Targeting the human double minute (HDM)-2 ubiquitin ligase as a strategy against non-Hodgkin’s lymphoma

14016 Background: The ubiquitin-proteasome pathway has been validated as a target for non-Hodgkin lymphoma (NHL) with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of an anti-apoptotic heat shock protein response, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor. Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule MI-63, an inhibitor of the HDM-2-p53 interaction, in both p53 wild-type and -mutant cell line models. Results: Treatment of wild-type p53 MCL, NHL, and acute lymphocytic leukemia (ALL) cell lines with MI-63 induced a dose- and time- dependent inhibition of proliferation, with an IC50 in the 1.0–5.0 μM range. This was associated with G1/S cell cycle arrest , and apoptosis mediated by caspase-3. MI-63 induced accumulation and phopshorylation of p53 and also enhanced MDM-2 levels. Multiple p53 target genes were induced, including p21Cip1 and p53-upregulated modulator of apoptosis (PUMA), resulting in cleavage of poly-ADP-ribose-polymerase (PARP). Cell lines expressing certain p53 mutants were sensitive to the effects of MI-63, resulting in activation of caspases 3, 8, 9 and apoptosis. Cells without p53 expression were resistant to MI-63, but at higher drug concentrations proliferation was still inhibited, indicating a possible impact on HDM-2-mediated but p53-independent cell death pathways. Combinations of MI-63 with other anti-tumor agents showed enhanced anti-proliferative effects that met the criteria for synergistic interactions. Conclusions: Inhibition of the HDM-2-p53 interaction is a promising approach both by itself, and in combination with currently used chemotherapeutics, against lymphoid malignancies, providing a rational for translation of this agent into the clinic. No significant financial relationships to disclose.

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Targeting the Human Double Minute (HDM)-2 p53 Ubiquitin Ligase as a Strategy Against Non-Hodgkin Lymphoma.

Blood ◽

10.1182/blood.v110.11.1374.1374 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1374-1374

Author(s):

Richard J. Jones ◽

Dajun Yang ◽

Nathalie Bruey-Sedano ◽

Robert Z. Orlowski

Keyword(s):

Hodgkin Lymphoma ◽

Cell Lines ◽

Human Umbilical Cord ◽

Wild Type ◽

Inhibition Of Proliferation ◽

P53 Expression ◽

Non Hodgkin Lymphoma ◽

Drug Concentrations ◽

Chemotherapy Agents ◽

The Impact

Abstract Background: The ubiquitin-proteasome pathway has been validated as a target for non-Hodgkin lymphoma (NHL) with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of an anti-apoptotic heat shock protein response, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One such attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor. Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule MI-63, an inhibitor of the HDM-2-p53 interaction, in both p53 wild-type and -mutant cell line models. Results: Treatment of wild-type p53 MCL, NHL, and acute lymphocytic leukemia (ALL) cell lines with MI-63 induced a dose- and time-dependent inhibition of proliferation, with an IC50 in the 1.0–5.0 μM range. This was associated with G1/S cell cycle arrest, and apoptosis mediated by caspases-3, 8 and 9. MI-63 induced accumulation and phosphorylation of p53 at serine 15 and 37, and also enhanced HDM-2 levels. Multiple p53 target genes were induced, including p21Cip1 and p53-upregulated modulator of apoptosis (PUMA), resulting in cleavage of poly-ADP-ribose-polymerase (PARP). MI-63 also decreased the levels of the ribonucleotide reductase subunit R2, and caused a corresponding increase in the R2p53 subunit. MI-63 also decreased the levels of E2F. Cell lines expressing certain p53 mutants were sensitive to the effects of MI-63, resulting in apoptosis. Cells without p53 expression were less sensitive to MI-63, but at higher drug concentrations proliferation was still inhibited, indicating a possible impact on HDM-2-mediated but p53-independent cell death pathways. Primary human umbilical cord vein endothelial cell growth was also inhibited and cells failed to recover after extended exposure to MI-63, whereas primary PBMC’s were unaffected by MI-63. Combinations of MI-63 with the molecularly targeted chemotherapy agents bortezomib and rapamycin were synergistic, with mean CI values of 0.88 and 0.6 respectively. The conventional chemotherapy agents doxorubicin and cisplatin were less effective at inducing synergism, with mean CI values of 1.06 and 0.9 respectively. Pretreatment of cells with MI-63 followed by chemotherapy was antagonistic with all agents used, while treatment with a chemotherapeutic first followed by MI-63 was additive to synergistic, indicating a sequence-dependent interaction. Conclusions: Inhibition of the HDM-2-p53 interaction is a promising approach both by itself, and in combination with currently used chemotherapeutics, against lymphoid malignancies, providing a rationale for translation of such agents into the clinic.

