The Hsp90 Inhibitor 17-AAG and FLT3 Kinase Inhibitor GTP14564 Synergistically Inhibit MLL Fusion Gene Leukemias with FLT3 Mutations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1167-1167
Author(s):  
Qing Yao ◽  
Ritsuo Nishiuchi ◽  
Toshio Kitamura ◽  
John H. Kersey
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Fusion Gene ◽  
Synergistic Effects ◽  
Leukemia Cells ◽  
G1 Arrest ◽  
Combined Effects ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Flt3 Mutations

Abstract 17-AAG, an inhibitor of the molecular chaperone Hsp90, results in apoptosis and inhibition of proliferation of certain leukemia cells. We have previously reported that responses to 17-AAG are highest in leukemia with FLT3 mutations, intermediate in leukemia with wild type FLT3 and lowest in FLT3 negative cells. 17-AAG operates primarily through reduction of total levels of FLT3 and other protein kinases, including RAF and AKT (Clin Cancer Res 2003, 4483). However, the inhibitory effects of 17-AAG on cells are partial. In this study we hypothesized that inhibition of FLT3 will enhance the effects of 17-AAG. GTP14564, an inhibitor of phosphorylation of FLT3 tyrosine kinase, was previously found to strongly inhibit proliferation of IL3-independent murine pro-B ITD-FLT3 Ba/F3 cells while proliferation of FLT3 ligand dependent Ba/F3 cells expressing wild type FLT3 was relatively resistant (JBC 2003, 32892). The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the mechanism of the combined effects in human cell lines with MLL fusion genes with or without FLT3 mutations. We first determined the importance of FLT3 mutations using small interfering RNA (siRNA) targeted to FLT3. Cell growth experiments showed that a FLT3-ITD human MLL-AF4 fusion gene cell line (MV4;11) was very sensitive to FLT3 siRNA, while the FLT3-wild type amplified SEMK2-M1 cell line was intermediately sensitive and FLT3-wild type RS4;11 cell line was resistant. FLT3 siRNA sensitivities paralleled inhibitory responses to GTP14564 in cell proliferation assays. Importantly, we observed synergistic inhibitory effects on cell proliferation when 17-AAG and GTP14564 were combined in FLT3-ITD cells (MV4;11 and MOLM-13). Synergistic effects were also observed in cells with amplified wild type FLT3 (SEMK2-M1) but not in those with normal level of wild type FLT3 (RS4;11). Cell cycle analysis of MV4;11, SEMK2-M1 and RS4;11 showed that 17-AAG inhibited cells in G0/G1 phase with a reduced S phase fraction after a 24 hrs treatment. Apoptosis, represented by <2N fraction and cleaved PARP (by Western blotting), was induced by 17-AAG only in MV4;11 and SEMK2-M1 cells, but not in RS4;11 cells. GTP14564 induced G0/G1 arrest and apoptosis in MV4;11 and SEMK2-M1 cells, but in contrast to 17-AAG had no effect on RS4;11 cells. Combined treatment with 17-AAG and GTP14564 enhanced G0/G1 arrest and apoptosis in MV4;11 and SEMK2-M1, but not in RS4;11 cells. By Western blotting, 17-AAG reduced total cellular quantities of FLT3 and AKT in all cells tested while GTP14564 did not change these quantities. Significantly, both 17-AAG and GTP14564 alone and in combination reduced the levels of phosphorylated FLT3 and phosphorylated STAT5 in MV4;11 and SEMK2-M1 cells, but not in RS4;11 cells. In summary, GTP14564 inhibits cell proliferation and induces apoptosis of FLT3 mutated (but not FLT3 wild-type) MLL fusion gene leukemia cells and that the addition of 17-AAG results in synergistic inhibitory effects. The mechanism of the synergistic effects was found to be the result of complementary inhibitory actions of 17-AAG and GTP14564 both on FLT3 and on downstream phospho-proteins in the RAS/RAF/AKT and STAT signal transduction pathways. 17-AAG, which is currently in clinical trials, combined with FLT3 kinase inhibitors such as GTP14564 has the potential to enhance therapeutic efficacy, particularly in high risk MLL fusion gene leukemias with FLT3 mutation.

