scholarly journals Single-Cell RNA Sequencing Suggests Novel Drivers of Chronic Lymphocytic Leukemia Patients with Ibrutinib Resistance

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1539-1539
Author(s):  
Hui Jin ◽  
Zijuan Wu ◽  
Huayuan Zhu ◽  
Lei Fan ◽  
Luqiao Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
Cell Line ◽  
Lymphocytic Leukemia ◽  
Blue Staining ◽  
Number Of Patients ◽  
Transcriptional Pattern ◽  
Ibrutinib Resistance

Abstract Objective: Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor. Results: ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolism Pathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I). Conclusion: In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-Cell RNA-seq Reveals LGALS1 and LAG3 as Novel Drivers of Ibrutinib Resistance in Chronic Lymphocytic Leukemia

2021 ◽  
Author(s):  
Hui Jin ◽  
Bin Huang ◽  
Zijuan Wu ◽  
Huayuan Zhu ◽  
Hanning Tang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Expression Profiles ◽  
Marker Gene ◽  
Lymphocytic Leukemia ◽  
Primary Cells ◽  
Ibrutinib Resistance

Abstract BackgroundChronic lymphocytic leukemia (CLL) is a highly heterogeneous malignant lymphoproliferative B-cell disorder that can be treated using ibrutinib, a Bruton’s tyrosine kinase inhibitor. However, the ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. MethodsHere, we identified lectin galactoside-binding soluble 1 (LGALS1) and lymphocyte-activating gene 3 (LAG3) as potential markers for ibrutinib-resistant CLL using single-cell RNA sequencing (scRNA-seq), and the results were validated in an ibrutinib-resistant CLL cell line (MEC1-IR) and primary cells from CLL patients. Marker-gene expression was detected while functional analyses were conducted with or without OTX008, a selective Galectin-1 inhibitor. ScRNA-seq revealed that the biological features, gene expression profiles, and clonal signatures of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-resistant CLL were distinct from those displayed by PBMCs from ibrutinib-sensitive patients.ResultsA close correlation between LGALS1 and LAG3 expression was observed and these factors were found to be highly expressed in ibrutinib-resistant CLL, with diagnostic and prognostic stratification, indicating that they may serve as drivers of ibrutinib-resistant CLL. Concordantly, LGALS1 and LAG3 expression was higher in ibrutinib-resistant CLL cells and primary cells, and OTX008 suppressed proliferation and induced apoptosis in both cells.ConclusionLGALS1 and LAG3 gene panel is promising indicator of ibrutinib-sensitivity and prognosis marker of CLL. LGALS1 inhibitor OTX008 is effective in CLL patients, both those naïve to and those resistant to ibrutinib.

scholarly journals Characterization of TET and IDH gene expression in chronic lymphocytic leukemia: comparison with normal B cells and prognostic significance

2016 ◽  
Vol 8 (1) ◽  
Author(s):  
Michaël Van Damme ◽  
Emerence Crompot ◽  
Nathalie Meuleman ◽  
Marie Maerevoet ◽  
Philippe Mineur ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia

Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1743-1743
Author(s):  
Chi-Ling Chiang ◽  
Frank W Frissora ◽  
Zhiliang Xie ◽  
Xiaomeng Huang ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Cell Line ◽  
Tumor Antigen ◽  
Targeted Delivery ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

scholarly journals Targeting PAK1 Overcomes Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1546-1546
Author(s):  
Zijuan Wu ◽  
LEI Fan ◽  
Luqiao Wang ◽  
Hanning Tang ◽  
Yi Miao ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
High Throughput ◽  
Combination Index ◽  
Lymphocytic Leukemia ◽  
Chromatin Conformation ◽  
Rna Seq ◽  
Ibrutinib Resistance

Abstract Objective: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance. Methods: An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining. Results: Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G). To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I). IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI<1) of this combinatorial therapy (Figure 1K). Conclusions: In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expressed barcodes enable clonal characterization of chemotherapeutic responses in chronic lymphocytic leukemia

2019 ◽  
Author(s):  
Aziz Al’Khafaji ◽  
Catherine Gutierrez ◽  
Eric Brenner ◽  
Russell Durrett ◽  
Kaitlyn E. Johnson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
Rna Sequencing ◽  
Genetic Alterations ◽  
Response To Therapy ◽  
Lymphocytic Leukemia ◽  
Single Cell Rna Sequencing ◽  
Cancer Cell Population