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The Impact of Chlorambucil and Valproic Acid on Cell Viability, Apoptosis and Expression of p21, HDM2, BCL2 and MCL1 Genes in Chronic Lymphocytic Leukemia

Cells ◽

10.3390/cells10051088 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1088

Author(s):

Katarzyna Lipska ◽

Agata Filip ◽

Anna Gumieniczek

Keyword(s):

Valproic Acid ◽

Chronic Lymphocytic Leukemia ◽

Cell Viability ◽

Antiepileptic Drug ◽

Drug Combination ◽

Target Genes ◽

Histone Deacetylation ◽

Lymphocytic Leukemia ◽

Malignant Cells ◽

The Impact

Malignant cells in chronic lymphocytic leukemia (CLL) show resistance to apoptosis, as well as to chemotherapy, which are related to deletions or mutations of TP53, high expression of MCL1 and BCL2 genes and other abnormalities. Thus, the main goal of the present study was to assess the impact of chlorambucil (CLB) combined with valproic acid (VPA), a known antiepileptic drug and histone deacetylation inhibitor, on apoptosis of the cells isolated from 17 patients with CLL. After incubation with CLB (17.5 µM) and VPA (0.5 mM), percentage of apoptosis, as well as expression of two TP53 target genes (p21 and HDM2) and two genes from Bcl-2 family (BCL2 and MCL1), were tested. As a result, an increased percentage of apoptosis was observed for CLL cells treated with CLB and VPA, and with CLB alone. Under the treatment with the drug combination, the expression of p21 gene was visibly higher than under the treatment with CLB alone. At the same time, the cultures under CLB treatment showed visibly higher expression of BCL2 than the cultures with VPA alone. Thus, the present study strongly suggests further investigations on the CLB and VPA combination in CLL treatment.

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CtBP1 Is a Novel and Essential Corepressor for BCL6 Autoregulation.

Blood ◽

10.1182/blood.v110.11.3581.3581 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3581-3581

Author(s):

Lourdes M. Mendez ◽

Jose Polo ◽

Melissa Krupski ◽

Jessica Yu ◽

Ari M. Melnick ◽

...

Keyword(s):