The Hsp90 Inhibitor, 17-AAG, and Topoisomerase ll Inhibitor, Etoposide, Synergistically Inhibit Leukemias with FLT3 Mutations through Inhibition of DNA Repair Proteins, Chk1 and Rad51.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2461-2461
Author(s):  
Qing Yao ◽  
Brenda Weigel ◽  
John Kersey
Keyword(s):  
Dna Repair ◽  
Cell Lines ◽  
Topoisomerase Ii ◽  
Leukemia Cells ◽  
G1 Arrest ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Inhibition Of Proliferation ◽  
Dna Repair Proteins ◽  
Flt3 Mutations

Abstract 17-AAG, an analogue of gelganamycin is an inhibitor of the molecular chaperone Hsp90, that results in apoptosis and inhibition of proliferation of myeloid or mixed lineage (MLL fusion gene induced) leukemia cells. The sensitivity to 17-AAG is highest in leukemia with FLT3 mutations, intermediate in wild type FLT3 and lowest in FLT3 negative leukemia cells. 17-AAG results in reduction of total levels of protein kinases FLT3, RAF, AKT and Chk1, a protein involved in DNA repair. Etoposide is a clinically effective agent in myeloid or mixed lineage leukemia therapy. Etoposide inhibits topoisomerase II and results in production of DNA double-strand breaks (DSBs). Normally these DSBs can be repaired by Rad51 via homologous recombination (HR). In DNA repair, Chk1 is required for HR through the interaction with Rad51. The effects of 17-AAG on Rad51 are unknown. Our current study evaluated the single and combined effects of 17-AAG and etoposide, and the mechanism of these effects in human leukemia cell lines with or without FLT3 mutations. Cell growth experiments using the MTT assay showed that the cell lines with MLL fusion genes and internal tandem duplication of FLT3 (ITD-FLT3) (Molm13 and MV4;11) were sensitive to both 17-AAG and etoposide. The IC50s for 17-AAG were 31 nM and 40 nM, respectively; the IC50s for etoposide were 37.7 nM and 45.6 nM, respectively. Wild-type FLT3 cells (HPB-Null and RS4;11) were less sensitive to both 17-AAG and etoposide; the IC50s for 17-AAG were 470 nM and 700 nM, respectively, and the IC50s for etoposide were 72.2 nM and 101.5 nM, respectively. The combination effects of 17-AAG and etoposide on cell proliferation were analyzed using Combination Index method (CalcuSyn software). Importantly, we observed synergistic inhibitory effects in FLT3-ITD cells (MV4;11 and Molm13) but only additive effects in wild type FLT3 cells (HPB-Null and RS4;11). Cell cycle analysis of MV4;11 and Molm13 cells showed that 17-AAG increased cells in G0/G1 phase after a 24 hrs treatment, while etoposide induced G2/M arrest only. Combined treatment with 17-AAG and etoposide results in Go/G1 arrest before the cells enter S phase, as with 17-AAG alone. Western-blotting showed that 17-AAG inhibited FLT3, Chk1 and in novel results Rad51 in ITD-FLT3 leukemia cells. To address the importance of FLT3 mutations on DNA repair proteins, Chk1 and Rad51, we used small interfering RNA (siRNA) targeted to FLT3. The results showed that Chk1 and Rad51 are dependent on constitutively activated FLT3 expression in ITD-FLT3 cells. In conclusion, the combination of 17-AAG with etoposide results in synergistic cellular inhibitory effects in ITD-FLT3 leukemia cells. The mechanism of the synergistic effects was found to be in part the result of inhibitory actions of 17-AAG on FLT3 dependent DNA repair proteins, Chk1 and in new findings Rad51 which are required for the repair of DNA damage induced by etoposide. 17-AAG, which is currently in clinical trials, combined with a topoisomerase II inhibitor, such as etoposide, has the potential to enhance therapeutic efficacy, particularly in high risk myeloid or mixed lineage leukemias with FLT3 mutation.

