Combinatorial Targeting of BCL6 and Anti-Apoptotic Proteins in Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 64-64
Author(s):  
Thibault Dupont ◽  
Zhenghong Dong ◽  
ShaoNing Yang ◽  
Ari Melnick ◽  
Leandro Cerchietti
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Constitutive Expression ◽  
B Cell Lymphoma ◽  
Cell Of Origin ◽  
Mutational Status ◽  
Apoptotic Genes ◽  
Apoptotic Proteins ◽  
The Impact

Abstract Abstract 64 BCL6 represents a survival factor in DLBCL and FL since specific BCL6 inhibitors (i.e: the peptidomimetic RI-BPI and the small molecule 79-6) kill DLBCL and transformed FL (tFL) cell lines. Our group showed that BCL2 and other anti-apoptotic genes are transcriptionally repressed by BCL6 and could be reactivated upon treatment with RI-BPI or 79-6. We also showed that BCL6 and BCL2 control distinct and non-overlapping survival pathways in these lymphomas. This suggests that blocking the function of anti-apoptotic proteins might overcome any resistance that these proteins might mediate in response to BCL6 inhibition. In addition, constitutive expression of BCL2 has been observed in DLBCL and FL cases. We therefore hypothesized that targeting both BCL6 and BCL2 would eliminate two of the most potent survival mechanisms and would translate in synergistic killing of these lymphomas. In order to test this hypothesis, we examined the proteome-wide consequence of BCL6 inhibition in a DLBCL cell line (SU-DHL6) transfected with siBCL6 by phospho-protein arrays. We found that 280 unique proteins changed their abundance after siBCL6, 40 of them related to (pro and anti) apoptosis signaling (p<0.001). By means of pathway analysis bioinformatic tools, we then identified anti-apoptotic proteins with increased abundance after siBCL6 that could be therapeutically targeted. Among these druggable proteins we found BCL2, BCL-XL, MCL-1, NEDD8, PARP1 and several ubiquitin ligases. We confirmed in independent experiments that BCL6 knockdown induced mRNA (by qPCR) and protein up-regulation (by immunobloting) of these genes in 2 additional DLBCL and 2 tFL cell lines. Treatment of these siBCL6-transfected cell lines with small molecules inhibitors of BCL2 family members (ABT-737 and oblatoclax), NEDD8 activating enzyme (MLN4924), PARP (olaparib) and proteasome (bortezomib) showed increased killing compared to each treatment alone. In order to identify rational combinatorial therapies that could be potentially translated for use in clinical trials, we performed additional studies with the BCL6 inhibitor RI-BPI that is being developed for clinical use. We first analyzed the impact of RI-BPI on the apoptosome in a panel of 6 DLBCL (SU-DHL6, Ly1, Ly7, Ly3, Ly10, SU-DHL4) and 4 tFL (DoHH2, WSU-DLCL2, Granta452, SC-1) cell lines. RI-BPI induced a profile of up-regulated pro and anti-apoptotic proteins similar to siBCL6. Because ABT-737 and obatoclax are active in DLBCL cells where apoptotic BH3 activators are neutralized by BCL2 or BCL-XL and RI-BPI treatment changes the stoichiometry of pro and anti-apoptotic proteins, we determined the post-RI-BPI BH3 profiling accordingly to the amount of BIM sequestration (by co-immunoprecipitation). Accordingly, sequential treatment of DLBCL and tFL cell lines with RI-BPI and ABT-737 or obatoclax synergistically killed BCL2/BCL-XL dependent cells (but not MCL-1 dependent cells). This effect was independent of the mutational status of BCL2, MCL1, BCL6, MYC and TP53. Olaparib was not tested in combination since most cell lines were resistant to clinically achievable concentrations of this drug. Bortezomib and MLN4924 were synergistic in most cell lines when combined with RI-BPI (as determined by isobologram analysis). The synergistic killing was associated with increase in caspase 7/3 activation (by a plate-based assay) and NFkB inhibition (by p65 DNA binding assay). This effect was independent of the cell of origin classification of the cell line (i.e. ABC vs. GCB). We then tested the combination of RI-BPI with ABT-737, MLN4924 or bortezomib in Ly1 xenograft models (n=10 mice per combination). Ly1 represents a DLBCL with 3q27 and t(14,18). We found that after 10 days of treatment, each combinatorial treatment was more effective than their individual components (p=0.02, p=0.01 and p<0.01 for RI-BPI with ABT-737, MLN4924 and bortezomib, respectively; T-test, day 10). Detailed toxicity studies revealed no toxicity excess with these combinations. In sum, our work shows that pharmacologic targeting of anti-apoptotic pathways induced by inhibition of BCL6 activity successfully sensitized DLBCL and tFL cells to apoptosis. This effect was evident in cells in which the apoptosis resistant mechanism evolved as response to BCL6 inhibition and gene de-repression as well as those with constitutive overexpression of anti-apoptotic genes. Disclosures: No relevant conflicts of interest to declare.

