scholarly journals NOTCH1 Mutated IGHV Unmutated Chronic Lymphocytic Leukemia Cells Are Characterized By a Constitutive Overexpression of Nucleophosmin-1 and Ribosome-Associated Components

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3308-3308
Author(s):  
Federico Pozzo ◽  
Tamara Bittolo ◽  
Erika Tissino ◽  
Francesca Maria Rossi ◽  
Riccardo Bomben ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Protein Expression ◽  
Ribosomal Proteins ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
Control Vector ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Notch1 Mutations

Abstract Background: stabilizing mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al, Blood, 2012; Del Poeta et al, Br J Haematol, 2013; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform. Aim: to identify molecular/biological features of NOTCH1 mutated CLL. Methods: the presence of NOTCH1 mutations was investigated by ARMS-PCR or by direct sequencing. The percentage of NOTCH1 mutated DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR) and/or by next-generation-sequencing. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4x44K platform. Specific gene/protein validations were performed by QRT-PCR, western blotting, flow cytometry and immunofluorescence. Cell proliferation was evaluated by Cell Trace Violet assay. CLL-like cell line MEC-1 was transfected with a vector either with a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or with a NICD carrying a missense mutation (c.5304G>A), generating a stop codon at the beginning of the sequence, as control. Results:i) in a cohort of 588 IGHV-UM CLL, 144/588 (24.5%) cases carried NOTCH1 mutations (NOTCH1-mut cases), with a percentage of NOTCH1 mutated DNA ranging from 1% to 37%. NOTCH1-mut cases (8 cases; 11%-37% of NOTCH1 mutated DNA) showed higher NOTCH1 protein expression than NOTCH1 wild type cases (NOTCH1-wt, 11 cases) employing monoclonal antibodies either recognizing the trans-membrane (mean fold-change=3.0) or the intracellular (mean fold-change=2.1) NOTCH1 domain. ii) A GEP comparing purified cells of 5 IGHV-UM NOTCH1-mut cases (15%-37% of NOTCH1 mutated DNA) and 5 IGHV-UM NOTCH1-wt cases selected nucleophosmin-1 (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-mut CLL. A higher expression of NPM1 and of the genes codifying for 4 ribosomal proteins in NOTCH1-mut cases was validated in a wider independent series of 34 cases by QRT-PCR (18 NOTCH1-mut cases).iii) Western blot in 19 cases (8 NOTCH1-mut cases) confirmed a higher NPM1 protein expression in NOTCH1-mut cases (1.3-5.2 range of fold-change) also at protein level. When intracellular distribution and fluorescent intensity of NPM1 immunostaining were evaluated, an additional cytoplasmic staining was visible in NOTCH1-mut cases, along with a clear nucleolar staining visible in both NOTCH1-mut and NOTCH1-wt cases. NPM1 protein expression was also determined by an intra-nuclear staining by flow cytometry. By using this approach, 2 NOTCH1-mut cases were sorted according to NPM1 expression; notably, the NPM1high subpopulation showed a higher NOTCH1 mutational load than the NPM1low subpopulation. iv) MEC-1 cells transfected with mutated NICD showed higher NOTCH1 transcript and protein levels than MEC-1 cells transfected with the control vector (increments over the control: NICD transcript =2.2, NICD protein =1.3). These NICD mutated MEC-1 cells were characterized by higher NPM1 transcript and protein levels than the MEC-1 cells transfected with the control vector (increments over the control: NPM1 transcript =2.0, NPM1 protein =1.5). By performing a cell-trace-violet proliferation assay, NICD mutated MEC-1 cells were characterized by higher proliferation rates than MEC-1 cells transfected with the control vector (p=0.018). Finally, when the transfected MEC-1 cells were treated with 0.5 microM etoposide for 48 hours, NICD mutated MEC-1 cells presented higher viability rates than the MEC-1 cells transfected with the control vector, as evaluated by Annexin V/7-AAD staining (percentage of viable cells: 70% vs. 50%). Conclusions: NPM1 was constitutively overexpressed in NOTCH1-mut IGHV-UM CLL together with several ribosome-associated components. An increased activity of the ribosomal machinery/DNA-repair mechanisms (Lindstrom MS. Biochem.Res.Int. 2011) in NOTCH1-mut CLL can confer proliferative advantages and resistance to chemotherapeutic agents. Disclosures No relevant conflicts of interest to declare.

