EVI1 RNAi knockdown. We will present the detailed results of these profiling studies as well as the results of data mining for identification of putative EVI1 regulated miRNAs. Further functional studies will be performed in order to select validated miRNA target genes and to asses their contribution to the leukemic phenotype. We anticipate that the discovery of such miRNAs may provide new targets for therapeutic intervention.

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Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis

Cancers ◽

10.3390/cancers13040642 ◽

2021 ◽

Vol 13 (4) ◽

pp. 642

Author(s):

Sierrah M. Grigsby ◽

Ann Friedman ◽

Jennifer Chase ◽

Bridget Waas ◽

James Ropa ◽

...

Keyword(s):

Cell Lines ◽

Protein Interactions ◽

Target Genes ◽

Leukemic Cell ◽

Therapeutic Index ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Rapid Reduction ◽

Hematopoietic Stem ◽

The Impact

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

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P04.20 The role of YAP in Glioblastoma cell lines

Neuro-Oncology ◽

10.1093/neuonc/noab180.074 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii22-ii23

Author(s):

G Casati ◽

L Giunti ◽

A Iorio ◽

A Marturano ◽

I Sardi

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Western Blotting ◽

Cytotoxic Effect ◽

Target Genes ◽

Hippo Pathway ◽

Combined Treatment ◽

Set Up ◽

The Hippo Pathway

Abstract BACKGROUND Glioblastoma (GBM) is a primary human malignant brain tumor, the most common in adults. Several studies have highlighted the Hippo-pathway as a cancer signalling network. The Hippo pathway is an evolutionarily conserved signal cascade, which is involved in the control of organ growth. Dysregulations among this pathway have been found in lung, ovarian, liver and colorectal cancer. The key downstream effector of the Hippo-pathway is the Yes-associated protein (YAP); in the nucleus, its function as transcription co-activator is to interact with transcription factors, resulting in the expression of target genes involved in pro-proliferating and anti-apoptotic programs. MATERIAL AND METHODS Using western blotting analysis, we determined the nuclear expression of YAP on three GBM cell lines (U87MG, T98G and A172). To investigate which inhibitors against the Hippo-pathway were the most efficient, we performed a cytotoxic assay: we treated all the three cell lines with different inhibitors such as Verteporfin (VP), Cytochalasin D (CIT), Latrunculin A (LAT), Dobutamine (DOB) and Y27632. Afterwards, we performed a treatment using Doxorubicin (DOX) combined with the inhibitors, evaluating its cytotoxic effect on our cell lines, through cell viability experiments. More western blotting experiments were performed to investigate the oncogenic role of YAP at nucleus level. Furthermore, preliminary experiments have been conducted in order to investigate the apoptosis, senescence and autophagy modulation due to the Hippo-pathway. RESULTS We showed our cell lines express nuclear YAP. We assessed the efficiency of the main inhibitors against Hippo-pathway, proving that VP, LAT A and CIT show a strong cytostatic effect, linked to time increase; plus we saw a cytotoxic effect on T98G. The association of DOX with selected inhibitors is able to reduce cell viability and nuclear YAP expression rate in all three GBM lines. Finally, preliminary experiments were set up to assess how and if the mechanisms of apoptosis, autophagy and senescence were affected by the Hippo-pathway. The combination of DOX with inhibitors promotes resistance to apoptosis. CONCLUSION Our results show that nuclear YAP is present in all tumor lines, thus confirming that this molecular pathway is functioning in GBM lines. Nuclear YAP is more highly expressed after DOX administration. Moreover, the combined treatment (DOX with Hippo-pathway inhibitors) reduces both cell proliferation and viability, and increases the rate of apoptosis. Preliminary experiments on senescence and autophagy were used to determine the best Hippo-pathway inhibitor. These data demonstrate that the Hippo-pathway plays a crucial role in GBM proliferation and resistance to apoptosis. Inhibiting this pathway and in particular the transcription factor YAP, in association with DOX, might be an excellent therapeutic target.

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