Distinct Drug Responses of CD8+CD4+ and CD8+CD4−leukemic Cell Subsets in Mice with Notch1-Induced T-ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1504-1504
Author(s):  
Yingchi Zhang ◽  
Chunlan Hua ◽  
Hui Cheng ◽  
Weili Wang ◽  
Xiaomin Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Treatment ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Double Positive ◽  
Rapamycin Treatment

Abstract Abstract 1504 Leukemic cells are heterogeneous, so defining the differences of leukemic cell sub-populations in response to drug treatment may ultimately help design better strategies to maximize the efficacy of chemotherapeutic agents for patients. We have begun to study this issue with Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in mice. The Notch1 signaling pathway plays an essential role in cell growth and differentiation, and activated mutations in the Notch1 gene are frequently observed in human T-ALL. Over-expression of the intracellular Notch1 domain (ICN1) in murine hematopoietic cells is able to induce robust T-ALL in mice. In our current study, we examined T-ALL cells, specifically the CD8 and CD4 double positive (CD8+CD4+) and CD8 single positive (CD8+CD4−) cell sub-populations, for their distinct sensitivities to drug treatment. We first treated T-ALL mice with a cell-cycle nonspecific chemical drug, cyclophosphamide (CTX), or a mTORC1 inhibitor, rapamycin, when the overall T-ALL cell population rose to more than 10% in mouse peripheral blood. We found that CTX treatment effectively reduced the T-ALL cells and led to an increase in the ratio of CD8+CD4+ versus CD8+CD4− T-ALL, indicating that CD8+CD4− T-ALL cells were more sensitive to CTX treatment. In contrast, rapamycin had the opposite effect. When compared with mice treated with rapamycin or CTX alone, the combination of the two drugs led to a significant improvement for T-ALL bearing mice with respect to leukemia initiation, progress and overall survival. We then set out to investigate the mechanism of different sensitivities of CD8+CD4+ and CD8+CD4− T-ALL cells upon CTX or rapamycin treatments. We found that CD8+CD4+ and CD8+CD4−T-ALL cell groups responded similarly to CTX treatment after 12 hours of treatment. However, CD8+CD4−T-ALL cells continued to undergo apoptosis 24 hours after CTX treatment. The expression of the anti-apoptosis gene Bcl-2 in CD8+CD4+ T-ALL cells was significantly higher than that in CD8+CD4− T-ALL cells, while the expression of the pro-apoptosis genes Bax, p53 and Noxa in CD8+CD4+ T-ALL cells was lower than that of CD8+CD4−T-ALL cells after 24 hour CTX treatment. This suggests that CD8+CD4+ and CD8+CD4− T-ALL cells activate apoptosis differently after CTX treatment. Interestingly, rapamycin treatment did not affect apoptosis in the same manner as CTX treatment, but instead, it arrested more T-ALL cells in the G0 phase. More importantly, only CD8+CD4+, but not CD8+CD4−T-ALL cells were sensitive to rapamycin treatment in arresting cell cycle at different time points. The expression levels of CDK2 and CDK4 were significantly lower in CD8+CD4+ T-ALL cells when compared with CD8+CD4− T-ALL cells, while p27 expression in CD8+CD4+ T-ALL cells was higher than that of CD8+CD4− T-ALL cells after rapamycin treatment for 3 or 10 days. These data provide a molecular basis for the distinct apoptotic and cell cycle arrest responses of CD8+CD4+ and CD8+CD4− T-ALL cell subsets to CTX or rapamycin treatment. Taken together, our study documents previously unappreciated, yet distinct properties of CD8+CD4+ and CD8+CD4−Notch1-induced T-ALL cells in the context of specific drug treatment and suggests a therapeutic potential of combining CTX and rapamycin treatment for T-ALL patients. This strategy may also help design better chemotherapeutical regimes for other types of leukemia and cancer. Disclosures: No relevant conflicts of interest to declare.

