Negative Myeloma Cell Research Articles

Class II ferroptosis inducers are a novel therapeutic approach for t(4;14)-positive multiple myeloma

AbstractMultiple myeloma (MM) is a clonal plasma cell malignancy that is characterized by genetic heterogeneity. The cytogenetic abnormality t(4;14) strongly predicts poor outcome in patients with MM, even in the era of novel drugs. Ferroptosis is a new approach to antitumor therapy, but the relationship between ferroptosis and MM cytogenetic abnormalities remains largely unclear. In this study, we show that t(4;14)-positive but not t(4;14)-negative MM cells are susceptible to class II ferroptosis inducers (FINs) in a preclinical setting, which is dependent on the significant upregulation of the MM SET domain-containing protein (MMSET). Mechanistically, MMSET upregulates acyl-coenzyme A synthetase long-chain family member 4 transcription by binding to its promoter region, leading to increased polyunsaturated fatty acid (PUFA) levels and enhanced sensitivity of t(4;14)-positive MM cells to ferroptosis. Supplementation with PUFAs efficiently restores the susceptibility of t(4;14)-negative MM cells to ferroptosis. In addition, combining class II FIN treatment with bortezomib in t(4;14)-positive MM cells attenuates cellular glutathione and induces both apoptosis and ferroptosis levels by inhibiting the increase in solute carrier family 7 member 11, demonstrating synergistic antitumor activity in vitro and in a xenograft model. Taken together, our findings suggest that targeting ferroptosis with class II FINs is a novel and promising therapeutic approach to improve the outcome of t(4;14)-positive patients with MM.

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation.

The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.

Receptor Targeted Delivery of the p38γ Inhibitor PIK-75 By Organic-Core Templated Lipid Nanoparticles in Cutaneous T Cell Lymphoma

Introduction: Patients with relapse/refractory cutaneous T cell lymphomas (CTCL) have limited therapeutic options. p38γ, part of the mitogen-activated protein kinases (MAPK) pathway, has been identified as a critical driver of CTCL. PIK-75, a known p110α inhibitor, was found to inhibit p38γ and induced cell death in CTCL cells in vitro and in vivo tumor xenograft models. However, PIK-75 is very hydrophobic and difficult to systemically deliver and achieve clinical efficacy. Therefore, designing a carrier to formulate and enable targeted cellular delivery of PIK-75 is a logical next step to facilitate translation. We, and others, have shown that scavenger receptor type B1 (SR-B1) is often overexpressed in hematologic malignancies and can be targeted by synthetic lipid nanoparticles for efficient drug delivery. Our group pioneered the use of inorganic and organic core (oc) "templates” to synthesize lipid nanoparticles (LNP) that target SR-B1. The ocLNPs are an ideal construct for the delivery of hydrophobic drugs to malignant cells. Therefore, we hypothesized that ocLNPs could be adapted to formulate PIK-75 and deliver this drug via SR-B1 to CTCL cells to induce cell death. Methods: A suite of organic core molecules were synthesized and tested for their ability to support the synthesis of PIK-75 loaded ocLNPs. The PIK-75 ocLNPs were synthesized by combining an organic core with PIK-75, phospholipids, and apolipoprotein A-I. The drug loaded ocLNPs were purified by dialysis and filtration. PIK-75 ocLNPs were characterized for size (size exclusion chromatography, dynamic light scattering and electron microscopy), surface charge (zeta potential) and drug loading. SR-B1 expression in primary CTCL samples was assessed by RNA sequencing. SR-B1 positive CTCL cell lines (HH and HuT78), and SR-B1 positive normal cells, HepG2 (hepatocytes) and THP-1 (macrophages), as well as SR-B1 negative PIK-75-sensitive U266B1 (myeloma) cells were used for these studies. Expression of SR-B1 in all cell lines was confirmed by western blot and flow cytometry. Cell viability was measured using the MTS assay. An SR-B1 blocking antibody was used to demonstrate the necessity of SR-B1 for PIK-75 ocLNP targeting and drug delivery. Results: Nineteen out of 49 primary patient lesional CTCL skin samples (38.8%) demonstrated SR-B1 over-expression, defined as an expression level greater than two standard deviations above the mean expression measured in normal, CD3+ peripheral T cells using RNAseq data. Fourteen of the 19 samples (74%) were from patients with advanced disease (stage III and IV). PIK-75 was successfully incorporated into ocLNPs. Data showed that ~16 PIK-75 molecules were loaded per ocLNP. PIK-75 ocLNPs induced significant cell death in SR-B1 positive CTCL cell lines HH (IC50 = 3.95 nM) and HuT 78 (IC50 = 6.33 nM), while treatment of an PIK-75 sensitive, SR-B1 negative myeloma cell line (U266B1) with PIK-75 ocLNPs resulted in minimal cell death. Treatment of HH cells with PIK-75 ocLNPs for a 2 hour "pulse” followed by 72 hours of additional culture was completed to test active SR-B1 targeting. Data reveal significant cell death, which was significantly reduced by the addition of an SR-B1 blocking antibody (10 nM PIK-75 ocLNPs, viability 31.6% ± 4.3% w/o blocking antibody vs viability 83.6% ± 8.4% w/ blocking antibody). Treatment of hepatocytes and macrophages, both known to express SR-B1, with PIK-75 ocLNPs revealed no cell death even after 72 hours of culture. Conclusions: ocLNPs can be successfully loaded with drug cargo, in this case hydrophobic PIK-75, enabling active delivery via SR-B1 in CTCL. PIK-75 ocLNPs potently induced cell death in CTCL cells while having minimal negative effects on hepatocytes or macrophages. These results provide proof-of-principle that ocLNPs can be successfully leveraged to deliver hydrophobic small molecule drugs by actively targeting SR-B1.

