Essential Role for the MAML1 Co-Activator In T-ALL

Abstract Abstract 2501 T cell acute lymphoblastic leukemia (T-ALL) is the most common malignancy in children and accounts for nearly one third of all pediatric cancers. In this type of leukemia, lymphoid progenitor cells that are responsible for the generation of mature lymphocytes become genetically altered, leading to deregulated proliferation, survival, and clonal expansion. Two common genetic alterations frequently associated with this disease are mutations in the NOTCH1 cell-surface receptor and aberrant expression of the TAL1 transcription factor, with each abnormality detected in more than half of human T-ALL patients. The mutations in the NOTCH1 gene result in the aberrant activation of Notch signaling, a highly conserved signal transduction pathway that is critical for lymphocyte growth, maturation and survival. The constitutive activation of Notch signaling induces leukemia in mouse models and is required for human T-ALL leukemic cell growth and survival. On the other hand, TAL1 is required for the functions of hematopoietic stem cells and is essential for the generation of the erythroid and myeloid lineages. The ectopic activation of the TAL1 gene deregulates normal hematopoietic stem cell renewal and differentiation, leading to leukemia in cooperation with other oncogenes. Therefore, Notch and TAL1 oncogenic activities are critical for the initiation and maintenance of T-ALL. In this study, we investigated the role of a transcriptional co-activator, MAML1, in regulating NOTCH1 and TAL1 transforming activities in leukemic cells. In addition to its known function in co-activating Notch signaling, we found that MAML1 is a novel interacting partner for TAL1. MAML1 also enhanced TAL1 transcriptional activities, suggesting a role for MAML1 in TAL1-regulated transcription and leukemogenesis. A subset of T-ALL leukemic cells exhibit aberrant activation in both the NOTCH1 and TAL1 activities; thus, it suggests that these two genetic alterations cooperate in promoting leukemic cell growth and survival. Indeed, we found that the combined inhibition of both the pathways (via the pharmacological blockade of Notch signaling and shRNA-mediated TAL1 knockdown) results in synergistic responses in leukemic cells that carry genetic alterations in both the NOTCH1 and TAL1 genes, indicating that the two pathways synergize in promoting T-ALL. Since MAML1 appears to be a common key regulator for both TAL1 and Notch1 pathways, we next determined whether MAML1 expression level affects leukemic cell growth and survival. Gene knockdown studies suggest that MAML1 is essential for leukemic cell growth and survival by possibly regulating NOTCH1 and TAL1-mediated transcription. Overall, our data reveals a novel common regulatory mechanism for both NOTCH1 and TAL1 oncogenic pathways, and suggest that the manipulation of MAML1 expression or functional activities will affect leukemia initiation and progression. Therefore, our current studies focus on assessing the MAML1 co-activator as a target for these two oncogenic pathways. Disclosures: Griffin: Novartis: Consultancy, Research Funding.

Download Full-text

Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival

Blood ◽

10.1182/blood-2004-09-3706 ◽

2005 ◽

Vol 105 (11) ◽

pp. 4477-4483 ◽

Cited By ~ 150

Author(s):

Peter S. Hammerman ◽

Casey J. Fox ◽

Morris J. Birnbaum ◽

Craig B. Thompson

Keyword(s):

Cell Growth ◽

Cell Size ◽

Ectopic Expression ◽

Hematopoietic Cells ◽

Tumor Formation ◽

Hematopoietic Cell ◽

Leukemic Cells ◽

Growth And Survival ◽

Critical Components ◽

Cell Growth And Survival

Abstract The Akt kinases promote hematopoietic cell growth and accumulation through phosphorylation of apoptotic effectors and stimulation of mTOR-dependent translation. In Akt-transformed leukemic cells, tumor growth can be inhibited by the mTOR inhibitor rapamycin, and clinical trials of rapamycin analogs for the treatment of leukemia are under way. Surprisingly, nontransformed hematopoietic cells can grow and proliferate in the presence of rapamycin. Here, we show that Pim-2 is required to confer rapamycin resistance. Primary hematopoietic cells from Pim-2– and Pim-1/Pim-2–deficient animals failed to accumulate and underwent apoptosis in the presence of rapamycin. Although animals deficient in Akt-1 or Pim-1/Pim-2 are viable, few animals with a compound deletion survived development, and those that were born had severe anemia. Primary hematopoietic cells from Akt-1/Pim-1/Pim-2–deficient animals displayed marked impairments in cell growth and survival. Conversely, ectopic expression of either Pim-2 or Akt-1 induced increased cell size and apoptotic resistance. However, though the effects of ectopic Akt-1 were reversed by rapamycin or a nonphosphorylatable form of 4EBP-1, those of Pim-2 were not. Coexpression of the transgenes in mice led to additive increases in cell size and survival and predisposed animals to rapid tumor formation. Together, these data indicate that Pim-2 and Akt-1 are critical components of overlapping but independent pathways, either of which is sufficient to promote the growth and survival of nontransformed hematopoietic cells.