PAX5/TEL Exhibits a Dominant-Negative Role by Inhibiting the Expression of Genes Involved in BCR Signalling and Suppressing the Binding of Wild Type PAX5 to Its Direct Target Gene CD79A (mb-1) in a human Pre-B ALL Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3455-3455
Author(s):  
Gabriela B. Iwanski ◽  
Nils Heinrich Thoennissen ◽  
PohYeen Lor ◽  
Norihiko Kawamata ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Fusion Product ◽  
Wild Type ◽  
Rt Pcr ◽  
Downstream Target

Abstract Abstract 3455 Poster Board III-343 Acute lymphoblastic leukemia (ALL), one of the most common malignancies in childhood, is a heterogeneous disease with individual leukemia subtypes differing in their response to chemotherapy. Recent findings suggest that disruptions of B cell receptor (BCR) signalling pathways may be involved in the development of ALL. The transcription factor PAX5 is essential for the commitment of lymphoid progenitors to the B-lymphocytic lineage. In 30% of childhood B-ALL cases, PAX5 is a frequent target of aberrancies, showing monoallelic loss, point mutations, or chromosomal translocations, whereas the role of these aberrancies is still poorly understood. Using high resolution SNP-chip analysis, we have recently identified several candidate partner genes fused to PAX5 in pediatric ALL, ETV6 (TEL), FOXP1, AUTS2, C20orf112, which bind to PAX5 recognition sequences as strongly as wild-type PAX5 (wt PAX5) suppressing its transcriptional activity in a dominant-negative fashion. In order to study the role of PAX5/TEL in leukemic evolution of B-ALL, we transfected the leukemic BCP cell line Nalm 6, which endogenously expresses PAX5, with a retroviral vector encoding PAX5/TEL and confirmed its expression by Western blotting and RT-PCR. Previously, the fusion gene PAX5/TEL has been cloned into the retroviral vector pMSCV-IRES-GFP (MIGR) from a patient diagnosed with B-cell precursor ALL (BCP) with t(9;12)(q11;p13). This fusion product consists of the 5′-end NH2 terminal region of the PAX5 gene and the almost whole sequence of the TEL gene. PAX5/TEL-MIGR expressing cells were sorted for GFP and analyzed by gene expression profiling on Affymetrix HG-U133 plus 2.0 Array in comparison to cells transfected with vector control (MIGR) and a MIGR vector encoding wt PAX5 (wtPAX5/MIGR). The probes were normalized with the Affymetrix MAS5.0 software. Probes were considered to be differentially expressed with a fold change ≤ 2 or ≥ 2, respectively. We identified a set of about 200 genes that were differentially expressed in the PAX5/TEL expressing cells, most of which were downregulated, compared to the controls. A subset of these genes encodes proteins important for BCR signalling: RAG1, one of two key mediators in the process of V(D)J recombination, VPREB3, which is involved in the early phase of pre-BCR assembly, the Runt domain transcription factor Runx1 (AML1) and FOXP1. The latter two genes are fusion partners of PAX5 in pediatric B-ALL and loss of FOXP1 leads to impaired DH–JH and VH–DJH rearrangement. Additionally, we found BACH2, which plays an important role during B-cell development, as well as protein kinase C-epsilon (PKCe) to be downregulated. PKCe is highly expressed in B cells linking the BCR to the activation of mitogen-activated protein kinases (MAPK). We confirmed the downregulation of the affected genes by RT-PCR. Strikingly, VPREB3 expression showed a significant downregulation of up to 170-fold, and RAG1 up to 90-fold. Loss of the RAG1/2 locus has been found in four precursor B-cell ALL cases, which indicates that defects in this process might contribute to leukemogenesis. We also detected a significant decrease in the expression of wt PAX5 as well as its direct downstream target CD79A (mb-1). CD79A (mb-1) encodes the B cell receptor component Ig-a and its early B cell-specific mb-1 promoter is a target for regulation by early B cell-specific transcription factors like E2A, early B cell factor (EBF), and PAX5. The latter is important for the activation of the mb-1 promoter by recruiting Ets proteins through protein-protein interactions. We investigated the binding efficiency of wt PAX5 to the promoter region of CD79A by chromatin-immunoprecipitation (ChIP). For the ChIP assay, we used a PAX5 antibody detecting the C-terminal region of PAX5 so that the antibody can bind the wt PAX5 but not the fusion product PAX5/TEL of which the C-terminal side is fused to TEL. Binding of wt PAX5 to the promoter region of CD79A was diminished by expression of the PAX5/TEL-fusion protein compared to the controls, leading to repression of CD79A, which we also confirmed by RT-PCR. In conclusion, we show that the expression of PAX5/TEL in a leukemic cell line has a repressor function on the expression of wt PAX5 as well as other genes important in BCR signalling. Also, we demonstrated that PAX5/TEL has a negative impact on the binding affinity of one of the direct downstream target genes of wt PAX5. Our results indicate a repressor role of the fusion gene PAX5/TEL including BCR signalling and point towards its contribution to leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1026-1026
Author(s):  
Erica A. Lehotzky ◽  
Mark Y. Chiang
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Leukemia Cells ◽  
Inhibitory Effects ◽  
Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

