Apoptosis Of Plasma Cells Research Articles

A Phase I/II Trial Combining High-Dose Melphalan and Autologous Transplant with Bortezomib for Multiple Myeloma: A Dose- and Schedule-Finding Study

We did a randomized phase I/II trial designed to evaluate the safety and efficacy of combining the proteasome inhibitor bortezomib with high-dose melphalan as the conditioning for high-dose therapy and autologous transplant for myeloma. Enrolled patients were limited to those who did not achieve a very good partial remission (VGPR) following one or more induction regimens, and were randomized to receive a single escalating dose of bortezomib (1.0, 1.3, or 1.6 mg/m(2)) either 24 hours before or 24 hours after high-dose melphalan. Dose escalation was based on the escalation with overdose control (EWOC), a Bayesian statistical model. Bone marrow aspirates were collected before initiation of therapy and at the time of transplant to evaluate which sequence resulted in maximal plasma cell apoptosis, and response to transplant was assessed by the International Myeloma Working Group criteria. Among 39 randomized patients, 20 received bortezomib after melphalan and 19 received bortezomib before melphalan. Toxicities and posttransplant hematopoietic recovery rates were similar between arms. The overall response rate for all patients was 87%, with 51% achieving a VGPR or better. Pharmacodynamic studies showed greater plasma cell apoptosis among patients who received bortezomib following melphalan. The use of bortezomib in conjunction with high-dose melphalan is safe, with data suggesting improved efficacy. A single dose of bortezomib administered after high-dose melphalan is the recommended dose and schedule for future clinical investigation.

CpG-Oligodeoxynucleotide Inhibits Smad-Dependent Bone Morphogenetic Protein Signaling: Effects on Myeloma Cell Apoptosis and In Vitro Osteoblastogenesis

The TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) with a phosphorothioate backbone (PTO-CpG-ODN) is evaluated in clinical trials as a vaccine adjuvant or as treatment of cancers. Bone morphogenetic proteins (BMPs) regulate growth and differentiation of several cell types, and also induce apoptosis of cancer cells. Cross-talk between BMP- and TLR-signaling has been reported, and we aimed to investigate whether CpG-ODN influenced BMP-induced osteoblast differentiation or BMP-induced apoptosis of malignant plasma cells. We found that PTO-CpG-ODN inhibited BMP-2-induced osteoblast differentiation from human mesenchymal stem cells. Further, PTO-CpG-ODN counteracted BMP-2- and BMP-6-induced apoptosis of the human myeloma cell lines IH-1 and INA-6, respectively. In contrast, PTO-CpG-ODN did not antagonize the antiproliferative effect of BMP-2 on hMSCs or IH-1 cells. Inhibition of Smad-signaling and p38 MAPK-signaling indicated that apoptosis of IH-1 cells is dependent on Smad-signaling downstream of BMP, whereas the antiproliferative effect of BMP-2 on IH-1 cells also involves p38 MAPK-signaling. Together, the data suggested a specific inhibition by PTO-CpG-ODN on BMP-Smad-signaling. Supporting this we found that PTO-CpG-ODN inhibited BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines. This effect appeared to be independent of TLR9 because GpC-ODN and other ODNs with the ability to form multimeric structures inhibited Smad-signaling as efficiently as PTO-CpG-ODNs, and because knockdown of TLR9 by small interfering RNA in INA-6 cells did not blunt the effect of PTO-CpG-ODN. In conclusion, our results demonstrate that PTO-CpG-ODN inhibits BMP-signaling, and thus might provoke unwanted TLR9-independent side effects in patients.

TG101209, a novel JAK2 inhibitor, has significant in vitro activity in multiple myeloma and displays preferential cytotoxicity for CD45+ myeloma cells

Interaction of myeloma cells with the bone marrow microenvironment is mediated in large part through different cytokines, especially VEGF and IL6. These cytokines, especially IL6, leads to upregulation of the JAK/STAT pathway in myeloma cell, contributing to increased proliferation, decreased apoptosis, and acquired drug resistance. Here, we examined the preclinical activity of a novel JAK2 inhibitor TG101209. TG101209 induced dose- and time-dependent cytotoxicity in a variety of multiple myeloma (MM) cell lines. The induction of cytotoxicity was associated with inhibition of cell cycle progression and induction of apoptosis in myeloma cell lines and patient-derived plasma cells. Evaluation of U266 cell lines and patient cells, which have a mix of CD45 positive and negative cells, demonstrated more profound cytotoxicity and antiproliferative activity of the drug on the CD45+ population relative to the CD45- cells. Exploring the mechanism of action of TG101209 indicated downregulation of pJak2, pStat3, and Bcl-xl levels with upregulation of pErk and pAkt levels indicating cross talk between signaling pathways. TG101209, when used in combination with the PI3K inhibitor LY294002, demonstrated synergistic cytotoxicity against myeloma cells. Our results provide the rationale for clinical evaluation of TG101209 alone or in combination with PI3K/Akt inhibitors in MM.