scholarly journals JAK2 Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 583-583
Author(s):  
Elisabeth M.P. Steeghs ◽  
Isabel S. Jerchel ◽  
Willemieke de Goffau-Nobel ◽  
Alex Q. Hoogkamer ◽  
Judith M. Boer ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Induced Resistance ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cell Precursor

Abstract Background In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines. Aim Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib. Methods This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels. Results JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS). Conclusion JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Therapeutic Implications of Ventx in Acute Lymphocytic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2457-2457
Author(s):  
Hong (Jenny) Gao ◽  
Xiaoming Wu ◽  
Zhenglun (Jerry) Zhu
Keyword(s):  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Cell Model ◽  
Lymphoblastic Leukemia ◽  
Wnt Signaling Pathway ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Therapeutic Implications ◽  
Novel Therapeutic

Abstract Abstract 2457 Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, representing about a third of all pediatric cancers. Despite significant progress in ALL treatment over the past decades, relapse occurs in about 20% cases, which are often resistant to conventional chemotherapy. The canonical Wnt/b-catenin signaling has been implicated in pathogenesis of ALL, and has been a target of intensive investigation for its potential application in cancer prevention and treatment. During our recent studies, using methods of reverse genetics and developmental modeling of Xenopus embryogenesis, we identified VentX as a novel Wnt antagonist and a putative tumor suppressor in lymphocyte leukemia. On the basis of our prior findings that VentX inhibit proliferation of lymphoblastic leukemic cells, our current studies are designed to explore the potential role of VentX as a novel therapeutic target of ALL and its underlying mechanisms. Using lymphoblastic cell model and real-time RT-PCR technique, we found that VentX expression can be induced by chemotherapeutic agents. We found that elevated expression of VentX caused a senescence phenotype in lymphoblastic leukemic cells and importantly, down-regulation of VentX expression by RNA interference technique led to strong attenuation of the cytotoxic effects of chemotherapeutic agents. Mechanistically, we found that VentX binds to the promoter and p53 and p16 and transactivates the p53/p21 and the p16/pRb senescence pathways, which have been implicated in pathogenesis of ALL. We found further that VentX control these two senescence pathways in primary B lymphocytes and that inactivation of both pathways rather than one is required to abolish the inhibitory effects of VentX on the proliferation of lymphoblastic leukemia cells. In summary, the results of our investigation suggest that VentX is a novel therapeutic target of ALL. We found that VentX poised as a pivotal link between the oncogenic Wnt signaling pathway and the tumor suppressing senescence pathway. Given the implications of Wnt and senescence pathway in leukemia stem cells, a resource of chemo-resistance and relapse, targeting VentX may present novel opportunities for managing refractory cases of ALL and the eventual cure of the diseases. Disclosures: No relevant conflicts of interest to declare.

The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1850-1850
Author(s):  
Naveed I. Khan ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Nuclear Translocation ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cancer Genes ◽  
Wnt Proteins

Abstract Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.

Rapid BCP-ALL Engraftment in NOD/SCID Mice Is Characterized by a High Leukemia Initiating Cell (LIC) Frequency.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2985-2985
Author(s):  
Luca Trentin ◽  
Manon Queudeville ◽  
Sarah M. Eckhoff ◽  
Felix Seyfried ◽  
Manuel Herrmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Late Onset ◽  
Lymphoblastic Leukemia ◽  
Cyclin B1 ◽  
Leukemic Cells ◽  
Disease Manifestation ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
Significant Difference