MLL-AF6 Mediated Transformation Is Dependent On the H3K79 Methyl-transferase Dot1l

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3502-3502
Author(s):  
Aniruddha J Deshpande ◽  
Liying Chen ◽  
Maurizio Fazio ◽  
Amit U. Sinha ◽  
Kathrin M Bernt ◽  
...  
Keyword(s):  
Cell Line ◽  
Target Genes ◽  
Fusion Gene ◽  
Histone Methyltransferase ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Employment Equity ◽  
Specific Antibodies ◽  
Equity Ownership ◽  
H3k79 Methylation

Abstract Abstract 3502 The t(6;11)(q27;q23) produces a chimeric MLL-AF6 oncogene, and is a recurrent chromosomal rearrangement observed in patients with diverse hematologic malignancies such as acute myelogenous leukemia (AML), as well as both B-cell and T-cell acute lymphoblastic leukemias (ALL). The presence of an MLL-AF6 translocation predicts a particularly poor prognosis. Of particular biological interest, the MLL-AF6 translocation is the most common fusion event in which MLL fuses to a predominantly cytoplasmic protein. Very little is known about the molecular mechanisms of transformation mediated by the MLL-AF6 fusion oncogene, forestalling the development of specific therapeutic strategies for t(6;11)(q27;q23) positive leukemias. Recent studies suggest that the histone methyltransferase DOT1L could be an important therapeutic target in MLL-rearranged leukemias. We sought to assess whether MLL-AF6 mediated transformation is also dependent on aberrant H3K79 methylation using genomic, genetic and pharmacological approaches. First, we performed chromatin immuno-precipitation using H3K79me2 specific antibodies followed by next generation sequencing (ChIP-seq) on murine MLL-AF6 leukemias as well as on ML2, the human myelomonocytic leukemia cell line bearing the MLL-AF6 fusion gene. We observed that in both murine and human MLL-AF6 leukemia cells, MLL-fusion target genes display markedly high levels of H3K79 dimethylation as compared to other highly expressed genes. We then investigated whether MLL-AF6-induced transformation was dependent on aberrant H3K79 methylation through genetic or pharmacologic inhibition of the Dot1l histone methyltransferase. Lineage negative/Sca-1 positive/Kit positive (LSK) cells from mice bearing homozygous Dot1l floxed alleles were immortalized by retroviral expression of the MLL-AF6 fusion gene. Cre-recombinase mediated excision of Dot1l from MLL-AF6 transformed bone marrow cells resulted in a significant reduction in H3K79 dimethylation at the promoters of the MLL-target genes Hoxa9, Hoxa10 and Meis1, with a concomitant decrease in their expression. Dot1l excision significantly diminished the clonogenic capacity, abrogated blast colony formation in methylcellulose based medium, and enhanced differentiation of MLL-AF6 transformed cells. We then sought to assess whether EPZ004777, a recently described specific small molecule inhibitor of DOT1L could show efficacy against murine and human MLL-AF6 transformed cells. Dot1l inhibition using EPZ004777 significantly diminished H3K79 dimethylation globally (as assessed by immunoblotting) as well as on MLL-target genes (as assessed by ChIP-qPCR) using H3K79me2 specific antibodies. Importantly, EPZ004777 treatment significantly impaired the proliferation of both murine MLL-AF6 transformed cells as well as the ML2 cell line, whereas the proliferation rates of Hoxa9-Meis1 transformed cells as well as the human MLL-germline cell line HL60 were unaffected despite a similar decrease in H3K79me2 levels. EPZ004777 treatment induced cell cycle arrest as well as increased apoptosis in MLL-AF6 positive, but not control leukemia cells, demonstrating a selective activity of the DOT1L inhibitor EPZ004777 on MLL-AF6 transformed cells. In summary, we demonstrate that the MLL-AF6 oncoprotein requires continued activity of the histone methyltransferase DOT1L for aberrant epigenetic activation of downstream target oncogenes. More studies are needed to understand the mechanisms by which DOT1L is recruited to MLL-target genes by the MLL-AF6 fusion, since AF6 is not believed to normally associate with DOT1L. Nevertheless, the demonstration that H3K79 methylation is important for MLL-AF6 mediated transformation indicates that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation. Disclosures: Olhava: Epizyme: Employment. Daigle:Epizyme, Inc.: Employment. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Armstrong:Epizyme: Consultancy.