Cell Differentiation ◽

B Cell ◽

Cell Lines ◽

Cell Biology ◽

Target Genes ◽

Point Mutations ◽

B Cell Lymphoma ◽

B Cell Differentiation ◽

Wild Type ◽

Negative Autoregulation

Abstract BCL6 is POZ/BTB transcription repressor that is required for the germinal center (GC)- stage of B cell development and its deregulated expression underlies the development of many GC-derived B cell lymphomas such as diffuse large B cell lymphoma (DLBCL). BCL6 carries out its biological function by repressing target genes involved in various aspects of B cell biology such as DNA damage response, cell-cycle regulation and plasma cell differentiation. Recent publications indicate that BCL6 differentially utilizes its corepressor partners to silence target genes involved in different biological processes. Negative autoregulation of BCL6 is likely to play an important role in B-cell differentiation, and is frequently disrupted in DLBCL due to translocation or point mutation of the BCL6 promoter. However, from a mechanistic standpoint, it is not known how BCL6 mediates negative autoregulation. BCL6 is reported to repress its target genes through binding of the SMRT, NCoR and BCoR corepressors to its N-terminal POZ domain and through binding of the MTA3 and HDAC2 corepressors to its second repression domain. However, a BCL6 mutant unable to bind these corepressors retained near wild-type repression activity on the BCL6 promoter. The expression of endogenous BCL6 was unchanged in DLBCL cell lines treated with BCL6 Peptide Inhibitor, which selectively disrupts the association between BCL6 and its POZ domain corepressors, or with MTA3 siRNA. This led us to consider the possibility that BCL6 autoregulation proceeds through a novel corepressor. Several POZ transcription factors can interact with CtBP as their corepressor. We found BCL6 and CtBP can interact in both the ectopically expressed and endogenous settings in DLBCL cells. Moreover, our ChIP experiments demonstrate that CtBP is present in the 5′UTR of BCL6 at sites that were previously shown by us and others to mediate BCL6 negative autoregulation. Nearly half of DLBCL patients are estimated to carry translocations and “activating” point mutations in the 5′UTR of BCL6 which allow negative autoregulation to be bypassed. In DLBCL cell lines carrying BCL6 promoter mutations or translocations, CtBP was preferentially bound to the wild-type BCL6 allele. Moreover, CtBP siRNA specifically derepressed the wild-type allele sparing the translocated BCL6 allele driven by heterologous promoters. This allelic analysis of BCL6 is consistent with a model in which BCL6 recruits CtBP to carry out negative autoregulation. Tiling ChIP-on-chip of BCL6 target genes showed colocalization of CtBP in a BCL6 repression complex at only a subset of target genes, including BCL6. However, the BCL6 locus was the only target dependent exclusively on CtBP for repression. In an effort to address the corepressor requirements of BCL6 autoregulation, we have uncovered a novel BCL6 corepressor, CtBP. Our results substantiate the growing body of evidence that BCL6’s mechanism of repression is dynamic, selectively calling upon corepressors to silence different cohorts of target genes perhaps reflecting segregation of biological functions. Our study provides new insight into normal BCL6-driven biology and also informs BCL6-targeted lymphoma therapies.

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Potent Anti-Cancer Activity of Anti-NTB-a Monoclonal Antibodies in Preclinical Leukemia and Lymphoma Models

Blood ◽

10.1182/blood.v112.11.4975.4975 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4975-4975

Author(s):

Wouter Korver ◽

Xiaoxian Zhao ◽

Shweta Singh ◽

Cecile Pardoux ◽

Ishita Barman ◽

...

Keyword(s):

B Lymphocytes ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

Therapeutic Potential ◽

Surface Protein ◽

Lymphocytic Leukemia ◽

Lymphoid Cells ◽

Complement Dependent Cytotoxicity ◽

Promising Target

Abstract NTB-A is a CD2-related cell surface protein expressed on lymphoid cells including B-lymphocytes from Chronic Lymphocytic Leukemia (CLL) and lymphoma patients. We have generated a series of mAbs against NTB-A and assessed their therapeutic potential in preclinical models. Selected mAbs to NTB-A were further tested in functional Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL and lymphoma patients. Potent cytotoxic activity was demonstrated against B cells isolated from CLL patients and B lymphoma cell lines. Chimeric anti-NTB-A mAbs demonstrated anti-tumor activity equal to rituximab against CA46 human lymphoma xenografts in nude mice at a low dose. In a chimpanzee safety study, a single dose of lead anti-NTB-A mAb 994.1 resulted in near-complete depletion of peripheral lymphocytes while having a minimal effect on T cell activation. Taken together, these results demonstrate NTB-A as a promising target with an acceptable safety profile for immunotherapy of leukemia and lymphomas.