Abstract Abstract 2985 Recently, we identified in a NOD/SCID human (hu) B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) mouse model two different engraftment phenotypes that we named time to leukemia (TTL) short (TTLshort) and TTL long (TTLlong) and that reflected the rapid or late onset of leukemia in the mice. We showed that the rapid and late leukemia engraftments in the mice highly correlated with the relapse free survival of patients initially stratified as standard risk at diagnosis. In order to analyze if the two distinct phenotypes are characterized by different frequency of leukemia initiating cells (LICs), we investigated the NOD/SCID repopulating activity of 4 individual BCP ALL samples. We performed a limiting dilution transplantation assay using freshly isolated spleen cells form 2 TTLshort and 2 TTLlong xenografted mice with full-blown leukemia. We created 5 groups of 8 mice each and transplanted intravenously 105, 104, 103, 102, 101 cells per mouse/group. Leukemia engraftment in the peripheral blood (PB) was routinely evaluated by flowcytometry and the mice were sacrificed at the onset of disease manifestation. We defined a mouse as engrafted when we could detect ≥ 1% CD19 positive cells in the PB. In 3 out of 4 samples, 100% of the mice transplanted with 105 and 104 cells engrafted; only in one sample (TTLlong) we had a 75% and 12,5% successful engraftment using 105 and 104 cells respectively. The percentage of engrafted mice using 103 cells was 87,5% and 75% for the 2 TTLshort samples and 37,5% and 0% for the 2 TTLlong specimens. No mice transplanted with the 102 cells isolated from the 2 TTLlong samples engrafted whereas 50% and 12,5% of the mice transplanted with the 102 cells isolated from the 2 TTLshort engrafted successfully. No one of the mice transplanted with 101 cells engrafted. The LIC frequency was calculated using the Poisson single-hit model; a higher LIC frequency was calculated in the 2 TTLshort (1/329 and 1/739) compared to the 2 TTLlong (1/2159 and 1/74028). As cells in the G0 phase of the cell cycle are considered as the reservoir for the new cycling cells and putative LICs, we analyzed by flowcytometry the percentage of G0 cells, defined as cells staining negative for the Ki-67 antigen, within TTLshort (N=8) and TTLlong (N=7). No statistical difference was found between the analyzed samples. Nevertheless, we observed that in both TTLshort and TTLlong almost all cells were actively progressing through the cell cycle. Aiming to further characterize distinctive biological features related to the different LIC frequency, we analyzed by flowcytometry the cell cycle profiles of 9 TTLshort and 8 TTLlong mice. We used a cell cycle analysis based on the simultaneous labeling of DNA (7-AAD) and RNA (Pyronin Y) that allows to distinguish between cells that are in G1 but have not yet actively entered the S phase of the cell cycle (named G1a cells) from cells that are actively progressing form G1 to S (named G1b cells). A higher percentage of G1a cells was detected in the TTLshort compared to TTLlong (p = 0.004 Mann-Whitney Test) suggesting the presence of a stand-by-cell fraction in the TTLshort almost ready to proceed through the cell cycle. On the other hand, a higher percentage of G1b cells was found in the TTLlong compared to TTLshort (p = 0.001, Mann-Whitney Test). No significant difference was found in the proportion of cells in the “S/G2/M subgroup”. We also analyzed by Western Blot CYCLIN B1 expression in TTLshort (N=5) and TTLlong (N=5): a statistically different CYCLIN B1 expression was found between the two groups (p = 0.009 Mann-Whitney Test). As this molecule is involved in the progression of cells from the G2 to M phase of the cell cycle, we analyzed by flowcytometry the mitotic cells fraction defined as the cells positive for the phosphorilation of the Ser10 on the histone H3 in 6 TTLshort and 5 TTLlong specimens. A higher proportion of P-H3(Ser10) positive cells was detected in the TTLshort compared to the TTLlong (p = 0.045, Mann-Whitney Test). All together these data indicate that the two NOD/SCID engraftment phenotypes are characterized by different frequency of LICs. Furthermore, our functional analyses reveal a distinctive progression of the leukemic cells through the cell cycle showing a higher number of cells (G1a cells) ready to progress through the G1-S phases in the TTLshort/poor prognosis leukemia. Disclosures: No relevant conflicts of interest to declare.

