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1.
Front Oncol ; 13: 1043694, 2023.
Article in English | MEDLINE | ID: mdl-37114129

ABSTRACT

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by an aberrant cytokine network that can support tumor growth by triggering janus kinase (JAK)/STAT pathways. Targeting cytokine-signaling should then be a rational therapeutic strategy but the JAK inhibitor ruxolitinib failed to control and seemingly accelerated the disease in clinical trials. Methods: The effect of ruxolitinib on primary human CLL cells was studied in vitro and in vivo. Results: Ruxolitinib increased phosphorylation of IRAK4, an important toll-like receptor (TLR)- signaling intermediate, in circulating CLL cells in vitro. It also enhanced p38 and NFKB1 phosphorylation while lowering STAT3 phosphorylation in CLL cells activated with TLR-7/8 agonists and IL-2. Among the cytokines made by activated CLL cells, high levels of IL-10 contributed strongly to STAT3 phosphorylation and inhibited TLR7 activity. Ruxolitinib limited TLR-mediated IL10 transcription and markedly reduced IL-10 production in vitro. It also decreased blood levels of IL-10 while increasing TNFα along with phospho-p38 expression and gene sets associated with TLR-activation in CLL cells in vivo. The bruton's tyrosine kinase inhibitor ibrutinib decreased IL-10 production in vitro but, in contrast to ruxolitinib, blocked initial IL10 transcription induced by TLR-signaling in vitro, decreased TNFα production, and deactivates CLL cells in vivo. Discussion: These findings suggest the possible benefits of inhibiting growth factors with JAK inhibitors in CLL are outweighed by negative effects on potential tumor suppressors such as IL-10 that allow unrestrained activation of NFκB by drivers such as TLRs. Specific inhibition of growth-promoting cytokines with blocking antibodies or infusing suppressive cytokines like IL-10 might be better strategies to manipulate cytokines in CLL.

2.
J Immunol ; 209(9): 1662-1673, 2022 11 01.
Article in English | MEDLINE | ID: mdl-36104109

ABSTRACT

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.


Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Humans , Agammaglobulinaemia Tyrosine Kinase , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Phosphoric Monoester Hydrolases , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Tyrosine , Interferons
3.
Front Immunol ; 12: 772304, 2021.
Article in English | MEDLINE | ID: mdl-34868034

ABSTRACT

In the past decade, aberrant O-GlcNAcylation has emerged as a new hallmark of cancer. O-GlcNAcylation is a post-translational modification that results when the amino-sugar ß-D-N-acetylglucosamine (GlcNAc) is made in the hexosamine biosynthesis pathway (HBP) and covalently attached to serine and threonine residues in intracellular proteins by the glycosyltransferase O-GlcNAc transferase (OGT). O-GlcNAc moieties reflect the metabolic state of a cell and are removed by O-GlcNAcase (OGA). O-GlcNAcylation affects signaling pathways and protein expression by cross-talk with kinases and proteasomes and changes gene expression by altering protein interactions, localization, and complex formation. The HBP and O-GlcNAcylation are also recognized to mediate survival of cells in harsh conditions. Consequently, O-GlcNAcylation can affect many of the cellular processes that are relevant for cancer and is generally thought to promote tumor growth, disease progression, and immune escape. However, recent studies suggest a more nuanced view with O-GlcNAcylation acting as a tumor promoter or suppressor depending on the stage of disease or the genetic abnormalities, proliferative status, and state of the p53 axis in the cancer cell. Clinically relevant HBP and OGA inhibitors are already available and OGT inhibitors are in development to modulate O-GlcNAcylation as a potentially novel cancer treatment. Here recent studies that implicate O-GlcNAcylation in oncogenic properties of blood cancers are reviewed, focusing on chronic lymphocytic leukemia and effects on signal transduction and stress resistance in the cancer microenvironment. Therapeutic strategies for targeting the HBP and O-GlcNAcylation are also discussed.