Combined measurement of plasma cell proliferative and apoptotic index in multiple myeloma defines patients with good and poor prognosis

We assessed plasma cell proliferation (by propidium iodide index, PC-PI) and apoptosis (by annexin-V index PC-AI) for the estimation of overall survival (OS) in multiple myeloma (MM) patients. 181 patients with newly diagnosed MM were assessed, treated using conventional chemotherapy. The values best discriminating patients with poor prognosis were PC-PI > 3.0% and PC-AI < 4.75%, and for good prognosis PC-PI ≤ 3.0% and PC-AI ≥ 4.75%. The median OS was 8 months vs 40 months, p = 0.0002. Our results suggest that combined measurement of plasma cell proliferation and apoptosis creates a unique strong prognostic factor based on growth characteristics of the tumor clone.

Abstract 1635: Combination of the HDAC inhibitor romidepsin with the MEK inhibitor U0126 results in synergistic plasma cell apoptosis

Abstract Histone deacetylase inhibitors such as vorinostat and panobinostat have been tested as single agents in patients with relapsed myeloma with nominal activity. Yet, when combined with the proteasome inhibitor bortezomib, clinical and laboratory evidence of synergy has been observed. We sought to evaluate the individual signaling effects of the HDACi romidepsin, with the intent of developing additional combinations that may lead to clues of mechanism of action on human myeloma cells. Single agent studies demonstrated IC50 values for romidepsin alone of between 10 and 30nm, further experiments demonstrate that growth inhibition resulting from romidepsin exposure includes apoptosis and cell cycle arrest. Cell death is associated with increased caspase 3, 8, 9 and PARP cleavage. Cell cycle analysis indicates that romidepsin induces G0/G1 cell cycle arrest which is correlated with p21 upregulation. Cell signaling assessment demonstrates that romidepsin inhibits AKT phosphorylation. Surprisingly, romidepsin increases Erk phosphorylation with its resultant effects on cell growth, development and differentiation. Combining romidepsin with the MEK inhibitor U0126 suppresses MEK/ERK signaling, and synergistically enhances the antitumor effect of romidepsin (combination index value &amp;lt;1). Annexin V staining demonstrates significant apoptosis following 18 hr of treatment with U0126 (5µM) and 10nM romidepsin. Western blot confirms the development of significantly increased procasepase and PARP cleavage, as well as substantial downregulation of Mcl-1, Bcl-xL. The combination can overcome the antiapoptotic effect of exogenous cytokines, such as IL-6 and IGF1 in vitro. Further studies evaluating the effect of bone marrow stromal cells will be presented in addition of data evaluating other potential targets for oncogene addiction that can be used for further novel combinations to gain more understanding of the biological context of this combination both in vitro and in vivo. This study will improve the design of effective clinical combination strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1635.

Advances in diagnosing and managing antibody-mediated rejection.

Antibody-mediated rejection (AMR) is a unique, significant, and often severe form of allograft rejection that is not amenable to treatment with standard immunosuppressive medications. Significant advances have occurred in our ability to predict patients at risk for, and to diagnose, AMR. These advances include the development of newer anti-human leukocyte antigen (HLA)-antibody detection techniques and assays for non-HLA antibodies associated with AMR. The pathophysiology of AMR suggests a prime role for antibodies, B cells and plasma cells, but other effector molecules, especially the complement system, point to potential targets that could modify the AMR process. An emerging and potentially larger problem is the development of chronic AMR (CAMR) resulting from de novo donor-specific anti-HLA antibodies (DSA) that emerge more than 100 days posttransplantation. Therapeutic options include: (1) High-dose intravenously administered immunoglobulin (IVIG), which has many potential benefits. (2) The use of IVIG + rituximab (anti-CD20, anti-B cell). (3) The combination of plasmapheresis (PP) + low-dose IVIG with or without rituximab. Data support the efficacy of all of the above approaches. Newer approaches to treating AMR include using the proteosome inhibitor (bortezomib), which induces apoptosis in plasma cells, and eculizumab (anti-C5, anticomplement monoclonal antibody).

Bortezomib in kidney transplantation.

Although current therapies for pretransplant desensitization and treatment of antibody-mediated rejection (AMR) have had some success, they do not specifically deplete plasma cells that produce antihuman leukocyte antigen (HLA) antibodies. Bortezomib, a proteasome inhibitor approved for the treatment of multiple myeloma (a plasma cell neoplasm), induces plasma cell apoptosis. In this paper we review the current body of literature regarding the use of this biological agent in the field of transplantation. Although limited experience with bortezomib may seem to show promise in the realm of transplant recipients desensitization and treatment of AMR, there is also experience that may suggest otherwise. Bortezomib's role in desensitization protocols and treatment of AMR will be defined better as more clinical data and trials become available.

A Phase I Clinical Trial Testing the Combination of Bortezomib and Tipifarnib in Relapsed/Refractory Multiple Myeloma.