A Novel Role for Bcl-xL in the Context of p210 BCR/ABL B Cell Acute Lymphoblastic Leukemia (B-ALL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 156-156
Author(s):  
Jason G. Harb ◽  
Brenda I. Chyla ◽  
Claudia S. Huettner
Keyword(s):  
Cell Cycle ◽  
Pleural Effusion ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
S Phase ◽  
Model Systems ◽  
Leukemic Cells ◽  
Wild Type ◽  
Significant Difference

Abstract While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in the chronic phase of chronic myeloid leukemia, these drugs are inefficient for BCR/ABL associated B-cell acute lymphocytic leukemia (B-ALL). Therefore, it is necessary to identify molecular targets downstream of BCR/ABL to develop additional therapeutic approaches. Cells transformed by BCR/ABL are resistant to a wide variety of apoptotic stimuli and therapeutic strategies aimed at reinstating the apoptotic pathway appear as an attractive concept. Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and studies employing cell lines, as well as primary cells have linked BCR/ABL expression with increased levels of Bcl-xL, resulting in resistance to chemotherapeutic agents. To define the role of Bcl-xL in BCR/ABL associated B-ALL, we generated two inducible transgenic mouse models. In the first model, BCR/ABL and loss of Bcl-x expression are co-induced, and in the second model, leukemia is induced with expression of Bcl-xL protein well above the levels found in wildtype lymphoblasts. Surprisingly, we found that deletion of Bcl-xL did not inhibit leukemogenesis or affect apoptosis. Bcl-x deficient B-ALL mice rapidly succumbed to a B-ALL like disease with Bcl-x deficient B-ALL animals being moribund as early as 17 days after induction. By day 28, all mice (n=10) had died or had to be euthanized. Necropsy of animals suffering from Bcl-x deficient leukemia revealed massive lymphadenopathy, pleural effusion, and splenomegaly. While loss of Bcl-x in our B-ALL model led to a more severe phenotype with considerable tumor burden, no statistically significant difference was found between the survival time in Bcl-x deficient and wild type B-ALL animals due to development of pleural effusion in both models. The most prominent difference was the presence of mitotic figures in the peripheral blood, lymph node, and spleen of Bcl-x deficient B-ALL animals, suggestive of increased proliferation of Bcl-x deficient lymphoblasts. Cell cycle analysis of leukemic cells isolated from pleural effusion and spleen of Bcl-x deficient B-ALL mice demonstrated a significant increase of cells in S/G2/M phase (p ≤ 0.05) compared to wildtype lymphoblasts. Thus, loss of Bcl-xL results in increased passage through the cell cycle, while expression of the protein limits the proliferation rate. To test this hypothesis, we generated a second model in which Bcl-xL is expressed at higher levels than in wild type lymphoblasts. Overexpression of Bcl-xL in BCR/ABL positive mice led to reduced proliferation as significantly fewer leukemic cells were present in the S phase than in controls substantiating a role for in Bcl-xL proliferation of lymphoblasts. Initial studies performed to determine the mechanisms by which loss of Bcl-x leads to increased proliferation suggest that the protein may indirectly regulate stability of p27Kip1. Our data show that cells from Bcl-x deficient B-ALL mice in G1 and S phase contain less p27, as a consequence of proteosomal degradation. Clearly, our model systems demonstrate an unexpected and novel role for Bcl-xL in the context of BCR/ABL associated B-ALL. Ongoing studies are aimed at the identification of the mechanism and molecules through which Bcl-xL is linked to cell cycle and proliferation of BCR/ABL transformed lymphoblasts.