Nucleophosmin-1 and Ribosome-Associated Components Are Constitutively Overexpressed in NOTCH1 Mutated IGHV Unmutated CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3880-3880
Author(s):  
Michele Dal Bo ◽  
Federico Pozzo ◽  
Riccardo Bomben ◽  
Antonella Zucchetto ◽  
Erika Tissino ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Amplification Refractory Mutation System ◽  
Qrt Pcr ◽  
Mutational Status ◽  
Frameshift Deletion ◽  
Uptake Assay ◽  
Notch1 Mutations

Abstract Abstract 3880 Background: activating mutations of NOTCH1 have been identified in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM) CLL, chemorefractory CLL and CLL in advanced disease phases. In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain and cause an accumulation of an active NOTCH1 isoform. Notably, about 80% of NOTCH1 mutations are represented by a CT frameshift deletion at nucleotides 7544–7545 (c.7544–7545delCT). Clinically, the presence of NOTCH1 mutations is an independent predictor of overall survival in CLL and identifies a subset of patients with particularly unfavourable prognosis (Rossi et al, Blood, 119, 521, 2012). Aim: to identify peculiar molecular and biological features of NOTCH1 mutated CLL in the context of IGHV-UM CLL. Methods: the presence of the c.7544–7545delCT NOTCH1 frameshift deletion was investigated by an ad-hoc amplification refractory mutation system (ARMS) PCR set up to obtain an amplicon specific for the NOTCH1 mutated form and a second amplicon as control. The percentage of NOTCH1 DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR), calculating the ratio between the amount of the specific NOTCH1 mutated amplicon and the amount of the control amplicon, the latter representing the total amount of NOTCH1 DNA irrespective of its mutational status. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4×44K Agilent platform. The differential expression of specific genes/proteins was validated by QRT-PCR, western blotting and immunohistochemistry. A BrdU uptake assay was used to evaluate proliferation of CLL cells by CpG/IL2 stimulation. Results: in a cohort of 380 IGHV-UM CLL, the c.7544–7545delCT NOTCH1 mutation was found in 83/380 (21.8%) cases. QRT-PCR revealed a percentage of NOTCH1 mutated DNA ranging from 1 to 37%. CLL cases carrying the c.7544–7545delCT NOTCH1 mutation (NOTCH1-Mut) showed higher NOTCH1 protein expression than CLL cases lacking NOTCH1-Mut employing monoclonal antibodies either recognizing the trans-membrane (mean fold increase=3) or the intra-citoplasmic (mean fold increase=2.1) NOTCH1 domain. A GEP comparing RNA from purified CLL samples of 5 NOTCH1-Mut CLL and 5 CLL lacking NOTCH1-Mut was performed, selecting the 5 NOTCH1-Mut cases among those with the higher percentages of NOTCH1 mutated DNA (percentages of NOTCH1 mutated DNA ranging from 15 to 37%). This approach selected the nucleophosmin 1 gene (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-Mut CLL cases. A higher expression of the above mentioned genes in NOTCH1-Mut CLL was validated in a wider series of 34 cases (18 NOTCH1-Mut cases; NPM1, p=0.03; RPS6, p=0.045; RPS10, p=0.048; RPS17, p=0.048; RPS28, p=0.049; RPSA, p=0.048; RPL7A, p=0.039; RPL18, p=0.041, respectively). Western blot analysis in 8 cases (4 NOTCH1-Mut cases) confirmed a higher NPM1 expression in NOTCH1-Mut cases (range of fold increase from 1.6 to 5.2) also at protein level. Consistently, lymph nodes preparations from NOTCH1-Mut CLL cases revealed a strong NPM1 staining both in nucleoli and cytoplasms. Finally, when stimulated in-vitro with the CpG/IL2 combination, NOTCH1-mut IGHV-UM CLL cells proliferated, as detected by a BrdU uptake assay (>10 fold increase over control), and up-regulated NPM1 both at transcript (mean fold increase=2.02 after 18 hours of CpG exposure, p=0.001) and protein (fold increase of 1.34 after 6 hours of CpG exposure) levels. Conclusion: NPM1 was identified as constitutively overexpressed in NOTCH1-Mut IGHV-UM CLL together with several ribosome-associated components. These findings are suggestive for an increased activity of the ribosomal machinery in NOTCH1-Mut IGHV-UM CLL as part of the molecular processes leading to control of CLL cell growth and survival in this clinically unfavourable disease subset. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutations at 3' Untranslated Region (3'UTR) of NOTCH1 Are Associated with Low CD20 Expression Levels in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 306-306
Author(s):  
Tamara Bittolo ◽  
Federico Pozzo ◽  
Riccardo Bomben ◽  
Tiziana D'Agaro ◽  
Vanessa Bravin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Untranslated Region ◽  
Lymphocytic Leukemia ◽  
Transcript Levels ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Trisomy 12 ◽  
Anti Cd20 ◽  
Notch1 Mutations