Subject(s)
Hematologic Neoplasms/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Acylation , Animals , Hematologic Neoplasms/immunology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , N-Acetylglucosaminyltransferases/metabolism , T-Lymphocytes/immunology
4.
Cancer Med ; 10(24): 8789-8798, 2021 12.
Article in English | MEDLINE | ID: mdl-34791813

ABSTRACT

Preclinical observations that killing of chronic lymphocytic leukemia (CLL) cells was dexamethasone (DEX) were enhanced by concomitant inhibition of Bruton's tyrosine kinase and janus kinases (JAKs) motivated a phase II trial to determine if clinical responses to ibrutinib could be deepened by DEX and the JAK inhibitor ruxolitinib. Patients on ibrutinib at 420 mg daily for 2 months or with abnormal serum ß2M levels after 6 months or with persistent lymphadenopathy or splenomegaly after 12 months were randomized to receive DEX 40 mg on days 1-4 of a 4-week cycle for six cycles alone (three patients) or with ruxolitinib 15 mg BID on days 1-21 of each cycle (five patients). Ruxolitinib dosing was based on a previous phase I trial. Steroid withdrawal symptoms and significantly decreased serum IgG levels occurred in all patients regardless of their exposure to ruxolitinib. A fatal invasive fungal infection was seen in a patient taking DEX without ruxolitinib. Complete responses anticipated with addition of ruxolitinib were not seen. Gene expression studies suggested ruxolitinib had turned off interferon signaling in CLL cells and turned on genes associated with the activation of NFκB by TNF-α. Ruxolitinib increased blood levels of TNF-α by cycle 3 and decreased the inhibitory cytokine IL-10. These results suggest ruxolitinib releases activating signals for CLL cells that persist in patients on ibrutinib. This inhibitory JAK signaling may contribute to the therapeutic activity of ibrutinib. Thus JAK inhibitors provide no added value with ibrutinib for disease control and should be used with caution in CLL patients. Combining glucocorticoids with ibrutinib may increase the risk of serious infects.


Subject(s)
Adenine/analogs & derivatives , Janus Kinases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Piperidines/therapeutic use , Adenine/pharmacology , Adenine/therapeutic use , Aged , Female , Humans , Male , Middle Aged , Piperidines/pharmacology
5.
J Immunol ; 205(10): 2629-2639, 2020 11 15.
Article in English | MEDLINE | ID: mdl-33067379

ABSTRACT

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has profound activity in chronic lymphocytic leukemia (CLL) but limited curative potential by itself. Residual signaling pathways that maintain survival of CLL cells might be targeted to improve ibrutinib's therapeutic activity, but the nature of these pathways is unclear. Ongoing activation of IFN receptors in patients on ibrutinib was suggested by the presence of type I and II IFN in blood together with the cycling behavior of IFN-stimulated gene (ISG) products when IFN signaling was blocked intermittently with the JAK inhibitor ruxolitinib. IFN signaling in CLL cells from human patients was not prevented by ibrutinib in vitro or in vivo, but ISG expression was significantly attenuated in vitro. ISGs such as CXCL10 that require concomitant activation of NF-κB were decreased when this pathway was inhibited by ibrutinib. Other ISGs, exemplified by LAG3, were decreased as a result of inhibited protein translation. Effects of IFN on survival remained intact as type I and II IFN-protected CLL cells from ibrutinib in vitro, which could be prevented by ruxolitinib and IFNR blocking Abs. These observations suggest that IFNs may help CLL cells persist and specific targeting of IFN signaling might deepen clinical responses of patients on ibrutinib.


Subject(s)
Adenine/analogs & derivatives , Interferon Type I/metabolism , Interferon-gamma/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Piperidines/pharmacology , Adenine/pharmacology , Adenine/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Apoptosis/immunology , Cell Survival/drug effects , Cell Survival/immunology , Drug Resistance, Neoplasm/immunology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Nitriles , Piperidines/therapeutic use , Primary Cell Culture , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines , Receptors, Interferon/antagonists & inhibitors , Receptors, Interferon/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Tumor Cells, Cultured
6.
Cancer Med ; 8(4): 1540-1550, 2019 04.
Article in English | MEDLINE | ID: mdl-30843659

