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1.
Blood Cancer J ; 5: e312, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25978432

ABSTRACT

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Histone Deacetylase Inhibitors/administration & dosage , Immunomodulation , Multiple Myeloma/drug therapy , Drug Synergism , Flow Cytometry , Humans , Hydroxamic Acids/administration & dosage , Immunoblotting , In Vitro Techniques , Lenalidomide , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Transfection , Vorinostat
2.
Leukemia ; 29(9): 1918-27, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25801913

ABSTRACT

Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.


Subject(s)
Endoplasmic Reticulum Stress , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Multiple Myeloma/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Gene Knockdown Techniques , Histone Deacetylases/genetics , Humans , Interleukin-6/metabolism , Isoenzymes , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Proteasome Inhibitors/pharmacology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction/drug effects , Stromal Cells/drug effects , Stromal Cells/metabolism
4.
Leukemia ; 28(3): 680-9, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23913134

ABSTRACT

Histone deacetylases (HDACs) represent novel molecular targets for the treatment of various types of cancers, including multiple myeloma (MM). Many HDAC inhibitors have already shown remarkable antitumor activities in the preclinical setting; however, their clinical utility is limited because of unfavorable toxicities associated with their broad range HDAC inhibitory effects. Isoform-selective HDAC inhibition may allow for MM cytotoxicity without attendant side effects. In this study, we demonstrated that HDAC3 knockdown and a small-molecule HDAC3 inhibitor BG45 trigger significant MM cell growth inhibition via apoptosis, evidenced by caspase and poly (ADP-ribose) polymerase cleavage. Importantly, HDAC3 inhibition downregulates phosphorylation (tyrosine 705 and serine 727) of signal transducers and activators of transcription 3 (STAT3). Neither interleukin-6 nor bone marrow stromal cells overcome this inhibitory effect of HDAC3 inhibition on phospho-STAT3 and MM cell growth. Moreover, HDAC3 inhibition also triggers hyperacetylation of STAT3, suggesting crosstalk signaling between phosphorylation and acetylation of STAT3. Importantly, inhibition of HDAC3, but not HDAC1 or 2, significantly enhances bortezomib-induced cytotoxicity. Finally, we confirm that BG45 alone and in combination with bortezomib trigger significant tumor growth inhibition in vivo in a murine xenograft model of human MM. Our results indicate that HDAC3 represents a promising therapeutic target, and validate a prototype novel HDAC3 inhibitor BG45 in MM.


Subject(s)
Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/drug effects , Multiple Myeloma/enzymology , Cell Division , Cell Line, Tumor , Gene Knockdown Techniques , Histone Deacetylases/genetics , Humans , Multiple Myeloma/pathology
6.
Blood Cancer J ; 3: e156, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24185503

ABSTRACT

Our goal is to develop a rapid and scalable system for functionally evaluating deregulated genes in multiple myeloma (MM). Here, we forcibly expressed human cMYC and KRAS12V in mouse T2 B cells (IgM(+)B220(+)CD38(+)IgD(+)) using retroviral transduction and transplanted these cells into lethally irradiated recipient mice. Recipients developed plasmacytomas with short onset (70 days) and high penetrance, whereas neither cMYC nor KRAS12V alone induced disease in recipient mice. Tumor cell morphology and cell surface biomarkers (CD138(+)B220(-)IgM(-)GFP(+)) indicate a plasma cell neoplasm. Gene set enrichment analysis further confirms that the tumor cells have a plasma cell gene expression signature. Plasmacytoma cells infiltrated multiple loci in the bone marrow, spleen and liver; secreted immunoglobulins; and caused glomerular damage. Our findings therefore demonstrate that deregulated expression of cMYC with KRAS12V in T2 B cells rapidly generates a plasma cell disease in mice, suggesting utility of this model both to elucidate molecular pathogenesis and to validate novel targeted therapies.

7.
Leukemia ; 27(12): 2366-75, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23807770

ABSTRACT

Small-molecule multi-targeted cyclin-dependent kinase (CDK) inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638's mode-of-action in MM cell lines with wild-type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638 triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA-binding activity. In addition, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1 and miR-21. Our data provide the rationale for the development of transcriptional CDKIs as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.


Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Multiple Myeloma/pathology , Pyrazoles/pharmacology , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/physiology , Urea/analogs & derivatives , Animals , Apoptosis/drug effects , Humans , Male , Mice , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Real-Time Polymerase Chain Reaction , Urea/pharmacology , Xenograft Model Antitumor Assays
8.
Oncogene ; 29(16): 2325-36, 2010 Apr 22.
Article in English | MEDLINE | ID: mdl-20101221

ABSTRACT

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.


Subject(s)
Apoptosis/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Multiple Myeloma/drug therapy , Piperidines/pharmacology , Pyrazoles/pharmacology , RNA Polymerase II/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Glycogen Synthase Kinase 3 beta , Humans , Male , Mice , Mice, SCID , Multiple Myeloma/pathology
9.
Leukemia ; 23(9): 1679-86, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19369963

ABSTRACT

Earlier studies have shown that ascorbic acid (vitamin C) inhibits bortezomib-induced cytotoxicity against cancer cells in vitro. However, the clinical significance of vitamin C on bortezomib treatment is unclear. In this study, we examined whether daily oral intake of vitamin C inhibits antimultiple myeloma (MM) activities of bortezomib. Vitamin C, at orally achievable concentrations, inhibited in vitro MM cell cytotoxicity of bortezomib and blocked its inhibitory effect on 20S proteasome activity. Specifically, plasma collected from healthy volunteers taking 1 g/day vitamin C reduced bortezomib-induced MM cell death in vitro. This antagonistic effect of vitamin C against proteasome inhibitors is limited to the boronate class of inhibitors (bortezomib and MG262). In vivo activity of this combination treatment was then evaluated using our xenograft model of human MM in SCID (severe combined immune-deficient) mice. Bortezomib (0.1 mg/kg twice a week for 4 weeks) significantly inhibits in vivo MM cell growth, which was blocked by oral vitamin C (40 mg/kg/day). Therefore, our results for the first time show that vitamin C can significantly reduce the activity of bortezomib treatment in vivo; and importantly, suggest that patients receiving treatment with bortezomib should avoid taking vitamin C dietary supplements.