The PAF Complex Regulation of Prmt5 Facilitates Leukemic Progression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3914-3914
Author(s):  
Justin Serio ◽  
Wei Chen ◽  
Maria Mysliwski ◽  
Lili Chen ◽  
James Ropa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Cytokine Signaling ◽  
Cycle Arrest ◽  
Acute Myeloid

Abstract Acute myeloid leukemias have been linked with dysregulated epigenetic landscapes sometimes attributed to altered functions of epigenetic regulators. The Polymerase-Associated Factor complex (PAFc) is an epigenetic regulator involved in transcriptional initiation, elongation and termination and directly interacts with the CTD of RNA Pol II. The complex is comprised of 6 subunits in human cells, Paf1, Cdc73, Ctr9, Leo1, Rtf1 and Ski8. Many of these subunits have key roles in a variety of cancers including acute myeloid leukemia (AML). We have previously shown the relevance of the PAFc in MLL-rearranged leukemias where its interaction with MLL fusion-proteins is required for leukemic progression in vitro and in vivo (Muntean et al. 2013 Blood, Muntean et al. 2010 Cancer Cell). However, little is known about the gene programs controlled by the PAFc and how these contribute to leukemogenesis. Here we identify Prmt5, an arginine methyltransferase, as a direct downstream target gene of the PAFc. Prmt5 is upregulated in variety of cancers and has been linked to cell cycle progression and activation of known oncoproteins. In addition, Prmt5 has been implicated in AML and is essential for normal hematopoiesis where loss of Prmt5 induces bone marrow aplasia due to impaired cytokine signaling (Tarighat et al. 2015 Leukemia, Liu et al. 2015 J Clin Invest). Our work establishes a major role for the PAFc in regulating Prmt5 expression in AML. We observe that excision of the Cdc73 subunit of the PAFc results in reduced proliferation, the induction of differentiation, cell cycle arrest, and a mild increase in apoptosis. Several key epigenetic marks are reduced globally upon loss of Cdc73 including H4R3me2s, a modification catalyzed by Prmt5. RNA sequencing and bioinformatics analysis using GSEA, revealed that loss of Cdc73 led to increased expression of a gene program associated with hematopoietic differentiation, in agreement with our cellular characterization. In addition, the downregulation of a methyltransferase gene program was detected upon Cdc73 excision. Included in this signature were several members of the Prmt family. Analysis of changes in expression following loss of Cdc73 and functional relevance in MLL-AF9 leukemic cells led us to Prmt5 as a gene critically important in AML cells and modulated by the PAFc. To interrogate the function of Prmt5 in AML cells, we performed shRNA knockdown experiments which resulted in reduced proliferation, reduced cell fitness, G1 cell cycle arrest and global reduction H4R3me2s. ChIP experiments revealed that the PAFc localizes to the Prmt5 locus in mouse and human derived leukemic cells. Further, preliminary data suggests the MLL-AF9 fusion protein also localizes to the Prmt5 locus and may enhance its transcriptional output. The enzymatic activity of Prmt5 is necessary for AML cell growth as wild type PRMT5 can rescue proliferation of Prmt5 knock-down cells while a catalytic dead mutant cannot. Furthermore, we have observed that knockdown of Prmt5 increases the disease latency of Hoxa9/Meis1 induced leukemia in vivo. Utilizing a commercially available inhibitor for Prmt5, EPZ015666 (Chan-Pembre et al. 2015 Nat Chem Bio), we show pharmacologic inhibition of PRMT5 reduces the growth of a spectrum of human leukemic cell lines, suggesting PRMT5 is important for multiple subtypes of AML. Overall, our findings elucidate the PAFc as a regulator of Prmt5 expression that is necessary for the maintenance of AML. Disclosures No relevant conflicts of interest to declare.

Upregulation of Mir-99a Is Associated with Poor Prognosis of Acute Myeloid Leukemia and Promotes Myeloid Leukemia Cell Expansion

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5086-5086
Author(s):  
Xiaohui Si ◽  
Xiaoyun Zhang ◽  
Xing Hao ◽  
Yunan Li ◽  
Zizhen Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Cycle Arrest ◽  
Myeloid Leukemia Cell