Abstract 3851Poster Board III-787 BackgroundPreclinical work from our group and others has demonstrated that the combination of a farnesyl transferase inhibitor (FTI) and the proteasome inhibitor bortezomib results in enhanced plasma cell apoptosis and is associated with AKT activation (David, Blood 2005). Sequence of bortezomib followed by a FTI synergistically inhibited cell growth compared to concurrent treatment. More recently, further preclinical data suggests that the mechanism responsible for this profound synergy is due to inhibition of HDAC6 with a resultant inhibition of both the proteasome and aggresome pathway (David ASH 2007). Based upon these observations, with the MMRC, we initiated a phase I trial combining the FTI tipifarnib with bortezomib to clinically evaluate the efficacy of this combination. MethodsPatients with relapsed or refractory myeloma were treated with bortezomib at 1.0 mg/m2 or 1.3mg/m2 given on days 1,4,8, and 11 in conjunction with escalating doses of tipifarnib (100-400mg PO BID) given on days 2-15 every 21 days, respectively. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control (EWOC)). Eligibility criteria included a serum creatinine of <2.5, normal liver function, ANC>500, and platelets >25. ResultsTwenty-six patients have been enrolled to date: bortezomib 1.0 mg/m2 with tipifarnib 100 mg (n=6), 200mg (n=5), 300mg (n=7), 400mg (n=2) and bortezomib 1.3mg/m2 with tipifarnib 300mg (n=6), 400mg to be enrolled. Median age is 63 (range 42-77). 19/26 patients had received prior high dose therapy. The average number of prior therapies was 4, and, of the 26 patients, 10 were refractory to bortezomib (relapsed on therapy or within 6 months) 11 were bortezomib naïve, and 5 were previously exposed to bortezomib but not refractory. Among these patients with advanced myeloma and refractory disease, 50% had stabilization of disease or better. Maximum number of cycles received was 10. Of note, among the patients achieving clinical benefit, 1 had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug-related toxicities were GI (17.0%) with nausea/vomiting occurring most frequently. Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many entered with platelet counts of 25-50. Additional grade 3 toxicities included renal insufficiency (related to PD), pneumonia and altered mental status which were all considered due to disease progression. There were no Grade 3- 5 non-hematologic drug related toxicities. There were no cardiac events or DVT, and 1 patient experienced grade 2 peripheral neuropathy who did not have pre- existing PN at baseline. ConclusionsThe combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among a group of patients with refractory and advanced myeloma. To date the optimal doses of both tipifarnib and bortezomib have yet to be defined, and additional patients will be enrolled to define the MTD by escalation of tipifarnib to 400 with 1.3mg/m2 of bortezomib. Correlative studies evaluating the effect of the combination on HDAC6 and plasma cell apoptosis will be presented. Disclosures:Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Kaufman:Millennium : Consultancy; Celgene: Consultancy, Research Funding; Genzyme: Consultancy; Merck: Research Funding.

Gold Compound Auranofin (RIDAURA®) Induces Apoptosis of Human Myeloma Cells by Targeting STAT3 and NF-κB Pathways, with Clinical Potential.

Abstract 3837Poster Board III-773Multiple myeloma (MM) is a plasma cell malignancy that remains incurable despite the use of conventional chemotherapy. Recent advances in the field of cell biology could lead to new therapeutic agents based upon molecular targeting, such as thalidomide and proteasome inhibitor bortezomib. These agents have remarkable activity against both newly diagnosed and refractory patients with MM, but prolonged exposure to them may result in the development of de novo drug resistance. Thus, it is necessary to identify and validate novel agents with less toxicity to overcome drug resistance and to improve the clinical outcome of MM. Progression and chemoresistance are thought to involve IL-6, whose expression is induced by NF-κB, through its regulation of the growth and survival of MM cells. IL-6 leads to constitutive activation of STAT3, which in turn results in the expression of high levels of Mcl-1. Thus, the constitutive activation of both NF-κB and STAT3 plays an important role in MM cell proliferation, and both molecules are important targets for the treatment of MM. Auranofin (AF: RIDAURA®; GSK) is a coordinated gold compound that has been widely used for the treatment of rheumatoid arthritis based on its anti-inflammatory properties through the inhibition of NF-κB activation. Therefore, assuming that AF has the potency to induce apoptosis in MM cells by interfering with NF-κB and STAT pathways, it may become a candidate for a novel therapeutic agent. To address our hypothesis, the effects of AF on inducing apoptosis of various MM cells were examined. Further, the molecular mechanism of AF-induced apoptosis in MM cells was investigated. AF inhibited the growth of U266 cells in a time- and dose-dependent manner with IC50 of 50 nM at 24 h. AF significantly induced cell cycle arrest at the G1 phase and subsequent apoptosis of U266 cells. AF-induced apoptosis in various human MM cell lines and CD138-positive plasma cells from patients with MM involved the activation of caspases-3, -8, and -9. Treatment with AF inhibited the constitutive and IL-6-induced activation of STAT3, and then downregulated the expression of Mcl-1 but not that of Bcl-2 or Bcl-xL proteins. To clarify the biological significance of Mcl-1 in AF-induced apoptosis of MM cells, Mcl-1 expression vector (pEGFP-hmcl-1) and control vector were introduced into U266 cells (designated as U266/mcl and U266/neo cells). Induction of apoptosis by AF was abrogated in U266/mcl, but not in U266/neo cells. We next examined the effect of AF on the DNA binding activity of STAT3. Electrophoretic mobility gel shift assay (EMSA) using U266 nuclear extracts demonstrated that IL-6-induced STAT3 binding activity was inhibited by the presence of AF. These results suggest that AF inhibits the IL-6-induced JAK/STAT pathway selectively and induces apoptosis in MM cells via the downregulation of Mcl-1. In addition, AF downregulated the activation of NF-κB in an IκB-independent manner, and also inhibited DNA binding activity in U266 cells. Although NF-κB inhibitory peptide (SN-50) did not directly induce the expression of Mcl-1, the combination of SN-50 with AF reduced the levels of Mcl-1, suggesting that inhibition of NF-κB potentiates the apoptotic effect of AF. Finally, our in vitro data prompted us to examine whether or not the effects of AF are equally valid in a clinical settings. The protocol for assessing the clinical benefit of oral administration of AF (RIDAURA®) in patients with relapsed or refractory MM was approved by the Ethics Committee of Saitama Medical University. Since 2008, 7 patients with refractory MM were treated with oral RIDAURA® 6 mg daily. The median follow-up for patients was 7.5 months. All 7 patients responded to AF, including one PR (partial response) and 6 SD (stable disease) with no progression. No adverse events have been observed to date. Details of the clinical outcome will be presented. In conclusion, gold compound AF inhibited constitutive activation of both STAT3 and NF-κB, resulting in the downregulation of anti-apoptotic Mcl-1 protein in MM cells with clinical relevance. A low pharmacological concentration (50 nM) of AF is widely employed for the treatment of rheumatoid arthritis without any side effects; therefore, it may be used to treat MM without the risk of severe toxicity. We propose that AF (RIDAURA®) may have potential use as a new molecular-targeted agent for the treatment of MM. Disclosures:Off Label Use: Auranofin in the treatment of multiple myeloma.