AbstractThe remarkable evolutionary capacity of cancer is a major challenge to current therapeutic efforts. Fueling this evolution is its vast clonal heterogeneity and ability to adapt to diverse selective pressures. Although the genetic and transcriptional mechanisms underlying these responses have been independently evaluated, the ability to couple genetic alterations present within individual clones to their respective transcriptional or functional outputs has been lacking in the field. To this end, we developed a high-complexity expressed barcode library that integrates DNA barcoding with single-cell RNA sequencing through use of the CROP-seq sgRNA expression/capture system, and which is compatible with the COLBERT clonal isolation workflow for subsequent genomic and epigenomic characterization of specific clones of interest. We applied this approach to study chronic lymphocytic leukemia (CLL), a mature B cell malignancy notable for its genetic and transcriptomic heterogeneity and variable disease course. Here, we demonstrate the clonal composition and gene expression states of HG3, a CLL cell line harboring the common alteration del(13q), in response to front-line cytotoxic therapy of fludarabine and mafosfamide (an analog of the clinically used cyclophosphamide). Analysis of clonal abundance and clonally-resolved single-cell RNA sequencing revealed that only a small fraction of clones consistently survived therapy. These rare highly drug tolerant clones comprise 94% of the post-treatment population and share a stable, pre-existing gene expression state characterized by upregulation of CXCR4 and WNT signaling and a number of DNA damage and cell survival genes. Taken together, these data demonstrate at unprecedented resolution the diverse clonal characteristics and therapeutic responses of a heterogeneous cancer cell population. Further, this approach provides a template for the high-resolution study of thousands of clones and the respective gene expression states underlying their response to therapy.

scholarly journals The IGLV3-21R110 Defines a Subset of Chronic Lymphocytic Leukemia with Intermediate Epigenetic Subtype and Poor Outcome

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Ferran Nadeu ◽  
Romina Royo ◽  
Guillem Clot ◽  
Martí Duran-Ferrer ◽  
Alba Navarro ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Rna Sequencing ◽  
Prognostic Value ◽  
Lymphocytic Leukemia ◽  
Whole Genome ◽  
Biological Features ◽  
Mutational Status ◽  
Bcr Signaling

Introduction: B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B-cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling conferring aggressive behavior. Epigenetic studies have defined three CLL subtypes based on methylation signatures reminiscent of pre- and post-germinal center B-cells named naïve-like (n-CLL), intermediate (i-CLL) and memory-like CLL (m-CLL) with different biological features. i-CLL carry a borderline IGHV mutational load and a significant higher usage of IGHV3-21/IGLV3-21. The integration of these factors might translate into novel insights in CLL pathogenesis with implications on the proposed stratification of the patients. Aim: To determine the clinical and biological features of the IGLV3-21R110 in CLL in the light of the epigenetic subtypes and immunogenetic, genomic and transcriptomic landscapes of the tumors. Methods: We characterized the immunoglobulin (IG) gene of 584 CLL cases from whole-genome/exome and RNA sequencing using our recently developed algorithm IgCaller (Nadeu et al., Nat. Commun. 2020) and MiXCR, respectively. The genomic makeup of the tumors was obtained from whole-genome/exome sequencing while RNA sequencing data for 369 cases was used for gene expression analyses. Expression levels of WNT5A and WNT5B were verified by quantitative PCR with reverse transcriptase. Primary end points were time to first treatment (TTFT) and overall survival (OS) calculated from the date of diagnosis. All patients gave written informed consent. The study was approved by the Ethics Committee of the Hospital Clínic of Barcelona. Results: The IGLV3-21R110 was detected in 6.5% of cases being similarly distributed between mutated (6.5%) and unmutated (6.6%) IGHV cases (P=0.56). In contrast, the IGLV3-21R110 was found in 30/79 (38%) i-CLL compared to only 5/291 (1.7%) m-CLL and 1/189 (0.5%) n-CLL (P<0.001). All stereotyped subset #2 cases carried IGLV3-21R110 while 62% of IGLV3-21R110 i-CLL had non-stereotyped IG genes. IGLV3-21R110 i-CLL had a borderline IGHV mutational status (median 97.7%) that was higher than i-CLL lacking the IGLV3-21R110 (median 96.2%, P=0.005). IGLV3-21R110 i-CLL had significantly higher number of SF3B1 and ATM mutations, and total number of driver alterations. Nonetheless, the R110 mutation was the sole alteration in one i-CLL case and accompanied only by del(13q) in three. Although composite regarding IGHV mutational status, IGLV3-21R110 i-CLL transcriptomically resembled naïve-like/unmutated IGHV CLL and had a specific expression signature of 64 genes with overexpression of WNT5A and WNT5B as hallmarks. No differences were observed in the expression profile of subset #2 and non-subset #2 IGLV3-21R110 i-CLL tumors. On the other hand, i-CLL lacking the IGLV3-21R110 phenotypically mirrored memory-like/mutated IGHV cases. In relation to prognosis, IGLV3-21R110 i-CLL had a short TTFT and OS similar to n-CLL/unmutated IGHV cases whereas non-IGLV3-21R110 i-CLL had a good prognosis similar to memory-like/mutated IGHV. Therefore, i-CLL cases, which have been associated with an intermediate prognosis between m-CLL and n-CLL in previous studies, can be divided in two subgroups of cases with opposed clinical evolutions based on the IGLV3-21R110. Indeed, the IGLV3-21R110 and n-CLL subtype retained independent prognostic value in multivariate analyses while the i-CLL lost its prognostic prediction both for TTFT and OS. The prognostic value of the IGLV3-21R110 was also independent of the IGHV mutational status. In terms of applicability in the clinics, all n-CLL cases were classified as unmutated IGHV and 98% of m-CLL were mutated IGHV. Thus, either a complete IG characterization (IGHV mutational status and IGLV3-21R110) or the integration of the n-CLL subtype and IGLV3-21R110 identified virtually the same subset of patients with aggressive disease. Conclusions: The IGLV3-21R110 defines a CLL subset with borderline IGHV mutations, specific driver alterations, a gene expression signature including WNT5A/B overexpression, and an unfavorable prognosis independent of the IGHV mutational status and epigenetic subtypes. Our findings support the identification of IGLV3-21R110 CLL as a particular subgroup of the disease with relevance in the risk stratification of the patients. Disclosures Nadeu: Janssen: Honoraria. Campo:NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America..