New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2641-2641
Author(s):  
Suning Chen ◽  
Stefan Nagel ◽  
Bjoern Schneider ◽  
Maren Kaufmann ◽  
Ursula R. Kees ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Small Sample ◽  
Upstream Region ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mir-155 Expression Raises the Apoptotic Threshold in Waldenström Macroglobulinemia By Inhibition of FOXO3a and Bim

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1671-1671 ◽  
Author(s):  
Brian T Gaudette ◽  
Lawrence H. Boise
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Therapeutic Index ◽  
Fold Increase ◽  
Malignant Cell ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Apoptotic Proteins ◽  
Induced Apoptosis

Abstract Waldenström Macroglobulinemia (WM) is a proliferative disorder of lymphoplasmacytic cells in the lymph nodes and bone marrow. Phenotypically, WM cells are CD19+, CD20+, CD22+, CD38+, CD138+/- and are lymphoid or plasmacytic in morphology. The disease is characterized by abundant secretion of monoclonal, IgM which causes much of morbidity associated with WM. The disease carries a high prevalence of activating somatic mutations in MyD88 (91%) and CXCR4 (28%), which have been shown to contribute to poor prognosis. These mutations involve signaling cascades that activate pathways known to enhance survival signaling including Bcl-xL. Generally, upregulation of pro-apoptotic Bcl-2 family proteins is observed as cancer cells break differentiation and proliferation checkpoints. To counter this, it becomes necessary for the cell to increase expression of anti-apoptotic Bcl-2 proteins making it dependent on a particular protein or set of proteins for survival. However, we have previously shown data that Bcl-2 family expression in WM is characterized by low expression of both pro- and anti-apoptotic proteins. To investigate a mechanism for this regulation, we examined the Bcl-2 family expression in three WM cell lines and observed that in two lines, BCWM.1 and MWCL-1, the pro-apoptotic BH3-only protein Bim was expressed at very low levels or absent, respectively, which corresponded with low sensitivity to inducers of Bim-dependent intrinsic apoptosis including ABT-737 and dexamethasone. These cell lines were sensitive to bortezomib which can induce apoptosis independent of Bim via a tBid-dependent mechanism. In the third WM cell line, RPCI-WM1, Bim was expressed at moderate levels but the pro-apoptotic proteins Bak and Bax were underexpressed and absent, respectively, which rendered the cell line completely apoptosis-deficient. Having ruled out genomic copy number variation at the loci corresponding to these genes and finding no evidence of epigenetic silencing by methylation, we examined the expression of microRNAs targeting these genes. We first examined the predicted targets of seven commonly dysregulated microRNAs in WM. Of these only one, miR-494, was found to have a moderately conserved target site in the 3’ UTR of Bim. However, the expression pattern of miR-494 did not correlate with the pattern of Bim expression in the WM cell lines. None of these microRNAs were predicted to target Bax or Bak. Therefore, we examined the expression of the remaining commonly dysregulated microRNAs and found that miR-155 was expressed at much higher levels in BCWM.1 and MWCL-1 than in RPCI-WM1 or the multiple myeloma (MM) cell line MM1.s. miR-155 is known to both directly and indirectly regulate FOXO3a, a transcription factor important in the induction of Bim. Confirming this, we observed low protein expression of FOXO3a in both BCWM.1 and MWCL-1 cells. To test this mechanism we stably expressed an anti-miR that targets miR-155 or a control anti-miR in all three WM cell lines and observed an increase in mRNA for FOXO3a and Bim as well as an increase in Bim protein in BWCM.1 and MWCL-1 cells expressing anti-miR-155, while no effect on Bim was observed in the RPCI-WM1 line that does not express miR-155 at high levels. This corresponded with a two-fold increase in ABT-737-induced apoptosis in both BWCM.1 and MWCL-1 in the absence of any additional death signal. As expected, miR-155 antagonism did not significantly increase bortezomib-induced apoptosis. These data indicate that miR-155 expression raises the apoptotic threshold in WM by limiting FOXO3a-mediated Bim expression. Cancer therapy relies on the ability to kill malignant cells at a lower dose than would kill healthy cells. This therapeutic index relies heavily on what is termed mitochondrial priming which is a measure of the expression of pro-apoptotic proteins in a cell. The malignant cell remains alive due to sequestration of these proteins by anti-apoptotic proteins, yet requires less death signaling to cause release of sufficient quantities of pro-apoptotic proteins to activate apoptosis. The data presented here indicate that increased expression of miR-155 raises the apoptotic threshold of WM cells by inhibiting Bim expression and thereby compromises the therapeutic index of many agents. Therefore, the sensitivity to a variety of apoptosis-inducing therapies would be increased by targeting miR-155 in combination as part of the treatment modality. Disclosures No relevant conflicts of interest to declare.