Combined Measurement of Plasma Cell Proliferation and Apoptosis in Multiple Myeloma.

Abstract 4884 BackgroundPlasma cell proliferation and apoptosis represent key factors in multiple myeloma (MM) expansion and tumor survival. Both the proliferative and the apoptotic index have been shown to be strong and independent prognosticators in MM. Moreover, they do not correlate with most standard prognostic factors, representing thus different inherent features of the myeloma clone. The aim of the study was to assess the combined measurement of proliferation and apoptosis, giving the best predictive value for long and short MM survivals. MethodsWe assessed a cohort of 181 patients with newly diagnosed multiple myeloma, treated using conventional chemotherapy (regimens MP, VBMCP, VAD, CyVAD). In all the patients we measured proliferative and apoptotic index at the time of diagnosis, before the start of treatment. The proliferative activity of myeloma plasmocytes in bone marrow aspirate was measured by flow- cytometry using propidium-iodide index (PC-PI), representing the percentage of plasma cells in S-phase of the cell cycle; apoptosis was assessed using flow cytometry using an annexin-V (PC-AI) index. Subsequent statistical analysis of the the Kaplan-Meier curves of overall survival was evaluated using the MATLAB routine. We assessed different PC-PI and PC-AI tresholds with the best separation of groups with good and poor prognosis, using the log rank procedure. Additional measures of performance were obtained looking at the success with which two groups selected using the PC-AI and PC-PI thresholds reflected short term and long term survivors. ResultsThe median follow-up of the group was 25 months (range 1 – 117 months). At the time of analysis, 137 patients had died (76%). Plasma cell proliferative index varied in the range 1.2 – 4.2, with median 2.5; apoptotic index was in the range 1.4 – 24.5 with median 4.3. Patients were divided into 4 groups based on PC-PI and PC-AI thresholds and then keeping only those two groups that demonstrated the worst and best overall survival based on the survival analysis of PC-PI and PC-AI individually. The best discriminating values for patients with poor prognosis (n=20) were PC-PI > 3.0% and PC-AI < 4.75%, and for patients with good prognosis (n = 71) PC-PI ' 3.0% and PC-AI ≥ 4.75%, with median overall survival 8 months versus 40 months respectively, p = 0.0002. The precision of the correct prediction of individual patient prognosis based on this grouping was in the patients with short survival 0.70 and in long survivors 0.58. Due to an imbalanced number of patients especially with high proliferation we were unable to create a single significant combined index across the entire patient group. Moreover, the patients outside the defined ranges substantially influenced the sensitivity and especially the specificity of the test, suggesting that defining of the extreme groups using PC-PI and PC-AI creates a better prognosticator than the assessment of the whole cohort based on a single combined index. ConclusionPlasma cell proliferation and apoptosis reflect different processes of MM kinetics with the growth characteristics on one hand, and survival on the other. Their assessment as single factors provides valuable information about the biology of the clone and contributes to the estimation of overall survival. Their combined measurement, however, significantly separates two extreme groups of patients with different prognosis and may thus become a valuable auxiliary parameter in MM patients stratification. DisclosuresNo relevant conflicts of interest to declare.