Abstract Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p<0.0001) and non-trisomy 12 CLL (mean MFI in 16 3'UTR NOTCH1-mut cases = 2033.50 vs. mean MFI in 468 NOTCH1-wt cases = 3294.07; p=0.0001), and comparable to coding NOTCH1-mut cases (trisomy 12 CLL, mean MFI in 34 coding NOTCH1-mut cases = 2570.73; p=0.8530; non-trisomy 12 CLL, mean MFI in 56 coding NOTCH1-mut cases = 2538.13; p=0.1500) (Fig. 1b). Similarly, in both trisomy 12 CLL and non-trisomy 12 CLL categories, MS4A1 transcript levels were lower in 3'UTR NOTCH1-mut cases than in NOTCH1-wt cases (p=0.0274 and p=0.0072, respectively), again with expression levels comparable with coding NOTCH1-mut cases (p=0.2874 and p=0.9610, respectively, Fig. 1c). iv) Susceptibility to anti-CD20 in vitro. In keeping with CD20 levels, 3'UTR NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab, in CDC assay, than NOTCH1-wt cells (7 3'UTR NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.67% vs. 25.56%; p=0.0349; mean relative lysis upon ofatumumab: 39.26% vs. 60.64%; p=0.0286). In the same manner, coding NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab than NOTCH1-wt cases (9 coding NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.53% vs. 25.56%; p=0.0339; mean relative lysis upon ofatumumab: 30.60% vs. 60.64%; p=0.0114; Fig. 1d). v) Modulation of CD20 expression by NOTCH1 inhibition. GSI treatment increased both CD20 protein and transcript levels in 3'UTR NOTCH1-mut cases, as previously reported for coding NOTCH1-mut cases (p=0.0376 and p=0.0326, respectively; Fig. 1e,Pozzo et al., Leukemia, 2016). Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

Cytotoxic Effect of R-Etodolac (SDX-101) in Combination with Purine Analogues or Monoclonal Antibodies on Ex-Vivo B-Cell Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2122-2122
Author(s):  
Pawel Robak ◽  
Anna Linke ◽  
Barbara Cebula ◽  
Lorenzo M. Leoni ◽  
Tadeusz Robak ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Protein Expression ◽  
Cytotoxic Effect ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Apoptotic Protein ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil. Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX). Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic (<0.8) interactions. Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p<0.03, vs. single drugs alone). The SDX-101+2-CdA combination reduced viability by 27.8% (p<0.006 vs. single drugs), SDX-101+RTX reduced viability by 22.3% (p<0.01 vs. single drugs), and that of SDX-101+ALT combination reduced it by 18.7% (n.s.). The CIs for those combinations were 0.89, 1.17, 0.95, and 1.25, respectively. Apoptosis analysis by sub-G1 fraction and caspase-3 activity confirmed the viability results for each tested combination. Analysis of apoptosis-regulating proteins showed, that SDX-101 combined with RTX or 2-CdA distinctly up-regulated Bax (p<0.02, vs. single drugs), whereas other combinations did not significantly increase its expression. The expression of p73 protein showed 2–4 fold increase after treatment with SDX-101 alone, and all combinations further up-regulated p73, especially that with RTX (up to 6-fold increase in p73 expression). Interestingly, although SDX-101 (400 μM at 24 hr) increased protein expression of Mcl-1, SDX-101 combination with RTX, 2-CdA or FA caused a 2-3-fold decrease of its expression. Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.

Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Leukemia (SLL) Exhibit Altered Cytokine Protein Levels in Peripheral Blood Serum

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2646-2646
Author(s):  
Laura A Paganessi ◽  
Robin R Frank ◽  
Sucheta Jagan ◽  
Reem Karmali ◽  
Yan Li ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Fold Change ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
Protein Levels ◽  
Healthy Donors ◽  
Cytokine Levels

Abstract Abstract 2646 Introduction: Chronic lymphocytic leukemia (CLL)/Small lymphocytic leukemia (SLL) is one of the most common forms of indolent lymphomas in elderly adults. Currently, CLL is not treated until it develops into later stage disease. An increase in the knowledge of the biology of CLL could aid in the development of new treatment strategies for early stage CLL, thus positively impacting disease progression. We therefore investigated the cytokine levels present in peripheral blood (PB) serum of CLL patients and compared them to levels in healthy donors. Methods: PB was obtained from 25 untreated CLL and 3 untreated SLL patients and 29 normal healthy donors under an IRB approved protocol. Serum was stored at −80°C until analyzed. 86% of the patients were Rai 0/1, 3.5% Rai stage 2, and 10.5% unknown at diagnosis. Risk stratification based on cytogenetics and FISH found of the enrolled patients, 47% good, 25% intermediate, and 14% poor risk. The risk factor was unknown in 14%. β-2 microglobulin and 54 cytokine protein levels in 2 different serum samples per patient (collected at least one year post diagnosis) were measured in duplicate using MILLIPLEX™, a multiplex Luminex based technology. Genespring 11.5.1 software was used to convert the data into base-2 logarithmic values and then apply a median baseline transformation across all samples. Data is grouped according to the source of the sample (CLL or normal PB serum). A Filter on Volcano Plot analysis is then done. This filter analysis performs an unpaired t-test, which yields a p-value as well as computes the fold change of each cytokine. Results: As expected, β-2 microglobulin was significantly higher in CLL patients compared to normal samples (p=5.17×10−7, 1.79 fold). Using a minimum of a 1.5 fold change and p-value≤0.05, 16 cytokines had significantly higher expression and 9 cytokines had significantly lower expression in CLL samples compared to normal samples (see Table 1). Conclusion: These changes in cytokine levels help provide insight to the peripheral blood microenvironment, in which circulating CLL cells reside. Some cytokines were substantially higher in CLL patients; soluble IL-2 receptor alpha (sIL-2Rα) and stem cell factor (SCF). The substantially higher levels of sIL-2Rα have also been observed in follicular lymphoma (FL) and appear to predict which FL patients will have a reduced survival (Yang et al., Blood 2011). Levels of sIL-2Rα correlating to survival may be a phenomenon seen in all B-cell lymphomas. SCF is thought to be important in early B-cell development but its receptor c-kit (CD117) has not been reported to be expressed on B-CLL cells. The chemokine receptors CXCR4, CXCR5 and CCR7 are expressed on B-CLL cells. Interestingly, their corresponding ligands, CXCL12, CXCL13, and CCL21 are significantly higher (Table 1) in the PB serum of CLL patients. It is likely that existing immunomodulatory therapies, such as IMiDs, lenalidomide or thalidomide alter the PB levels of the cytokine milieau. Other successful hematological malignancy therapies involve targeted monoclonal antibodies. Therefore, a deeper understanding of the cytokine microenvironment will provide guidance in the development of future targeted therapies or highlight current therapies for other malignancies that could be promising for CLL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NOTCH1 Mutations Are Associated with Low CD20 Expression in Chronic Lymphocytic Leukemia: Evidences for a NOTCH1-Mediated Epigenetic Regulatory Mechanism

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 296-296
Author(s):  
Federico Pozzo ◽  
Tamara Bittolo ◽  
Pietro Bulian ◽  
Erika Tissino ◽  
Francesca Maria Rossi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Hdac Inhibitor ◽  
Lymphocytic Leukemia ◽  
Transcript Levels ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Mutational Burden ◽  
Cd20 Expression ◽  
Anti Cd20 ◽  
Notch1 Mutations

