38 results on '"William Matsui"'
Search Results
2. Loss of p53 enhances the tumor-initiating potential and drug resistance of clonogenic multiple myeloma cells
- Author
-
Yu-Tai Chang, Ian T. Chiu, Qiuju Wang, Jorge Bustamante, Wenxuan Jiang, Kiera Rycaj, S. Stephen Yi, Joey H. Li, Jeanne Kowalski-Muegge, and William Matsui
- Subjects
Hematology - Abstract
Tumor relapse and drug resistance are major factors that limit the curability of multiple myeloma (MM). New regimens have improved overall MM survival rates, but patients with high-risk features continue to have inferior outcomes. Chromosome 17p13 deletion (del17p) which includes the loss of the TP53 gene is a high-risk cytogenetic abnormality and is associated with poor clinical outcomes due to relatively short remissions and the development of pan-drug resistant disease. Increased relapse rates suggest that del17p enhances clonogenic growth, and we found that the loss of p53 increased both the frequency and drug resistance of tumor-initiating MM cells (TICs). Subsequent RNA sequencing (RNA-seq) studies demonstrated significant activation of the Notch signaling pathway and upregulation of inhibitor of DNA binding (ID1/ID2) genes in p53-knock out (p53-KO) cells. We found that the loss of ID1 or HES-1 expression or treatment with a gamma-secretase inhibitor (GSI) significantly decreased the clonogenic growth of p53-KO, but not p53 wild-type cells. GSI treatment in a small set of MM specimens also reduced the clonogenic growth in del17p samples but not in non-del17p samples. This effect was specific as overexpression of the Notch intracellular domain (NICD) rescued the effects of GSI treatment. Our study demonstrates that the Notch signaling and ID1 expression are required for TIC expansion in p53-KO MM cells. These findings also suggest that GSI may be specifically active in patients with p53 mutant MM.
- Published
- 2023
3. Prospective study of nonmyeloablative, HLA-mismatched unrelated BMT with high-dose posttransplantation cyclophosphamide
- Author
-
Ephraim J. Fuchs, Douglas E. Gladstone, Yvette L. Kasamon, Ivan Borrello, Lode J. Swinnen, Robert A. Brodsky, Javier Bolaños-Meade, Richard J. Jones, Richard F. Ambinder, Leo Luznik, Gary L. Rosner, William Matsui, Marianna Zahurak, Mark J. Levis, and Carol Ann Huff
- Subjects
medicine.medical_specialty ,Cyclophosphamide ,chemical and pharmacologic phenomena ,Gastroenterology ,Umbilical cord ,03 medical and health sciences ,0302 clinical medicine ,immune system diseases ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Transplantation ,business.industry ,Hematology ,Total body irradiation ,Tacrolimus ,Fludarabine ,surgical procedures, operative ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Sirolimus ,Immunology ,Bone marrow ,business ,Busulfan ,030215 immunology ,medicine.drug - Abstract
Allogeneic blood or marrow transplantation (BMT) candidates may lack HLA-matched, related haploidentical, and unrelated umbilical cord options. Barriers to partially HLA-mismatched, unrelated donor (mMUD) BMT include excess graft-versus-host disease (GVHD), graft failure, and death. We prospectively studied nonmyeloablative (NMA) mMUD BMT with high-dose posttransplantation cyclophosphamide (PTCy) for patients with hematologic malignancies. Three transplants were performed with busulfan/fludarabine conditioning, with subsequent change to fludarabine/Cy/total body irradiation (flu/Cy/TBI). Twenty mMUD transplants are reported using flu/Cy/TBI, T-cell replete bone marrow grafts, and PTCy, mycophenolate mofetil, and sirolimus or tacrolimus (1 patient) for GVHD prophylaxis. The median patient age was 56. Ofthese unrelated grafts, 45% had ≥2 mismatched HLA loci, 25% had ≥3 mismatched loci, and 50% had HLA-C mismatches. No graft failure or grades 3-4 acute GVHD occurred. The median times to neutrophil recovery (≥500/μL) and platelet recovery (≥20 000/μL) were 19 days and 31 days, respectively. Full-donor chimerism was achieved in 95% of evaluable patients by day 60. The 180-day probability of grades 2-4 acute GVHD (all grade 2) was 25%, and the 1-year probability of any chronic GVHD was 16% (none severe). The 2-year nonrelapse mortality probability was 6%. With 4-year median follow-up, the 1-year progression-free and overall survival probabilities were 65% and 75%, respectively. NMA, T-cell replete mMUD BMT is thus a potentially viable option for patients without other suitable donors. This trial was registered at www.clinicaltrials.gov as #NCT01203722.
- Published
- 2017
4. Risk-stratified outcomes of nonmyeloablative HLA-haploidentical BMT with high-dose posttransplantation cyclophosphamide
- Author
-
Douglas E. Gladstone, Gabrielle T. Prince, William Matsui, Keith W. Pratz, Amy E. DeZern, Hua Ling Tsai, Robert A. Brodsky, Shannon R. McCurdy, Ivana Gojo, Jennifer A. Kanakry, Lode J. Swinnen, Heather J. Symons, Javier Bolaños-Meade, Mark J. Levis, Richard J. Jones, Ephraim J. Fuchs, Christopher G. Kanakry, Margaret M. Showel, Karlo Perica, Carol Ann Huff, Yvette L. Kasamon, Leo Luznik, Ivan Borrello, Michael A. McDevitt, Richard F. Ambinder, Gary L. Rosner, and B. Douglas Smith
- Subjects
Male ,Transplantation Conditioning ,Graft vs Host Disease ,Biochemistry ,Gastroenterology ,Postoperative Complications ,immune system diseases ,hemic and lymphatic diseases ,Bone Marrow Transplantation ,hemic and immune systems ,Hematology ,Middle Aged ,Prognosis ,Combined Modality Therapy ,Survival Rate ,surgical procedures, operative ,Hematologic Neoplasms ,Histocompatibility ,Female ,Risk assessment ,medicine.drug ,Adult ,medicine.medical_specialty ,Adolescent ,Cyclophosphamide ,Immunology ,chemical and pharmacologic phenomena ,Risk Assessment ,Young Adult ,Internal medicine ,medicine ,Humans ,Transplantation, Homologous ,Antineoplastic Agents, Alkylating ,Survival rate ,Aged ,Neoplasm Staging ,Retrospective Studies ,Transplantation ,Dose-Response Relationship, Drug ,business.industry ,Retrospective cohort study ,Cell Biology ,Tacrolimus ,Surgery ,business ,Follow-Up Studies - Abstract
Related HLA-haploidentical blood or marrow transplantation (BMT) with high-dose posttransplantation cyclophosphamide (PTCy) is being increasingly used because of its acceptable safety profile. To better define outcomes of nonmyeloablative (NMA) HLA-haploidentical BMT with PTCy, 372 consecutive adult hematologic malignancy patients who underwent this procedure were retrospectively studied. Risk-stratified outcomes were evaluated using the refined Disease Risk Index (DRI), developed to stratify disease risk across histologies and allogeneic BMT regimens. Patients received uniform conditioning, T-cell–replete allografting, then PTCy, mycophenolate mofetil, and tacrolimus. Six-month probabilities of nonrelapse mortality and severe acute graft-versus-host disease were 8% and 4%. With 4.1-year median follow-up, 3-year probabilities of relapse, progression-free survival (PFS), and overall survival (OS) were 46%, 40%, and 50%, respectively. By refined DRI group, low (n = 71), intermediate (n = 241), and high/very high (n = 60) risk groups had 3-year PFS estimates of 65%, 37%, and 22% (P < .0001), with corresponding 3-year OS estimates of 71%, 48%, and 35% (P = .0001). On multivariable analyses, the DRI was statistically significantly associated with relapse, PFS, and OS (each P < .001). This analysis demonstrates that the DRI effectively risk stratifies recipients of NMA HLA-haploidentical BMT with PTCy and also suggests that this transplantation platform yields similar survivals to those seen with HLA-matched BMT.
- Published
- 2015
5. Mantle cell lymphoma activation enhances bortezomib sensitivity
- Author
-
Sarah Brennan, Jeanne Kowalski, Brooke Meade, William Matsui, Qiuju Wang, and Akil Merchant
- Subjects
Proteasome Endopeptidase Complex ,Cellular differentiation ,Plasma Cells ,Immunology ,Lymphoma, Mantle-Cell ,Biology ,Lymphocyte Activation ,Biochemistry ,Aldehyde Dehydrogenase 1 Family ,Bortezomib ,Mice ,Cancer stem cell ,hemic and lymphatic diseases ,Cell Line, Tumor ,Plasma cell differentiation ,medicine ,Animals ,Humans ,Clonogenic assay ,Cell Proliferation ,Lymphoid Neoplasia ,Retinal Dehydrogenase ,Cell Differentiation ,Cell Biology ,Hematology ,Aldehyde Dehydrogenase ,Boronic Acids ,Clone Cells ,Isoenzymes ,Oligodeoxyribonucleotides ,CpG site ,Drug Resistance, Neoplasm ,Pyrazines ,Toll-Like Receptor 9 ,Unfolded Protein Response ,Proteasome inhibitor ,Cancer research ,Drug Screening Assays, Antitumor ,Stem cell ,Proteasome Inhibitors ,medicine.drug - Abstract
Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH+ cells that were highly clonogenic. Moreover, ALDH+ MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9, and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH+ MCL cells, induced terminal plasma cell differentiation, and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH+ cells as well as improve the activity of proteasome inhibitors in MCL.
- Published
- 2010
6. The paradox of response and survival in cancer therapeutics
- Author
-
B. Douglas Smith, William Matsui, Richard J. Jones, and Carol Ann Huff
- Subjects
Oncology ,medicine.medical_specialty ,Immunology ,Population ,Biochemistry ,Cancer stem cell ,Neoplasms ,Internal medicine ,medicine ,Humans ,education ,Survival rate ,Multiple myeloma ,education.field_of_study ,business.industry ,Complete remission ,Cancer ,Cell Biology ,Hematology ,Prognosis ,medicine.disease ,Survival Rate ,Treatment Outcome ,Cancer cell ,Stem cell ,business ,Perspectives - Abstract
Although most patients with cancer respond to therapy, few are cured. Moreover, objective clinical responses to treatment often do not even translate into substantial improvements in overall survival. For example, patients with indolent lymphoma who achieved a complete remission with conventional-dose therapies in the prerituximab era did not experience a survival advantage over similar patients treated with a “watch and wait” approach. Several studies have also shown that neither the magnitude nor the kinetics of clinical response has an impact on survival in multiple myeloma. Recent data suggesting many malignancies arise from a rare population of cells that exclusively maintains the ability to self-renew and sustains the tumor (ie, “cancer stem cells”) may help explain this paradox that response and survival are not always linked. Therapies that successfully eliminate the differentiated cancer cells characterizing the tumor may be ineffective against rare, biologically distinct cancer stem cells. New methods for assessing treatment efficacy must also be developed, as traditional response criteria measure tumor bulk and may not reflect changes in rare cancer stem cell populations. In this article, we discuss the evidence for cancer stem cells in hematologic malignancies and possible ways to begin targeting these cells and measuring clinical effectiveness of such treatment approaches.