A phase I MMRC clinical trial testing the combination of bortezomib and tipifarnib in relapsed/refractory multiple myeloma

8597 Background: Preclinical work from our group has demonstrated that the combination of a farnesyl transferase inhibitor and bortezomib results in enhanced plasma cell apoptosis, and more recently, we have suggested that the mechanism for this synergy is due to inhibition of HDAC6, and inhibition of the proteasome and aggresome pathway. Methods: Patients with relapsed or refractory myeloma were treated with bortezomib at 1.0 or 1.3 mg/m2 on a standard schedule in conjunction with escalating doses of tipifarnib (100–400mg/BID) given on days 2–15 every 21 days. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control). Results: 22 patients have been enrolled, of which 18 are evaluable, into respective tipifarnib dose levels 100 mg (n=6), 200mg (n=5) and 300mg (n=5) and 400mg (n=2). Median age for the enrolled patients is 59 and median time from myeloma diagnosis was 4.7 years. The average number of prior therapies was 4.5. Among these patients with advanced myeloma and refractory disease, stabilization of disease or better was seen among 8/10 patients with 2 of the 8 achieving an MR. Among the patients achieving clinical benefit, 1 patient had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug related side effect was was Gr2 diarrhea (23.5%). Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many were entered onto study with platelet counts between 25 and 50. Additional grade 3 toxicities included renal insufficiency (related to PD), pneumonia and altered mental status which were all considered not related to study drug but to progression of disease. There were no Grade 3-5 drug related toxicities. One patient experienced grade 2 peripheral neuropathy who did not have pre existing PN at baseline. Conclusions: The combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among 10 of 18 patients with refractory myeloma. Optimal dose of tipifarnib and bortezomib have yet to be defined, and additional patients are being enrolled with 1.3mg/m2 of bortezomib, and escalating doses of tipifarnib. [Table: see text]

Proteasome Inhibition Causes Apoptosis of Normal Human Plasma Cells Preventing Alloantibody Production

Antibody production by normal plasma cells (PCs) against human leukocyte antigens (HLA) can be a major barrier to successful transplantation. We tested four reagents with possible activity against PCs (rituximab, polyclonal rabbit antithymocyte globulin (rATG), intravenous immunoglobulin (IVIG) and the proteasome inhibitor, bortezomib) to determine their ability to cause apoptosis of human bone marrow-derived PCs and subsequently block IgG secretion in vitro. IVIG, rituximab and rATG all failed to cause apoptosis of PCs and neither rituximab nor rATG blocked antibody production. In contrast, bortezomib treatment led to PC apoptosis and thereby blocked anti-HLA and antitetanus IgG secretion in vitro. Two patients treated with bortezomib for humoral rejection after allogeneic kidney transplantation demonstrated a transient decrease in bone marrow PCs in vivo and persistent alterations in alloantibody specificities. Total IgG levels were unchanged. We conclude that proteasome activity is important for PC longevity and its inhibition may lead to new techniques of controlling antibody production in vivo.

Multiple Myeloma: Biology, Standard Therapy, and Transplant Therapy

The understanding of the pathogenesis of multiple myeloma has undergone a major transformation over the past eight years. New insights into the microenvironment of the plasma cell as well as elucidation of signaling pathways that prevent plasma cell apoptosis are leading to rapid new drug development. The introduction of novel agents has led to a significant increase in survival. Combinations of novel agents are expected to provide higher complete response rate with anticipated prolongation of relapse free and overall survival. Autologous and allogeneic stem cell transplantation remains an integral part of therapy further improving the outcomes following induction with novel agents.

Laryngeal Amyloidosis Causing Hoarseness and Airway Obstruction

Amyloidosis constitutes a fraction of 1% of benign localized laryngeal tumors and may occasionally be associated with systemic disease. A chronic, insidious, progressive, recurrent disease characterized by hoarseness, dyspnea, and stridor, it is caused by extracellular deposition of insoluble, abnormal tissue injurious fibrils. Submucosal lesions occur frequently in the vestibular folds and ventricles, less commonly in the subglottis and aryepiglottic folds and least on the vocal folds. Apple green birefrigence under polarized light after Congo red staining, electron microscopic fibrillar structure, and a beta-pleated sheet structure observed by x-ray diffraction are confirmatory. Two presented cases add to the small literature review of similar patients. Case 1 was a 70-year-old man with severe hoarseness, incomplete glottic closure, ovoid concentric stenosis of the inferior glottis and subglottis, who initially was not diagnosed by several laryngologists and speech therapists. He required multiple microlaryngoscopic excisions and dilations. Because low dose radiation induces plasma cell apoptosis in other diseases, external beam radiation therapy (EBRT) was hypothesized to eliminate amyloidogenic plasma cells. Case 2 was a 46-year-old welder with progressive dyspnea for 2-3 years and hoarseness, voice loss, and stridor over 6-7 months. Masses caused airway obstruction of the anterior commissure, vestibular, and vocal folds, with extension to the subglottis. Two phonomicrosurgical CO(2) laser-assisted resections relieved upper airway obstruction and restored voice. Conservative surgical intervention and long-term followup are essential. Further studies are needed to determine if a radiation dose response relationship exists to control laryngeal amyloidosis.