scholarly journals Combined Genetic Lesions in TP53 and CDKN2A/CDKN2B Drive B Cell Receptor-Dependent/Costimulatory Signal-Independent Proliferation in Richter Syndrome

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Supriya Chakraborty ◽  
Claudio Martines ◽  
Fabiola Porro ◽  
Ilaria Fortunati ◽  
Alice Bonato ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Board Of Directors ◽  
Combined Treatment ◽  
Cell Receptor ◽  
Leukemia Cells ◽  
Wild Type ◽  
Advisory Committees ◽  
Richter Syndrome

B cell receptor (BCR) signals play a critical role in the pathogenesis of chronic lymphocytic leukemia (CLL), but their role in regulating CLL cell proliferation has still not been firmly established. Unlike normal B cells, CLL cells do not proliferate in vitro upon engagement of the BCR, suggesting that CLL cell proliferation is regulated by other signals from the microenvironment, such as those provided by Toll-like receptor (TLR) ligands or T cells. However, the rapid reduction in the percentage of proliferating CLL cells in patients treated with a BTK or SYK inhibitor suggests that the BCR may be more directly involved, at least in a subset of cases. To further address this issue, we investigated the expression of cell cycle regulatory proteins in human and Eμ-TCL1-derived murine CLL cells stimulated with immobilized anti-IgM or cognate antigen, respectively. In both cases, BCR stimulation induced the expression of the positive cell cycle regulators MYC, CCND1, CCND2 and CDK4, consistent with G1 cell cycle entry, but also induced the expression of the negative regulators CDKN1A, CDKN2A and CDKN2B, which block cell cycle progression. Since combined deficiency of these negative regulators occurs in approximately one quarter of Richter syndrome cases because of deletion of the CDKN2A/CDKN2B locus and inactivating genetic lesions of the CDKN1A regulator TP53, we introduced these defects by CRISPR/Cas9 in autoreactive murine Eμ-TCL1 leukemia cells and investigated the effects on leukemia behavior. Adoptive transfer experiments showed that combined targeting of TP53, CDKN2A and CDKN2B results in accelerated leukemia growth and morphological changes consistent with Richter's transformation, including more diffuse infiltration, larger and more pleomorphic cells, and a higher proliferation rate. Moreover, in vitro experiments showed that cells with combined TP53/CDKN2A/2B deficiency had acquired the capacity for spontaneous proliferation, in contrast to control, TP53- or CDKN2A/2B-targeted cells which died after a couple of weeks in culture. Nucleotide sequencing of the TP53/CDKN2A/2B-targeted cells showed complete disappearance of the wild type alleles, suggesting that biallelic loss of all three genes is required for spontaneous growth in vitro. Combined disruption of TP53, CDKN2A and CDKN2B in two other autoreactive Eμ-TCL1 leukemias yielded the same results. To determine whether the spontaneous in vitro proliferation is dependent on BCR signals, the TP53/CDKN2A/2B-deficient cells were transfected with Cas9 ribonucleoprotein complexes targeting the IgM heavy chain constant region (IGHM) gene or were treated with the BCR inhibitors ibrutinib, idelalisib and fostamatinib. Disruption of the IGHM gene or treatment with a BCR inhibitor resulted in almost complete block of proliferation. In contrast, knockdown of the TLR-adaptor protein MyD88 had no effect. Considering that T cells were not present in the culture conditions, these experiments establish that proliferation of Eμ-TCL1 leukemia cells with biallelic TP53/CDKN2A/2B disruption is BCR-dependent but independent of costimulatory signals. To validate these findings in a human setting, we performed experiments with two recently established Richter syndrome patient-derived xenografts, one with biallelic inactivation/deletion of TP53, CDKN2A and CDKN2B (RS9737), and one with wild type TP53, CDKN2A and CDKN2B (RS1316). BrdU incorporation experiments showed that only RS9737 cells proliferate in vitro following BCR stimulation, whereas both RS9737 and RS1316 cells proliferate following stimulation with CpG + IL-15 or CD40L + IL-4 + IL-21. Finally, we tested the activity of combined treatment with a BCR inhibitor and the CDK4/6 inhibitor palbociclib against the murine and human TP53/CDKN2A/2B-deficient Richter syndrome models. Combined treatment showed synergistic activity in vitro and significantly prolonged mouse survival in vivo compared to single agent treatment (n = 10 mice/group, P<0.001). In conclusion, these data provide evidence that BCR signals are directly involved in regulating CLL cell proliferation and suggest that frequently co-occurring genetic lesions in TP53 and CDKN2A/2B contribute to Richter transformation by allowing for BCR dependent/costimulatory signal independent proliferation, which can be therapeutically targeted with a BCR and CDK4/6 inhibitor combination. Disclosures Deaglio: Verastem: Research Funding; Heidelberg Pharma: Research Funding. Laurenti:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Efremov:Janssen-Cilag International: Speakers Bureau.