- Published
- 2006
7. Association of Foxp3 regulatory gene expression with graft-versus-host disease
- Author
-
Georgia B. Vogelsang, Tahiro Shin, Elizabeth C. Matsui, Christopher J. Thoburn, Yuji Miura, Richard J. Jones, Allan D. Hess, Emilie C. Bright, Michele Phelps, Ephraim J. Fuchs, Sally Arai, and William Matsui
- Subjects
Adult ,CD4-Positive T-Lymphocytes ,Male ,Time Factors ,Adolescent ,Transcription, Genetic ,T-Lymphocytes ,Immunology ,Graft vs Host Disease ,Bone Marrow Cells ,chemical and pharmacologic phenomena ,Thymus Gland ,CD8-Positive T-Lymphocytes ,Biology ,Biochemistry ,Immune tolerance ,Immune system ,immune system diseases ,medicine ,Humans ,RNA, Messenger ,Immunologic Tolerance ,Aged ,Bone Marrow Transplantation ,Regulation of gene expression ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Membrane ,FOXP3 ,Forkhead Transcription Factors ,Receptors, Interleukin-2 ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,DNA-Binding Proteins ,Transplantation ,surgical procedures, operative ,Graft-versus-host disease ,Gene Expression Regulation ,Leukocytes, Mononuclear ,Female ,Cytokine storm ,Stem Cell Transplantation ,Thymidine - Abstract
Graft-versus-host disease (GVHD) is characterized by an impairment of mechanisms that underlie the development of immunologic tolerance. Although the cytokine storm associated with GVHD leads to expression of cell surface markers on both effector and regulatory T cells, regulatory CD4+ T cells that play an instrumental role in the maintenance of tolerance appear to uniquely express the Foxp3 transcriptional repressor. Foxp3 mRNA expression was significantly decreased in peripheral blood mononuclear cells from patients with either allogeneic GVHD or autologous GVHD compared with patients without GVHD. Expression of Foxp3 negatively correlated with the severity of GVHD but positively correlated with recent thymic emigrants. The results suggest that defective thymic function contributes to the impaired reconstitution of immune regulatory mechanisms following transplantation. The decrease in regulatory mechanisms after transplantation appears to provide an environment permissive to the development of GVHD. (Blood. 2004;104:2187-2193)
- Published
- 2004
8. Characterization of clonogenic multiple myeloma cells
- Author
-
Carol Ann Huff, Richard J. Jones, Curt I. Civin, William Matsui, James Barber, Yvette C. Tanhehco, Qiuju Wang, Matthew T Malehorn, and B. Douglas Smith
- Subjects
Syndecans ,Immunology ,Antigens, CD34 ,Antineoplastic Agents ,Mice, SCID ,Biochemistry ,Article ,Immunophenotyping ,Antibodies, Monoclonal, Murine-Derived ,Mice ,Antigen ,Mice, Inbred NOD ,immune system diseases ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Clonogenic assay ,B-Lymphocytes ,Severe combined immunodeficiency ,Membrane Glycoproteins ,CD40 ,biology ,Stem Cells ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,Antigens, CD20 ,medicine.disease ,Molecular biology ,Clone Cells ,Cell culture ,biology.protein ,Proteoglycans ,Syndecan-1 ,Stem cell ,Antibody ,Multiple Myeloma ,Rituximab ,Biomarkers ,Neoplasm Transplantation - Abstract
The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138+ plasma cells. CD138- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138+ cells were not. Furthermore, CD138- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM “stem cells” are CD138- B cells with the ability to replicate and subsequently differentiate into malignant CD138+ plasma cells.
- Published
- 2004
9. Bone Marrow Niche - Multiple Myeloma Cross-Talk Generates Bortezomib Resistance
- Author
-
Gabriel Ghiaur, Salvador Alonso, Richard J. Jones, and William Matsui
- Subjects
education.field_of_study ,Stromal cell ,Immunology ,Cell ,Population ,Mesenchymal stem cell ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,medicine.anatomical_structure ,Stroma ,Plasma cell differentiation ,medicine ,Proteasome inhibitor ,Bone marrow ,education ,medicine.drug - Abstract
Bortezomib (BTZ), a proteasome inhibitor that induces accumulation of misfolded proteins and apoptosis in highly secretory cells, has improved survival in multiple myeloma (MM) but fails to achieve cure. It has been postulated that a population of MM cells, phenotypically similar to B-cells, are intrinsically resistant to BTZ, and contribute to minimal residual disease (MRD) and relapse (Matsui et al., 2008). Bone marrow (BM) stroma expresses CYP26 enzymes and creates a retinoic acid (RA)-low environment that prevents differentiation of normal and malignant cells (Meng et al., 2015). Since RA promotes plasma cell differentiation, Ig secretion (Ertesvag et al., 2007) and thus, ER-stress (Xu et al., 2007), we tested if the BM niche induces BTZ resistance by promoting a B-cell program in MM. Consistent with this, MM cells (H929 and MM1S) cultured under low RA conditions (co-cultured with BM stroma or treated with AGN194310, a pan-RA receptor inhibitor) up regulated B-cell markers (BCL6), and down regulated genes associated with plasma cell differentiation and BTZ sensitivity (XBP1s, Blimp, CHOP) (Fig 1A). In addition, inhibition of stromal CYP26 via R115866 reversed these gene expression changes in stroma co-culture conditions (Fig 1A). To verify whether a low-RA environment promotes BTZ resistance, we incubated MM cells with BM mesenchymal cells for 5 days. Then, MM cells were separated from stroma, and treated with BTZ. Incubation with BM stroma induced BTZ resistance of MM cells, which was completely overcome by CYP26 inhibition or by the CYP26 resistant retinoid IRX5183 (Fig 1B). Moreover, MM cells pretreated with AGN for 5 days were also resistant to BTZ (Fig 1B). Interestingly, this BTZ resistant phenotype was preserved for up to 48h upon removal of MM cells from stromal co-culture (Fig 1C). Recent studies suggest that malignant cells remodel their microenvironments to build a more protective niche (Schepers et al., 2013). Consistent with this, we found that stromal CYP26A1 was highly upregulated after co-culture with MM cells. Supernatant from MM cells had similar effects, implicating a soluble factor on this interaction. Since secretion of SHH was implicated in chemotherapy resistance in MM (Liu et al., 2014), we tested if MM-induced CYP26 up-regulation was dependent on SHH signaling. As hypothesized, treatment with the Smo antagonist cyclopamine, or the use of SMO knockout (KO) stromal cells, overcame CYP26A1 up regulation by MM cells (Fig 1D) To test if cell extrinsic SHH signaling in stromal niche contributes to BTZ resistance by modulating RA pathway in MM cells, we modeled MM-BM stroma interactions in a xenograft setting. Each mouse carried two subcutaneous tumors consisting of Luciferase expressing MM1S cells and SmoFl/Fl BM stroma cells, transduced with either a control vector (WT stroma) or Cre-recombinase (Smo KO stroma) (Fig E). Mice were treated with IRX5183, BTZ, or the combination. Consistent with our in vitro data, tumors with Smo KO stroma showed a significant response to BTZ, while those with WT stroma were refractory, as determined by exponential increase in bioluminescence. However, the combination of IRX+BTZ resulted in a dramatic and equivalent response regardless of the phenotype of the stromal compartment (Fig 2A, 2B). Moreover, tumors with Smo KO stroma, or treated with IRX, showed decrease expression of B-cell markers, and up-regulation of genes associated with plasma cell differentiation and BTZ sensitivity. In conclusion, we show that the BM niche promotes a B-cell program in MM, characterized by a low ER-stress and BTZ resistance. This niche-induced program is maintained even after displacement from the BM, which may have important implications for the use of mobilization-sensitization strategies. Finally, we propose the existence of a bi-directional crosstalk between MM cells and their respective niches. In our model, MM cells via secretion of SHH modify BM stroma to create RA-low environments, induce BTZ resistance and contribute to MRD. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
- Published
- 2015
10. High-dose cyclophosphamide for severe aplastic anemia: long-term follow-up
- Author
-
B. Douglas Smith, Ephraim J. Fuchs, Carol Ann Huff, Allen R. Chen, Robert A. Brodsky, Steven N. Goodman, William Matsui, Richard F. Ambinder, Donna Dorr, Richard J. Jones, and Leo Luznik
- Subjects
Adult ,Male ,medicine.medical_specialty ,Time Factors ,Cyclophosphamide ,Adolescent ,Anemia ,Clinical Trials and Observations ,Immunology ,Drug Resistance ,Biochemistry ,Gastroenterology ,chemistry.chemical_compound ,Young Adult ,Refractory ,Internal medicine ,Cause of Death ,medicine ,Humans ,Aplastic anemia ,Child ,Survival analysis ,Aged ,Hematology ,Dose-Response Relationship, Drug ,business.industry ,Bone marrow failure ,Anemia, Aplastic ,Cell Biology ,Middle Aged ,medicine.disease ,Survival Analysis ,Nitrogen mustard ,Surgery ,Hematopoiesis ,Treatment Outcome ,chemistry ,Child, Preschool ,Female ,business ,medicine.drug ,Follow-Up Studies - Abstract
Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder that can be treated with bone marrow transplantation, immunosuppressive therapy, and high-dose cyclophosphamide. Here, we report long-term follow-up on 67 SAA patients (44 treatment-naive and 23 refractory) treated with high-dose cyclophosphamide. At 10 years, the overall actuarial survival was 88%, the response rate was 71% with the majority being complete, and the actuarial event-free survival was 58% in 44 treatment-naive SAA patients. Patients with refractory SAA fared less well after high-dose cyclophosphamide therapy; at 10 years, overall actuarial survival, response, and actuarial event-free survival rates were 62%, 48%, and 27%, respectively. High-dose cyclophosphamide is highly effective therapy for severe aplastic anemia. Large randomized controlled trials will be necessary to establish how results of high-dose cyclophosphamide compare with either bone marrow transplantation or standard immunosuppressive regimens, such as antithymocyte globulin and cyclosporine.
- Published
- 2010
11. Circulating clonotypic B cells in classic Hodgkin lymphoma
- Author
-
Milada S. Vala, Brandy Perkins, Lan L. Gellert, James Barber, Jonathan M. Gerber, Carole B. Miller, Sarah Brennan, Mark J. Siedner, Richard F. Ambinder, Richard J. Jones, William Matsui, M. Victor Lemas, Yvette L. Kasamon, and Christopher D. Gocke
- Subjects
Adult ,Male ,Immunology ,Population ,Immunoglobulins ,Stem cell marker ,Biochemistry ,Antigen ,Cell Movement ,Cell Line, Tumor ,hemic and lymphatic diseases ,medicine ,Humans ,education ,Lymph node ,Aged ,education.field_of_study ,B-Lymphocytes ,Lymphoid Neoplasia ,biology ,business.industry ,Cell Biology ,Hematology ,Middle Aged ,medicine.disease ,Hodgkin Disease ,Lymphoma ,medicine.anatomical_structure ,Cell culture ,Cancer research ,biology.protein ,Female ,Antibody ,Stem cell ,business - Abstract
Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells, they are unlike any normal cells of that lineage. Moreover, the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago, the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation, however, has never been corroborated, and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27+ALDHhigh B cells, sharing immunoglobulin gene rearrangements with lymph node HRS cells, were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear, these data suggest they may be the initiating cells for HL.
- Published
- 2009
12. The multiple myeloma–associated MMSET gene contributes to cellular adhesion, clonogenic growth, and tumorigenicity
- Author
-
William Matsui, Abde M. Abukhdeir, Qiuju Wang, Joseph P. Garay, Josh Lauring, Hiroyuki Konishi, John P. Gustin, Robert J. Arceci, and Ben Ho Park
- Subjects
Immunology ,Transplantation, Heterologous ,Down-Regulation ,Mice, Nude ,Biology ,Biochemistry ,Translocation, Genetic ,Colony-Forming Units Assay ,Mice ,Cell Line, Tumor ,Cell Adhesion ,Animals ,Humans ,Receptor, Fibroblast Growth Factor, Type 3 ,Clonogenic assay ,Alleles ,Regulation of gene expression ,Chromosomes, Human, Pair 14 ,Gene knockdown ,Neoplasia ,Cell Cycle ,Gene targeting ,Cell Biology ,Hematology ,Histone-Lysine N-Methyltransferase ,Cell cycle ,Fibroblast growth factor receptor 3 ,Prognosis ,Molecular biology ,Transplantation ,Gene Expression Regulation, Neoplastic ,Repressor Proteins ,Cell culture ,Gene Targeting ,Female ,RNA Interference ,Chromosomes, Human, Pair 4 ,Multiple Myeloma ,Neoplasm Transplantation - Abstract
Multiple myeloma (MM) is an incurable hematologic malignancy characterized by recurrent chromosomal translocations. Patients with t(4;14)(p16;q32) are the worst prognostic subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves 2 potential target genes: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally overexpressed in t(4;14) MM, whereas FGFR3 expression is lost in one-third of cases. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Down-regulation of MMSET expression in MM cell lines by RNA interference and by selective disruption of the translocated MMSET allele using gene targeting dramatically reduced colony formation in methylcellulose but had only modest effects in liquid culture. In addition, MMSET knockdown led to cell-cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Finally, MMSET knockdown and knockout reduced tumor formation by MM xenografts. These results provide the first direct evidence that MMSET plays a significant role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.
