ABSTRACT
Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2-/-Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Leukemia, Myeloid, Acute/metabolism , Protein Phosphatase 2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Animals , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Knockout , Protein Phosphatase 2/genetics , Proto-Oncogene Proteins c-myc/geneticsABSTRACT
Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody-based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.
Subject(s)
Hematologic Neoplasms , Immunoconjugates , Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoma, Mantle-Cell , Animals , Antibodies, Monoclonal , Lymphoma, Mantle-Cell/drug therapy , Lymphoma, Mantle-Cell/genetics , MiceABSTRACT
BACKGROUND: Qualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML. METHODS: Bone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity. RESULTS: Upon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden. DISCUSSION: These data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.
Subject(s)
Antibodies, Monoclonal/pharmacology , Cell Membrane/metabolism , Cytotoxicity, Immunologic/drug effects , Immunoglobulin Fc Fragments/metabolism , Interleukins/metabolism , Killer Cells, Natural/immunology , Leukemia, Myeloid, Acute/immunology , Sialic Acid Binding Ig-like Lectin 3/metabolism , Aged , Animals , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Cell Line, Tumor , Cell Membrane/drug effects , Female , Humans , Interleukin-2/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/transplantation , Leukemia, Myeloid, Acute/therapy , Lymphocyte Activation/drug effects , Male , Mice , Middle Aged , Protein Binding/drug effects , Protein Engineering , Vidarabine/analogs & derivatives , Vidarabine/pharmacologyABSTRACT
Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.
Subject(s)
Breast Neoplasms/drug therapy , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , Killer Cells, Natural/immunology , Leukemia, Myeloid, Acute/drug therapy , Melanoma, Experimental/drug therapy , Protein Kinase Inhibitors/pharmacology , Animals , Apoptosis , Breast Neoplasms/enzymology , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Proliferation , Cytotoxicity, Immunologic/immunology , Female , Humans , Killer Cells, Natural/drug effects , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Melanoma, Experimental/enzymology , Melanoma, Experimental/immunology , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Phosphorylation , STAT1 Transcription Factor/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.
Subject(s)
Cell Cycle Checkpoints/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , MicroRNAs/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/antagonists & inhibitors , Animals , Biomarkers, Tumor , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , DNA Methylation , Disease Models, Animal , Epigenesis, Genetic , Humans , Immunoconjugates/administration & dosage , Immunoconjugates/chemistry , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , MicroRNAs/administration & dosage , MicroRNAs/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Survival Rate , Theranostic Nanomedicine , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
Prostate apoptosis response 4 (Par-4) is a tumor suppressor that prevents proliferation and induces cell death in several solid tumors. However, its role in B-cell malignancies has not been elucidated. To describe the role of Par-4 in chronic lymphocytic leukemia (CLL) pathogenesis, we developed a B-cell-specific human Par-4-overexpressing mouse model of CLL using the TCL1 leukemia model. While Par-4 transgenic mice did not display any obvious defects in B-cell development or function, disease burden as evidenced by abundance of CD19+CD5+ B cells in the peripheral blood was significantly reduced in Par-4 × TCL1 mice compared with TCL1 littermates. This conferred a survival advantage on the Par-4-overexpressing mice. In addition, a B-cell-specific knockout model displayed the opposite effect, where lack of Par-4 expression resulted in accelerated disease progression and abbreviated survival in the TCL1 model. Histological and flow cytometry-based analysis of spleen and bone marrow upon euthanasia revealed comparable levels of malignant B-cell infiltration in Par-4 × TCL1 and TCL1 individuals, indicating delayed but pathologically normal disease progression in Par-4 × TCL1 mice. In vivo analysis of splenic B-cell proliferation by 5-ethynyl-2-deoxyuridine incorporation indicated >50% decreased expansion of CD19+CD5+ cells in Par-4 × TCL1 mice compared with TCL1 littermates. Moreover, reduced nuclear p65 levels were observed in Par-4 × TCL1 splenic B cells compared with TCL1, suggesting suppressed NF-κB signaling. These findings have identified an in vivo antileukemic role for Par-4 through an NF-κB-dependent mechanism in TCL1-mediated CLL-like disease progression.
Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Carcinogenesis/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Neoplasms, Experimental/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Animals , Carcinogenesis/genetics , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , Mice, Transgenic , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Proto-Oncogene Proteins/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Diseases with clonal hematopoiesis such as myelodysplastic syndrome and acute myeloid leukemia have high rates of relapse. Only a small subset of acute myeloid leukemia patients are cured with chemotherapy alone. Relapse in these diseases occurs at least in part due to the failure to eradicate leukemic stem cells or hematopoietic stem cells in myelodysplastic syndrome. CD123, the alpha chain of the interleukin-3 receptor heterodimer, is expressed on the majority of leukemic stem cells and myelodysplastic syndrome hematopoietic stem cells and in 80% of acute myeloid leukemia. Here, we report indiscriminate killing of CD123+ normal and acute myeloid leukemia / myelodysplastic syndrome cells by SL-401, a diphtheria toxin interleukin-3 fusion protein. SL-401 induced cytotoxicity of CD123+ primary cells/blasts from acute myeloid leukemia and myelodysplastic syndrome patients but not CD123- lymphoid cells. Importantly, SL-401 was highly active even in cells expressing low levels of CD123, with minimal effect on modulation of the CD123 target in acute myeloid leukemia. SL-401 significantly prolonged survival of leukemic mice in acute myeloid leukemia patient-derived xenograft mouse models. In addition to primary samples, studies on normal cord blood and healthy marrow show that SL-401 has activity against normal hematopoietic progenitors. These findings indicate potential use of SL-401 as a "bridge-to-transplant" before allogeneic hematopoietic cell transplantation in acute myeloid leukemia / myelodysplastic syndrome patients.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Interleukin-3 Receptor alpha Subunit/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Myelodysplastic Syndromes/drug therapy , Recombinant Fusion Proteins/pharmacology , Animals , Cell Line, Tumor , Heterografts , Humans , Interleukin-3 Receptor alpha Subunit/analysis , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , Recombinant Fusion Proteins/therapeutic use , Tumor Cells, CulturedABSTRACT
Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eµ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)-approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Gene Expression Regulation, Leukemic , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Cell Cycle , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Deletion , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice, Inbred C57BL , Mice, Inbred NOD , Receptors, Antigen, B-Cell/metabolism , Signal Transduction , Up-RegulationABSTRACT
Acute myeloid leukemia (AML) is the second most common type of leukemia in adults. Incidence of AML increases with age with a peak incidence at 67 years. Patients older than 60 years have an unfavorable prognosis due to resistance to conventional chemotherapy. Volasertib (BI 6727) is a cell-cycle regulator targeting polo-like kinase which has been evaluated in clinical trials in AML. We evaluated effects of volasertib in primary patient samples and NK cells. At equivalent doses, volasertib is cytotoxic to AML blasts but largely spares healthy NK cells. We then evaluated the effect of volasertib treatment in combination with BI 836858 on primary AML blast samples using antibody-dependent cellular cytotoxicity (ADCC) assays. Volasertib treatment of NK cells did not impair NK function as evidenced by comparable levels of BI 836858 mediated ADCC in both volasertib-treated and control-treated NK cells. In summary, volasertib is cytotoxic to AML blasts while sparing NK cell viability and function. Higher BI 836858 mediated ADCC was observed in patient samples pretreated with volasertib. These findings provide a strong rationale to test combination of BI 836858 and volasertib in AML.
ABSTRACT
Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS.
ABSTRACT
Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing Notch1 mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including MYC and HES1, and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
Subject(s)
Carcinogenesis , Gene Expression Regulation, Leukemic , MafB Transcription Factor/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptor, Notch1/genetics , Signal Transduction , Animals , Disease Models, Animal , Female , Humans , MafB Transcription Factor/genetics , Mice , Mice, Inbred C57BL , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Protein c-ets-2/genetics , Proto-Oncogene Protein c-ets-2/metabolism , Receptor, Notch1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, CulturedABSTRACT
Acute myeloid leukemia (AML) is the most common type of acute leukemia, affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence, novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (gemtuzumab ozogamicin) have been shown to improve overall survival, validating CD33 as a target for antibody-based therapy of AML. Here, we report the in vitro efficacy of BI 836858, a fully human, Fc-engineered, anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine, 2 hypomethylating agents that show efficacy in older patients, did not compromise BI 836858-induced NK-cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a 10-day course of DAC (pre-DAC, days 4, 11, and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared with pre-DAC treatment. Analysis of ligands to activating receptors (NKG2D) showed significantly increased NKG2D ligand [NKG2DL] expression in day 28 post-DAC samples compared with pre-DAC samples; when NKG2DL receptor was blocked using antibodies, BI 836858-mediated ADCC was significantly decreased, suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Azacitidine/analogs & derivatives , Killer Cells, Natural/drug effects , Leukemia, Myeloid, Acute/therapy , Sialic Acid Binding Ig-like Lectin 3/immunology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Azacitidine/administration & dosage , Azacitidine/pharmacology , Cells, Cultured , Combined Modality Therapy , Cytotoxicity, Immunologic , Decitabine , Drug Synergism , HL-60 Cells , Humans , Immunoglobulin Fc Fragments/administration & dosage , Immunoglobulin Fc Fragments/pharmacology , Killer Cells, Natural/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacologyABSTRACT
Mantle-cell lymphoma (MCL) remains incurable despite numerous therapeutic advances. OSU-2S, a novel nonimmunosuppressive FTY720 (Fingolimod) derivative, exhibits potent cytotoxicity in MCL cell lines and primary cells. OSU-2S increased the surface expression of CD74, a therapeutic antibody target in MCL cells. OSU-2S, in combination with anti-CD74 antibody milatuzumab, enhanced cytotoxicity in MCL. Moreover, MCL tumor antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeted immunonanoparticle-carrying OSU-2S (2A2-OSU-2S-ILP)-mediated selective cytotoxicity of MCL in vitro, as well as activity in a xenografted mouse model of MCL in vivo. The newly developed OSU-2S delivery using ROR1-directed immunonanoparticles provide selective targeting of OSU-2S to MCL and other ROR1(+) malignancies, sparing normal B cells.