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Research Resource: Comparison of Gene Profiles From Wild-Type ERα and ERα Hinge Region Mutants

Molecular Endocrinology ◽

10.1210/me.2014-1122 ◽

2014 ◽

Vol 28 (8) ◽

pp. 1352-1361 ◽

Cited By ~ 9

Author(s):

Katherine A. Burns ◽

Yin Li ◽

Liwen Liu ◽

Kenneth S. Korach

Keyword(s):

Cell Line ◽

Cell Lines ◽

Nuclear Export ◽

Target Genes ◽

Nuclear Translocation ◽

Biological Activities ◽

Hinge Region ◽

Wild Type ◽

Estrogen Response ◽

Rapid Action

We showed previously that the hinge region of estrogen receptor (ER) α is involved in mediating its actions. The hinge 1 (H1) ERα mutant has disrupted nuclear localization and has lost interaction with c-JUN, but retains estrogen response element (ERE)–mediated functions. The hinge 2 + nuclear export sequence (H2NES) ERα mutant does not maintain nuclear translocation with hormone and no longer activates ERE target genes but does retain a nongenomic, nonnuclear, rapid-action response. Herein, we used the human endometrial cancer Ishikawa stable cell lines (Ishikawa/vector, Ishikawa/wild-type [WT] ERα, Ishikawa/H1 ERα, or Ishikawa/H2NES ERα) to characterize the biological activities of these 2 ERα hinge region mutants. We confirmed by confocal microscopy increased cytoplasmic ERα in the H1 ERα cell line and full cytoplasmic ERα localization in the H2NES ERα cell line. Luciferase assays using the 3xERE reporter showed activation of H1 ERα and H2NES ERα by estradiol (E2) treatment, but using the endogenous pS2 reporter, luciferase activity was only seen with the H1 ERα cell line. Examining cell proliferation revealed that only the WT ERα and H1 ERα cell lines increased proliferation after treatment. Using microarrays, we found that WT ERα and H1 ERα cluster together, whereas vector and H2NES ERα are most similar and cluster independently of E2 treatment. These studies revealed that the nongenomic activities of ERα are unable to mediate proliferative changes or the transcriptional profile after treatment and demonstrate the importance of genomic action for ERα/E2-mediated responses with the nongenomic actions of ERα being complementary to elicit the full biological actions of ERα.

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Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽

10.1182/blood-2021-149442 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 248-248

Author(s):

Alice Bonato ◽

Riccardo Bomben ◽

Supriya Chakraborty ◽

Giulia Felician ◽

Claudio Martines ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Pi3k Inhibitor ◽

Leukemia Cells ◽

Wild Type ◽

Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P<0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P<0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P<0.001, with 1.0 μM IBR: 28% vs 53%, P<0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with >10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

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Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

10.21203/rs.3.rs-51227/v3 ◽

2020 ◽

Author(s):

Shejuan An ◽

Jeannie Camarillo ◽

Tina Huang ◽

Daphne Li ◽

Juliette Morris ◽

...

Keyword(s):

Cell Lines ◽

Radiation Treatment ◽

Diffuse Intrinsic Pontine Glioma ◽

Response To Treatment ◽

Wild Type ◽

Post Translational Modification ◽

Pontine Glioma ◽

Epigenetic Signatures ◽

The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

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The influence of IONPs core size on their biocompatibility and activity in in vitro cellular models

Scientific Reports ◽

10.1038/s41598-021-01237-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Natalia Janik-Olchawa ◽

Agnieszka Drozdz ◽

Damian Ryszawy ◽

Maciej Pudelek ◽

Karolina Planeta ◽

...

Keyword(s):