scholarly journals MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 462-462 ◽  
Author(s):  
Svetlana S Itskovich ◽  
Jason Clark ◽  
James C. Mulloy ◽  
Matthew D Disney ◽  
Ashish R Kumar
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Fusion Gene ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Wild Type

Abstract Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals HB9 Represses Hematopoietic Stem Cell Proliferation and Induces Senescence

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1539-1539
Author(s):  
Deborah Ingenhag ◽  
Franziska Auer ◽  
Arndt Borkhardt ◽  
Julia Hauer
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Line ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Oncogenic Potential ◽  
Stem Cell Proliferation ◽  
Senescent Phenotype

Abstract Introduction: HB9 is a transcription factor encoded by homeobox gene B9 (HLXB9). It is physiologically expressed during early embryonic development as well as in pancreatic beta- and motor neuronal cell development. Ectopic HB9 expression is found in infant acute myeloid leukemia with translocation t(7;12), accounting for up to one third of infant AML cases with a poor 3-year EFS of 0% irrespective of the treatment approach. We previously showed that HB9 regulates cell-cell interaction/adhesion (Wildenhain et al. Leukemia, 2010) in hematopoietic cells and influences the prostaglandin signalling pathway (Wildenhain & Ingenhag et al. JBC, 2012). In this study we focussed on the oncogenic potential of HB9 in hematopoiesis. Methods: To investigate the oncogenic influence of HB9 expression on hematopoiesis, we developed an in vivo murine transplantation model. HB9-transduced lineage negative (Lin-) murine HSCs were transplanted into lethally irradiated wild-type mice and we monitored hematopoietic reconstitution and leukemia emergence by serial retroorbital bleedings for up to one year. Final analysis included comprehensive flow cytometric analysis of all hematopoietic compartments, with respect to dissemination of blast cells and cellular distribution. In vitro studies included proliferation as well as cell cycle analysis. Senescent phenotype was characterized by senescence-associated beta-galactosidase staining and cellular morphology. Knockdown of p53 was obtained via transfection of siRNA. Results: Transplantation of HB9- or mock-transduced murine Lin- cells into lethally irradiated wild-type recipient mice (n=10) showed >80% donor chimerism and HB9-transduced Lin- cells gave rise to all hematopoietic lineages (B-lineage: CD19+, T-lineage: CD3+, NK-lineage: Nk1.1+, granulocytic lineage: Gr-1+, Monocytic lineage: CD11b+) in the peripheral blood, indicating no lineage-related preference of HB9-expressing HSCs. Reconstitution of peripheral blood cell compartments in HB9 transplanted mice, however, was significantly decreased in all three lineages (CD3+: 9.5-fold, CD19+: 34.7-fold , Gr+: 1.8-fold) compared to the control group with respect to copy number, mRNA and protein expression. We did not observe an accumulation of hematopoietic stem (LT-HSC, ST-HSC, MPP) and precursor cells subsets (CLP, MEP, CMP, GMP) in the bone marrow of mice transplanted with HB9-positive Lin- cells. Finally, mice transplanted with HB9-transduced Lin- cells did not develop leukemia after 12 months follow-up. The decreased reconstitution capacity of HB9 expressing HSCs led us to the assumption that HB9 represses cellular proliferation in vivo. Thus we performed proliferation studies in vitro. Ectopic expression of HB9 in the murine NIH3T3 cell line revealed a complete inhibition of cell proliferation