Abstract Background. NOTCH1 mutations are found in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases and in Richter Syndrome transformations (Rossi et al. Blood, 2013). In CLL, NOTCH1 mutations have been recently associated with clinical resistance to anti-CD20 immunotherapy (Stilgenbauer et al. Blood, 2014, Dal Bo et al. AHO, 2014). In lymphoproliferative disorders, susceptibility to rituximab is determined by CD20 levels (Golay et al. Blood, 2001), which are in turn epigenetically modulated via HDAC (Shimizu et al., Leukemia, 2010). Aim. To investigate whether NOTCH1 mutations could affect CD20 expression in CLL. Methods. NOTCH1 mutations and NOTCH1 mutational burden were evaluated by ARMS PCR, QRT-PCR, conventional and next generation sequencing. NOTCH1 protein levels were evaluated by western blot. CD20 expression was evaluated by flow cytometry. Transcript levels of MS4A1, encoding for CD20 protein, were evaluated by QRT-PCR. CLL cells and CLL-like cell line MEC-1 cells were treated with the HDAC inhibitor valproic acid (VPA) at a concentration of 3mM. Susceptibility to rituximab was evaluated by complement dependent cytotoxicity (CDC) assay. MEC-1 cells were transfected with a vector containing a NOTCH1 intracellular domain (NICD) carrying either the 7544-7545delCT or a stop codon at the beginning of NICD sequence (c.5304G>A), as control. Results. i) In a cohort of 452 CLL, 54 cases carried NOTCH1 mutations. NOTCH1-mutated cases (NOTCH1-mut) with high mutational burden showed 3.0- fold higher transmembrane NOTCH1 and 2.1- fold higher NICD protein expression compared to NOTCH1 wild-type cases (NOTCH1-wt). NOTCH1-mut showed a lower Mean Fluorescent Intensity (MFI) of CD20 expression than NOTCH1-wt both in trisomy 12 (tris12, 18 NOTCH1-mut vs. 44 NOTCH1-wt, p<0.001) and in non-tris12 (36 NOTCH1-mut vs. 354 NOTCH1-wt, p=0.005) cases. MS4A1 transcript levels were lower in NOTCH1-mut than in NOTCH1-wt (tris12 CLL, p=0.024; non-tris12 CLL, p=0.002). By performing cell sorting to isolate CD20low and CD20high subpopulations in 4 cases with subclonal NOTCH1 mutations (range 26%-45%), the CD20low subpopulation always showed an enrichment in NOTCH1 mutational burden when compared to the CD20high counterpart, along with lower MS4A1 expression levels. NICD mutated MEC-1 cells, expressing higher NICD transcript (2.2 over the control) and NICD (1.3 over the control) protein, were characterized by lower CD20 (0.1 over the control) and MS4A1 transcript (0.77 over the control) expression. ii) In keeping with CD20 levels, primary NOTCH1-mut CLL cells showed lower relative lysis induced by rituximab than NOTCH1-wt CLL cells (mean relative lysis: NOTCH1-mut, 5% vs. NOTCH1-wt, 30%, p=0.008). This phenomenon was confirmed by using the MEC-1 cell model (mean relative lysis: NICD mutated, 3% vs. control, 30%, p=0.006). iii) NICD mutated MEC-1 cells expressed higher levels of both HDAC1 (2.0 over the control) and HDAC2 (1.4 over the control) transcripts. Moreover, in co-immunoprecipitation experiments, NICD mutated MEC-1 cells were characterized by lower levels of HDAC1/HDAC2 bound to the transcriptional factor CBF-1, due to a competition for the binding to CBF1 with the high NICD levels present in these cells, and suggesting a nuclear interplay between NOTCH1 and HDAC where CBF-1 is the connecting element. The dependency of low CD20 expression by HDAC activity was demonstrated by the capability of VPA to increase CD20 expression in primary NOTCH1-mut CLL cells (1.3 over the control) and NICD mutated MEC-1 transfectants (1.4 over the control). Conclusions. CLL cases carrying NOTCH1 mutations are characterized by low CD20 expression levels that may confer resistance to anti-CD20 immunotherapy. The low CD20 expression, at least in part due to HDAC-dependent repression mechanism(s), can be reverted by HDAC inhibitor therapy. Disclosures No relevant conflicts of interest to declare.

BCL2 expression in chronic lymphocytic leukemia: lack of association with the BCL2 −938A>C promoter single nucleotide polymorphism

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 874-877 ◽  
Author(s):  
Aneela Majid ◽  
Olga Tsoulakis ◽  
Renata Walewska ◽  
Stefan Gesk ◽  
Reiner Siebert ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Chronic Lymphocytic Leukemia ◽  
Protein Expression ◽  
Lymphocytic Leukemia ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Protein Levels ◽  
Bcl2 Protein ◽  
High Level ◽  
Bcl2 Expression