A Phase I MMRC Clinical Trial Testing the Combination of Bortezomib and Tipifarnib in Relapsed/Refractory Multiple Myeloma

Background: Preclinical work from our group and others has demonstrated that the combination of a farnesyl transferase inhibitor (FTI) and the proteasome inhibitor bortezomib results in enhanced plasma cell apoptosis and is associated with AKT activation (David, Blood 2005). More recently, further preclinical data suggests that the mechanism responsible for this profound synergy is due to inhibition of HDAC6 with a resultant inhibition of both the proteasome and aggresome pathway (David ASH 2007). Based upon these observations, with the MMRC we initiated a phase I trial combining the FTI tipifarnib with bortezomib to clinically evaluate the efficacy of this combination.Methods: Patients with relapsed or refractory myeloma were treated with bortezomib at 1.0 mg/m2 given on days 1,4,8, and 11 in conjunction with escalating doses of tipifarnib (100–400mg/BID) given on days 2–15 every 21 days. Dose escalation was accomplished using an adaptive phase I design (Escalation With Overdose Control (EWOC)). Eligibility criteria included a serum creatinine of <2.5, normal liver function, ANC>500, and platelets >25. If dose escalation is able to proceed to 400mg of tipifarnib with 1.0 mg/m2, the tipifarnib dose escalation will restart with bortezomib given at 1.3 mg/m2.Results: Sixteen patients have been enrolled to date into respective tipifarnib dose levels 100 mg(n=6),200mg (n=5) and 300mg (n=5). Median age for the enrolled patients is 59 (range 43–76) and median time from myeloma diagnosis was 4.7 years. 15/16 patients had received prior high dose therapy. The average number of prior therapies was 4.5, and of the16 patients, 8 were refractory to prior bortezomib (relapsed on therapy or within 6 months) 4 were bortezomib naïve, and 4 were previously exposed to bortezomib but not known to be refractory. Among these patients with advanced myeloma and refractory disease, stabilization of disease or better was seen among 7/16 patients with 2 of the 7 achieving an MR. Of note, among the patients achieving clinical benefit, 1 patient had a stable M-protein, but experienced an 80% reduction in circulating plasma cells while on therapy, and another has had a 75% reduction in the free light chain assay. The most common drug related side effects were was Gr2 diarrhea (23.5%). Hematologic toxicities were difficult to ascertain as patients had advanced myeloma and many were entered onto study with platelet counts between 25 and 50. Additional grade 3 toxicities included renal insufficiency (related to progression), pneumonia and altered mental status which were all considered unrelated to study drug, but were associated with progression of disease. There were no Grade 3 –5 drug related toxicities. There were no cardiac events or DVT, and 1 patient experienced grade 2 peripheral neuropathy who did not have pre existing PN at baseline.Conclusions: The combination of bortezomib and tipifarnib is supported by preclinical rationale and has produced stable disease or better among a group of patients with refractory and advanced myeloma. To date the optimal dose of both tipifarnib and bortezomib have yet to be defined, and additional patients will be enrolled to define the MTD for tipifarnib with 1.0mg/m2 of bortezomib, followed by escalation of tipifarnib with 1.3mg/m2 of bortezomib. Correlative studies evaluating the effect of the combination on HDAC6 and plasma cell apoptosis will be presented.

The Immunotoxin HM1.24-ETA′ Is a Potent Inducer of Apoptosis in Malignant Plasma Cells

Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma (MM) still needs improvement. Antibody-based targeted therapies are increasingly used for tumor therapy, and may represent interesting options for MM patients. HM1.24 is a surface molecule that is over expressed on malignant plasma cells and efficiently internalized from the cell surface. It may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the development and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of an HM1.24-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and purified to homogeneity by affinity chromatography. Specific binding to HM1.24 was demonstrated by immunofluorescence staining and flow cytometry using antigen positive and negative cells. HM1.24-ETA′ efficiently inhibited growth of IL-6 dependent and IL-6 independent myeloma cell lines (INA-6, RPMI8226, U266). Half maximal growth inhibition was observed at low nanomolar concentrations. Further analyses demonstrated that target cell killing occurred via induction of apoptosis, as evidenced by Annexin V/propidium iodide staining and detection of PARP cleavage. The cytotoxic effect was completely blocked by adding excess of unconjugated parental antibody, demonstrating that the effect was antigen-specific and not mediated by non-specific uptake of the immunotoxin. Importantly, HM1.24-ETA′ efficiently triggered apoptosis (>80% Annexin V positive cells) of freshly isolated plasma cell leukemia cells within 48h. In conclusion, HM1.24-ETA′ efficiently triggered apoptosis of multiple myeloma cell lines as well as freshly isolated tumor cells. These results indicate that HM1.24 may represent a promising target structure for efficient antigen-specific delivery of cytotoxic compounds to plasma cell tumors.