ABSTRACT

Methods to deepen clinical responses to ibrutinib are needed to improve outcomes for patients with chronic lymphocytic leukemia (CLL). This study aimed to determine the safety and efficacy of combining a janus kinase (JAK)-inhibitor with ibrutinib because JAK-mediated cytokine-signals support CLL cells and may not be inhibited by ibrutinib. The JAK1/2 inhibitor ruxolitinib was prescribed to 12 CLL patients with abnormal serum beta-2 microglobulin levels after 6 months or persistent lymphadenopathy or splenomegaly after 12 months on ibrutinib using a 3 + 3 phase 1 trial design (NCT02912754). Ibrutinib was continued at 420 mg daily and ruxolitinib was added at 5, 10, 15, or 20 mg BID for 3 weeks out of five for seven cycles. The break was mandated to avoid anemia and thrombocytopenia observed with ruxolitinib as a single agent in CLL. The combination was well-tolerated without dose-limiting toxicities. Cyclic changes in platelets, lymphocytes, and associated chemokines and thrombopoietic factors were observed and partial response criteria were met in 2 of 12 patients. The results suggest that JAK-signaling helps CLL cells persist in the presence of ibrutinib and ruxolitinib with ibrutinib is well-tolerated and may be a useful regiment to use in combination therapies for CLL.


Subject(s)
Janus Kinases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Protein Kinase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidines/therapeutic use , Signal Transduction/drug effects , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers , Cytokines/metabolism , Female , Humans , Janus Kinase Inhibitors/administration & dosage , Janus Kinase Inhibitors/adverse effects , Janus Kinase Inhibitors/therapeutic use , Janus Kinases/antagonists & inhibitors , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Lymphocyte Count , Male , Middle Aged , Nitriles , Piperidines , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/adverse effects , Pyrazoles/administration & dosage , Pyrazoles/adverse effects , Pyrimidines/administration & dosage , Pyrimidines/adverse effects , Treatment Outcome
7.
Cell Death Dis ; 10(2): 117, 2019 02 11.
Article in English | MEDLINE | ID: mdl-30741932

ABSTRACT

The ETS transcription factor Fli-1 controls the expression of genes involved in hematopoiesis including cell proliferation, survival, and differentiation. Dysregulation of Fli-1 induces hematopoietic and solid tumors, rendering it an important target for therapeutic intervention. Through high content screens of a library of chemicals isolated from medicinal plants in China for inhibitors of a Fli-1 transcriptional reporter cells, we hereby report the identification of diterpenoid-like compounds that strongly inhibit Fli-1 transcriptional activity. These agents suppressed the growth of erythroleukemic cells by inducing apoptosis and differentiation. They also inhibited survival and proliferation of B-cell leukemic cell lines as well as primary B-cell lymphocytic leukemia (B-CLL) isolated from 7 patients. Moreover, these inhibitors blocked leukemogenesis in a mouse model of erythroleukemia, in which Fli-1 is the driver of tumor initiation. Computational docking analysis revealed that the diterpenoid-like compounds bind with high affinity to nucleotide residues in a pocket near the major groove within the DNA-binding sites of Fli-1. Functional inhibition of Fli-1 by these compounds triggered its further downregulation through miR-145, whose promoter is normally repressed by Fli-1. These results uncover the importance of Fli-1 in leukemogenesis, a Fli-1-miR145 autoregulatory loop and new anti-Fli-1 diterpenoid agents for the treatment of diverse hematological malignancies overexpressing this transcription factor.


Subject(s)
DNA/metabolism , Diterpenes/chemistry , Proto-Oncogene Protein c-fli-1/metabolism , Animals , Apoptosis/drug effects , Binding Sites , Carcinogenesis/drug effects , Cell Line, Tumor , DNA/chemistry , Diterpenes/pharmacology , Diterpenes/therapeutic use , Gene Expression Regulation, Neoplastic/drug effects , Humans , Kaplan-Meier Estimate , Leukemia/drug therapy , Leukemia/mortality , Leukemia/pathology , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Docking Simulation , Promoter Regions, Genetic , Protein Structure, Tertiary , Proto-Oncogene Protein c-fli-1/antagonists & inhibitors , Proto-Oncogene Protein c-fli-1/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA, Small Interfering/therapeutic use
8.
Leukemia ; 33(7): 1650-1662, 2019 07.
Article in English | MEDLINE | ID: mdl-30696950