- Published
- 2008
13. Inhibiting Oncogenic RAS in Multiple Myeloma By Targeting Scaffold-ERK Interactions
- Author
-
William Matsui, Qiuju Wang, Ross McMillan, Christian B. Gocke, Vesselin R. Penchev, Julien Sage, and Paul A. Khavari
- Subjects
MAPK/ERK pathway ,Severe combined immunodeficiency ,Cell cycle checkpoint ,biology ,Cell growth ,Immunology ,Cell Biology ,Hematology ,Signal transduction inhibitor ,medicine.disease ,Biochemistry ,WW domain ,IQGAP1 ,biology.protein ,medicine ,Cancer research ,Clonogenic assay - Abstract
The introduction of novel therapeutic agents over the past decade has significantly improved the outcomes of patients with newly diagnosed multiple myeloma (MM). Despite these advances, virtually all patients relapse and develop drug-resistant disease that carries a dismal prognosis. In addition to chemoresistance, relapse and tumor regrowth are dependent on self-renewal to maintain clonogenic growth potential over time. A better understanding of the factors responsible for tumor self-renewal and regrowth may lead to novel therapies and improved survival rates. RAS mutations are common in MM and have been demonstrated to increase in frequency with relapse and disease progression. These findings implicate RAS in chemoresistance, self-renewal, and tumor regrowth upon relapse. We have begun to directly examine the functional role of mutant RAS signaling in MM by stably transducing KRASmut MM cells with a tetracycline-inducible shRNA and found that KRAS knockdown decreases MM clonogenic growth in vitro. Although inhibiting oncogenic RAS may be beneficial for treating relapsed disease it remains an elusive drug target. Recently, our collaborators have developed a novel small peptide RAS signaling inhibitor that mimics the WW domain of IQGAP1 which competes for ERK binding to scaffolds. The WW peptide specifically targets solid tumors containing RAS (or BRAF) mutations and is well tolerated in mouse models. Thus, by targeting only tumorigenic cells it may demonstrate decreased toxicity compared to existing small molecule inhibitors of MAPK signaling. We analyzed gene expression profiling data from the University of Arkansas (GSE2658) and found that elevated IQGAP1 expression levels (top 12.5%) are associated with increased disease-related mortality in MM (log-rank p = 0.0002, HR 4.3, 95% CI 2.0-9.1). This suggests that IQGAP1 may play an important biological role in MM disease progression and that inhibition of IQGAP1 and RAS signaling with the WW peptide may have efficacy and specificity in MM patients. We have treated RAS mutated MM cell lines with the WW peptide or a scrambled (SCR) control peptide and found that the WW peptide effectively decreases pERK levels after 24 hours at doses as low as 20 nM. The WW peptide also decreases cell growth in RAS mutated but not wild-type MM cells by inducing a cell cycle arrest (by BrdU/7-AAD staining). Importantly, treatment of RAS mutated MM cell lines with the WW peptide significantly decreased colony formation in vitro and led to a reduction in the frequency of highly clonogenic CD138neg MM cells. Finally, an immunodeficient mouse xenograft model was used to determine the effect of the WW peptide on engraftment potential. We treated NCI-H929NRAS(G13D) cells with SCR or WW peptide and injected them into NOD/SCID/IL2γchainKO (NSG) mice. Engraftment as detected by kappa light chain ELISA was significantly reduced in the WW peptide treated group. These data further implicate RAS signaling as well as MAPK signaling in MM self-renewal and identify a potential novel therapy for treatment of relapsed/refractory MM. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
14. Aberrant Hedgehog Pathway Activity Marks Clinical MDS Progression and Accelerates Leukemic Transformation in Vivo
- Author
-
Peter D. Aplan, Amy E. DeZern, Yiting Lim, William Matsui, Lukasz P. Gondek, Qiuju Wang, Hideki Makishima, and Jaroslaw P. Maciejewski
- Subjects
education.field_of_study ,Immunology ,Population ,CD34 ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Haematopoiesis ,Leukemia ,medicine.anatomical_structure ,Cancer research ,medicine ,Bone marrow ,Progenitor cell ,Stem cell ,education - Abstract
Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML. Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039). In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. Figure 1 Figure 1. Disclosures Maciejewski: Alexion: Speakers Bureau; Celgene: Speakers Bureau.
- Published
- 2014
15. Targeting PYK2 Mediates Microenvironment-Specific Myeloma Cell Death
- Author
-
Lori A. Hazlehurst, Jennifer Gemmer, Ismael Rosado-Lopez, Linda Mathews, Tuan Nguyen, Conor C. Lynch, Jonathan A. Pachter, Bin Fang, Jennifer E. Ring, John M. Koomen, Kenneth H. Shain, Liang Nong, Robert A. Macleod, William Matsui, and Mark B. Meads
- Subjects
Tumor microenvironment ,Stromal cell ,biology ,Chemistry ,Immunology ,Integrin ,Cell Biology ,Hematology ,Biochemistry ,Focal adhesion ,Cell killing ,Cancer stem cell ,Bone marrow neoplasm ,Cancer research ,biology.protein ,Stem cell - Abstract
Multiple myeloma (MM) remains an incurable malignancy, at least in part, due to minimal residual disease (MRD). It has been hypothesized that MRD is influenced by the survival promoting aspects of the bone marrow tumor microenvironment (TME) and populations of putative MM cancer stem cells. Therefore, identification of survival determinants specific to the TME and putative MM stem cells is critical for the rational design of therapy and elimination of MRD1. Within the context of the TME, we have shown that collaborative signaling between β1 integrin-mediated adhesion to fibronectin (FN) and Interleukin-6 (IL-6) confers a more malignant phenotype via amplification of STAT3 activation2. We have validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in MM patient specimens by flow cytometry. Further characterization of the events modulated under these culture conditions with quantitative phospho-tyrosine profiling identified 193 differentially phosphorylated peptides after adhesion to FN, treatment with IL-6, and the combination. Phosphopeptides represented proteins involved in signal transduction, cytoskeleton assembly/adhesion, transcription, RNA processing, metabolism, and the cell cycle. Seventy-seven phosphorylations were up-regulated upon adhesion, including PYK2/FAK2, paxillin, CASL, and p130CAS consistent with focal adhesion (FA) formation. Western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN or bone marrow stromal cells (BMSC), correlating with the amplification of IL-6-induced STAT3 and JAK1. We hypothesized that the collaborative signaling between β1 integrin and the IL-6 signal transducer, gp130, was mediated by FA formation and PYK2. Although PYK2 has been linked to downstream signaling events of other JAK-dependent cytokine receptors, it has not been associated with a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families3-6. Assessing downstream gene expression, we also demonstrated that adhesion to FN alone or co-stimulation with FN and IL-6 upregulated mRNA and protein expression of the MM survival-associated gene, deptor7, and this enhanced expression was abrogated by RNAi knockdown of STAT3 or PYK2. Both pharmacological and molecular targeting of PYK2 attenuated the amplification of STAT3 phosphorylation in adhered cells. Importantly, MM cells co-cultured with patient BMSCs showed similar gp130 and β1 integrin-specific enhancement of PYK2, JAK1, STAT3 signaling when adhered to patient BMSCs, demonstrating that PYK2 modulates co-stimulation specific STAT3 signaling in more complex, clinically relevant models of the microenvironment. Interestingly, targeting PYK2 with antisense oligonucleotides (ISIS Pharmaceuticals) or with the dual PYK2/FAK (Focal adhesion kinase) tyrosine kinase inhibitors VS-4718, VS-6062, and VS-6063 (Verastem, Inc.) induced preferential MM cell killing in the context of adhesion to patient BMSCs, but not in monoculture or under transwell co-culture conditions. We confirmed that this cell death correlated with inhibition of BMSC-induced PYK2 and STAT3 phosphorylation. Further analysis similarly demonstrated a preferential reduction in colony formation in BMSC-adherent MM cell lines and patient specimens treated with VS-6063 relative to MM cells in the absence of BMSCs. Lastly, we examined the effects of targeting PYK2 with VS-6063 in ALDH+ MM cancer stem cells. We demonstrated that co-culture with BMSCs increased the percentage of ALDH+ MM cancer stems relative to monoculture. VS-6063 decreased the percentage of the ALDH+ cells associated with BMSC to greater degree than MM cells without BMSC. These data identify a novel PYK2-mediated survival pathway activated within the context of multiple microenvironmental cues, suggesting that PYK2 is a putative TME-specific therapeutic target. Further, putative MM cancer stem cells are sensitive to PYK2 inhibition by the FAK/PYK2 inhibitor VS-6063. As such, these data indicate that PYK2 may be critical target for the eradication of MRD by targeting both MM cells in the context of TME and MM cancer stem cells. These results suggest that MM represents a compelling clinical development path for FAK/PYK2 inhibitors, such as VS-4718 and VS-6063, in clinical trials focusing on eradication of MRD. Disclosures Shain: Onyx/Amgen: Research Funding; Celgene: Research Funding; Envision/Celgene: Speakers Bureau; L&M Healthcare/Onyx/Amgen: Speakers Bureau. Ring:Verastem: Employment. Pachter:Verastem Inc.: Employment, Equity Ownership.
- Published
- 2014
16. Outcomes Of Allogeneic Blood Or Marrow Transplantation (AlloBMT) In Multiple Myeloma With Post-Transplantation Cyclophosphamide (PTCy)
- Author
-
Carol Ann Huff, Jennifer A. Kanakry, William Matsui, Mark J. Levis, Nilanjan Ghosh, Richard F. Ambinder, Javier Bolaños-Meade, Richard J. Jones, Xiaobu Ye, Douglas E. Gladstone, Robert A. Brodsky, Lode J. Swinnen, Ephraim J. Fuchs, Ivan Borrello, Leo Luznik, and Yvette L. Kasamon
- Subjects
medicine.medical_specialty ,Cyclophosphamide ,Bortezomib ,business.industry ,Immunology ,Hazard ratio ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Surgery ,Transplantation ,medicine.anatomical_structure ,Median follow-up ,medicine ,Bone marrow ,business ,Multiple myeloma ,medicine.drug ,Cause of death - Abstract
Background AlloBMT is a potentially curative treatment for multiple myeloma (MM). However, its effectiveness has been compromised by high transplant related mortality (TRM) and acute and/or chronic graft-versus host disease (GvHD). Our development of PTCy has reduced the incidence of GvHD allowing safe and effective related haploidentical alloBMT. PTCy promotes tolerance in both alloreactive host and donor T cells, leading to suppression of both graft rejection and GVHD after alloBMT. Patients and Methods As part of an IRB-approved study we performed a retrospective analysis on all patients with MM who underwent alloBMT followed by PTCy. A total of 39 patients who were transplanted between 2003 and 2011 were identified. All patients received 2 doses of PTCy 50mg/kg on days 3 and 4 after alloBMT. High risk MM was defined as having any one of the following: del(13q) by cytogenetics or t(4;14), t(14;16), t(14;20), -17p ,+1q21 on FISH/cytogenetics. International Staging System (ISS) stage was based on beta2 microglobulin and albumin at diagnosis of symptomatic MM. The definitions of progression, stable disease and response were as per the International Myeloma Working Group criteria. Survival probability was determined using Kaplan-Meier method and differences assessed with the log-rank test. Cox regression was used to model prognostic factors with respect to progression-free (PFS) and overall survival (OS) rates. Results The median follow-up for living patients was 80 months (22-115). The minimum follow up was 23 months. The median age at BMT was 54 (range 37-70). 30 patients (77%) received a transplant from a matched sibling donor. Of the remaining 9 patients, 7 (18%) received a transplant from a haploidentical donor and 2 (5%) from a matched unrelated donor. 28 patients (72%) were in > PR at the time of alloBMT. Only 1 patient (3%) was in a CR prior to alloBMT. Thirty patients (77%) received reduced intensity conditioning and the remaining 9 patients (23%) received myeloablative conditioning. 28 patients (72%) received unmanipulated bone marrow, while remaining 11 patients (28%) received mobilized peripheral blood cells. The median time between diagnosis and alloBMT was 10.6 months (4.1-178.7). Cytogenetics and FISH for evaluating high risk MM were available for 26 patients (66.6%) and of those 15 patients (58%) had high risk features. 28 patients (72%) were evaluable for ISS at diagnosis and of those 13 patients (46%) had ISS III at diagnosis. 36 patients (92%) had been treated with either bortezomib based or immunomodulatory (Imid) based therapy prior to alloBMT. 7 patients (18%) had received an autologous transplant prior to alloBMT. 20 patients (51%) are alive after a median follow up of > 6 years after alloBMT. Only 1 (2.5%) patient died from complications related to alloBMT. At last follow up, 6 patients (15%) are in sustained first complete remission after alloBMT. Of the 9 patients who received myeloablative prep, 6 are alive and 3 are in sustained CR. At 6 months following alloBMT, 12 patients (31%) were in a deeper response compared to their pre-transplant status. 15 patients (38%) developed > grade 2 acute GvHD at a median of 1.5 (range 0.6-4.5) months. Only 3 patients (8%) developed grade 3 acute GvHD and there was no grade 4 acute GVHD. 14 (36%) patients received systemic treatment for acute GvHD. 5 (13%) patients developed chronic GvHD. The median OS was 96 months, and the median PFS was 14 months (95% CI 6.2-32.8). Chronic GvHD was significantly associated with PFS, with a median PFS of 90 months in patients who developed cGvHD compared to 11 months in patients who did not (hazard ratio = 0.3, 95% CI 0.07-1.25). The major cause of death was disease progression. Conclusion The use of PTCy led to a very low TRM in MM, including with related haploidentical donors. Although only a minority of patients maintained long-term PFS, the prolonged OS allows incorporation of novel post-alloBMT strategies to improve disease control. Disclosures: Off Label Use: Outcomes of Allogeneic Blood Or Marrow Transplantation (AlloBMT) In Multiple Myeloma With Post-Transplantation Cyclophosphamide (PTCy).
