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1.
Cancer Immunol Res ; 11(9): 1222-1236, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37378662

ABSTRACT

The receptor tyrosine kinase AXL is a member of the TYRO3, AXL, and proto-oncogene tyrosine-protein kinase MER family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.


Subject(s)
Neoplasms , Receptors, Chimeric Antigen , Humans , Axl Receptor Tyrosine Kinase , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases/genetics
2.
Blood Cancer J ; 12(7): 99, 2022 07 01.
Article in English | MEDLINE | ID: mdl-35778390

ABSTRACT

Constitutively activated B cell receptor (BCR) signaling is a primary biological feature of chronic lymphocytic leukemia (CLL). The biological events controlled by BCR signaling in CLL are not fully understood and need investigation. Here, by analysis of the chromatin states and gene expression profiles of CLL B cells from patients before and after Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib treatment, we show that BTKi treatment leads to a decreased expression of APOBEC3 family genes by regulating the activity of their enhancers. BTKi treatment reduces enrichment of enhancer marks (H3K4me1 and H3K27ac) and chromatin accessibility at putative APOBEC3 enhancers. CRISPR-Cas9 directed deletion or inhibition of the putative APOBEC3 enhancers leads to reduced APOBEC3 expression. We further find that transcription factor NFATc1 couples BCR signaling with the APOBEC3 enhancer activity to control APOBEC3 expression. We also find that enhancer-regulated APOBEC3 expression contributes to replication stress in malignant B cells. In total we demonstrate a novel mechanism for BTKi suppression of APOBEC3 expression via direct enhancer regulation in an NFATc1-dependent manner, implicating BCR signaling as a potential regulator of leukemic genomic instability.


Subject(s)
APOBEC Deaminases , Leukemia, Lymphocytic, Chronic, B-Cell , Receptors, Antigen, B-Cell , APOBEC Deaminases/biosynthesis , APOBEC Deaminases/genetics , APOBEC Deaminases/metabolism , Chromatin , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/metabolism
3.
Blood Adv ; 6(11): 3458-3471, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35395072

ABSTRACT

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study, hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration, indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed, HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters naïve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary, HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME.


Subject(s)
Extracellular Vesicles , Multiple Myeloma , Humans , Intercellular Signaling Peptides and Proteins , Proteomics , Tumor Microenvironment
4.
Noncoding RNA ; 8(1)2022 Feb 05.
Article in English | MEDLINE | ID: mdl-35202088

ABSTRACT

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

5.
Blood ; 138(26): 2810-2827, 2021 12 30.
Article in English | MEDLINE | ID: mdl-34407545

ABSTRACT

E1912 was a randomized phase 3 trial comparing indefinite ibrutinib plus 6 cycles of rituximab (IR) to 6 cycles of fludarabine, cyclophosphamide, and rituximab (FCR) in untreated younger patients with CLL. We describe measurable residual disease (MRD) levels in E1912 over time and correlate them with clinical outcome. Undetectable MRD rates (<1 CLL cell per 104 leukocytes) were 29.1%, 30.3%, 23.4%, and 8.6% at 3, 12, 24, and 36 months for FCR, and significantly lower at 7.9%, 4.2%, and 3.7% at 12, 24, and 36 months for IR, respectively. Undetectable MRD at 3, 12, 24, and 36 months was associated with longer progression-free survival (PFS) in the FCR arm, with hazard ratios (MRD detectable/MRD undetectable) of 4.29 (95% confidence interval [CI], 1.89-9.71), 3.91 (95% CI, 1.39-11.03), 14.12 (95% CI, 1.78-111.73), and not estimable (no events among those with undetectable MRD), respectively. In the IR arm, patients with detectable MRD did not have significantly worse PFS compared with those in whom MRD was undetectable; however, PFS was longer in those with MRD levels <10-1 than in those with MRD levels above this threshold. Our observations provide additional support for the use of MRD as a surrogate end point for PFS in patients receiving FCR. In patients on indefinite ibrutinib-based therapy, PFS did not differ significantly by undetectable MRD status, whereas those with MRD <10-1 tended to have longer PFS, although continuation of ibrutinib would very likely be necessary to maintain treatment efficacy.


Subject(s)
Adenine/analogs & derivatives , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Neoplasm, Residual/diagnosis , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Adenine/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cyclophosphamide/therapeutic use , Female , Humans , Male , Middle Aged , Prognosis , Progression-Free Survival , Rituximab/therapeutic use , Treatment Outcome , Vidarabine/analogs & derivatives , Vidarabine/therapeutic use
6.
Blood ; 137(10): 1365-1376, 2021 03 11.
Article in English | MEDLINE | ID: mdl-32992344

ABSTRACT

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.