ABSTRACT

Glucocorticoid (GC) receptor (GR) phosphorylation and signature genes were studied in chronic lymphocytic leukemia (CLL) cells to help place GCs within modern treatment algorithms. In contrast to normal B and T cells, transcription of GC-regulated genes was not rhythmic and the synthetic GC dexamethasone (DEX) could not inhibit toll-like receptor (TLR)-responses in CLL cells. This intrinsic GC-resistance was associated with aberrant GR-phosphorylation on activating Ser211 and inhibitory Ser226 sites. Ibrutinib increased transcription of the GR-signature gene GILZ in circulating CLL cells along with GR(pS211)/GR(pS226) ratios and lytic sensitivity to DEX that were not reversed by the competitive antagonist mifepristone in vitro. However, ibrutinib could not improve GR-responses in circulating CLL cells activated with IL2 and TLR7/8 agonists to mimic conditions in pseudofollicle microenvironments. Addition of the janus kinase inhibitor ruxolitinib to block ibrutinib-insensitive signals increased GILZ transcription in pseudofollicle conditions in vitro and in a clinical trial (NCT02912754), and also increased GR(S211)/GR(S226) ratios and DEX-mediated killing in patient samples in vitro. These observations suggest that intrinsic resistance to endogenous GCs is characteristic of CLL cells and ibrutinib may help increase the therapeutic activity of GCs by non-canonical activation of GR.


Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Glucocorticoid/metabolism , Tumor Microenvironment/drug effects , Adenine/analogs & derivatives , Biomarkers, Tumor/genetics , Circadian Rhythm , Drug Therapy, Combination , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Nitriles , Phosphorylation , Piperidines , Receptors, Glucocorticoid/genetics , Toll-Like Receptors/metabolism , Transcriptional Activation , Tumor Cells, Cultured
9.
EBioMedicine ; 35: 222-232, 2018 Sep.
Article in English | MEDLINE | ID: mdl-30174282

ABSTRACT

The intrinsic humoral immunodeficiency of chronic lymphocytic leukemia (CLL) is often managed with immunoglobulin replacement therapy (IgRT) to maintain IgG levels in the low-normal range (6-8 g/L) but optimal targets for IgG and timing to commence IgRT are unclear. IgG levels fell near 6 g/L at rates of -0.85±0.14 g/L/year in 51 patients who required treatment for CLL within 4.5±0.4 years from initial diagnosis and - 0.27±0.04 g/L/year in 40 patients with progressive disease who remained untreated after 8.5±0.5 years. In contrast, endogenous IgG levels remained above 8 g/L in patients with highly indolent disease (n = 25) and TNFα and beta-2-microglobulin (ß2M) in blood decreased when IgRT was used to increase IgG levels over 9 g/L. At 15 g/L but not 5 g/L, the IgRT product Hizentra® inhibited B cell receptor (BCR)-activation, TNFα production, and survival in vitro, particularly of CLL cells that spontaneously made little TNFα. These findings suggest deterioration of the humoral immune system is associated with progressive CLL and altering the dosing of IgRT to achieve higher than conventional IgG target levels may have therapeutic activity.


Subject(s)
Disease Progression , Immunoglobulin G/blood , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Tumor Necrosis Factor-alpha/blood , Adenine/analogs & derivatives , Cell Death/drug effects , Humans , Immunoglobulin G/pharmacology , Injections, Subcutaneous , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Piperidines , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Time Factors , beta 2-Microglobulin/blood
10.
Leuk Res ; 66: 28-38, 2018 03.
Article in English | MEDLINE | ID: mdl-29353760

ABSTRACT

The tumor microenvironment (TME) is critical to the longevity of tumor B cells in chronic lymphocytic leukemia (CLL). Bone marrow mesenchymal stem cells (BMMSCs) and the cytokines they produce including IL-6 are important components of the TME in CLL. We found BMMSCs supported the survival of CLL cells in vitro through an IL-6 dependent mechanism. IL-17 which induces IL-6 generation in a variety of cells increased production of IL-6 both in CLL cells and BMMSCs in vitro. In a xenograft CLL mouse model, BMMSCs and the culture supernatant of BMMSCs increased engraftment of CLL cells through an IL-6 mediated mechanism with human recombinant IL-6 showing similar effects in vivo. Human recombinant IL-17 treatment also increased CLL engraftment in mice through an IL-6 mediated mechanism. Plasma of CLL patients showed elevated levels of both IL-6 and IL-17 by ELISA compared with healthy controls, with levels of IL-6 linearly correlated with IL-17 levels. CLL patients requiring fludarabine based chemotherapy expressed higher levels of IL-6 and IL-17, while CLL patients with the lowest levels of IgA/IgM had higher levels of IL-6, but not IL-17. These data imply an important role for the IL-17/IL-6 axis in CLL which could be therapeutic targets.