Abstract Acute myeloid leukemia (AML) is a hereditarily and clinically heterogeneous disease driven by a subpopulation of leukemic stem cells (LSCs) that generate the bulk of leukemic cells. LSCs can resist available treatments and result in disease progression and relapse. High number of LSCs or expression of a LSC related gene signature is independently associated with poor prognosis in AML, supporting the notion for LSCs as important targets for therapeutic intervention. It is now evident that miRNAs contribute significantly to the leukemogenic phenotype. To dissect the miRNA expression profile of LSCs, we performed miRNA array analysis in enriched LSCs cells and non-LSCs (leukemic blast cells) from bone marrow (BM) cells of the same AML patient at different disease stages. We identified that miR-99a was significantly upregulated in the LSCs obtained at relapse compared to the LSCs collected at the time of initial diagnosis. We also found that miR-99a were upregulated in LSCs compared to non-LSCs in a larger cohort of AML patients. The cohort were divided into two groups based on the median expression level of miR-99a in LSCs reference to non-LSCs, and correlation analysis indicated that higher expression levels of miR-99a in LSCs were significantly correlated with worse overall survival and event-free survival in these AML patients. Compared with cord blood CD34+ HSPCs, miR-99a was significantly highly expressed in LSCs from AML patients. The expression level of miR-99a was also higher in K562/A02 (multidrug resistant counterpart of K562 cell line) and K562/G01 (Gleevec resistant counterpart of K562 cell line) than the parental K562 cells. These results point to the importance of a novel role of miR-99a in LSC activity and disease progression. By gain-of-function studies via stable lentiviral transduction, we demonstrated that miR-99a functionally regulated the growth and survival of leukemic cells in vitro and in vivo. Enforced miR-99a expression significantly increased the clonogenic capacity in primary AML LSCs. Further studies in myeloid leukemia cell lines revealed that ectopic miR-99a led to accelerated cell-cycle progression and cell proliferation. MiR-99a overexpression (OE) also promotes leukemic cell survival after exposure to chemotherapeutic agents in AML cell lines. In vivo study in a subcutaneous xenograft model showed that a significant lower tumor inhibition rate was observed overtime in the miR-99a/OE cell injected mice compared to the Ctrl cell injected mice when treated with Ara-C. All these results demonstrated that miR-99a, the miRNA enriched from LSCs at relapse stage, plays an important role in myeloid leukemic cell survival and expansion. To explore the mechanism underlying the enhanced proliferation and chemoresistance meditated by miR-99a, we identified differential gene expression profiles by microarray analysis. The results provide a potential regulatory pathway that links the downregulated targets of miR-99a (i.e., PPP3CA) to the dysregulated key genes associated with leukemic cell expansion. Overexpression of miR-99a led to upregulation of CCNE1, and downregulation of CDKN2A which mediate G1 cell cycle arrest by stabilizing the structure of p53 or inducing p53 activation. All these results suggest an important role of miR-99a in cell cycle entry and progression, leading to the accelerated proliferation and overcoming of chemotherapeutic-agents mediated cell cycle arrest. Collectively, our study provides strong evidence that miR-99a plays an important role in altering biological properties, which may lead to the pathogenesis of myeloid leukemia and chemoresistance induction. The levels of miR-99a may be used as a biomarker for the prognosis and progression of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals FOXM1 Is Overexpressed in B-Acute Lymphoblastic Leukemia (B-All) and Its Inhibition Sensitizes B-All Cells to Chemotherapeutic Drugs

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2245-2245 ◽  
Author(s):  
Francesca Consolaro ◽  
Giuseppe Basso ◽  
Giampietro Viola ◽  
Eric W-.F. Lam
Keyword(s):  
Cell Cycle ◽  
Cell Viability ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Chemotherapeutic Drugs ◽  
Glucocorticoid Treatment