scholarly journals Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Miguel Quijada Álamo ◽  
Maria Hernandez-Sanchez ◽  
Ana E. Rodriguez ◽  
Claudia Pérez Carretero ◽  
Marta Martín Izquierdo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Depth Analysis ◽  
The Impact

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P &lt; 0.001; P &lt; 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

ERCC1, KRAS mutation, and oxaliplatin sensitivity in colorectal cancer: Old dogs and new tricks.

2012 ◽  
Vol 30 (4_suppl) ◽  
pp. 489-489 ◽  
Author(s):  
Armando Orlandi ◽  
Mariantonietta Di Salvatore ◽  
Michele Basso ◽  
Cinzia Bagalà ◽  
Antonia Strippoli ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Predictors Of Response ◽  
Mutational Status ◽  
Retrospective Clinical Study ◽  
Significant Difference ◽  
Before And After ◽  
The Impact

489 Background: Oxaliplatin is widely used in metastatic colorectal cancer, but currently there are not valid predictors of response to this drug. In our recent retrospective clinical study we have shown a greater efficacy of Oxaliplatin in patients with metastatic colorectal cancer with mutated (mt) K-RAS. We hypothesized that the mutational status of K-RAS could influence the expression of ERCC1, one of the main mechanisms of Oxaliplatin resistance. Methods: We used four cell lines of colorectal cancer: two K-RAS wild type (wt) (HCT-8 and HT-29) and two K-RAS mt (SW620 and SW480). We evaluated the sensitivity of these cell lines to Oxaliplatin by MTT-test and the ERCC1 levels before and after 24 h exposure to Oxaliplatin by Real-Time PCR. We silenced K-RAS in a K-RAS mt cell line to evaluate the impact on Oxaliplatin sensitivity and ERCC1 levels. We also silenced ERCC1 in order to confirm the importance of this protein as a Oxaliplatin resistance factor. Results: The K-RAS mt cell lines were more sensitive to Oxaliplatin (OR 2.68; IC 95% 1.511-4.757 p<0.001). The basal levels of ERCC1 did not show significant differences between K-RAS mt and wt cell line, however, after 24 h exposure to Oxaliplatin, only the K-RAS wt lines showed the ability to induce ERCC1, with a statistically significant difference (OR 42.9 IC 95% 17.260-106.972 p<0.0005). The silencing of K-RAS in K-RAS mt cell lines demonstrated to reduce sensitivity to Oxaliplatin associated with the acquisition of the ability to induce ERCC1. The silencing of ERCC1 in K-RAS wt cell lines enhance the sensibility to Oxaliplatin. Conclusions: The K-RAS mutated cell lines were more sensitive to Oxaliplatin. This feature seems to be secondary to the inability of these cells to induce ERCC1 after exposure to Oxaliplatin. K-RAS can thus be a predictor of response to Oxaliplatin in colorectal cancer representing a surrogate for ability to induce ERCC1.

The Allosteric AKT Inhibitor MK-2206 Demonstrates Potent Antiproliferative Activity in Lymphoma Cells and Synergizes with the HDAC Inhibitor Vorinostat,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3729-3729
Author(s):  
Daniela Buglio ◽  
Manuela Lemoine ◽  
Jaymie Estrella ◽  
Sattva S. Neelapu ◽  
Richard Eric Davis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Antiproliferative Activity ◽  
Hdac Inhibitor ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Parp Cleavage ◽  
Cell Of Origin ◽  
Akt Inhibitor ◽  
Lymphoma Cells

Abstract Abstract 3729 The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma. Disclosures: No relevant conflicts of interest to declare.