B-Cell Scaffold Protein with Ankyrin Repeats (BANK1) Is Differentially Expressed in ZAP-70 Negative Compared to ZAP-70 Positive Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2938-2938
Author(s):  
Frank Dicker ◽  
Susanne Schnittger ◽  
Claudia Schoch ◽  
Alexander Kohlmann ◽  
Wei-Min Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Differentially Expressed Genes ◽  
Expression Profiling ◽  
Lymphocytic Leukemia ◽  
Differentially Expressed ◽  
Cell Scaffold

Abstract The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity > 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.

The Mevalonate Pathway As a Metabolic Target to Circumvent Multidrug-Resistance in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 735-735
Author(s):  
Marta Coscia ◽  
Micol Rigoni ◽  
Chiara Riganti ◽  
Ivana Campia ◽  
Candida Vitale ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Line ◽  
Stromal Cell ◽  
Rho Kinase ◽  
Lymphocytic Leukemia ◽  
Mdr1 Gene ◽  
Mdr1 Gene Expression ◽  
Stromal Cell Line

Abstract Abstract 735 The mutational status of the tumor immunoglobulin heavy chain variable region (IGHV) is a very reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. We have recently shown that UM CLL cells are more dependent on microenvironment-mediated signals for survival (Coscia M. et al, Leukemia, 2011), but it is currently unknown whether this also translates into an enhanced chemotherapy resistance. Multi drug resistance (MDR) is mediated by the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. The Pgp protein is the product of the mdr1 gene whose regulation is under the positive control of the mevalonate (Mev) metabolic pathway, the downstream Ras/RhoA dependent signaling pathways and the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α). The aim of this study was to investigate the MDR phenotype and signaling cascade in M and UM CLL cells under basal conditions and after exposure to prosurvival microenvironment-mediated signals. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from untreated CLL patients. More than 90% pure CLL cells were co-cultured with the murine stromal cell line M2-10B4. Culture conditions included: medium alone, Zoledronic acid (ZA) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L) and the HIF-1α inhibitor YC-1 (10 μmol/L). The level of activity of the Mev pathway was evaluated by measuring the intermediate metabolite farnesyl pyrophosphate (FPP) and the final product cholesterol (Chol) as previously described (Coscia M. et al, J Cell Mol Med, 2010). The Ras and RhoA activity, was evaluated by measuring the GTP binding. Rho kinase activity was measured using the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano). The following antibodies were used for Western Blot (WB) analyses: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2; anti-ERK 1/2; anti-HIF-1α; anti-Pgp; anti-GAPDH. The activity of HIF-1 in the nuclear extracts was assessed by the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). Mdr1 gene expression was evaluated by RT-PCR. Intracellular Doxorubicine (Doxo) accumulation was evaluated by cytofluorimetric analyses. We have found that the Mev pathway is significantly accelerated in freshly isolated UM vs M CLL cells. This hypermetabolic activity translates into a higher activation of the Ras/ERK-1/2 and RhoA/Rho kinase signaling pathways, and to a significantly more active HIF-1α in UM compared to M CLL cells. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to Pgp expression and more effective Doxo extrusion in these cells. The co-culture of CLL cells with the murine stromal cell line M2-10B4 further accelerated the Mev pathway and activated the HIF-1α/mdr1/PgP axis leading to even lower intracellular Doxo retention. This stroma-mediated effect was particularly evident in UM CLL cells. Targeting of the Mev pathway with ZA and statins, and targeting of ERK-1/2 and HIF-1α with specific inhibitors (PD85 and YC1-10, respectively) significantly reduced basal and stroma-induced activity of HIF-1α/mdr1/PgP axis, and significantly increased the intracellular concentrations of Doxo in UM CLL cells. These data indicate that the pharmacological inhibition of the Mev pathway and of the downstream HIF-1α/mdr1/PgP axis can be regarded as a novel potential strategy to circumvent basal and environment-mediated chemoresistance of CLL cells. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding, scientific adviser board.

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