- Published
- 2013
17. Dissecting The Potential Role Of Prevnar As An Anti-Myeloma Vaccine
- Author
-
Ivan Borrello, Ervin Griffin, Lee Susan, William Matsui, Carol Ann Huff, Nilanjan Ghosh, Amy Sidorski, Anna Ferguson, Andrea Casildo, Kimberly A. Noonan, and Lakshmi Rudraraju
- Subjects
business.industry ,T cell ,Immunology ,Pneumococcal 7-Valent Conjugate Vaccine ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Vaccination ,medicine.anatomical_structure ,Immune system ,Antigen ,Conjugate vaccine ,medicine ,business ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
Background Prevnar, is a multi-valent conjugate vaccine given to children and adults over 50 for the prevention of Streptococcus pneumonia, otis media and pneumococcal pneumonia. The conjugate in Prevnar is a CRM-197 protein molecule which is a nontoxic recombinant Diphtheria toxin. Prevnar serves as an excellent tool in monitoring overall immune response changes in myeloma patients’ pre and post treatment. Humoral B-cell responses can be measured by antibody responses to the pneumococcal antigens, while T cell responses to CRM-197. Clinical Study We previously conducted a study to determine the efficacy of lenalidomide to augment vaccine specific responses in patients with myeloma. Two cohorts of patients were studied. In cohort A (N=10), the first Prevnar vaccine was given two weeks prior to starting lenalidomide and the second vaccine on day 14 of cycle 2 of lenalidomide. In cohort B (N=7), both Prevnar vaccines were given on lenalidomide (day 14 of cycle 2 and 4). As we previously reported patients in cohort B had an overall better B and T cell response to Prevnar compared to cohort A. These responses were due to an overall change in B and T cell phenotype attained with lenalidomide therapy. Results Prospectively, patients in cohort B also had an unexpected overall increase in disease response and in response duration. In Cohort A only 10% of patients responded to therapy while 60% of patients in Cohort B had a clinical response. The patients with a measurable clinical response had a 5-fold increase in the percentage of tumor specific bone marrow (BM) T cells after two vaccinations with Prevnar whereas the non-responding patients had no increase in tumor specific BM T cells. Parelleling the anti-tumor response, responders showed a 15 fold increase in CRM-197 specific BM T cells after the second vaccination. Patients with no clinical response showed minimal CRM-197 T cell immunity. CRM-197 is a specific inhibitor of HB-EGF; syndecan-1 (CD138) is an HB-EGF co-receptor as well as a marker for myeloma plasma cells. We hypothesized that HB-EGF specific responses produced by vaccination with the Prevnar vaccine, and CRM-197 specifically, may have contributed to the overall increased clinical responses in our clinical trial. Responding patients had a 5-fold increase in HB-EGF specific BM T cells after vaccine 2 while clinical non-responders had no increase in HB-EGF specific BM T cells. T cells specificity for purified HB-EGF correlated with both CRM-197 and tumor specific responses. Finally the myeloma cell lines U266, H929, KMS-11 and KMS-12 co-stained for CD138 and HB-EGF with 47% of CD138+ myeloma cells co-expressing HB-EGF. Conclusions We hypothesize that the CRM-197 moiety of the Prevnar vaccine can prime T cell responses against HB-EGF on plasma cells. This immune response, in turn, weakens the tumor stromal interactions in the tumor microenvironment and potentially enhances the anti-tumor efficacy of immunomodulatory drugs such as lenalidomide. Therefore, Prevnar may possibly serve as a candidate anti-myeloma vaccine. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
18. Response: Hodgkin lymphoma stem cells
- Author
-
Richard J. Jones, Christopher D. Gocke, William Matsui, and Richard F. Ambinder
- Subjects
Letter to the editor ,business.industry ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Correspondence ,Cancer research ,Medicine ,Hodgkin lymphoma ,Lymph ,Stem cell ,business - Abstract
In his letter to the editor, Dr Kuppers calls attention to additional support that clonotypic B cells are present in Hodgkin lymphoma (HL) and may represent HL stem cells. We were unaware of Jansen and colleagues' prior work describing such cells in HL lymph nodes,[1][1],[2][2] and we thank Dr Ku
- Published
- 2009
19. The Telomerase Inhibitor, Imetelstat, Rapidly Reduces Myeloma Cancer Stem Cells (CSCs) in a Phase II Trial
- Author
-
Stephen M. Kelsey, Ashraf Badros, Quiju Wang, Kevin Nishimoto, Anita Reddy, Richard J. Jones, Carol Ann Huff, William Matsui, and Stuart Monic J
- Subjects
Oncology ,medicine.medical_specialty ,Telomerase ,Bortezomib ,business.industry ,Immunology ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Transplantation ,Imetelstat ,Internal medicine ,medicine ,Proteasome inhibitor ,business ,Multiple myeloma ,medicine.drug ,Lenalidomide - Abstract
Abstract 4898 Despite improved response rates with the use of novel agents, the vast majority of patients with multiple myeloma (MM) experience disease relapse and progression. This clinical pattern suggests the cells responsible for tumor regrowth persist following treatment, and we previously demonstrated that highly tumorigenic and self-renewing MM cells are relatively resistant to a wide range of anti-myeloma agents. These MM CSCs are present in the peripheral blood circulation and express memory B cell surface antigens (CD19 and CD27) and the stem cell marker aldehyde dehydrogenase (ALDH). We have also studied cellular mechanisms regulating self-renewal and found that MM CSCs express increased levels of telomerase activity (TA). In preclinical models, the novel telomerase inhibitor imetelstat has been found to inhibit CSCs from a wide range of tumor types, and we reported that imetelstat reduced TA in MM CSCs resulting in the loss of self-renewal and clonogenic growth potential. In an open label phase II clinical trial initiated in February 2011, previously treated MM patients (pts) (n=6) with stable but detectable disease following treatment with an iMid or proteasome inhibitor received single agent imetelstat (7.5–9.4 mg/kg IV on days 1 and 8 every 21–28 days). Additional pts (n=4) receiving lenalidomide maintenance and whose disease was stable for at least three months have also been enrolled. Pts received imetelstat therapy until evidence of disease progression or toxicity. Median age is 62.5 years (range 53–68); median number of prior therapies is 1 (range 1–4) including 4 pts with prior autologous transplant and 1 pt with prior mini-allogeneic transplant; 8 pts with prior bortezomib use; 9 pts with prior iMiD use; and 4 pts with concurrent lenalidomide use with receipt of lenalidomide for a median of 11.38 months (range 4.51–19.21 months) prior to initiation of imetelstat. As of July 30, 2012, 6 pts remain on study. Four pts were discontinued from imetelstat therapy after receiving a median of 7 doses of imetelstat either due to disease progression (n=2) or hematologic toxicity (n=2). Cytopenias were the most frequently reported toxicity with 8 of 10 pts demonstrating grade 3–4 thrombocytopenia and neutropenia during cycle 2, often requiring dose reductions or holds in subsequent cycles. The frequency of circulating MM CSCs (CD19+CD27+ALDH+) was quantified by flow cytometry. Circulating MM CSCs could be detected prior to the initiation of imetelstat (mean 10.7 × 10e3 cells/ml, range 17–53 × 10e3) in 8 of the 9 pts who have completed at least 2 cycles of treatment with imetelstat. In the single remaining patient, circulating CSCs were assessed from Cycle 2 onwards. Over the course of treatment, the frequency of MM CSCs decreased significantly, on average 2 fold every 30 days (Fig 1), in 8 of the 9 patients studied despite no upgrades in clinical response as per IWG criteria. In two pts who received 4 and 6 cycles of single agent imetelstat respectively, standard responses detected as decreasing or plateaued serum M protein or light chain levels were sustained over 4 months following discontinuation (Fig 2). Moreover, these delayed responses occurred in the absence of any additional therapy. These findings demonstrate that imetelstat rapidly decreases circulating MM CSCs. In addition, several patients experienced delayed, but sustained, clinical responses as measured by standard criteria. Therefore, imetelstat may have therapeutic implications for MM and other malignancies driven by CSCs. Figure 1. Serial measurement of CSCs in patients (n=9) treated with imetelstat Figure 1. Serial measurement of CSCs in patients (n=9) treated with imetelstat Figure 2. Clinical response in Pts 003 and 004 based on changes in paraprotein level. Dashed line represents off study evaluation. Figure 2. Clinical response in Pts 003 and 004 based on changes in paraprotein level. Dashed line represents off study evaluation. Disclosures: Huff: Celgene: Scientific Advisory Board Other; Novartis: Consultancy. Jones:Cytomedix: Royalties, Royalties Patents & Royalties. Reddy:Geron Corp.: Employment. Nishimoto:Geron Corp: Employment. Stuart:Geron Corp: Consultancy, Employment; OncoMed Pharmaceuticals: Consultancy. Kelsey:Geron Corp: Employment. Matsui:Geron Corp.: Research Funding.