Cell Lines ◽

Therapeutic Potential ◽

Proliferation Rate ◽

Core Size ◽

Phagocytic Cells ◽

Migration Activity ◽

Core Diameter ◽

Cellular Models ◽

The Core ◽

The Impact

AbstractAlthough the key factor affecting the biocompatibility of IONPs is the core size, there is a lack of regular investigation concerning the impact of the parameter on the toxicity of these nanomaterials. Therefore, such studies were carried out in this paper. Their purpose was to compare the influence of PEG-coated-magnetite NPs with the core of 5, 10 and 30 nm on six carefully selected cell lines. The proliferation rate, viability, metabolic activity, migration activity, ROS levels and cytoskeleton architecture of cells have been evaluated for specified incubation periods. These were 24 and 72-h long incubations with IONPs administered in two doses: 5 and 25 µg Fe/ml. A decrease in viability was observed after exposure to the tested NPs for all the analyzed cell lines. This effect was not connected with core diameter but depended on the exposure time to the nanomaterials. IONPs increased not only the proliferation rate of macrophages—being phagocytic cells—but also, under certain conditions stimulated tumor cell divisions. Most likely, the increase in proliferation rate of macrophages contributed to the changes in the architecture of their cytoskeleton. The growth in the level of ROS in cells had been induced mainly by the smallest NPs. This effect was observed for HEK293T cells and two cancerous lines: U87MG (at both doses tested) and T98G (only for the higher dose). This requires further study concerning both potential toxicity of such IONPs to the kidneys and assessing their therapeutic potential in the treatment of glioblastoma multiforme.

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Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01773-x ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Shejuan An ◽

Jeannie M. Camarillo ◽

Tina Yi-Ting Huang ◽

Daphne Li ◽

Juliette A. Morris ◽

...

Keyword(s):

Cell Lines ◽

Radiation Treatment ◽

Diffuse Intrinsic Pontine Glioma ◽

Response To Treatment ◽

Wild Type ◽

Post Translational Modification ◽

Pontine Glioma ◽

Epigenetic Signatures ◽

The Impact

Abstract Background Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles. Methods We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7). Results We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

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Bioluminescence Imaging of Spontaneous Lymphomas and Contribution of Distinct NFκB Pathways.

Blood ◽

10.1182/blood.v108.11.2377.2377 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2377-2377

Author(s):

Lee Ratner ◽

Leon Bernal-Mizrachi ◽

Daniel Rauch ◽

John Harding ◽

Shimon Gross ◽

...

Keyword(s):

Cell Lines ◽

Target Genes ◽

Enzyme Inhibitor ◽

Bone Destruction ◽

Lymphocytic Leukemia ◽

Stable Form ◽

Death Domain ◽

Small Interfering Rnas ◽

Primary Tumors

Abstract The nuclear factor κB (NFκB) pathways have been implicated in tumorigenesis in several lymphoid malignancies, but the responsible mechanisms remain to be fully defined. EBV and HTLV-1 immortalized lymphoma cell lines with constitutively active NFκB were found to be resistant to inducers of the extrinsic and intrinsic apoptosis pathways. Several NFκB-target genes were over-expressed in these cell lines, including Bcl-xL, Fas-associated death domain-like IL-1β converting enzyme inhibitor protein (FLIP), and inhibitors of apoptosis (CIAP, XIAP, and survivin). Inhibition of the canonical or non-canonical NFκB pathways with small interfering RNAs (siRNA) or adenovirus expressing a stable form of inhibitor of NFκB (IκB) enhanced sensitivity to apoptosis inducers, and resulted in lower levels of expression these NFκB target genes. Repression of XIAP with siRNA was particularly potent in enhancing apoptosis sensitivity. These findings demonstrate an important role of both NFκB pathways in mediating resistance to apoptosis, and distinctive anti-apoptotic downstream target gene profiles responsible for this effect. In order to utilize HTLV-1 Tax activity as an in vivo biomarker for tumorigenesis, we generated transgenic mice with firefly luciferase driven by the HTLV-1 long terminal repeat (LTRLUC) and bred them with the GzBTax animals to produce double transgenic LTRLUC/GzBTax (LLT) mice which develop large granular lymphocytic leukemia, splenomegaly, localized bone destruction, and solid tumors of the ears, legs, nose, and tail. We found that the bioluminescence of the LLT mice serves as a reporter for spontaneous tumorigenesis in vivo, is transferable as a useful tumor marker in cell culture and allograft models derived from primary tumors, is inducible by immunomodulatory agents, and is a sensitive indicator for even the earliest events in tumor development.

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