Download Full-text

IL-17A-Mediated Notch Signaling in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3434.3434 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3434-3434

Author(s):

Rao H. Prabhala ◽

Teru Hideshima ◽

Mariateresa Fulciniti ◽

Sophia Adamia ◽

Rajya Lakshmi Bandi ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Cell Growth ◽

Protein Expression ◽

Notch Signaling ◽

Cellular Protein ◽

Full Length ◽

Growth And Survival ◽

Cell Growth And Survival

Abstract Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽

10.1182/blood.v77.1.49.bloodjournal77149 ◽

1991 ◽

Vol 77 (1) ◽

pp. 49-54

Author(s):

H Kodama ◽

M Iizuka ◽

T Tomiyama ◽

K Yoshida ◽

M Seki ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Leukemic Cell ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Leukemic Cell Lines ◽

Hematopoietic Factors ◽

In Vitro Cell Lines

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

Download Full-text

Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽

10.1182/blood.v77.1.49.49 ◽

1991 ◽

Vol 77 (1) ◽

pp. 49-54 ◽

Cited By ~ 5

Author(s):

H Kodama ◽

M Iizuka ◽

T Tomiyama ◽

K Yoshida ◽

M Seki ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Leukemic Cell ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Leukemic Cell Lines ◽

Hematopoietic Factors ◽

In Vitro Cell Lines

Abstract Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

Download Full-text

Sensitizing Leukemic Cells to NF-κB Inhibitor Treatment by Repressing TNFα-Mediated, NF-κB-Independent Survival Signaling

Blood ◽

10.1182/blood.v120.21.1878.1878 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1878-1878

Author(s):

Andrew Volk ◽

Yechen Xiao ◽

Junping Xin ◽

Dewen You ◽

Rachel Schmidt ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Growth ◽

Small Molecule ◽

Leukemic Cell ◽

Leukemic Cells ◽

Inhibition Mechanism ◽

Mechanism Analysis ◽

Hematopoietic Stem ◽

Inhibitor Treatment

Abstract Abstract 1878 NF-κB activation is essential for leukemic cell and stem cell (LSC) survival and self-renewal, but is significantly less essential for similar functions in normal bone marrow hematopoietic stem/progenitor cells (HSPCs). As a result, LSCs are more sensitive to both pharmacologic and genetic NF-κB inhibition than HSPCs. These sensitivities suggest that NF-κB signaling could be a potential therapeutic target in the treatment of leukemia. However, high doses of NF-κB inhibitor treatment are also associated with significant inflammation-mediated toxicity to liver, skin and other tissues. Therefore, new approaches are needed that will be able to protect normal tissues while simultaneously enhancing the effects of NF-κB inhibition on leukemic cells. By utilizing genetic knock-out HSPC/leukemia models in combination with small molecule inhibitors, we searched for factors that could sensitize leukemic cells to NF-κB inhibition while simultaneously protecting HSPCs. We demonstrated that targeted inhibition of TNFα induced NF-κB-independent signaling would be a useful approach to treat leukemia in combination with NF-κB inhibition. We found that deactivating TNFα signaling either by genetic deletion of its receptors or through neutralizing the ligand with an antibody can significantly enhance NF-κB inhibition-induced leukemic cell elimination. In contrast, deactivation of TNFα signaling can significantly protect normal HSPCs from NF-κB inhibitor-induced death. Mechanistic studies revealed that TNFα stimulates several similar signals in both leukemic cells and HSPCs, including NF-κB, ERK, AKT, p38 and JNK. In order to determine which of these signals would best augment NF-κB inhibition, we performed biochemical analyses and searched for candidate survival signals activated downstream of TNFα and that operated independently of NF-κB. This analysis revealed that TNFα-induced ERK and AKT signals are NF-κB dependent, while TNFα-induced p38 and JNK signals are NF-κB independent. Inhibition of p38 enhanced leukemic cell growth, and was therefore ruled out as a candidate. Our analyses showed that JNK was activated by TNFα stimulation, operated independently of NF-κB activation, and also repressed leukemic cell growth. Further study confirmed that TNFα-dependent JNK activation has opposite functions in HSPCs and leukemic cells: JNK acts by promoting cell survival in leukemic cells while inducing cell death in HSPCs. We confirmed this result by inactivating the JNK signal via small molecule inhibitor, and found that we could significantly sensitize leukemic cells to NF-κB inhibition while protecting normal HSPCs from TNFα-mediated cell death associated with NF-κB inhibition. Mechanism analysis suggested that TNFα represses the growth of HSPCs by a JNK-RIP1/RIP3-dependent necroptosis mechanism, whereas TNFα promotes the expansion of leukemic cells by inducing the parallel activation of NF-κB-dependent AKT/ERK signaling and NF-κB-independent JNK signaling. In conclusion, our studies suggest the simultaneous inhibition of both NF-κB and TNFα-induced NF-κB-independent signals like JNK might provide a more comprehensive approach for targeted treatment of leukemias that also protects against deleterious inflammation in the bone marrow and other tissues. Disclosures: Nand: Celgene: Research Funding.

Download Full-text