Role of B-Cell Maturation Antigen (BCMA) Targeted Immunotherapies in Relapsed and Refractory Multiple Myeloma- a Systematic Review

Introduction Multiple Myeloma (MM) is a plasma cell dyscrasia characterized by neoplastic proliferation of plasma cells. Despite the recent advances in disease management, it still contributes to significant morbidity and mortality, thus highlighting the unmet need for new treatment options. B-cell maturation antigen (BCMA) is a transmembrane glycoprotein in the tumor necrosis factor receptor superfamily 17 and has been shown to have high expression on plasma cells. Preclinical studies in MM have shown significant efficacy for BCMA targeted immunotherapies, thus providing a potential therapeutic option to overcome the obstacle of myeloma relapse and recurrence. In this comprehensive review, we have reported the role of anti-BCMA therapies in the treatment of relapse and refractory Multiple Myeloma (RRMM). Methods Systematic review was performed according to PRISMA statement. PubMed, Ovid Embase, Web of Science, Cochrane Library & Clinicaltrails.gov were searched, without any filters, using the medical search terms (MeSH) "Multiple Myeloma" AND "B Cell Maturation Antigen". Most recent reports of original phase I/II/III studies reporting safety and efficacy measures of anti-BCMA immunotherapies in RRMM were considered for inclusion. After a detailed screening of 854 studies, 10 phase-1 studies (Ali, SA. 2016, Fan, F. 2017, Brudno, JN. 2018, Zhao, WH. 2018, Trudel, S. 2018, Topp, MS. 2018, Liu, Y. 2018, Cohen AD. 2019, Raje, N. 2019 & Xu, J. 2019) were considered for inclusion. Results (Table-1) A total of 294 patients with RRMM who had received a median range of 3-7 previous lines of therapies were included. Out of these, 36.7% (n= 108) patients received bispecific T-cell engager (BiTE) antibodies against BCMA antigen while rest of patients received anti-BCMA chimeric antigen receptor T-cell (CART) therapy. Patients with CART therapy received autologous derived in-vitro modified stem cells. These patients subsequently developed higher incidence of grade 3-4 cytopenias owing to myeloablation with fludarabine and cyclophosphamide. The dosage ranges for both therapies were heterogenous owing to phase I dose escalation designs. All responses were evaluated according to IMWG response criteria. The cumulative overall response rate (ORR) for studied population was 59.86% (n= 176), while individual ORR was 26.85% (n= 29) for BiTE therapy and 79% (n=147) for CART therapy. Majority of responders (82%, n= 145) received ≥VGPR (very good partial response) status. But relapse/progression of disease after achieving response was a significant issue as 42.5% (n= 125) patients relapsed or progressed and developed BCMA negative myeloma cells in their bone marrow. The progression free survival and survival data is premature at this stage and requires further elaboration in phase II/III clinical trials. Toxicity profile was consistent with use of myeloablation and with the standard toxicity profile seen in other B-cell malignancies except for the incidence of grade ≥ neuropathy which was not much common (1.7%, n= 5). Regarding the grade ≥ 3 cytopenias, incidence of neutropenia was 20% (n=59), anemia 21.4% (n=63), thrombocytopenia 19.4% (n= 57). Grade ≥ 3 cytokine release syndrome (CRS) seen in 11.2% (n= 33) patients which responded well to Tocilizumab and corticosteroids except for death in two cases. All studies showed a significant correlation of CART cell dose and myeloma tumor burden with intensity of cytokine release syndrome (CRS). In each dose escalation trial, sustained and better responses were achieved at higher doses with a parallel enhanced incidence of grade 3-4 toxicities. The responses were not found to be influenced by previous lines of therapy, and patients with higher risk cytogenetics showed a reduced response compared to patients with standard risk cytogenetics in all studies. Conclusion BCMA targeted immunotherapies provide a promising option for highly refractory multiple myeloma patients. But small patient population and phase-I design limit the projection of results to real world setting. Furthermore, relapse/progression risk needs to be weighed against the significant toxicity profile. So, more controlled clinical trials with longer follow up periods are needed to delineate the efficacy and survival outcomes of this therapeutic option. Disclosures Anwer: In-Cyte: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Limitations of bystander killing in Th1/M1 immune responses against a secreted tumor antigen