Subject(s)
Antineoplastic Agents/antagonists & inhibitors , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Boronic Acids/antagonists & inhibitors , Pyrazines/antagonists & inhibitors , Animals , Bortezomib , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Mice , Multiple Myeloma/drug therapy , Proteasome Inhibitors
10.
Bone Marrow Transplant ; 33(9): 881-9, 2004 May.
Article in English | MEDLINE | ID: mdl-14990986

ABSTRACT

SUMMARY: In all, 55 patients at high risk or ineligible for a conventional allogeneic hematopoietic stem cell transplant (HSCT) received a regimen consisting of extracorporeal photopheresis, pentostatin, and reduced dose total body irradiation. The median age was 49 years (18-70 years); 44 received a sibling and 11 an unrelated HSCT; 44% were over the age of 50 years and 31% had undergone a prior HSCT. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate. Full donor chimerism was documented in 98% by day +100. The 1000-day nonrelapse mortality was 11%. The median follow-up is 502 days (154-1104 days). The 1- and 2-year overall survival (OS) and event-free survival (EFS) are 67, 58 and 55%, and 47%, respectively. Patients who had not received a prior HSCT or had less than three prior chemotherapy regimens had a 71% OS and 67% EFS at 1 year. Greater than grade II aGVHD developed in 9% and chronic GVHD (cGVHD) in 43%, and extensive in 12% and limited in 31%. Of the patients, 86% who engrafted had a disease response, 72% had complete and 14% partial responses. This novel reduced intensity preparative regimen was well tolerated and associated with a low incidence of transplant-related mortality and serious acute and cGVHD.


Subject(s)
Graft vs Host Disease/prevention & control , Hematologic Neoplasms/therapy , Hematopoietic Stem Cell Transplantation/methods , Transplantation Chimera , Transplantation Conditioning/methods , Adolescent , Adult , Aged , Cyclosporine/pharmacology , Disease-Free Survival , Female , Humans , Male , Methotrexate/pharmacology , Middle Aged , Pentostatin/therapeutic use , Time Factors , Transplantation, Homologous , Treatment Outcome , Whole-Body Irradiation
11.
Bone Marrow Transplant ; 29(9): 719-25, 2002 May.
Article in English | MEDLINE | ID: mdl-12040467

ABSTRACT

Despite significant advances in stem cell manipulation and post-transplant immunosuppression, chronic graft-versus-host disease (cGVHD) remains a cause of major long-term morbidity in survivors of allogeneic stem cell transplantation. Extracorporeal photopheresis (ECP) is a novel therapeutic intervention which has demonstrated efficacy in patients with refractory acute and chronic GVHD. Clinical responses have been reported in skin and visceral GVHD. While the long-term immunomodulatory effects of ECP in cGVHD are unknown, recent studies of patients undergoing a 6- to 12-month course of ECP treatment demonstrated an attenuation of Th1-mediated cytokine secretion by activated T-helper cells, a shift in the DC1/DC2 ratio favoring plasmacytoid rather than monocytoid dendritic cell profiles, and a decrease in antigen responsiveness by dendritic cells. The implications of these immunomodulatory effects of ECP on pathogenesis and clinical outcome remains a fertile area for future research.


Subject(s)
Graft vs Host Disease/therapy , Photopheresis , Bone Marrow Transplantation/adverse effects , Chronic Disease , Graft vs Host Disease/etiology , Graft vs Host Disease/immunology , Humans , Immune System/cytology , Immune System/metabolism
12.
Blood ; 98(5): 1622-5, 2001 Sep 01.
Article in English | MEDLINE | ID: mdl-11520818

ABSTRACT

Extracorporeal photochemotherapy (ECP) has been associated with clinical improvement in several patients with acute and chronic graft-versus-host disease (cGVHD) after allogeneic bone marrow transplantation, but the mechanism of action is unknown. This study tested the hypothesis that in patients with cGVHD, ECP modulates alloreactivity by affecting activated lymphocyte populations or by altering the interaction between effector lymphocytes and antigen-presenting cells (APCs). Ten patients who had refractory cGVHD were treated with ECP, and the clinical response to and immunologic effects of this therapy were assessed. Seven patients had a response and 3 had no change in clinical manifestations of cGVHD. One patient died from catheter-related sepsis. Immunologic effects observed after ECP included normalization of inverted ratios of CD4 to CD8 cells, an increase in the number of CD3-CD56+ natural killer (NK) cells, and a decrease in CD80+ and CD123+ circulating dendritic cells. The results suggest that ECP modulates both NK cells and APC populations in patients with cGVHD.


Subject(s)
Extracorporeal Circulation , Graft vs Host Disease/drug therapy , PUVA Therapy , Adult , Antigen Presentation/drug effects , Bone Marrow Transplantation/adverse effects , CD4-CD8 Ratio , Chronic Disease , Combined Modality Therapy , Dendritic Cells/drug effects , Drug Resistance , Female , Hematologic Neoplasms/therapy , Humans , Immunosuppressive Agents/therapeutic use , Killer Cells, Natural/drug effects , Lymphocyte Activation/drug effects , Male , Middle Aged , Models, Biological , T-Lymphocytes, Cytotoxic/drug effects
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