- Published
- 2012
20. Phase I/II Study of Marrow Infiltrating Lymphocytes (MILs) Generates Measurable Myeloma-Specific Immunity in the Autologous Stem Cell Transplant (SCT) Setting
- Author
-
Jeffrey Bitzan, Janice M Davis Sproul, Jonathan D. Powell, Richard J. Jones, Javier Bolaños-Meade, Susan Fiorino, Carol Ann Huff, M. V. Lemas, Lakshmi Rudraraju, Ivan Borrello, William Matsui, Kimberly A. Noonan, and Leo Luznik
- Subjects
medicine.medical_specialty ,education.field_of_study ,business.industry ,T cell ,Immunology ,Population ,CD28 ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Graft-versus-host disease ,Immune system ,medicine.anatomical_structure ,Internal medicine ,medicine ,Bone marrow ,Stem cell ,business ,education ,CD8 - Abstract
Abstract 997 Adoptive T cell therapy requires the infusion of highly tumor specific T cells capable of trafficking to the tumor site, killing the tumor and persisting over time. MILs (as compared to peripheral blood T cells) possess many of these features (Noonan, Ca, Res. 2006). We here report the initial results from our Phase I/II trial. 22 patients were transplanted with a Melphalan-200. 19 with a CD34-selected product, 1 unselected autologous stem cells and 2 auto-BMT. 12 underwent SCT as initial consolidation and 10 patients with relapsed disease that were heavily pre-treated with an average of 3.2 prior therapies. Mean ISS score was 2.0. Average age was 56 (29–71). MILs were expanded with anti-CD3/CD28 beads for 7 days (avg. fold expansion 48.5 and avg. cell dose 2.8×10e7 CD3/kg). MILs were infused on day +3. Patients in a CR were ineligible for this study. The best clinical response included 6CRs (27%), 10PRs (45%). Autologous GVHD which was observed in 32% of patients, was limited to the skin and required no treatment. Development of this syndome did not correlate with improved clinical outcomes. Lymphoid recovery was rapid with a mean absolute lymphocyte count (ALC) on day 15 of 886 cells/ul. The laboratory immune monitoring studies were only performed in the bone marrow – the disease site and not in the peripheral blood. The percent CD3+ T cells in the BM was 13% at baseline with a CD4:CD8 ratio of 1:3. By day 60, BM T cell reconstitution was complete and showed an inversion of the CD4:8 ratio of 0.39 that persisted through day 360. At baseline, there were more CD4 effector memory (CD4EM) (CD62L−/CD45RO+) than CD4 central memory (CD4CM) (CD62L+/CD45RO+) (43% vs 27%). The CD4CM population peaked on day 60 at 43% and persisted through 1 year, CD4EM remained unchanged, and the CD4Effector decreased from 19.8% to 5.6%. The CD8 subpopulations remained unchanged from pre-SCT to 1 year post-SCT. Treg numbers doubled from harvest to post-SCT consistent with previous studies showing a greater number of Tregs in healthy BM compared to MM-BM. Activated T cells (CD69+) doubled from pre- to post-SCT. IFNγ production in both CD4 and CD8 cells more than doubled compared to pre-SCT and was maintained at 1 year suggesting the persistence of the infused MILs. The immune monitoring in the BM based on clinical responses revealed that patients achieving a CR/PR showed greater CD8 numbers at day +60 compared to stable disease (SD) or progressive disease (PD)(14.5% vs. 7.6%, respectively) and inversion of the CD4/CD8 ratio at 1 year (0.55 vs 1.26). In patients with SD or PD, the immune infiltrate in the BM was characterized by a large numbers of effector and effector-memory T cells and few CD8CM at baseline. CR patients possessed the fewest CD8Effector and the most CD8CM with persistence of CD8CM out to one year. These patients also showed a greater expansion in IFNγ cells as well as a greater amount of IFNγ production at each time point. Importantly, tumor-specific IFNγ production of CD3 cells in the BM was predictive of a clinical response. Patients achieving a CR showed twice the antigen-specific IFNγ production compared to all other groups at day 180 which persisted through 1 year. Analyses of additional immune subsets will be discussed in greater detail. This is the first reported trial utilizing activated MILs as a source of tumor specific T cells. We have demonstrated the ability to effectively expand MILs ex vivo and provide an analysis of the parameters of immune reconstitution demonstrating that the clinical outcome correlated with the robustness of the immune response in the BM. Disclosures: Luznik: Otsuka Pharmaceuticals: Research Funding.
- Published
- 2011
21. The TGF- β Family Member Growth Differentiation Factor 15 (GDF15) Regulates the Self-Renewal of Multiple Myeloma Cancer Stem Cells
- Author
-
Jasmin Roya Agarwal, Qiuju Wang, William Matsui, Toshihiko Tanno, and Akil Merchant
- Subjects
Stromal cell ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cytokine ,medicine.anatomical_structure ,Cancer stem cell ,medicine ,Cancer research ,Bone marrow ,GDF15 ,Stem cell ,Antigen-presenting cell ,Transforming growth factor - Abstract
Abstract 2954 Multiple myeloma (MM) cancer stem cells (CSCs) possess both enhanced tumorigenic potential and relative drug resistance suggesting they play a major role in disease relapse and progression. Therefore, a better understanding of the processes regulating MM CSCs may lead to the development of novel therapies that prevent tumor regrowth and improve long-term outcomes. Normal stem cells are tightly regulated by factors within the local microenvironment that include both soluble factors and direct contact with accessory cells. However, external factors regulating MM CSCs have not been identified. Recent studies have demonstrated that stromal cells in the MM bone marrow microenvironment secrete growth differentiation factor 15 (GDF15), a member of the TGF-b family. We initially studied the role of this cytokine in the pathogenesis of MM by examining circulating GDF15 levels in MM patients. Compared to healthy volunteers, we found that median GDF15 levels were significantly increased in MM patients (821 vs. 390 pg/ml; n=16; p Disclosures: No relevant conflicts of interest to declare.
- Published
- 2011
22. Targeting Brouton's Tyrosine Kinase with PCI-32765 Blocks Growth and Survival of Multiple Myeloma and Waldenström Macroglobulinemia Via Potent Inhibition of Osteoclastogenesis, Cytokines/Chemokine Secretion, and Myeloma Stem-Like Cells in the Bone Marrow Microenvironment
- Author
-
Betty Y. Chang, Yu-Tzu Tai, Laurence Elias, Michele Cea, William Matsui, Guang Yang, Nikhil C. Munshi, Sun-Young Kong, Michael A. Sellitto, Ruben D. Carrasco, Paul G. Richardson, Steven P. Treon, Jianjun Zhao, Zachary R. Hunter, Yolanda Calle, Joseph J. Buggy, Michelle C. Chen, Jianhong Lin, Mariateresa Fulciniti, Yiguo Hu, Kenneth C. Anderson, and Antonia Cagnetta
- Subjects
Myeloid ,Stromal cell ,biology ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Plasma cell ,Biochemistry ,Molecular biology ,medicine.anatomical_structure ,Cytokine ,Cancer cell ,Chemokine secretion ,medicine ,biology.protein ,Cancer research ,Bruton's tyrosine kinase ,Bone marrow - Abstract
Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
- Published
- 2011
23. Combination Therapy with Entinostat (MS-275) and GM-CSF to Enhance Differentiation In Myeloid Malignancies
- Author
-
Cho Eunpi, William Matsui, B. Douglas Smith, Jeanne Kowalski, Hua-Ling Tsai, and Richard J. Jones
- Subjects
Oncology ,medicine.medical_specialty ,education.field_of_study ,Myeloid ,Combination therapy ,Entinostat ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,chemistry.chemical_compound ,Leukemia ,Platelet transfusion ,medicine.anatomical_structure ,Maintenance therapy ,chemistry ,Internal medicine ,medicine ,education ,business ,Progressive disease - Abstract
Abstract 3312 Background: Histone actylases (HAC and histone deacetylases (HDAC) are two important enzymes in epigenetic control that can affect transcription of important regulatory transcription factors. Entinostat is a HDAC inhibitor that has been shown in vivo and in vitro to have anti-proliferative effects on many cancer cell types (Abujamra Leukemia Res 2009). When administered at low concentration to leukemic cell lines, entinostat induced p21-mediated growth arrest and expression of differentiation markers; higher concentrations led to marked increase in reactive oxygen species, mitochondrial damage, caspase activation and apoptosis (Rosato Cancer Res 2003). A Phase I study using entinostat as a single agent in relapsed and refractory leukemia showed in vivo differentiation potential with several patients showing significant increases in their mature granulocyte population and increased acetylation of the CD34+ blast population (Gojo Blood 2006). GM-CSF has been shown to enhance the differentiation potential of various agents such as interferon-alpha, all-trans-retinoic acid, bryostatin, and numerous other anti-neoplastic agents. The effects of combination therapy with GM-CSF and entinostat in patients with high-risk MDS or refractory and/or relapsed AML are presented here. Methods: A Phase II study was conducted to assess the safety and efficacy of combination therapy with GM-CSF and entinostat in patients with high-risk MDS and relapsed or refractory AML who are not eligible for allogeneic bone marrow transplant (BMT). The combination of entinostat and GM-CSF was administered in 6-week (42 day) cycles for at least 2 cycles. Entinostat was originally give at 8 mg/m2 weekly but was eventually adjusted to 4 mg/m2 weekly for the first 4 out of 6 weeks due to toxicity. GM-CSF was given at a single dose of 125 micrograms/m2/day for days 1–35 in the cycles 1, 2, 4 and 6 and days 1–42 in cycles 3 and 5. Patients who tolerated two cycles of 4 mg/m2 were assessed for response through measurements of peripheral blood, bone marrow aspirate and biopsies. Transfusion requirements and adverse events (AE) were recorded on all subjects throughout the study period. Clinical responses for AML and MDS were measured according to International Working Group definitions of complete response (CR), partial response (PR), stable disease (SD), hematologic improvement, and progressive disease (PD). Results: A total of 24 patients met the eligibility criteria for response assessment. Median age was 71 (range 52–84) years and 15 (63%) were male. Of the 19 patients with AML, 8 had relapsed/refractory disease, 7 had AML arising from MDS, 3 had therapy-related AML, and 1 had de novo AML. The remaining 5 patients had a primary diagnosis of MDS. 10 patients (42%) completed 2 or more cycles at the 4 or 6 mg/m2 dose of MS-275. These patients completed a total of 33 cycles, 1 resulting in CR, 4 in PR, 24 in SD, and 4 in PD. In addition to these standard endpoints, improvements were also noted in peripheral neutrophil counts (p Conclusion: Although treatment with entinostat and GM-CSF did not result in durable remissions, there were notable improvements in absolute neutrophil and platelet counts without negatively impacting the blast percentage. These findings suggests that therapy with entinostat and GM-CSF differentially promotes growth of mature myeloid cells and appears associated with better marrow function by minimizing the need for platelet transfusions. Such strategies may be most effective when applied to patients with low disease burdens or as maintenance therapy for patients with high risk disease in remission. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.
- Published
- 2010
24. Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM
- Author
-
Theodore L. DeWeese, Yiting Lim, William Matsui, Akil Merchant, Mohammad Hedayati, and Yonggang Zhang
- Subjects
Myeloid ,business.industry ,Immunology ,Bone marrow failure ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,In vivo ,Chloroquine ,medicine ,Cancer research ,Bone marrow ,Progenitor cell ,business ,medicine.drug - Abstract
Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.
- Published
- 2010
25. Origin of the Myeloma Stem Cell
- Author
-
William Matsui
- Subjects
Cell type ,business.industry ,Immunology ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Cancer stem cell ,Cancer research ,medicine ,Progenitor cell ,Stem cell ,Clonogenic assay ,business ,Multiple myeloma ,Adult stem cell - Abstract
Abstract SCI-4 Increasing evidence suggest that most human cancers consist of phenotypically and functionally heterogeneous cell types. Moreover, in some tumors, phenotypic-functional relationships exist and the capacity for clonogenic growth and self-renewal may be restricted to specific cell types. In multiple myeloma, malignant plasma cells make up the vast majority of tumor cells, phenotypically characterize the disease, and are responsible for clinical symptoms. However, myeloma plasma cells also appear terminally differentiated similar to their normal counterparts and incapable of long-term proliferation. Similar to other laboratories, we have identified minor populations of B cells that share the identical immunoglobulin gene rearrangements as the malignant plasma cells in most myeloma patients. We have further studied the functional capacity of these clonotypic B cells and found that they have ability to produce ectopic tumor growth both in vitro and in immunodeficient mice that can recapitulate human disease.1,2 Moreover, these cells are relatively resistant to several agents currently used to treat multiple myeloma suggesting that they have the capacity to persist following treatment and are responsible for tumor regrowth and disease relapse. In order to inhibit these drug resistant cancer stem cells, we have studied cellular pathways that regulate the self-renewal of normal adult stem cells and found that several, including the developmental pathway Hedgehog, are potential therapeutic targets.3 Finally, as these concepts are translated to the clinic, several challenges have emerged including the ability to detect activity against myeloma stem cells that make up only a minority of the overall tumor burden.4 To this end, we have begun to develop biomarker strategies to serially quantify myeloma stem cells in patients undergoing treatment and will examine their utility as clinical endpoints. 1. Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332–2336. 2. Matsui W, Wang Q, Barber JP, et al. Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance. Cancer Research. 2008;68:190–197. 3. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proceedings of the National Academy of Sciences, USA. 2007;104:4048–4053. 4. Huff CA, Matsui W, Smith BD, Jones RJ. The paradox of response and survival in cancer therapeutics. Blood. 2006;107:431–434. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding. Off Label Use: Rituximab in multiple myeloma.