T-cell recognition of tumor antigens presented on tumor-infiltrating macrophages (TAMs) induces a tumoricidal M1-like phenotype. Resultant indirect immune responses could eliminate not only antigen secreting (AgPOS), but also antigen negative (AgNEG) tumor cells via bystander killing. Such broad-spectrum response could eliminate antigenically heterogeneous tumors. Using an in vivo model of CD4+ T-cell mediated immunity against MHC II negative myeloma cells, bystander killing of AgNEG cells was ineffective due to strict spatial constraints of Th1-induced TAM cytotoxicity.

Characterization of Mechanisms for Acquiring Resistant Phenotype Using a Novel Screening Strategy for Detecting Rituximab-Resistant Mutations

AbstractAbstract 1550 Background:We previously reported that mutations of CD20 gene were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be involved in relapse/resistance after rituximab containing therapy. Most of the patients that had mutation in the C-terminal leagion were diagnosed as CD20 negative by immunohistochemistry using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. So at first we determined the binding site of L26 antibody on CD20 protein. Then we developed novel diagnostic antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods:To determine the epitope of L26 antibody, we established six sub-lines expressing various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. Then we carried out epitope-mapping using these cell lines. To detect comprehensive CD20 molecules including that having mutation in C-terminal region, we developed antibodies that recognize near the amino terminus of CD20 molecules (CD20N antibody). CD20N antibody is the only monoclonal antibody that recognizes N-terminal region of CD20 so far. Using these antibodies, we screened the specimens of the cases diagnosed as CD20 negative determined by L26-based immunohistochemistry. Results:The epitope-mapping revealed that L26 recognizes near the C-terminus of CD20. This suggested that most of CD20 molecules with the C-terminal deletion mutation or frame-shift mutation could not be recognized by L26. Then we screened previously diagnosed specimens and found several cases that having the cells stained by our novel antibody but not by L26. Genetic analysis revealed that all these cells had a mutation in the C-terminal cytoplasmic region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells with mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids with premature stop at the amino acid position 250. Interestingly, mutant CD20 molecule expressed adjacent to the plasma membrane, but rituximab could not bind to these cells. DNA sequencing study about genome and mRNA of CD20 gene suggested that the lymphoma cells of this patient had one normal and one mutated CD20 allele. Discussions:The C-terminal region of CD20 may undertake a pivotal role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of CD20 in C-terminal cytoplasmic region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. The resistance caused by gene mutation thought to be irreversible. And it should be discriminated from transient downregulation of antigen expression. We propose here that immunohistochemical screening using CD20N antibody is very rapid and effective screening stategy that find out irreversible rituximab resistant cases. Disclosures:No relevant conflicts of interest to declare.