Subject(s)
Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Gene Frequency , Gene Rearrangement , Humans , Somatic Hypermutation, Immunoglobulin
8.
Oncotarget ; 10(1): 60-75, 2019 Jan 01.
Article in English | MEDLINE | ID: mdl-30713603

ABSTRACT

Predicting disease progression in chronic lymphocytic leukemia (CLL) remains challenging particularly in patients with Rai Stage 0/I disease that have an unmutated immunoglobulin heavy chain variable region (UM IGHV). Even though patients with UM IGHV have a poor prognosis and generally require earlier treatment, not all UM IGHV patients experience more rapid disease progression with some remaining treatment free for many years. This observation suggests biologic characteristics other than known prognostic factors influence disease progression. Alterations in long non-coding RNA (lncRNA) expression levels have been implicated in diagnosis and prognosis of various cancers, however, their role in disease progression of early Rai stage UM CLL is unknown. Here we use microarray analysis to compare lncRNA and mRNA profiles of Rai 0/I UM IGHV patients who progressed in <2 years relative to patients who had not progressed for >5 years. Over 1,300 lncRNAs and 940 mRNAs were differentially expressed (fold change ≥ 2.0; p-value ≤ 0.05). Of interest, the differentially expressed lncRNAs T204050, NR_002947, and uc.436+, have known associated genes that have been linked to CLL. Thus, our study reveals differentially expressed lncRNAs in progressive early stage CLL requiring therapy versus indolent early Rai stage UM CLL. These lncRNAs have the potential to impact relevant biological processes and pathways that influence clinical outcome in CLL.

9.
Exp Hematol ; 57: 42-49.e1, 2018 01.
Article in English | MEDLINE | ID: mdl-29030084

ABSTRACT

The genetic abnormalities underlying multiple myeloma (MM) are notoriously complex and intraclonal heterogeneity is a common disease feature. In the current study, we describe the establishment of a monoclonal immunoglobulin A (IgA) kappa (κ) MM cell line designated MC-B11/14. Cytogenetic and fluorescence in situ hybridization analyses of the original and relapse patient samples revealed that the MM clone was nonhyperdiploid and possessed an 11;14 chromosomal translocation. The MC-B11/14 cell line, established from the relapse sample, is tetraploid and houses the t(11;14) abnormality. Given our long-standing interest in Ig function and secretion, we next used CRISPR technology to knock out IgA heavy-chain expression in the MC-B11/14 cells to assess the biological consequences of converting this cell line to one only expressing κ light chains. As expected, secretion of intact IgA was undetectable from MC-B11/14IgA- cells. Sensitivity to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, MC-B11/14IgA- cells were found to be significantly more resistant to bortezomib treatment. This study describes the establishment of a new human MM cell line tool with which to study disease biology and the use of CRISPR technology to create a potentially useful model with which to study MM light-chain escape.


Subject(s)
CRISPR-Cas Systems , Cell Line, Tumor , Gene Knockout Techniques , Genes, Immunoglobulin , Immunoglobulin A/genetics , Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/pathology , Amino Acid Sequence , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Base Sequence , Bone Marrow Transplantation , Bortezomib/administration & dosage , Bortezomib/pharmacology , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 11/ultrastructure , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 14/ultrastructure , Combined Modality Therapy , Fatal Outcome , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoglobulin kappa-Chains/biosynthesis , Immunoglobulin kappa-Chains/genetics , Immunophenotyping , Male , Middle Aged , Multiple Myeloma/drug therapy , Multiple Myeloma/therapy , Myeloma Proteins/biosynthesis , Myeloma Proteins/genetics , Sequence Alignment , Tetraploidy , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Translocation, Genetic
10.
Am J Hematol ; 92(8): 759-763, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28402581