- Published
- 2010
26. Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response
- Author
-
Akil Merchant, Sarah Brennan, William Matsui, Ping Zhang, Qiuju Wang, Giselle Joseph, Shyam Biswal, and Anju Singh
- Subjects
medicine.medical_treatment ,Immunology ,Null (mathematics) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,Cytokine ,medicine ,Null cell ,Bone marrow ,Stem cell ,Cytokine receptor - Abstract
Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
27. Hedgehog Signaling Regulates HSC Proliferation
- Author
-
Akil Merchant, Giselle Joseph, and William Matsui
- Subjects
Myeloid ,integumentary system ,Immunology ,Cell Biology ,Hematology ,Granulocyte ,Biology ,medicine.disease ,Biochemistry ,Hedgehog signaling pathway ,Haematopoiesis ,Leukemia ,medicine.anatomical_structure ,Cancer cell ,Cancer research ,medicine ,Bone marrow ,Progenitor cell - Abstract
Hedgehog (Hh) signaling is essential for normal development and is dysregulated in many cancers. Hh signaling is active in normal bone marrow and the majority of acute myeloid leukemias, however, the precise role of Hh signaling and its positive effector Gli1 in normal or malignant hematopoiesis is not known. We have analyzed the bone marrow of Gli1 null mice to understand the role of this transcription factor in normal hematopoiesis in order to gain insight into its potential role in leukemia. Gli1 null mice develop normally and have normal peripheral blood counts but the bone marrow shows skewing of the c-Kit+Sca1+Lin-neg (KSL) progenitor compartment with increased CD34negKSL long-term HSC (LT-HSC) and decreased 34+KSL short-term HSC (ST-HSC). An analogous difference was observed in the c-Kit+Sca1negLinneg (KL) myeloid progenitor compartment with an increase in FcRγlowCD34+KL common myeloid progenitors (CMP) and decrease in the FcRγhighCD34+KL granulocyte monocyte progenitors (GMP). We speculated that these differences could be due to impaired cell cycle since both the ST-HSC and GMP are more proliferative than LT-HSC and CMP, respectively. Cell cycle analysis by DNA content and BrdU pulse labeling (100mg/kg IP 14 hours prior to analysis) revealed a marked decrease of proliferation in the LT-HSC, ST-HSC, CMP, and GMP compartments of Gli1 null mice. We supported this conclusion by demonstrating that the bone marrow of Gli1 null mice are relatively radio-resistant. Mice exposed to 400 cGy of total body irradiation followed with serial blood counts revealed less severe nadir, but delayed rebound of white blood cells in Gli1 null mice. We further hypothesized that although Gli1 appears to be dispensable for steady-state peripheral hematopoiesis, it might be necessary for rapid proliferation of progenitors needed during stressed hematopoiesis. In brain development, where Hh signaling is much better understood, active Hh signaling is critical for regulating proliferation of neural stem cells and Gli1 activity significantly increases after depletion of neural progenitors with chemotherapy (Bai et al., Development, 2002). To extend this observation to hematopoiesis, we treated Gli1 null mice and wild-type litter-mates with 5-fluorouracil (5-FU) at 100mg/kg and measured serial blood counts. Gli1 null mice had a delayed recovery of total white blood cells and neutrophil counts at 6 days after 5-FU, but this difference normalized by 20 days after treatment. To confirm that this difference was due to impaired proliferation and not increased sensitivity to 5-FU, we treated Gli1 null and wild-type mice with G-CSF (10mcg/kg/day) for three days to stimulate neutrophil proliferation. Confirming our hypothesis, we observed an attenuated neutrophil response in G-CSF stimulated Gli1 null mice. In summary, we have demonstrated that Gli1 loss leads to decreased HSC and myeloid progenitor proliferation, which has important functional consequences for stress hematopoiesis. These data suggest that abnormal Hh activity in leukemia may be important for driving the uncontrolled proliferation of cancer cells. Gli1 null mice were a kind gift from Alexandra Joyner, Memorial Sloan-Kettering Cancer Center
- Published
- 2008
28. GPI-Anchored Protein Deficiency Confers Resistance to Apoptosis in PNH
- Author
-
Allan D. Hess, William Matsui, Richard Jones, Linzhao Cheng, James Barber, Guiben Chen, Rong Hu, Galina L. Mukhina, Robert A. Brodsky, Christopher J. Thoburn, and William J. Savage
- Subjects
Myeloid ,medicine.diagnostic_test ,Immunology ,Clone (cell biology) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Flow cytometry ,Haematopoiesis ,Immune system ,medicine.anatomical_structure ,Apoptosis ,medicine ,Tumor necrosis factor alpha ,Progenitor cell - Abstract
OBJECTIVE: Investigate the contribution of PIG-A mutations to clonal expansion in paroxysmal nocturnal hemoglobinuria (PNH). INTRODUCTION: Whether PNH cells are inherently less susceptible to apoptosis remains controversial. Studies using PNH mouse models and lymphoid cell lines have failed to find a difference in apoptosis, but reports using K562 cells have found a relative resistance. Studies using primary CD34+ cells from PNH patients have also found a relative resistance of PNH cells, but not in comparison to CD34+ cells from normal controls. We designed experiments 1) to control for genetic heterogeneity among PNH and control cell lines, 2) to address the potential methodological issues associated with studying lymphocytes, which are rarely affected in PNH, and 3) address inconsistency comparing normal and PNH progenitors that have experienced different in vivo environments. METHODS: GPI-anchored protein (GPI-AP) positive and negative primary CD34+ hematopoietic progenitors from PNH patients were assayed for annexin V positivity by flow cytometry in a cell-mediated killing assay using autologous effectors from PNH patients or allogeneic effectors from healthy controls. To specifically assess the role of the PIG-A mutation in the development of clonal dominance and address confounders of secondary mutation and differential immune selection in vivo, we established an inducible PIG-A CD34+ myeloid cell line, TF-1. Using a doxycycline-inducible wild type PIG-A tet-SUPER transgene expression system, in which GPI-AP− and GPI-AP+ cells are isogenic, we assessed apoptosis resistance and clonal expansion in response to various pro-apoptotic stimuli. Apoptosis resistance was assessed after exposure to allogeneic effectors, NK92 effectors, TNF-α, and γ-irradiation. Apoptosis was measured by annexin V/PI staining (effector experiments) or caspase 3/7 activity (TNF-α and γ-irradiation experiments). Blocking experiments of NK92-mediated killing utilized mAb to ULBP1 and ULBP2, as per Hanaoka et al. Clonal competition experiments tracked wild type and PIG-A mutant TF-1 cells using flow cytometry after exposure to TNF-α as a surrogate for immune attack. RESULTS: In PNH patients, GPI-AP− CD34+ hematopoietic progenitors were less susceptible than GPI-AP+ CD34+ precursors to autologous (8% versus 49%, p CONCLUSIONS: PIG-A mutations contribute to the clonal expansion in PNH by conferring a survival advantage to hematopoietic progenitors under pro-apoptotic stresses. Lack of GPI-anchored ULBP1 and ULBP2 expression contributes to the apoptosis resistance in a cell-mediated killing model but is not solely responsible for the apoptosis resistance. The global apoptosis resistance translated into expansion of the PNH clone in an in vitro model of immune attack.
- Published
- 2008
29. Cancer Stem Cell Activity in Mantle Cell Lymphoma Is Inhibited by TLR9 Activation
- Author
-
George J. Weiner, William Matsui, Sarah Brennan, Brooke Meade, and Qiuju Wang
- Subjects
Chemistry ,CpG Oligodeoxynucleotide ,Immunology ,TLR9 ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Lymphoma ,medicine.anatomical_structure ,immune system diseases ,Cancer stem cell ,hemic and lymphatic diseases ,medicine ,Cancer research ,Mantle cell lymphoma ,Stem cell ,Clonogenic assay ,B cell - Abstract
Patients with Mantle Cell Lymphoma (MCL) often respond to initial cytotoxic therapy, but invariably relapse. This clinical pattern suggests that standard therapies fail to eliminate a population of chemoresistant cells responsible for tumor regrowth and disease relapse. Studies in myeloid leukemias and multiple myeloma have suggested that cancer stem cells (CSC) are relatively quiescent and that this property promotes drug resistance. Therefore, the induction of CSC proliferation may increase their susceptibility to anti-cancer treatment. Unmethylated CpG DNA sequences induce the activation and differentiation of normal B cells through toll like receptor 9 (TLR9), and synthetic CpG oligonucleotides (ODN) are currently undergoing clinical trials in MCL and other B cell non-Hodgkin’s lymphomas. We initially examined the effects of TLR9 agonists on the clonogenic growth of the human MCL cell lines Granta 519, Jeko-1, and Rec-1 and found that the unmethylated CpG ODN 2006 (5ug/mL, 48 hours) significantly inhibited the ability of each of these cell lines to form colonies in methylcellulose (p
- Published
- 2008
30. Hedgehog Signaling in Normal and Malignant Hematopoiesis
- Author
-
Micheal McDevitt, Akil Merchant, Craig D. Peacock, B. Doug Smith, Judith E. Karp, William Matsui, David N. Watkins, Evan Jones, Tara L. Lin, and Giselle Joseph
- Subjects
Myeloid ,Cyclopamine ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Hedgehog signaling pathway ,Haematopoiesis ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Cancer stem cell ,medicine ,Cancer research ,Stem cell ,Smoothened - Abstract
The Hedgehog (Hh) signaling pathway is critical for normal development and dictates the self-renewal, proliferation and differentiation of normal stem cells and progenitors. Aberrant reactivation of Hh signaling has been described in a wide variety of human cancers and its role in normal stem cells suggest that pathway dysregulation contributes to oncogenesis and influences the cell fate decisions in cancer stem cells (CSC). Like their normal counterparts, CSC appear to undergo self-renewal as well as give rise to differentiated progeny, and these properties implicate that CSC are responsible for continual tumor cell production that underlies the initiation, maintenance and progression of clinical disease. Myeloid leukemias have long served as the model system for human CSC, but the cellular processes responsible for regulating these rare biologically distinct cell populations have remained unclear. We hypothesized that Hh pathway activation contributes to the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and studied Hh signaling in these settings. Using both RT-PCR for pathway components and a Gli1 reporter assay, we have found that Hh signaling is active in several human AML derived cell lines (Kasumi-1, KG1, KG1a) and primary AML and MDS samples. Approximately 80% (19/24) of primary AML samples tested express the downstream effectors GLI1 or GLI2 indicative of active Hh signaling. Furthermore, inhibition of Hh signaling with the naturally derived SMOOTHENED antagonist cyclopamine reduces the clonogenic growth of KG1 cells implicating the pathway in self-renewal. In contrast, cyclopamine failed to affect colony growth in the HL-60 cell line that lacks expression of Hh pathway signaling components, confirming that the effect of Hh inhibition is specific. In addition, the ectopic expression of Gli1 in KG1 cells partially rescued the effect of cyclopamine on colony formation further demonstrating the specific nature of this compound. We also studied normal CD34+ bone marrow cells and found that they expressed components of Hh pathway by RT-PCR. However, in contrast to KG1 cells, cyclopamine had little effect on the recovery of either normal hematopoietic progenitors or stem cells in an in vitro long-term culture assay. Therefore, it appears that Hh inhibition may preferentially inhibit myeloid leukemias. We further studied the role of Hh pathway activation on normal hematopoiesis and developed a transgenic mouse model in which SMOOTHENED is conditionally over-expressed in the myeloid lineage via Cre recombinase activity regulated by the Lysozyme promoter. Analysis of these mice demonstrated only subtle changes in peripheral blood counts, but further analysis of cells expressing the transgene revealed a significant reduction in the number of mature myeloid cells. This was confirmed by analyzing blood cells for the granulocyte marker Gr1 and pan-myeloid marker Mac1, both of which were significantly reduced in the SMOOTHENED over-expressing cells. These defects are reminiscent of MDS and further suggest that the Hh signaling pathway plays a role in normal hematopoiesis. Therefore, aberrant Hh pathway activation is a feature of myeloid leukemias and inhibitors such as cyclopamine may have a therapeutic role in the treatment of AML and MDS.