Subject(s)
Drug Delivery Systems , Interleukin-17 , Interleukin-6 , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Neoplasm Proteins , Signal Transduction , Adult , Aged , Aged, 80 and over , Animals , Female , Heterografts , Humans , Interleukin-17/antagonists & inhibitors , Interleukin-17/metabolism , Interleukin-6/antagonists & inhibitors , Interleukin-6/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Tumor Cells, Cultured
11.
EBioMedicine ; 15: 24-35, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27932296

ABSTRACT

Recent studies suggest there is a high incidence of elevated low-density lipoprotein (LDL) levels in Chronic Lymphocytic Leukemia (CLL) patients and a survival benefit from cholesterol-lowering statin drugs. The mechanisms of these observations and the kinds of patients they apply to are unclear. Using an in vitro model of the pseudofollicles where CLL cells originate, LDLs were found to increase plasma membrane cholesterol, signaling molecules such as tyrosine-phosphorylated STAT3, and activated CLL cell numbers. The signaling effects of LDLs were not seen in normal lymphocytes or glycolytic lymphoma cell-lines but were restored by transduction with the nuclear receptor PPARδ, which mediates metabolic activity in CLL cells. Breakdown of LDLs in lysosomes was required for the amplification effect, which correlated with down-regulation of HMGCR expression and long lymphocyte doubling times (LDTs) of 53.6±10.4months. Cholesterol content of circulating CLL cells correlated directly with blood LDL levels in a subgroup of patients. These observations suggest LDLs may enhance proliferative responses of CLL cells to inflammatory signals. Prospective clinical trials are needed to confirm the therapeutic potential of lowering LDL concentrations in CLL, particularly in patients with indolent disease in the "watch-and-wait" phase of management.


Subject(s)
Cytokines/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lipoproteins, LDL/metabolism , Signal Transduction , Cell Line, Tumor , Humans , Hydroxymethylglutaryl CoA Reductases/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Lipid Metabolism/drug effects , Lymphocytes/metabolism , Lysosomes/metabolism , Phosphorylation , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Vitamin E/metabolism
12.
Oncotarget ; 7(45): 72608-72621, 2016 Nov 08.
Article in English | MEDLINE | ID: mdl-27579615

ABSTRACT

Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors.


Subject(s)
Glucocorticoids/pharmacology , Janus Kinases/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Drug Resistance, Neoplasm , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Signal Transduction , Tumor Cells, Cultured
13.
Blood ; 128(7): 934-47, 2016 08 18.
Article in English | MEDLINE | ID: mdl-27297795

ABSTRACT

Novel agents such as the Bcl-2 inhibitor venetoclax (ABT-199) are changing treatment paradigms for chronic lymphocytic leukemia (CLL) but important problems remain. Although some patients exhibit deep and durable responses to venetoclax as a single agent, other patients harbor subpopulations of resistant leukemia cells that mediate disease recurrence. One hypothesis for the origin of resistance to venetoclax is by kinase-mediated survival signals encountered in proliferation centers that may be unique for individual patients. An in vitro microenvironment model was developed with primary CLL cells that could be incorporated into an automated high-content microscopy-based screen of kinase inhibitors (KIs) to identify agents that may improve venetoclax therapy in a personalized manner. Marked interpatient variability was noted for which KIs were effective; nevertheless, sunitinib was identified as the most common clinically available KI effective in overcoming venetoclax resistance. Examination of the underlying mechanisms indicated that venetoclax resistance may be induced by microenvironmental signals that upregulate antiapoptotic Bcl-xl, Mcl-1, and A1, which can be counteracted more efficiently by sunitinib than by ibrutinib or idelalisib. Although patient-specific drug responses are common, for many patients, combination therapy with sunitinib may significantly improve the therapeutic efficacy of venetoclax.


Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/therapeutic use , Sulfonamides/therapeutic use , Adenine/analogs & derivatives , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cellular Microenvironment/drug effects , Dose-Response Relationship, Drug , Humans , Imaging, Three-Dimensional , Indoles/pharmacology , Mutation/genetics , Piperidines , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Quinazolinones/pharmacology , Reproducibility of Results , Signal Transduction/drug effects , Stromal Cells/drug effects , Stromal Cells/pathology , Sulfonamides/pharmacology , Sunitinib , Up-Regulation/drug effects , bcl-X Protein/metabolism
14.
PLoS One ; 11(4): e0152073, 2016.
Article in English | MEDLINE | ID: mdl-27111430

ABSTRACT

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.