Abstract B-lymphoblastic leukaemic (B-ALL) patients that respond poorly to glucocorticoid therapy are predicted to relapse. An understanding of the biological mechanism underlying this poor responsiveness is therefore crucial for the development of more effective diagnostics and therapies. Forkhead box protein M1 (FOXM1) is a key transcriptional factor that regulates the expression of several genes that promote cell cycle progression, proliferation, DNA repair. Its expression is up-regulated in most cancer cells and is often linked to high proliferation rates and poor responsiveness to the therapy. In this context, we studied the role of FOXM1 in B-lymphoblastic leukaemia (B-ALL) in order to understand if FOXM1 could be a key target for leukaemia therapy. Our results showed that FOXM1 expression is higher in both B-ALL patients and cell lines compared to PBMC or CD19+ cells from healthy donors (Figure 1 A, 1 B). Figure 1A: FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1A:. FOXM1 overexpression in both B-ALL patients (A) and cell lines (B). Figure 1B Figure 1B. Furthermore FOXM1 protein levels were higher in glucocorticoid-resistant cell lines (REH, MHH-CALL2, SEM) when compared to their glucocorticoid-sensitive counterparts (RS4;11, NALM-6), suggesting that FOXM1 may have a role in mediating chemotherapeutic drug sensitivity and resistance in B-ALL. Furthermore, depletion of FOXM1 activity in B-ALL cell lines by either transient knockdown or treatment with a FOXM1 inhibitor, thiostrepton, significantly decreases the cell viability of cells that poorly respond to glucocorticoid treatment (REH). The decrease of cell viability was accompanied by an induction of G2/M arrest of the cell cycle along with a reduction of the S phase. Moreover thiostrepton synergises with common chemotherapeutic agents used in B-ALL therapy increasing their efficiency and overcoming drug resistance. All this data suggest that FOXM1 could be an important therapeutic target for overcoming the resistance to the conventional chemotherapeutic drugs. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Gain of Function Mutation in the NSD2 Histone Methyltransferase Drives Glucocorticoid Resistance Via Blocking Receptor Auto-Induction and BIM/Bmf Expression in ALL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3758-3758
Author(s):  
Jianping Li ◽  
Catalina Troche ◽  
Julia Hlavka Zhang ◽  
Jonathan Shrimp ◽  
Jacob S. Roth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Mutant Allele ◽  
Gene Editing ◽  
Conflicts Of Interest ◽  
Chemotherapeutic Agents ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Pediatric All