ER Stress and Inhibition of Key Apoptotic Caspases Regulate the Life Span of Short-Lived Plasma Cells

Plasma cells (PCs) are the terminally differentiated effector cells of the humoral immune system. The majority of PCs are short-lived and undergo programmed cell death in the form of apoptosis after a few days of intensive immunoglobulin secretion. Despite potentially wide-ranging implications for infection control, auto-immunity, and PC dyscrasias, the mechanisms that govern the initiation and execution of PC apoptosis are poorly understood. We used two well-established murine systems of PC differentiation and immunohistochemistry of human lymphoid tissue sections to study the regulation of PC apoptosis. IgM-secreting post-mitotic CD138+B220− PCs were differentiated in vitro from primary mouse splenic B cells using cytokines and LPS and purified by magnetic selection. Murine I.29mu+ B lymphoma cells were induced to undergo plasmacytic differentiation by stimulation with LPS. In both systems, terminal PC differentiation is followed by spontaneous apoptosis of half of the PCs within 48h, similar to PC apoptosis in vivo. We found that a sharp increase in endoplasmatic reticulum (ER) stress, which is caused by an imbalance between secretory load and capacity in the ER, occurs in PCs that have completed differentiation and begin to undergo apoptosis. In parallel, susceptibility specifically to ER stress-induced apoptosis but not to other apoptotic stimuli increases substantially in differentiated PCs, despite an ongoing ER stress response and expansion of the secretory machinery. Caspase-12, which has been linked specifically to ER stress-induced apoptosis, is activated and processed during programmed PC death. Using the specific inhibitor of caspase-12, zATADfmk, we found that caspase-12 mediates apoptotic DNA fragmentation and chromatin condensation in PCs undergoing apoptosis but not in B cells undergoing tunicamycin-induced apoptosis. In contrast, the major apoptotic effector caspases (caspase-9, caspase-3, caspase-7) downstream of the mitochondria become resistant to activation by apoptotic ER stress during terminal PC differentiation and are not activated during PC apoptosis. We observed that he pan-caspase inhibitor, zVADfmk, completely blocks tunicamycin-induced apoptosis in B cells but does not inhibit PC apoptosis or tunicamycin-induced cell death in PCs. Using the small molecule PAC-1, which specifically activates caspase-3 by targeting a “safety-catch” amino acid sequence that keeps caspase-3 inactive, we found that caspase-3 is stabilized in its inactive form in PCs and human myeloma cell lines, but not in B cells. Immunohistochemistry of human lymphoid tissue sections demonstrated that most primary reactive PCs and extramedullary myeloma cells undergo spontaneous apoptosis in vivo without activation of caspase-3. Thus, ER stress plays a major role in limiting the life span of short-lived PCs and activates caspase-12, which mediates nuclear apoptosis specifically in PCs. The major apoptotic effector caspases, however, become resistant to activation during terminal PC differentiation, and PC apoptosis is largely independent of caspases downstream of the mitochondria. These observations lead us to propose that developmentally regulated inhibition of key apoptotic caspases, which rapidly execute apoptosis in most cells, has evolved in PCs as a means to delay apoptosis under conditions of increasing ER stress linked to immunoglobulin secretion. Overwhelming ER stress ultimately limits the life span of short-lived PCs by inducing apoptosis using alternative mechanism involving caspase-12, which is redundant for the execution of ER stress-induced apoptosis in cells that can activate the classical effector caspases.

In Vitro Efficacy of Agonistic Antibody to TRAIL-R1 (Mapatumumab) and Low Dose Bortezomib in Multiple Myeloma

The proteasome inhibitor Bortezomib (Bz) is a potent inducer of plasma cell apoptosis via suppression of NFkB activity. Bz is also associated with the dose limiting induction of thrombocytopenia as well as inhibition of dendritic cell (DC) function, which may limit the induction of autologous T cell responses to myeloma antigens in vivo. We hypothesized that the dose, and therefore the adverse effects of Bz, may be reduced by the addition of the agonistic anti-TRAIL-R1 antibody Mapatumumab (Mp). A dose of Bz that did not result in detectable apoptosis was determined for each of the cultured human plasma cell lines (RPMI8226, U266, LP-1, NCI-H929, OPM-2 and JJN3) via titration experiments in which each cell line was cultured for 24–48 hrs in Bz concentrations from 0.1–10nM. Cells were stained with annexin V-FITC and the viability dye 7-AAD and analyzed on a BD LSRII flow cytometer. Plasma cell surface expression of TRAIL-R-1 and -2 were also assessed and correlated with sensitivity to Mp induced apoptosis. Bz monotherapy at 10nM produced 85–98% apoptosis in all plasma cell lines. In contrast 4/6 cell lines were sensitive to Mp alone, RPMI8226 at 0.06ug/ml, U266 and OPM-2 at 1ug/ml and LP-1 at 10ug/ml. When non-apoptosis inducing Bz doses were used in combination with titrated doses of Mp (from 0.01 to 50ug/ml) apoptosis of RPMI8226, U266 and OPM-2 was enhanced by 32%, 30% and 15% respectively compared to Mp alone (p=<0.05 for each cell line). The particular sensitivity of RPMI8226 to Bz/Mp therapy correlated with this cell line's higher level of TRAIL-R 1 surface expression in comparison to U266. In contrast, the addition of Bz to Mp did not result in any additional apoptosis induction over Mp alone in LP-1, JJN3 and NCI-H929. Conversely, 24 hrs of Bz pretreatment resulted in subsequent enhanced sensitivity of LP-1 to Mp but not NCI-H929. In conclusion, the effective dose of Bz may be significantly reduced when used in combination with Mp. The Bz+Mp combination therapy may be more efficacious than Bz alone and allow dose modification of Bz. Reduction in Bz dose may in-turn reduce side-effects whilst allowing the preservation or promotion of endogenous immune responses. [Display omitted]