Subject(s)
Antigens, CD/metabolism , ADAM Proteins/genetics , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Gene Silencing , HEK293 Cells , Humans
16.
Leuk Lymphoma ; 57(4): 797-802, 2016.
Article in English | MEDLINE | ID: mdl-26325342

ABSTRACT

The incidence of hypercholesterolemia and its possible relationship with clinical course were determined by reviewing the records of 231 consecutive patients presenting to a specialized Chronic Lymphocytic Leukemia (CLL) clinic. Evidence for elevated cholesterol was found in up to 174/231 patients (75%) based on existing use of statins (107 patients) or non-fasting low-density lipoprotein cholesterol levels greater than 2.5 mM. Excluding patients with 17p deletions, time to first treatment (TFT) was prolonged if patients were taking cholesterol-lowering statins (57.5 (IQR = 32, 77) vs 36 (IQR = 11, 100) months, p < 0.02). If patients were prescribed statins after being diagnosed with CLL, TFT was longer than if they were taking statins before the diagnosis. These observations suggest there is a high incidence of hypercholesterolemia in CLL patients and cholesterol-lowering may impact the disease course.


Subject(s)
Hypercholesterolemia/complications , Hypercholesterolemia/epidemiology , Leukemia, Lymphocytic, Chronic, B-Cell/complications , Leukemia, Lymphocytic, Chronic, B-Cell/epidemiology , Aged , Biomarkers , Cholesterol, LDL/blood , Cohort Studies , Female , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypercholesterolemia/drug therapy , Hypercholesterolemia/metabolism , Incidence , Kaplan-Meier Estimate , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Middle Aged , Risk Factors , Treatment Outcome
17.
Blood ; 126(6): 766-78, 2015 Aug 06.
Article in English | MEDLINE | ID: mdl-26041742

ABSTRACT

The regulation of toll-like receptor (TLR) signaling in a tumor microenvironment is poorly understood despite its importance in cancer biology. To address this problem, TLR7-responses of chronic lymphocytic leukemia (CLL) cells were studied in the presence and absence of a human stromal cell-line derived from a leukemic spleen. CLL cells alone produced high levels of tumor necrosis factor (TNF)-α and proliferated in response to TLR7-agonists. A signal transducer and activator of transcription 3 -activating stromal factor, identified as interleukin (IL)-6, was found to upregulate microRNA (miR)-17 and miR-19a, target TLR7 and TNFA messenger RNA, and induce a state of tolerance to TLR7-agonists in CLL cells. Overexpression of the miR-17-92 cluster tolerized CLL cells directly and miR-17 and miR-19a antagomiRs restored TLR7-signaling. Inhibition of IL-6 signaling with antibodies or small-molecule Janus kinase inhibitors reversed tolerization and increased TLR7-stimulated CLL cell numbers in vitro and in NOD-SCIDγc (null) mice. These results suggest IL-6 can act as tumor suppressor in CLL by inhibiting TLR-signaling.


Subject(s)
B-Lymphocytes/immunology , Gene Expression Regulation, Leukemic , Interleukin-6/immunology , MicroRNAs/immunology , Stromal Cells/immunology , Animals , Antibodies, Neutralizing/pharmacology , B-Lymphocytes/drug effects , B-Lymphocytes/pathology , Cell Line, Tumor , Coculture Techniques , Humans , Immune Tolerance , Interleukin-6/genetics , Janus Kinase 1/genetics , Janus Kinase 1/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , Mice, Inbred NOD , Mice, SCID , MicroRNAs/genetics , Oligonucleotides/pharmacology , RNA, Long Noncoding , RNA, Messenger/genetics , RNA, Messenger/immunology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , Signal Transduction , Stromal Cells/drug effects , Stromal Cells/pathology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/immunology , Tumor Microenvironment , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolism
18.
J Clin Oncol ; 33(6): 540-9, 2015 Feb 20.
Article in English | MEDLINE | ID: mdl-25154820

ABSTRACT

PURPOSE: T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies. PATIENTS AND METHODS: We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells. RESULTS: Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/µL. CONCLUSION: This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.