Outcomes Following Allogeneic Stem Cell Transplantation (AlloSCT) in Patients with Primary Mediastinal (PMBL), Germinal Center B (GCB) and Non-GCB Cell-like Diffuse Large B Cell Lymphomas (DLBCL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2563-2563 ◽  
Author(s):  
Issa F. Khouri ◽  
Rima M Saliba ◽  
Zijun Y. Xu-Monette ◽  
Martin Korbling ◽  
Gabriela Rondon ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cell ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
B Cell Lymphoma ◽  
Cell Of Origin ◽  
Bulky Disease ◽  
Stem Cell Source ◽  
The Impact

Abstract Background: Primary mediastinal (PMBL), GCB and non-GCB B-cell lymphoma are three major subtypes of DLBCL with distinct clinical outcomes. PMBL and DLBCL of GCB immunophenotype have a higher chance of cure than patients with non-GCB DLBCL when treated with conventional chemotherapy. In the relapsed/refractory setting, there is paucity of data regarding the impact of histological classification of DLBCL on survival following alloSCT in patients who were not eligible for or who have failed a prior autologous SCT (ASCT). Herein, we compare outcomes of alloSCT in these patients. Methods and Patients: We identified 101 de novo DLBCL patients who were treated at our center from January 1, 1998 to December 31, 2011. Seventeen (17%) tumors were PMBL. We determined the cell-of-origin in the remaining patients, based upon the Visco/Young and Choi immunohistochemical algorithms, and classified 62 (61%) as GCB and 22 (22%) as non-GCB tumors. GCB patients were older than non-GCB [median (range): 54 (21-70) vs 48(24-58) years, p=0.003)] and PMBL [26(19-48), P <0.001)]. Patients with PMBL, however, were more likely to have bulky disease at study entry (SE) than other subtypes [24% (PMBL) vs 5% (GCB) and 5% (non-GCB); P= 0.04]. Stage III-IV at SE in GCB, non-GCB and PMBL was present in 42%, 45% (P=0.8) and 35% (P=0.6), respectively; the number of prior chemotherapies received was 4 in both GCB and non-GCB and 5 in PMBL (P=0.3). The proportion of patients who have failed a prior ASCT was comparable in all three groups (32%, 32% and 47%, P=0.2). Refractory disease was present in 35% of GCB and PMBL patients at SE compared to 14% in non-GCB patients (P=0.03). Distribution of IPI, LDH, and proportion of PET+ patients (47%, 45% and 47%) were not statistically different in all 3 groups. A larger proportion of GCB patients (44%, reference) received non-myeloablative conditioning than non-GCB (23%, P=0.07), or PMBL (12%, P=0.3); the remaining patients received reduced-intensity conditioning (RIC). The proportion of patients who received a matched sibling donor in GCB, non-GCB and PML were 69% (reference), 64% (P=0.6) and 41% (P=0.03), respectively. The remaining patients received matched unrelated donors. More GCB patients received peripheral blood as stem cell source than non-GCB (89% vs 73%, P=0.08) or PMBL (47%, P=0.001) patients. Median year of transplant was 2006 in both GCB and non-GCB and 2004 in PMBL. Results: Median (range) follow-up months in surviving GCB, non-GCB and PML patients were 63 (5-157), 29 (7-117) and 52 (10-133) months respectively. The 3-year cumulative OS were 52% (reference), 18% (P=0.09) and 46% (P=0.6). The 3-year cumulative PFS were 39%, 12% (P=0.1) and 41% (P=0.9) (Figure). When we restricted the comparison to patients who had chemosensitive disease and received RIC, the 3-year OS rates for GCB, non-GCB and PMBL were 47%, 25% (HR=0.6, P=0.3) and 44% (HR=0.9, P=0.9), respectively. The 3-year PFS rates were 37%, 10% (HR=0.6, P=0.2) and 44% (HR=0.7, P=0.8). Non-relapse mortality at 3-years was 29% (reference), 47% (P=0.1), and 35% (P=0.6). The incidence of acute II-IV GVHD was 24%, 27% and 29%. The incidence of extensive chronic GVHD was 34%, 54% (P=0.07) and 41% (P=0.5). The major cause of death in all 3 subgroups was disease progression (38%, 38%, and 33%), followed by acute GVHD 14%, 13%, and 33%. Conclusions: Our results suggest a tendency for inferior survival after alloSCT in non-GCB when compared to GCB and PMBL subtypes of DLBCL. This occurred despite the younger age in the non-GCB versus GCB group. Innovative approaches are needed to improve outcomes in non-GCB patients after alloSCT. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Identification of Novel Epigenetic Therapies for ALK-Driven Haematological Malignancies

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1483-1483
Author(s):  
Stephen P. Ducray ◽  
Ricky Trigg ◽  
Andrew J. Bannister ◽  
Raymond Lai ◽  
Gerda Egger ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Haematological Malignancies ◽  
Front Line ◽  
Dnmt Inhibitor ◽  
Anaplastic Lymphoma