compared to mock control (n=3). The same effect was observed in human HT1080 cell line. Cell cycle analysis revealed a significant decrease of the S-phase (2-fold, p<0.05), stalling the cells in G1 and G2 phase of the cell cycle. In both cell line models HB9-transduced cells developed a senescent phenotype being multinuclear, flattened and enlarged. Staining for senescence-associated β-galactosidase activity was positive in HB9-transduced cells in contrast to complete absence in mock-transduced cells. Immunoblot analysis revealed that the HB9 dependent cell cycle arrest was mediated via p53-induced upregulation of p21. Knockdown experiments using p53-targeting siRNAs confirmed that the p53-signalling is responsible for the growth arrest because p53-knockdown was able to reverse the effect. Conclusion:In our study HB9 represses hematopoietic stem cell proliferation in vivo and induces a senescent phenotype in vitro. Senescence is an evasion mechanism in response to aberrant oncogene expression and induction of senescence is the first evidence for an oncogenic potential of HB9. Future studies elucidating the signal pattern of HB9-induced senescence will shed new light on the pathomechanism and potential therapeutic targets in the treatment of translocation t(7;12) positive AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals T lymphocyte-derived differentiation-inducing factor inhibits proliferation of leukemic and normal hemopoietic cells

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1333-1338 ◽  
Author(s):  
U Gullberg ◽  
E Nilsson ◽  
MG Sarngadharan ◽  
I Olsson
Keyword(s):  
Cell Line ◽  
T Lymphocyte ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Leukemia Cells ◽  
Gel Chromatography ◽  
Wild Type ◽  
Clonogenic Growth

Abstract A differentiation-inducing factor (DIF) for the promyelocytic HL-60 cell line is constitutively produced by the malignant T lymphocyte line HUT-102. DIF was highly purified from HUT-102-conditioned media by means of diethylaminoethanol (DEAE)-chromatography, gel chromatography, and high-resolution, ion-exchange chromatography on a MonoQ column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition to inducing differentiation of wild-type HL-60 cells, resulting in secondary inhibition of growth, DIF, at a tenfold lower concentration, inhibited the growth of some clones of the monoblastic U- 937 cell line as well as that of subclones of HL-60. The latter effect was most likely a primary growth inhibition and not secondary to differentiation; 50% inhibition of clonogenic growth in agar was seen at approximately 1.0 pmol/L of DIF. In addition, the clonogenic growth of fresh leukemia cells from 10 of 12 patients with acute myeloid leukemia (AML) was inhibited with 50% inhibition at approximately 10 pmol/L of DIF. The growth of normal granulocyte-macrophage colonies was inhibited at a similar concentration, whereas early erythroid colonies were much more resistant. DIF and interferon-gamma (gamma-IFN) were shown to be separate molecules inasmuch as a neutralizing antibody for gamma-IFN did not abolish the DIF effect. The differentiation effect on wild-type HL-60 and the proliferation inhibitory effect on leukemic and normal myeloid cells cochromatographed through all purification steps suggest that both activities are exhibited by identical polypeptides. DIF may have a role in regulating normal hemopoiesis. The growth inhibitory effect of DIF and the ability to induce differentiation of some leukemia cells may suggest a clinical utility in the treatment of leukemia.

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