- Published
- 2007
31. The MMSET Gene Contributes to the Growth, Adhesion, and Tumorigencitiy of t(4;14) Multiple Myeloma Cells
- Author
-
Abde M. Abukhdeir, Hiroyuki Konishi, Joseph P. Garay, Josh Lauring, Ben Ho Park, Robert J. Arceci, William Matsui, Qiuju Wang, and John P. Gustin
- Subjects
Gene knockdown ,Cell adhesion molecule ,Immunology ,Gene targeting ,Chromosomal translocation ,Cell Biology ,Hematology ,Biology ,Fibroblast growth factor receptor 3 ,biology.organism_classification ,Biochemistry ,Molecular biology ,Nude mouse ,Cell culture ,RNA interference - Abstract
Multiple myeloma (MM) is an incurable hematological malignancy characterized by recurrent chromosomal translocations. The t(4;14)(p16;q32) is associated with the worst prognosis of any patient subgroup in MM, although the basis for this poor prognosis is unknown. The t(4;14) is unusual in that it involves two potential target genes on chromosome 4: fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET). MMSET is universally over-expressed in t(4;14) MM, whereas FGFR3 expression is lost in one third of cases, suggesting a role for MMSET in myeloma pathogenesis. Nonetheless, the role of MMSET in t(4;14) MM has remained unclear. Here we demonstrate a role for MMSET in t(4;14) MM cells. Using homologous recombination-mediated gene targeting, we disrupted the N-terminal and full-length isoforms of MMSET in t(4;14)+ KMS-11 MM cells. Disruption of the translocated MMSET allele revealed that this allele accounts for most of the MMSET transcription in t(4;14) MM cells. Accordingly, selective targeting of the translocated allele, but not the non-translocated allele, led to reduced colony formation in methylcellulose and reduced tumor formation in nude mouse xenografts. Down-regulation of MMSET expression in t(4;14) MM cell lines by stable RNA interference (RNAi) led to a slower growth in liquid culture, a significant reduction in colony formation in methylcellulose, and decreased tumorigenicity in vivo. Additionally, MMSET knockdown led to partial cell cycle arrest of adherent MM cells and reduced the ability of MM cells to adhere to extracellular matrix. Cells with targeted disruption or knockdown of MMSET exhibited changes in expression levels of potential target genes, including several adhesion molecules. These results provide the first direct evidence that translocation-mediated overexpression of MMSET plays a critical role in t(4;14) MM and suggest that therapies targeting this gene could impact this particular subset of poor-prognosis patients.
- Published
- 2007
32. Clonotypic B Cells Circulate in Hodgkin’s Lymphoma (HL)
- Author
-
Lan Lin, Richard F. Ambinder, Richard J. Jones, James Barber, Christopher D. Gocke, Mark J. Siedner, William Matsui, Yvette L. Kasamon, and Kortney Hensley
- Subjects
CD20 ,biology ,Immunology ,Germinal center ,Cell Biology ,Hematology ,Plasma cell ,Biochemistry ,Molecular biology ,CD19 ,medicine.anatomical_structure ,Cancer stem cell ,Cell culture ,hemic and lymphatic diseases ,biology.protein ,medicine ,Stem cell ,B cell - Abstract
Reed-Sternberg (RS) cells, the hallmark of HL, are clonal B lineage cells generally characterized by their co-expression of CD15 and CD30. RS cells also occasionally express the plasma cell (PC) cell surface antigen CD138 (syndecan-1), and we thus hypothesized that RS may represent aberrant PC differentiation. We found that RS cells from the L428 HL cell line exhibited an expression profile similar to normal PC and those from multiple myeloma (MM): high expression of the PC transcription factor Blimp-1 and low expression of the germinal center B cell markers Pax-5 and Bcl-6. We recently demonstrated that CD138+ MM PC, like their normal counterparts, have limited replicative potential; rather, self-renewing CD138neg clonotypic B cells appear to be responsible for the initiation and maintenance of MM, giving rise to the differentiated CD138+ MM PC (Matsui et al Blood103: 2332–2336, 2004). To determine if RS cells similarly were differentiated progeny of rare HL cancer stem cells, we studied HL cell lines (L428, KM-H2) and found that each line contained a small (
- Published
- 2006
33. Cancer Stem Cell Targeting in Multiple Myeloma by GRN163L, a Novel and Potent Telomerase Inhibitor
- Author
-
Carol Ann Huff, Calvin B. Harley, Milada S. Vala, William Matsui, B. Douglas Smith, Qiuju Wang, James Barber, Robert J. Tressler, Alan K. Meeker, and Richard J. Jones
- Subjects
Telomerase ,Chemistry ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,Molecular biology ,Telomere ,Cancer stem cell ,Cell culture ,Stem cell ,Progenitor cell ,Telomerase inhibitor GRN163L ,Clonogenic assay - Abstract
Telomerase activity (TA) is required within normal cells capable of long-term replication, including stem cells and is upregulated in many cancers. In the absence of TA or presence of TA inhibitors, the progressive shortening of telomeres ultimately results in cellular senescence and/or apoptosis. These observations support that TA inhibitors represent a novel class of anti-tumor agents. Much evidence suggests that human cancers display a hierarchical cellular organization that mirrors normal tissues. Cancer stem cells (CSC) are derived from the malignant transformation of normal stem cells and progenitors and retain the capacity to self-renew. Moreover, CSC give rise to differentiated tumor cells that form the bulk of the tumor mass, but have little or no capacity for long-term proliferation. We recently demonstrated that the malignant CD138+ plasma cells in multiple myeloma (MM) have limited replicative potential; instead they arise from the differentiation of clonogenic CSC that resemble normal memory B cells (CD138negCD19+CD27+). In addition, several groups have demonstrated that telomerase inhibitors are active against human MM cell lines in vitro and in vivo. We examined TA in CD138+ plasma cells and CD138neg precursors, and studied the effects of telomerase inhibition against both cell populations. We isolated CD138+ and CD138neg cells by FACS from three human MM cell lines (RPMI 8225, NCI-H929, and U266) and measured TA using a PCR-based assay of activity. For each cell line, TA was detectable within both the CD138neg and CD138+ cell populations. GRN163L is a lipid conjugated 13 nucleotide thio-phosphoramidate oligonucleotide that acts as a potent and specific active site inhibitor of telomerase. We found that treatment with GRN163L (0.1–5μM) markedly reduced TA within 48 hours. To examine the effects of telomerase inhibition on clonogenic growth, we continuously cultured CD138+ and CD138neg RPMI 8226 cells with GRN163L (1μM). Cells were collected weekly, washed to remove GRN163L, and then plated in methylcellulose to assess colony formation. We found that GRN163L was active against both CD138+ and CD138neg cells and eliminated the colony forming potential of both by 5 weeks. Similarly, we found that GRN163L inhibited the in vitro clonogenic growth of CD138neg MM CSC isolated from the bone marrow aspirates of patients with MM. These data demonstrate that TA is detectable within both immature MM CSC and mature MM plasma cells, and that CSC from both cell lines and primary clinical samples are targeted by the telomerase inhibitor GRN163L. Therefore, this agent may offer a novel therapeutic approach to myeloma as well as other diseases in which CSC have been identified.
- Published
- 2006
34. Differentiation Therapy for Poor Risk Myeloid Malignancies: Results of a Dose Finding Study of Bryostatin-1 (BRYO) + GM-CSF
- Author
-
William Matsui, Steven Piantadosi, Sharyn D. Baker, B. Douglas Smith, Judith E. Karp, Richard J. Jones, Ming Zhao, Steven D. Gore, Douglas E. Gladstone, Milada S. Vala, and Carol Ann Huff
- Subjects
Acute promyelocytic leukemia ,medicine.medical_specialty ,Myeloid ,Bryostatin 1 ,Combination therapy ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Leukemia ,medicine.anatomical_structure ,Refractory ,Internal medicine ,Toxicity ,medicine ,Bone marrow ,business - Abstract
Clinically effective differentiating therapy has been limited to the use of all-trans-retinoic acid in patients (pts) with acute promyelocytic leukemia. We recently demonstrated that the in vitro activity of many pharmacologic differentiating agents, including the protein kinase C agonist bryo, was dependent on their ability to block cell cycle and maximized by the addition of growth factors. We studied the safety and efficacy of continuous infusion bryo in combination with GM-CSF (125 ug/m2/day SC) in patients (pts) with refractory and poor risk myeloid malignancies. Bryo was dose escalated using the Continual Reassessment Method from 6ug/m2/day to 20ug/m2/day for 14 days monthly and followed by 16ug/m2/day to 20ug/m2/day over 21 days each month. The treatment plan called for 2 cycles of combination therapy prior to response evaluation. A total of 32 patients with refractory or poor risk myeloid malignancies (9 relapsed or refractory AML, 1 therapy-related AML, 2 poor risk AML age >70, 19 poor risk MDS, 1 progressive PNH) were treated. The median age of subjects was 65 (range 23–75) yrs and a total of 73 (58 full + 15 partial) cycles were administered. The dose-limiting toxicity of the combination mirrored results from previous trials of bryo; grade 3–4 myalgias and arthralgias were observed in 12 of 14 pts at the highest dose levels (18 and 20ug/m2/day x 14 days, 16ug/m2/day x 21 days). Three pts discontinued therapy within a week of cycle 1 and were not considered evaluable for response. Eighteen (62%) pts responded (2 CRs and 16 PRs), 6 (21%) had stable disease, and 5 (17%) progressed. The PRs included 15 lineage responses and 1 bone marrow response (50% marrow blasts) who received a single cycle @ 16ug/m2/day x 21 days and remains in CR at 5 mos. Both pts refused further courses due to myalgias and arthralgias, and interestingly, the CRs were not achieved until 4–6 weeks after completion of therapy. Our newly developed assay (sensitivity 50 pg/ml) detected measurable bryo only at the highest doses which supported the correlative in vitro assays that detected in vivo differentiation at the same doses. Bryo + GM-CSF has remarkable clinical activity in refractory myeloid malignancies, but is intolerable at biologically active doses. These results do suggest that strategies utilizing differentiating agents in conjunction with growth factors warrant further investigation.