Despite improvements in chemotherapy that have increased the 5-year survival rates of pediatric ALL to close to 90%, 15-20% of patients may relapse with a very poor prognosis. Pediatric ALL patients, particularly those in relapse can harbor a specific point mutation (E1099K) in NSD2 (nuclear receptor binding SET domain protein 2) gene, also known as MMSET or WHSC1, which encodes a histone methyl transferase specific for H3K36me2. To understand the biology of mutant NSD2, we used CRISPR-Cas9 gene editing to disrupt the NSD2E1099K mutant allele in B-ALL cell lines (RCH-ACV and SEM) and T-ALL cell line (RPMI-8402) or insert the E1099K mutation into the NSD2WT T-ALL cell line (CEM) and B-ALL cell line (697). Cell lines in which the NSD2E1099K mutant allele is present display increased global levels of H3K36me2 and decreased H3K27me3. NSD2E1099Kcells demonstrate enhanced cell growth, colony formation and migration. NSD2E1099K mutant cell lines assayed by RNA-Seq exhibit an aberrant gene signature, mostly representing gene activation, with activation of signaling pathways, genes implicated in the epithelial mesenchymal transition and prominent expression of neural genes not generally found in hematopoietic tissues. Accordingly, NSD2E1099K cell lines showed prominent tropism to the central neural system in xenografts. To understand why this NSD2 mutations are identified prominently in children who relapse early from therapy for ALL, we performed high-throughput screening in our isogenic cell lines with the National Center for Advancing Translation Science (NCATS) Pharmaceutical Collection and other annotated chemical libraries and found that NSD2E1099K cells are resistant to glucocorticoids (GC) but not to other chemotherapeutic agents used to treat ALL such as vincristine, doxorubicin, cyclophosphamide, methotrexate, and 6-mercaptopurine. Accordingly, patient-derived-xenograft ALL cells with NSD2E1099K mutation were resistant to GC treatment. Reversion of NSD2E1099K mutation to NSD2WT restored GC sensitivity to both B- and T-ALL cell lines, which was accompanied by cell cycle arrest in G1 and induced-apoptosis. Furthermore, knock-in of the NSD2E1099K mutation conferred GC resistance to ALL cell lines by triggering cell cycle progression, proliferation and anti-apoptotic processes. Mice with NSD2E1099K xenografts were completely resistant to GC treatment while treatment of mice injected with isogenic NSD2WT cells led to significant tumor reduction and survival benefit. To illustrate these biological phenotypes and understand the molecular mechanism of GC resistance driven by NSD2E1099Kmutation, we investigated the GC-induced transcriptome, GC receptor (GR) binding sites and related epigenetic changes in isogenic ALL cell lines in response to GC treatment. RNA-Seq showed that GC transcriptional response was almost completely blocked in NSD2E1099K cells, especially in T-ALL cell lines, correlating with their lack of biological response. GC treatment activated apoptotic pathways and downregulated cell cycle and DNA repair pathways only in NSD2WT cells. The critical pro-apoptotic regulators BIM and BMF failed to be activated by GC in NSD2E1099K cells but were prominently activated when the NSD2 mutation was removed. Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that, the NSD2E1099K mutation blocked the ability of GR and CTCF to bind most GC response elements (GREs) such as those within BIM and BMF. While GR binding in NSD2WT cells was accompanied by increased H3K27 acetylation and gene expression, this failed to occur in NSD2 mutant cells. Furthermore, we found that GR RNA and protein levels were repressed in ALL cells expressing NSD2E1099K and GC failed to induce GR expression in these cells. Paradoxically, while H3K27me3 levels were generally decreased in NSD2E1099K cells, we saw increased levels of H3K27me3 at the GRE within the GR gene body where GR itself and CTCF normally bind, suggesting a novel role for the polycomb repressive complex 2 and EZH2 inhibitors for this form of GC resistance. In conclusion, these studies demonstrate that NSD2E1099K mutation may play an important role in treatment failure of pediatric ALL relapse by interfering with the GR expression and its ability to bind and activate key target genes. Gene editing screens are being performed to understand how to overcome this resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Effect of L-Asparaginase on the Nucleic Acid Metabolism and Cell Cycle of Human Leukemia Cells

Blood ◽  
1972 ◽  
Vol 39 (4) ◽  
pp. 575-580 ◽  
Author(s):  
E. Fred Saunders
Keyword(s):  
Cell Cycle ◽  
Nucleic Acid ◽  
Dna Synthesis ◽  
Rna Synthesis ◽  
Lymphoblastic Leukemia ◽  
Buffy Coat ◽  
Leukemic Cells ◽  
Nucleic Acid Metabolism ◽  
Rapid Decline ◽  
Human Leukemia

Abstract The effect of L-asparaginase on the cell cycle and nucleic acid synthesis of leukemic cells was studied in five children with acute lymphoblastic leukemia. Following an intravenous infusion of the drug, serial marrow samples were obtained for buffy coat volume, mitotic index, and autoradiographic assessment of DNA and RNA synthesis using tritiated thymidine and tritiated uridine, respectively. A rapid decline in buffy coat volume indicated a lytic effect on lymphoblasts. There was a greater kill of proliferative (blasts in the cell cycle) than nonproliferative (G0) leukemic cells. Mitotic indices changed little until 24 hr; in contrast, thymidine labeling indices decreased markedly to less than 50% of control by 6 hr. The changes in labeling indices prior to changes in mitotic indices indicated that L-asparaginase blocked the entrance of cells into the DNA synthesis period of the cell cycle. Cells already in DNA synthesis appeared to continue into mitosis. Uridine labeling indices decreased progressively in all patients. Uridine uptake was inhibited equally in both proliferative and nonproliferative blasts. Therefore, inhibition of RNA synthesis by L-asparaginase was independent of the proliferative activity of the marrow.

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