Subject(s)
Cytotoxins/pharmacology , Drug Delivery Systems , Lymphoma, Mantle-Cell/metabolism , Propylene Glycols/pharmacology , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Sphingosine/analogs & derivatives , Animals , Antibodies, Monoclonal, Humanized/pharmacology , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , Antigens, Neoplasm/metabolism , Fingolimod Hydrochloride , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Humans , Lymphoma, Mantle-Cell/genetics , Mice , Mice, Inbred NOD , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Sphingosine/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The nuclear export protein XPO1 is overexpressed in cancer, leading to the cytoplasmic mislocalization of multiple tumor suppressor proteins. Existing XPO1-targeting agents lack selectivity and have been associated with significant toxicity. Small molecule selective inhibitors of nuclear export (SINEs) were designed that specifically inhibit XPO1. Genetic experiments and X-ray structures demonstrate that SINE covalently bind to a cysteine residue in the cargo-binding groove of XPO1, thereby inhibiting nuclear export of cargo proteins. The clinical relevance of SINEs was explored in chronic lymphocytic leukemia (CLL), a disease associated with recurrent XPO1 mutations. Evidence is presented that SINEs can restore normal regulation to the majority of the dysregulated pathways in CLL both in vitro and in vivo and induce apoptosis of CLL cells with a favorable therapeutic index, with enhanced killing of genomically high-risk CLL cells that are typically unresponsive to traditional therapies. More importantly, SINE slows disease progression, and improves overall survival in the Eµ-TCL1-SCID mouse model of CLL with minimal weight loss or other toxicities. Together, these findings demonstrate that XPO1 is a valid target in CLL with minimal effects on normal cells and provide a basis for the development of SINEs in CLL and related hematologic malignancies.
Subject(s)
Acrylates/pharmacology , Karyopherins/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Receptors, Cytoplasmic and Nuclear/metabolism , Triazoles/pharmacology , Acrylates/chemistry , Acrylates/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Crystallography, X-Ray , Humans , Immunoblotting , Interleukin-10/metabolism , Interleukin-6/metabolism , Karyopherins/chemistry , Karyopherins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Mice , Mice, SCID , Mice, Transgenic , Microscopy, Confocal , Models, Molecular , Molecular Structure , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Triazoles/chemistry , Triazoles/metabolism , Exportin 1 ProteinABSTRACT
PURPOSE: During cell-cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression. EXPERIMENTAL DESIGN: Mantle cell lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. RESULTS: Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nmol/L silvestrol at 16 hours, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As showed in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P < 0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. CONCLUSIONS: These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies.
Subject(s)
Cyclin D1 , E2F1 Transcription Factor , Lymphoma, Mantle-Cell , Triterpenes/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cyclin D1/genetics , Cyclin D1/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathology , Lymphoma, Mantle-Cell/therapy , Metabolic Networks and Pathways , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Signal Transduction/drug effects , Transplantation, HeterologousABSTRACT
Tetraspanins are commonly believed to act only as "molecular facilitators," with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions: one lies in the N-terminal domain of CD37 in a predicted "ITIM-like" motif and mediates SHP1-dependent death, whereas the second lies in a predicted "ITAM motif" in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.