CD32B Expression Reflects Phenotypic and Functional Heterogeneity in Multiple Myeloma (MM)

Intraclonal heterogeneity (IH) in MM may provide a basis for the selection of resistance to therapy that occurs over time. Model systems to study IH in MM have not been defined so we focused on the expression of CD32B, the low-affinity inhibitory Fcγ-receptor that is a key participant in normal plasma cell apoptosis and a potential target for monoclonal antibody therapy. At diagnosis, CD138+ MM marrow cells are largely CD32B+ (n=72; median 94%, range 1–100) while at relapse the CD32B+ MM subpopulation is significantly smaller in both bone marrow (n=25, median 83%, (10–100), P = 0.02) and extramedullary sites (n=8, median 17% (0–78); P << 0.01). Among human MM cell lines only two of ten tested expressed cell-surface CD32B. We hypothesize that the CD32B phenotype of MM subpopulations contributes to the functional heterogeneity that is critical to the selection of resistance, and sought a model system to study CD32B-related IH. The KMS-12BM cell line (BM), a human MM line from marrow, has CD32B+ and CD32B− subpopulations as MM marrows do, while the KMS-12PE cell line (PE), from the pleural fluid of the same patient, is completely CD32B negative, as is often the case in extramedullary MM. Both BM and PE cells share t(11;14), del 17p and other cytogenetic abnormalities, and both contain intact CD32B genes (FCGR2B). We used 3 populations, BM CD32B+, BM CD32B− and PE (CD32B−), as well as sorted patient MM cells, to study the IH associated with CD32B. RNAi studies demonstrated that the expression of FCGR2B in BM cells is dependent on the Ets family transcription factor PU.1, while PE cells do not express PU.1. RT-PCR of patient MM cells sorted into paired CD32B+ and CD32B− fractions showed PU.1 and FCGR2B (CD32B) expression only in CD138+/CD32B+ cells. Cell-tracking studies with CFSE showed that BM CD32B+ cells gave rise to BM CD32B− cells but that the reverse did not occur. Cell-culture studies showed that BM CD32B+ cells have significantly lower apoptotic rates than BM CD32B− cells, while the latter display greater dependence on insulin for survival and on IGF-1 and MIP-1α for proliferation. In order to better understand the differences between CD32B+ and CD32B− populations, we performed transcript profiling. BM CD32B+ cells have significantly higher expression of the activin inhibin, while BM CD32B− cells overexpress insulin-response genes such as the kinase Akt2. Compared to BM cells, PE cells overexpress germ-cell transcription factors and inflammasome-related genes, while BM cells overexpress tetraspanins (CD9, CD37, CD53) and insulin-response genes. Of interest, there are also significant differences in the expression of BMP and Wnt-related genes (but not Hh or Notch genes). BM cells overexpress BMPR1A, FZD2 and Wnt5A while PE cells overexpress CRIM1, FZD1 and LEF1. These data indicate that functional IH exists in MM, that the KMS-12 cell lines provide a useful model for its study, and that MM may originate in a CD32B+ cell that begets CD32B− heirs. We continue to study PU.1 and the generation of CD32B− cells by CD32B+ cells; to validate transcript data using sorted patient specimens; and to assess with RNAi the contributions of BMP, Wnt and insulin-response genes to the survival and proliferation of CD32B+ and CD32B− cells, seeking novel therapeutic targets on which selection of resistance may depend.

Defective B‐cell response to T‐dependent immunization in lupus‐prone mice

Lupus anti-nuclear Ab show the characteristics of Ag-driven T-cell-dependent (TD) humoral responses. If autoAg elicit the same response as exogenous Ag, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone C57BL/6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin (NP-KLH) compared with total Ig, including a smaller percentage of B cells participating in the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells (PC) in the bone marrow. B6.TC PC expressed reduced levels of FcgammaRIIb, which suggests that reduced apoptosis in resident PC prevents the establishment of newly formed NP-specific PC in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous Ag and suggest that low FcgammaRIIb, hypergammaglobulinemia, and high autoAb production would be predictive of a poor response to immunization in lupus patients.