ABSTRACT

Akt is a downstream target of B cell receptor signaling and is a central regulator of CLL cell survival. We aim to investigate the safety and efficacy of the Akt inhibitor MK-2206 in combination with bendamustine and rituximab (BR) in relapsed and/or refractory CLL in a phase I/II study. A standard phase I design was used with cohorts of three plus three patients to determine the maximum tolerated dose (MTD) of MK-2206 in combination with BR in relapsed CLL. Single-agent MK-2206 (weekly dosed) was administered one-week in advance before BR on cycle 1 and subsequently was given with BR at the same time for cycle 2-6. Phase II employed the MTD of MK-2206 with BR to evaluate safety and efficacy of this study combination. Thirteen relapsed/refractory CLL were treated for maximal 6-cycle of therapy. The maximum tolerated dose of MK-2206 was 90 mg by mouth once weekly. The most common grade 3/4 adverse events were neutropenia (46%), febrile neutropenia (23%), rash (15%), diarrhea (15%), and thrombocytopenia (15%). Overall response rate was 92% with a median progression free survival and treatment free survival of 16 and 24 months, respectively. Five patients (38%) achieved complete remission or complete remission with incomplete count recovery, two of whom were MRD negative. The efficacy and tolerability of this combination indicates that Akt inhibition combined with chemoimmunotherapy is a promising novel treatment combination in CLL and deserves further prospective clinical trial.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Bendamustine Hydrochloride/administration & dosage , Female , Follow-Up Studies , Heterocyclic Compounds, 3-Ring/administration & dosage , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Male , Middle Aged , Neoplasm Staging , Recurrence , Retreatment , Rituximab/administration & dosage , Survival Analysis , Treatment Outcome
11.
Clin Chem ; 62(1): 243-51, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26430073

ABSTRACT

BACKGROUND: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions. METHODS: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment. RESULTS: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%). CONCLUSIONS: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration.


Subject(s)
Immunoglobulin Light Chains/blood , Multiple Myeloma/blood , Multiple Myeloma/diagnosis , Neoplasm, Residual/blood , Neoplasm, Residual/diagnosis , Peptides/blood , Bone Marrow/pathology , Bone Marrow Examination , Complementarity Determining Regions/blood , Complementarity Determining Regions/genetics , Humans , Immunoglobulin Light Chains/genetics , Multiple Myeloma/pathology , Neoplasm, Residual/pathology , Peptides/genetics , RNA, Messenger/blood , RNA, Messenger/genetics , Suction
12.
J Proteome Res ; 14(4): 1957-67, 2015 Apr 03.
Article in English | MEDLINE | ID: mdl-25734799

ABSTRACT

Immunoglobulin light chain (LC) amyloidosis (AL) is caused by deposition of clonal LCs produced by an underlying plasma cell neoplasm. The clonotypic LC sequences are unique to each patient, and they cannot be reliably detected by either immunoassays or standard proteomic workflows that target the constant regions of LCs. We addressed this issue by developing a novel sequence template-based workflow to detect LC variable (LCV) region peptides directly from AL amyloid deposits. The workflow was implemented in a CAP/CLIA compliant clinical laboratory dedicated to proteomic subtyping of amyloid deposits extracted from either formalin-fixed paraffin-embedded tissues or subcutaneous fat aspirates. We evaluated the performance of the workflow on a validation cohort of 30 AL patients, whose amyloidogenic clone was identified using a novel proteogenomics method, and 30 controls. The recall and negative predictive values of the workflow, when identifying the gene family of the AL clone, were 93 and 98%, respectively. Application of the workflow on a clinical cohort of 500 AL amyloidosis samples highlighted a bias in the LCV gene families used by the AL clones. We also detected similarity between AL clones deposited in multiple organs of systemic AL patients. In summary, AL proteomic data sets are rich in LCV region peptides of potential clinical significance that are recoverable with advanced bioinformatics.


Subject(s)
Amyloid/metabolism , Amyloidosis/diagnosis , Amyloidosis/metabolism , Immunoglobulin Light Chains/metabolism , Immunoglobulin Variable Region/metabolism , Peptides/isolation & purification , Proteomics/methods , Cohort Studies , Computational Biology , Humans , Peptides/metabolism
13.
Blood ; 125(3): 492-8, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25377784

ABSTRACT

Recent high-throughput sequencing and microarray studies have characterized the genetic landscape and clonal complexity of chronic lymphocytic leukemia (CLL). Here, we performed a longitudinal study in a homogeneously treated cohort of 12 patients, with sequential samples obtained at comparable stages of disease. We identified clonal competition between 2 or more genetic subclones in 70% of the patients with relapse, and stable clonal dynamics in the remaining 30%. By deep sequencing, we identified a high reservoir of genetic heterogeneity in the form of several driver genes mutated in small subclones underlying the disease course. Furthermore, in 2 patients, we identified convergent evolution, characterized by the combination of genetic lesions affecting the same genes or copy number abnormality in different subclones. The phenomenon affects multiple CLL putative driver abnormalities, including mutations in NOTCH1, SF3B1, DDX3X, and del(11q23). This is the first report documenting convergent evolution as a recurrent event in the CLL genome. Furthermore, this finding suggests the selective advantage of specific combinations of genetic lesions for CLL pathogenesis in a subset of patients.