Subject(s)
Antigens, Neoplasm/metabolism , Apoptosis , B-Lymphocytes/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Signal Transduction , Tetraspanins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/pathology , Bcl-2-Like Protein 11 , Cell Line, Tumor , Cell Survival , Chromatography, Liquid , Forkhead Box Protein O3 , Forkhead Transcription Factors/metabolism , HEK293 Cells , Humans , Immunoglobulin G/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Membrane Microdomains/metabolism , Membrane Potential, Mitochondrial , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Nanotechnology , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation , Protein Transport , Proteomics/methods , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Tandem Mass Spectrometry , Tetraspanins/chemistry , Tetraspanins/genetics , Time Factors , Transfection , TyrosineABSTRACT
Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a short median survival despite multimodal therapy. FTY720, an immunosuppressive drug approved for the treatment of multiple sclerosis, promotes MCL cell death concurrent with down-modulation of phospho-Akt and cyclin D1 and subsequent cell-cycle arrest. However, the mechanism of FTY720-mediated MCL cell death remains to be fully clarified. In the present study, we show features of autophagy blockage by FTY720 treatment, including accumulation of autolysosomes and increased LC3-II and p62 levels. We also show that FTY720-induced cell death is mediated by lysosomal membrane permeabilization with subsequent translocation of lysosomal hydrolases to the cytosol. FTY720-mediated disruption of the autophagic-lysosomal pathway led to increased levels of CD74, a potential therapeutic target in MCL that is degraded in the lysosomal compartment. This finding provided rationale for examining combination therapy with FTY720 and milatuzumab, an anti-CD74 mAb. Treatment of MCL cell lines and primary tumor cells with FTY720 and milatuzumab resulted in statistically significant enhanced cell death, which was synergistic in blastic variant MCL cell lines. Significant in vivo therapeutic activity of combination treatment was also demonstrated in a preclinical, in vivo model of MCL. These findings support clinical evaluation of this combination in patients with MCL.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antigens, Differentiation, B-Lymphocyte/metabolism , Histocompatibility Antigens Class II/metabolism , Lymphoma, Mantle-Cell/drug therapy , Propylene Glycols/pharmacology , Sphingosine/analogs & derivatives , Animals , Antibodies, Monoclonal, Humanized/administration & dosage , Antigens, Differentiation, B-Lymphocyte/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Autophagy/drug effects , Cell Death/drug effects , Cell Line, Tumor , Drug Synergism , Female , Fingolimod Hydrochloride , Gene Expression Regulation, Neoplastic/drug effects , Histocompatibility Antigens Class II/genetics , Humans , Immunoblotting , Immunosuppressive Agents/pharmacology , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathology , Lysosomes/metabolism , Mice , Mice, SCID , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Propylene Glycols/administration & dosage , Protein Transport/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Sphingosine/administration & dosage , Sphingosine/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.
Subject(s)
Antineoplastic Agents/pharmacology , Burkitt Lymphoma/pathology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Tyrosine/analogs & derivatives , p38 Mitogen-Activated Protein Kinases/physiology , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , B-Lymphocytes/drug effects , Burkitt Lymphoma/enzymology , Caspase 3/metabolism , Cell Death/drug effects , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Drug Evaluation, Preclinical/methods , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitor of Apoptosis Proteins/biosynthesis , Inhibitor of Apoptosis Proteins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Phosphorylation/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Survivin , Tumor Cells, Cultured , Tyrosine/administration & dosage , Tyrosine/pharmacologyABSTRACT
PURPOSE: Despite the progress that has been made in the treatment of mantle cell lymphoma (MCL), all patients invariably relapse with the currently available therapies. Because of the absence of curative therapy for MCL, we explored FTY720 as a novel agent against MCL. EXPERIMENTAL DESIGN: The cytotoxic effect of FTY720 in primary MCL tumor cells and cell lines were evaluated in vitro. The effects of FTY720 on caspase activation, generation of reactive oxygen species, and modulation of Cyclin D1 and Akt, which are implied in the pathogenesis of MCL, were investigated. The in vivo efficacy of FTY720 was evaluated in a Jeko-severe combined immunodeficient xenograft model of human MCL. RESULTS: FTY720 mediated time- and dose-dependent cytotoxicity in primary MCL tumor cells and MCL cell lines in vitro. FTY720-induced cytotoxicity occured independent of caspase activation but dependent on the generation of ROS in MCL. In addition, FTY720 treatment resulted in the time-dependent downmodulation of Cyclin D1 and accumulation of cells in G(0)-G(1) and G(2)-M phases of the cell cycle with concomitant decrease in S-phase entry. Furthermore, concentrations of FTY720 that induced cytotoxicity led to decreased phospho-Akt in primary MCL cells and cell lines. Most importantly, the in vivo therapeutic activity of FTY720 was shown in severe combined immunodeficient mice engrafted with the Jeko MCL cell line. CONCLUSIONS: These results provide the first evidence for a potential use of FTY720 in targeting key pathways that are operable in the pathogenesis of MCL and warrant further investigation of FTY720 in clinical trials to treat patients with MCL.