Subject(s)
Antigens, CD19/immunology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Immunotherapy, Adoptive/methods , Lymphoma, B-Cell/therapy , Lymphoma, Large B-Cell, Diffuse/therapy , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/transplantation , Adult , Aged , Combined Modality Therapy , Cyclophosphamide/administration & dosage , Female , Humans , Lymphoma, B-Cell/drug therapy , Lymphoma, B-Cell/immunology , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/immunology , Male , Middle Aged , T-Lymphocytes/immunology , Transplantation Conditioning/methods , Vidarabine/administration & dosage , Vidarabine/analogs & derivatives
19.
Blood ; 122(6): 969-80, 2013 Aug 08.
Article in English | MEDLINE | ID: mdl-23814018

ABSTRACT

High-dose glucocorticoids (GCs) can be a useful treatment for aggressive forms of chronic lymphocytic leukemia (CLL). However, their mechanism of action is not well understood, and resistance to GCs is inevitable. In a minimal, serum-free culture system, the synthetic GC dexamethasone (DEX) was found to decrease the metabolic activity of CLL cells, indicated by down-regulation of pyruvate kinase M2 (PKM2) expression and activity, decreased levels of pyruvate and its metabolites, and loss of mitochondrial membrane potential. This metabolic restriction was associated with decreased size and death of some of the tumor cells in the population. Concomitant plasma membrane damage increased killing of CLL cells by DEX. However, the nuclear receptor peroxisome proliferator activated receptor α (PPARα), which regulates fatty acid oxidation, was also increased by DEX, and adipocyte-derived lipids, lipoproteins, and propionic acid protected CLL cells from DEX. PPARα and fatty acid oxidation enzyme inhibitors increased DEX-mediated killing of CLL cells in vitro and clearance of CLL xenografts in vivo. These findings suggest that GCs prevent tumor cells from generating the energy needed to repair membrane damage, fatty acid oxidation is a mechanism of resistance to GC-mediated cytotoxicity, and PPARα inhibition is a strategy to improve the therapeutic efficacy of GCs.


Subject(s)
Drug Resistance, Neoplasm , Fatty Acids/metabolism , Glucocorticoids/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , PPAR alpha/metabolism , Adipocytes/cytology , Animals , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Culture Media, Conditioned , Dexamethasone/pharmacology , Gene Expression Regulation, Leukemic , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lipid Metabolism , Membrane Potential, Mitochondrial , Membrane Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Oxygen/metabolism , Phosphorylation , Propionates/chemistry , Thyroid Hormones/metabolism , Thyroid Hormone-Binding Proteins
20.
Int J Oncol ; 43(1): 169-76, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23677281

ABSTRACT

Identification of retrovirus integration sites is a powerful method to identify cancer-related genes. This approach led to the discovery of the Friend murine leukemia virus (F-MuLV) integration site-1 (fli-1). Viral insertion at the fli-1 locus induces erythroleukemia in susceptible strains of mice. Our recent data demonstrated that, F-MuLV-infected SCID mice, in contrast to wt CB17 controls, developed a non­erythroleukemic leukemia without viral integration at the fli-1 locus. Using ligation-mediated polymerase chain reaction (LM-PCR) approach we identified a total of 15 viral integration sites in F-MuLV-infected SCID mice. One of the identified insertion sites was located about 62 kb upstream of the myeloblastosis (myb) gene. While integration within or surrounding the myb gene has been reported before for murine leukemia viruses, the location of the viral integration site identified in F-MuLV­infected SCID mice is novel and has never been reported. Using PCR analysis we showed that viral integration at the myb locus occurs with a frequency of 35% and therefore is considered as a common integration site. Integration of F-MuLV in this locus resulted in upregulation of the MYB protein. Flow cytometry analysis and methylcellulose culture of leukemic cells isolated from tumors with viral integration close to the myb indicated tumors of myeloid origin. Our findings indicate that, in contrast to wt CB17 mice, F-MuLV-infected SCID mice display viral integration within myeloid specific gene loci that result in the development of myelogenous leukemia.


Subject(s)
Friend murine leukemia virus/genetics , Leukemia, Myeloid/genetics , Leukemia, Myeloid/virology , Proto-Oncogene Protein c-fli-1/genetics , Virus Integration/genetics , Animals , Disease Models, Animal , Friend murine leukemia virus/pathogenicity , Humans , Leukemia, Experimental/genetics , Leukemia, Myeloid/pathology , Mice , Mice, SCID , Mutagenesis, Insertional/genetics , Oncogenes , Proto-Oncogene Proteins c-myb/genetics , Trans-Activators/genetics
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