Subject(s)
Biomarkers, Tumor/genetics , Evolution, Molecular , Genetic Heterogeneity , High-Throughput Nucleotide Sequencing , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/genetics , Case-Control Studies , Chromosome Aberrations , Disease Progression , Follow-Up Studies , Humans , Longitudinal Studies , Prognosis
14.
J Proteome Res ; 13(11): 5198-205, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25134970

ABSTRACT

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.


Subject(s)
Agammaglobulinemia/blood , Hypergammaglobulinemia/blood , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Case-Control Studies , Humans , Immunoglobulin Light Chains/blood , Immunoglobulin lambda-Chains/blood , Immunoglobulin lambda-Chains/chemistry , Linear Models , Molecular Weight , Phenotype , Reference Values
15.
Oncotarget ; 5(14): 5686-99, 2014 Jul 30.
Article in English | MEDLINE | ID: mdl-25015330

ABSTRACT

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells within the bone marrow. There is a growing literature that tumor cells release biologically active microvesicles (MVs) that modify both local and distant microenvironments. In this study, our goals were to determine if MM cells release MVs, and if so, begin to characterize their biologic activity. Herein we present clear evidence that not only do both patient MM cells and human MM cell lines (HMCLs) release MVs, but that these MVs stimulate MM cell growth. Of interest, MM-derived MVs were enriched with the biologically active form of CD147, a transmembrane molecule previously shown by us to be crucial for MM cell proliferation. Using MVs isolated from HMCLs stably transfected with a CD147-GFP fusion construct (CD147GFP), we observed binding and internalization of MV-derived CD147 with HMCLs. Cells with greater CD147GFP internalization proliferated at a higher rate than did cells with less CD147GFP association. Lastly, MVs obtained from CD147 downregulated HMCLs were attenuated in their ability to stimulate HMCL proliferation. In summary, this study demonstrates the significance of MV shedding and MV-mediated intercellular communication on malignant plasma cell proliferation, and identifies the role of MV-enriched CD147 in this process.


Subject(s)
Basigin/biosynthesis , Multiple Myeloma/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cell Proliferation/physiology , Humans , Immunophenotyping , Multiple Myeloma/genetics , Multiple Myeloma/pathology
16.
J Proteome Res ; 13(4): 1905-10, 2014 Apr 04.
Article in English | MEDLINE | ID: mdl-24552626

ABSTRACT

Multiple myeloma is a disease characterized by a clonal expansion of plasma cells that secrete a monoclonal immunoglobulin also referred to as an M-protein. In the clinical laboratory, protein electrophoresis (PEL), immunofixation electrophoresis (IFE), and free light chain nephelometry (FLC) are used to detect, monitor, and quantify an M-protein. Here, we present an alternative method based on monitoring a clonotypic (i.e., clone-specific) peptide from the M-protein heavy chain variable region using LC-MS/MS. Tryptic digests were performed on IgG purified serum from 10 patients with a known IgG M-protein. Digests were analyzed by shotgun LC-MS/MS, and the results were searched against a protein database with the patient specific, heavy chain variable region gene sequence added to the database. In all 10 cases, the protein database search matched multiple clonotypic peptides from each patient's heavy chain variable region. The clonotypic peptides were then used to quantitate the amount of M-protein in patient serum samples using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The response for the clonotypic peptide observed by SRM correlated with the M-protein observed by PEL. In addition, the clonotypic peptide was clearly observed by SRM in samples that were negative by IFE and FLC. Monitoring clonotypic peptides using SRM has the capacity to redefine clinical residual disease because of its superior sensitivity and specificity compared with current analytical methods.