Introduction Through conserved signalling pathways, Anaplastic Lymphoma Kinase (ALK) is well-described in driving haematological malignancies including Anaplastic Large Cell Lymphoma (ALCL) and Diffuse Large B-Cell Lymphoma (DLBCL) and as such presents itself as an amenable therapeutic target. Hence, directed therapeutics (ALK tyrosine kinase inhibitors; TKI) are being used in the treatment of ALK-driven cancers. Unfortunately, findings in the clinic and clinical research studies have taught us that resistance to ALK inhibitors can develop through the activation of ALK signalling bypass tracks. As such there is a need for the development of novel front-line, dual-combination, as well as second-line therapies. Methods A large-scale epigenetic targeted drug library consisting of approximately 300 FDA-approved drugs and novel agents was applied to a number of cell lines representing ALK-driven haematological malignancies: ALCL cell lines (DEL, JB-6, KARPAS-299, SU-DHL-1, SUP-M2) and the DLBCL cell line LM-1. Drugs which caused a &gt;75% decrease in cell viability were classified as 'candidate drugs' and studied further. Results Several of the validated drugs have previously been used/trialled in the clinic for the treatment of various cancers, e.g. aurora kinase (XL-228), topoisomerase (Mitoxantrone HCl) and HDAC (Romidepsin) inhibitors - these functioned as internal controls for the drug screens. However, an assortment of novel drugs was also identified that have not previously been described in the context of the treatment of ALK-driven haematological malignancies; including the FLT3 inhibitor KW2449 which caused a &gt;75% decrease in viability in all the tested cell lines and as such may serve as a novel front line therapy. In addition, a novel DNA methyltransferase (DNMT) inhibitor was identified which is efficacious and resulted in a &gt;90% decrease in viability in all cell lines treated across both disease entities. Furthermore, we investigated the combinatorial potential of the identified DNMT inhibitor with ALK TKIs such as Brigatinib and observed the inhibitor acting synergistically (as per Bliss-Independence calculations) resulting in a further decrease in cell viability. Several cell lines that are resistant to ALK TKIs were also assessed for their sensitivity to the DNMT inhibitor and were shown to be susceptible to this drug, as demonstrated by a significant decrease in cell viability. Figure 1: (A) Viability following drug screen in a representative cell line. Those drugs which led to a &gt;75% change in viability were taken forward for validation, as shown in (B). (C) Candidate drugs identified for each of the cell lines tested, grouped according to their molecular target. Conclusion In conclusion, an epigenetic drug library has been employed to identify novel therapeutic agents for the treatment of ALK-driven haematological malignancies including ALCL and DLBCL. Data reveal a potent inhibitor of DNA methylation as a candidate drug that suppresses the growth of ALK-driven malignancies both alone and in combination with ALK TKIs. Significantly, this identified drug also inhibits the growth of cell lines resistant to directed therapeutics such as ALK TKIs suggesting it has potential clinical use. 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 A Novel Analysis Method for Evaluating the Interplay of Oxygen and Ionizing Radiation at the Gene Level

2021 ◽  
Vol 12 ◽  
Author(s):  
Jeannette Jansen ◽  
Patricia Vieten ◽  
Francesca Pagliari ◽  
Rachel Hanley ◽  
Maria Grazia Marafioti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Radiation Treatment ◽  
Hierarchical Cluster ◽  
Specific Gene ◽  
Analysis Method ◽  
Whole Genome Analysis ◽  
Gene Level ◽  
The Impact

Whilst the impact of hypoxia and ionizing radiations on gene expression is well-understood, the interplay of these two effects is not. To better investigate this aspect at the gene level human bladder, brain, lung and prostate cancer cell lines were irradiated with photons (6 Gy, 6 MV LINAC) in hypoxic and normoxic conditions and prepared for the whole genome analysis at 72 h post-irradiation. The analysis was performed on the obtained 20,000 genes per cell line using PCA and hierarchical cluster algorithms to extract the most dominant genes altered by radiation and hypoxia. With the help of the introduced novel radiation-in-hypoxia and oxygen-impact profiles, it was possible to overcome cell line specific gene regulation patterns. Based on that, 37 genes were found to be consistently regulated over all studied cell lines. All DNA-repair related genes were down-regulated after irradiation, independently of the oxygen state. Cell cycle-dependent genes showed up-regulation consistent with an observed change in cell population in the S and G2/M phases of the cell cycle after irradiation. Genes behaving oppositely in their regulation behavior when changing the oxygen concentration and being irradiated, were immunoresponse and inflammation related genes. The novel analysis method, and by consequence, the results presented here have shown how it is important to consider the two effects together (oxygen and radiation) when analyzing gene response upon cancer radiation treatment. This approach might help to unrevel new gene patterns responsible for cancer radioresistance in patients.

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