- Published
- 2005
35. Multiple Myeloma Stem Cells and Plasma Cells Display Distinct Drug Sensitivities
- Author
-
Qiuju Wang, Carol Ann Huff, B. Douglas Smith, William Matsui, James Barber, and Richard J. Jones
- Subjects
medicine.diagnostic_test ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Flow cytometry ,Haematopoiesis ,medicine.anatomical_structure ,Side population ,Cell culture ,medicine ,Stem cell ,Progenitor cell ,Clonogenic assay ,B cell - Abstract
We recently demonstrated that multiple myeloma (MM) is organized in a hierarchical manner in which clonogenic MM progenitors or stem cells resembling post-germinal B cells give rise to MM plasma cells (PC). To study the potential biologic differences between MM stem cells and MM PC, we examined each cellular subset for characteristics found in normal stem cells as well as their responses to various antitumor agents. The human MM cell lines RPMI 8226 and NCI-H929 were initially studied as we previously found that they recapitulate clinical MM specimens and consist of distinct cell populations based on the expression of the PC surface antigen CD138; CD138+ cells resemble typical MM PC, whereas CD138neg cells express B cell surface antigens and have greater clonogenic capacity. Examination of these cellular subpopulations by flow cytometry demonstrated that CD138neg cells were smaller and less granular by light scatter than CD138+ PC and expressed higher levels of the intracellular enzyme aldehyde dehydrogenase that is present in normal hematopoietic progenitors with self-renewal potential. Furthermore, cells expressing the side population phenotype after staining with the DNA binding dye Hoechst 33342 were exclusively CD138neg. We also investigated the effects of different clinically applicable agents on CD138+ and CD138neg cells. CD138+ and CD138neg cells isolated from RPMI 8226 and NCI-H929 cells by fluorescence activated cell sorting were treated with dexamethasone (dex, 100nM), bortezomib (velcade, 10nM), CC5013 (revlimid, 1μM), rituximab (10μg/ml) or alemtuzumab (campath,10μg/ml) for 72 hours followed by plating in methylcellulose to assess clonogenic capacity. CD138+ PC were significantly inhibited by dex (27 ± 11% recovery compared to untreated control cells), velcade (14 ± 6%) and revlimid (44 ± 27%), whereas rituximab (92 ± 25%) and campath (97 ± 18%) had little activity. In contrast, clonogenic growth of CD138neg cells was not significantly inhibited by dex (82 ± 19%), velcade (88 ± 29%), or revlimid (91 ± 14%), but was significantly decreased by rituximab (63 ± 22%) and campath (47 ± 27%). Similarly, clonogenic MM growth of CD138neg cells from 4 clinical MM samples was not affected by dex (84 ± 9%), velcade (82 ± 24%), or revlimid (93 ± 11%), but was significantly inhibited by rituximab (19 ± 7%) or campath (15 ± 11%). Clonogenic MM precursors may be distinguished from MM PC by a variety of biological parameters typically expressed by normal stem cells. Furthermore, these cellular subsets have different susceptibilities to a variety of clinical agents, and agents with activity against MM PC may be ineffective against MM stem cells. Moreover, agents without activity agasint MM PC may have major activity against MM stem cells. The divergent sensitivities of MM stem cells and PC may explain the dramatic, but transient, responses seen with many agents. Therapeutic strategies that result in long-term remissions may require the inhibition of both MM PC to reduce clinical symptoms and MM stem cells responsible for relapse.
- Published
- 2004
36. T Cell Activation and Regulation in Graft-Versus-Host Disease: Integral Role of CD28, CTLA4 and GITR Splice Variants
- Author
-
Elizabeth C. Matsui, Richard J. Jones, Allan D. Hess, William Matsui, Emilie C. Bright, Yuji Miura, and Christopher J. Thoburn
- Subjects
Lymphocyte ,T cell ,Immunology ,CD28 ,chemical and pharmacologic phenomena ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Immune tolerance ,medicine.anatomical_structure ,Graft-versus-host disease ,Lymphocyte costimulation ,medicine ,IL-2 receptor ,CD8 - Abstract
Graft-versus-host disease (GVHD) is a serious, life-threatening complication that occurs following allogeneic (allo) bone marrow transplantation (BMT). The use of non-specific immunosuppression or T cell depletion has reduced the incidence of GVHD but at the expense of increased rates of infection and leukemic relapse. Modulation of the major costimulatory pathway (CD28/CTLA4:B7) involved in T cell activation and regulation may lead to specific immune tolerance in the absence of global non-specific immunosuppression. The identification of mRNA splice variants encoding for soluble forms of CD28, CTLA4 and GITR suggests that costimulation of T cells is complex and is not limited to cell-cell contact. The present studies examined the hypothesis that the onset of GVHD and the re-establishment of immune tolerance correlate with the expression levels of these costimulatory molecules. mRNA transcript levels for the soluble (s) and full-length (fl; cell surface associated) variants assessed by quantitative PCR, were temporally examined in peripheral blood lymphocytes (PBLs) from patients undergoing alloBMT (n=38) or autologous (auto) BMT (n=39) with the induction of autoGVHD by cyclosporin A treatment post-transplant. Levels of s and fl CD28 mRNA transcripts in PBLs were significantly increased (>1.5 fold, P2.3-fold, P2.1-fold). sCTLA4 expression in patients with alloGVHD was significantly decreased than patients without alloGVHD. Interestingly, temporal analysis revealed that the levels for sCTLA4 paralleled the recovery from GVHD implicating an active process in the establishment of non-responsiveness. CD28, CTLA4 and GITR s and fl mRNA levels in CD4+CD25+ T regulatory (Treg) cells from allo and autoBMT patients were significantly increased (7-, 41- or 22-fold, P4 fold reduction of 3H-thymidine incorporation). However, pretreatment of the Treg subset with short interfering RNA (siRNA) to knockdown sCTLA4 gene (confirmed by quantitative PCR) significantly reduced the ability of these cells to suppress the response (minimal suppression was detected, 6%). In vitro siRNA studies also indicated that Treg cells with inhibited sCTLA4 expression were unable to suppress the response of IL-2-stimulated autoreactive CD8+ T cells. Taken together, the results indicate that increased expression of CTLA4 (soluble and cell-surface associated) and the “negative” signal delivered by this molecule to the T cell may regulate the development of GVHD and help to re-establish self tolerance after BMT. Defining the role of costimulation and the modulation of this pathway on immune recognition and regulation not only provides opportunities to enhance the re-establishment of tolerance but also may help to intensify anti-tumor immunotherapeutic strategies.
- Published
- 2004
37. Gm-Csf Improves Activity of Interferon as Primary Therapy for CML
- Author
-
Richard J. Jones, William Matsui, B. Douglas Smith, Douglas E. Gladstone, and Kathleen M. Murphy
- Subjects
Cytopenia ,medicine.medical_specialty ,Myeloid ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Hematologic Response ,Clinical trial ,medicine.anatomical_structure ,Imatinib mesylate ,Internal medicine ,Toxicity ,Medicine ,Bone marrow ,Progenitor cell ,business - Abstract
Not only does IFN prolong the survival of CML patients, but the 10-20% of patients who achieve a complete cytogenetic remission (CCyR) with IFN have a median survival >10 yrs and some of these patients may actually be cured. Imatinib mesylate (IM) has replaced IFN as front line therapy for newly diagnosed patients due to its favorable toxicity profile and superior initial response rate. Recent laboratory and clinical data suggest the impact of IM on CML stem cells may be limited, raising the question of durability of IM responses. Conversely, our data suggest that IFN is directly toxic to CML stem cells, at least in part through induction of CML stem cell terminal differentiation that also requires myeloid growth factors. IFN’s primary activity against CML stem cells rather than mature CML progenitors may explain the slow, but often durable, responses seen in IFN-treated patients, while the rapid responses induced by IM are likely to be a consequence of its impressive activity against mature CML progenitors. We report results of our clinical trial adding GM-CSF to IFN as upfront therapy to improve cytogenetic responses over IFN alone in patients with newly diagnosed CML. Patients received IFN with a goal of 5 x 106 units/m2/day and dose reductions for cytopenias. Upon achieving a hematologic response, pts started GM-CSF at 125μg/m2/day for 6 months. GM-CSF was initially dose-reduced for WBC > 10,000/μL; this parameter was changed to 20,000/μL after 2 of the first 3 pts were removed from study because of persistently elevated WBC despite 2 dose reductions of GM-CSF. No further pts have been removed from the study due to increased WBC. Response was monitored every 3 months by peripheral blood FISH for bcr-abl and every 6 months by bone marrow cytogenetics and RT-PCR. A total of 58 pts were enrolled on the trial with 51 considered evaluable for response (2 unevaluable due to persistently elevated WBC and 5 pts withdrew consent before the initial 3 month evaluation) and all 58 pts for toxicity. The combination of IFN and GM-CSF was well tolerated and 12/58 (20%) of enrolled pts withdrew consent due to treatment-related toxicity. However, many pts withdrew consent prior to achieving landmark responses, including 15/58 before achieving a MCR and 15/58 prior to a CCyR. Unfortunately, these groups received a historically short course of IFN-based therapy (median 14, range 3–45, mos) in part due to the availability of IM during this trial. Interestingly, 3 pts withdrew consent despite achieving a CCyR on the combination. Only 2 pts came off study due to progression (>35% Ph+). 35 (69%) of 51 evaluable patients obtained a MCR (Ph+ < 35%) at a median of 3 (range 3 – 15) months with over 1/3 of them achieving a CCyR at a median of 9 (range 3–21) months. An additional 13 pts in a MCR on IFN + GM-CSF achieved a CCyR after switching to IM because of patient preference. Thus, 51% (26/51) of evaluable patients on this trial are currently in a CCyR at a median follow-up of 4.3 (range 3–6) years. IFN + GM-CSF appears to be more active in CML than IFN alone, inducing a MCR in nearly 70% of patients. The incidence of CCyR with this combination is difficult to determine because many of the patients switched to IM while still responding to IFN. Because IFN + GM-CSF appears to target CML stem cells, while IM may not, this combination should be considered as part of the treatment algorithm for CML.
- Published
- 2004
38. Chronic Myeloid Leukemia Stem Cells and Their Differentiated Progeny Display Divergent Drug Sensitivities to Imatinib Mesylate and Interferon-Alpha
- Author
-
Anita L. Hawkins, B. Douglas Smith, Richard J. Jones, William Matsui, James Barber, Milada S. Vala, Carol Ann Huff, Constance A. Griffin, and Greg R. Angstreich
- Subjects
Myeloid ,Immunology ,CD33 ,Myeloid leukemia ,Imatinib ,Cell Biology ,Hematology ,Biology ,Colony-stimulating factor ,Biochemistry ,medicine.anatomical_structure ,Imatinib mesylate ,hemic and lymphatic diseases ,medicine ,Progenitor cell ,Stem cell ,neoplasms ,medicine.drug - Abstract
Imatinib has largely replaced interferon-alpha (IFN) as front-line therapy in the treatment of chronic myeloid leukemia (CML) because of its favorable toxicity profile and superior initial response rate. However, recent laboratory data demonstrating that CML stem cells may have limited susceptibility to imatinib brings into question the potential durability of these responses. In contrast, responses to IFN often take place over months or years, but extensive long-term clinical data indicate that they are often long lasting. In order to determine if the differences in clinical response kinetics could be explained by the activity of imatinib and IFN against CML stem cells or their differentiated progeny, we examined the effects of each agent on distinct cellular subsets in vitro. CD34+ CML progenitors were isolated from 4 patients with newly diagnosed chronic phase CML and incubated with IFN (1000U/ml) or imatinib (10μM) for 96 hours followed by long-term liquid culture over 1-3 weeks to assay more primitive CML stem cells. The effects of each drug on CML progenitors was determined by calculating the number of CFU-GM positive for BCR-ABL by FISH and comparing these values to the bcr-abl+ CFU-GM formed by untreated control cells. Initially, the CML CFU-GM recovery immediately following 96 hours of drug treatment was 62.7 ± 8% and 9.7 ± 2% from IFN and imatinib-treated groups, respectively. After 3 weeks of long-term culture, CML CFU-GM recovery was 23.4 ± 2.6% following IFN treatment and 57.3 ± 22% after imatinib. Thus, imatinib was significantly more toxic to committed CML progenitors than primitive CML progenitors responsible for the maintenance of long-term liquid cultures (p = 0.04). Conversely, IFN had significantly greater activity against primitive CML progenitors than committed progenitors (p = 0.05). Although IFN has diverse biological properties, the mechanisms responsible for its antileukemic activity are unknown. Treatment of the human CML cell line KT-1 with IFN resulted in increased expression of the myeloid antigens CD33 and myeloperoxidase as well as the inhibition of clonogenic growth demonstrating that IFN induced terminal differentiation. Furthermore, several groups have shown that myeloid growth factors also induce differentiation of CML cells in vitro and clinically enhance the activity of IFN; accordingly, the addition of GM-CSF (200U/ml) augmented the differentiation of KT-1 cells. Moreover, myeloid growth factors were required for differentiation as neutralizing antibodies against GM-CSF and IL-3 inhibited the activity of IFN. The addition of GM-CSF to IFN produced similar effects on clinically derived CD34+ CML cells. Although imatinib is a potent inhibitor of committed CML progenitors, it is relatively ineffective against more primitive CML stem cells. In contrast, IFN appears to act primarily against CML stem cells by inducing terminal differentiation that is dependent on the activity of myeloid growth factors. Imatinib and IFN have divergent effects on CML progenitors at different stages of maturation that may correlate with clinical response kinetics and durability.
- Published
- 2004
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.