Subject(s)
Immunoglobulins/blood , Immunoglobulins/chemistry , Multiple Myeloma/blood , Proteomics/methods , Tandem Mass Spectrometry/methods , Chromatography, Liquid , Humans , Immunoglobulin Heavy Chains , Immunoglobulin Variable Region , Peptides
17.
Br J Haematol ; 163(3): 326-33, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24032430

ABSTRACT

Detection of a 17p13.1 deletion (loss of TP53) or 11q22.3 deletion (loss of ATM), by fluorescence in situ hybridization (FISH), in chronic lymphocytic leukaemia (CLL) patients is associated with a poorer prognosis. Because TP53 and ATM are integral to the TP53 pathway, we hypothesized that 17p13.1- (17p-) and 11q22.3- (11q-) occurring in the same cell (clonal 17p-/11q-) would confer a worse prognosis than either 17p- or 11q-. We studied 2184 CLL patients with FISH (1995-2012) for the first occurrence of 17p-, 11q-, or clonal 17p-/11q-. Twenty (1%) patients had clonal 17p-/11q-, 158 (7%) had 17p- (including 4 with 17p- and 11q- in separate clones), 247 (11%) had 11q-, and 1759 (81%) had neither 17p- nor 11q-. Eleven of 15 (73%) tested patients with clonal 17p-/11q- had dysfunctional TP53 mutations. Overall survival for clonal 17p-/11q- was significantly shorter (1·9 years) than 17p- (3·1 years, P = 0·04), 11q- (4·8 years, P ≤ 0·0001), or neither 17p- nor 11q- (9·3 years, P ≤ 0·0001). Clonal 17p-/11q- thus conferred significantly worse prognosis, suggesting that loss of at least one copy of both TP53 and ATM causes more aggressive disease. Use of an ATM/TP53 combination FISH probe set could identify these very-high risk patients.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 11/ultrastructure , Chromosomes, Human, Pair 17/ultrastructure , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Adult , Aged , Ataxia Telangiectasia Mutated Proteins/deficiency , Ataxia Telangiectasia Mutated Proteins/genetics , Clone Cells/ultrastructure , Female , Genes, Neoplasm , Genes, Tumor Suppressor , Genes, p53 , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Leukemia, Lymphocytic, Chronic, B-Cell/classification , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Lymphocytes/ultrastructure , Male , Middle Aged , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Prognosis , Retrospective Studies , Tumor Suppressor Protein p53/deficiency
18.
Oncotarget ; 3(4): 502-13, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22522905

ABSTRACT

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.


Subject(s)
Complementarity Determining Regions/genetics , Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/genetics , Plasma Cells/metabolism , Sequence Analysis, DNA/methods , Clone Cells , DNA/genetics , Gene Library , Genomic Library , Humans , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Plasma Cells/pathology , V(D)J Recombination/genetics
19.
PLoS One ; 6(10): e26056, 2011.
Article in English | MEDLINE | ID: mdl-21998751

ABSTRACT

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.


Subject(s)
B-Lymphocytes/cytology , Cell Differentiation , Gene Expression Regulation, Neoplastic , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Lymphoid Enhancer-Binding Factor 1/metabolism , Aged , Aged, 80 and over , B-Lymphocytes/drug effects , B-Lymphocytes/metabolism , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/immunology , Cytokines/pharmacology , Cytoplasm/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/immunology , Humans , Immunoglobulin Light Chains/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Oligodeoxyribonucleotides/pharmacology , Positive Regulatory Domain I-Binding Factor 1 , Repressor Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Wnt Proteins/metabolism
20.
Cancer Genet Cytogenet ; 203(2): 161-8, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21156228

ABSTRACT

To better understand the implications of genomic instability and outcome in B-cell chronic lymphocytic leukemia (CLL), we sought to address genomic complexity as a predictor of chemosensitivity and ultimately clinical outcome in this disease. We used array-based comparative genomic hybridization (aCGH) with a one-million probe array and identified gains and losses of genetic material in 48 patients treated on a chemoimmunotherapy clinical trial. We identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14, and 17. Higher genomic complexity, as a mechanism favoring clonal selection, was associated with shorter progression-free survival, and predicted a poor response to treatment. Of interest, CLL cases with loss of p53 surveillance showed more complex genomic features and were found both in patients with a 17p13.1 deletion and in the more favorable genetic subtype characterized by the presence of 13q14.1 deletion. This aCGH study adds information on the association between poor trial response and increasing genetic complexity as CLL progresses.


Subject(s)
Comparative Genomic Hybridization , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Aged , Antineoplastic Agents/pharmacology , Chromosomes, Human, Pair 13 , Chromosomes, Human, Pair 17 , Cohort Studies , Disease-Free Survival , Drug Therapy/methods , Female , Gene Deletion , Genes, p53 , Humans , Immunotherapy/methods , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Male , Middle Aged , Models, Genetic , Oligonucleotide Array Sequence Analysis , Prognosis
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