High-level BCL2 expression is seen in most patients with chronic lymphocytic leukemia (CLL) in the absence of BCL2 chromosomal translocation. A single nucleotide polymorphism (SNP; −938C>A) within an inhibitory region of the BCL2 promoter has been reported to regulate BCL2 protein expression and to be associated with adverse prognostic features in CLL. We screened 276 patients with CLL for this SNP and 100 patients by quantitative Western blot for BCL2 expression. In contrast to the previous report, we found no association with BCL2 protein levels or with any clinical or laboratory parameters. BCL2 protein levels remained constant in 10 individual patients at different time points. A total of 19 patients with the lowest levels of BCL2 protein expression were biologically and clinically heterogeneous; 5 patients exhibited high-level BCL2 RNA expression and 4 were fludarabine resistant. BCL2 protein levels in CLL reflect a complex interplay of transcriptional and posttranscriptional controls, but do not appear to be associated with the −938C>A promoter SNP.

scholarly journals Protein expression analysis of chromosome 12 candidate genes in chronic lymphocytic leukemia (CLL)

Leukemia ◽  
2005 ◽  
Vol 19 (7) ◽  
pp. 1211-1215 ◽  
Author(s):  
D Winkler ◽  
C Schneider ◽  
A Kröber ◽  
L Pasqualucci ◽  
P Lichter ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Protein Expression ◽  
Candidate Genes ◽  
Expression Analysis ◽  
Lymphocytic Leukemia ◽  
Chromosome 12

scholarly journals A pilot study of lower doses of ibrutinib in patients with chronic lymphocytic leukemia

Blood ◽  
2018 ◽  
Vol 132 (21) ◽  
pp. 2249-2259 ◽  
Author(s):  
Lisa S. Chen ◽  
Prithviraj Bose ◽  
Nichole D. Cruz ◽  
Yongying Jiang ◽  
Qi Wu ◽  
...  
Keyword(s):  
Pilot Study ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Downstream Signaling ◽  
Protein Levels ◽  
Lower Drug ◽  
Dose Dependent ◽  
Initial Cycle ◽  
Different Doses ◽  
Dose Reductions

Abstract Ibrutinib is highly efficacious and used at 420 mg/d for treatment of chronic lymphocytic leukemia (CLL). We previously demonstrated a decline in Bruton’s tyrosine kinase (BTK) protein levels in CLL cells after 1 cycle of ibrutinib, suggesting ibrutinib dose could be lowered after the first cycle without loss of biological effect. To test this postulate, a pilot study (NCT02801578) was designed to systematically reduce ibrutinib dosing within the same patient with CLL over the course of three 28-day cycles. After an initial cycle of 420 mg/d, the dose was reduced to 280 mg/d in cycle 2, and then to 140 mg/d in cycle 3. Eleven patients began study treatment, and 9 completed the 3 cycles. Plasma and intracellular pharmacokinetics (PK), BTK occupancy, and pharmacodynamic (PD) response at different doses of ibrutinib were compared. Plasma and intracellular levels of ibrutinib were dose-dependent, and even the lowest dose was sufficient to occupy, on average, more than 95% of BTK protein. In concert, BTK downstream signaling inhibition was maintained with 140 mg/d ibrutinib in cycle 3, and there were comparable reductions in total and phospho-BTK (Tyr223) protein levels across 3 cycles. Reductions of plasma chemokine CCL3 and CCL4 levels, considered to be biomarkers of ibrutinib response, were similar during the 3 cycles. These PK/PD data demonstrate that after 1 cycle of ibrutinib at the standard 420 mg/d dose, the dose can be reduced without losing biological activity. Clinical efficacy of lower doses needs to be systematically evaluated. Such dose reductions would lower drug cost, lessen untoward toxicity, and facilitate rationale-based combinations. This trial was registered at www.clinicaltrials.gov as #NCT02801578.

Detection of t(14;18)(q32;q21) in B-Cell Chronic Lymphocytic Leukemia

2005 ◽  
Vol 129 (3) ◽  
pp. 410-411
Author(s):  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Susanne Schnittger ◽  
Claudia Schoch
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Chronic Lymphocytic Leukemia ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Lymphoma Therapy ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Cytomorphologic testing and multiparameter flow cytometry are the mainstays in diagnosing B-cell chronic lymphocytic leukemia, whereas fluorescence in situ hybridization that targets the translocation t(14;18)(q32;q21) often is used to identify follicular lymphoma. Therapy is highly diverse between both diseases. We describe a case with cytomorphologically and immunologically proven B-cell chronic lymphocytic leukemia in which t(14;18)(q32;q21) was found.

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