PU.1-Induced Growth Arrest and Apoptosis in Classical Hodgkin Lymphoma Cells

Abstract 2628PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineage cells. We have previously shown that PU.1 is down-regulated in various myeloma cell lines and myeloma cells from a subset of myeloma patients. In such cell lines, the promoter and the upstream regulatory element (URE) located in 17 kb 5'-upstream of the PU.1 gene are highly methylated. Furthermore, conditionally expressed PU.1 induces both cell growth arrest and apoptosis in PU.1-low to -negative myeloma cell lines, U266 and KMS12PE. Therefore, we concluded that the down-regulation of PU.1 is necessary for myeloma cell growth.In another B cell malignancy, classical Hodgkin lymphoma, it has been reported that PU.1 is also down-regulated through methylation of its promoter. To evaluate whether down-regulation of PU.1 is essential for growth of classical Hodgkin lymphoma cells, we conditionally expressed PU.1 in two classical Hodgkin lymphoma cell lines, L428 and KMH2, using the tet-off system (designated as L428tetPU.1 and KMH2tetPU.1 cells, respectively). Up-regulation of PU.1 by tetracycline removal induced complete growth arrest in L428tetPU.1 and KMH2tetPU.1 cells. Annexin V staining revealed that up-regulation of PU.1 induced apoptosis in both cell lines. Furthermore, BrdU staining analysis revealed that PU.1 induced G0/G1 arrest in those cells. L428tetPU.1 and KMH2tetPU.1 cells expressing PU.1 showed morphological changes that included the enlargement cytosol and the appearance of various sizes of vacuoles. We next injected L428tetPU.1 and KMH2tetPU.1 cells to immunodeficiency mice (Rag2−/− Jak3−/− bulb/c) subcutaneously. Tumor formation was observed in all those mice with continuous administration of tetracycline (0.5 g/l) in the drinking water. After enlargement of tumor to 1–2 cm diameter, we removed tetracycline in half of the mice. Tetracyclin withdrawal resulted in tumor regression or stable disease, whereas all the mice continuously receiving tetracycline had continuous tumor growth and finally died. These data strongly suggest that PU.1 induced growth arrest and apoptosis of classical Hodgkin lymphoma cells both in vitro and vivo.We next performed DNA microarray analysis to compare gene expression levels of L428tetPU.1 cells before and after PU.1 expression to elucidate the mechanisms of growth arrest and apoptosis induced by PU.1. Among genes related to cell cycle and apoptosis, p21 (CDKN1A) was highly up-regulated in L428tetPU.1 cells after PU.1 induction, and this was also confirmed by mRNA and protein levels. Finally, to clarify the role of p21 up-regulation by PU.1, we stably introduced p21 siRNA in L428tetPU.1 cells. Such stably expressed p21 siRNA rescued L428tetPU.1 cells from growth arrest induced by PU.1, suggesting that the growth arrest in L428tetPU.1 cells by PU.1 should be at least partially dependent on p21 up-regulation. These data suggested that up-regulation of PU.1 by demethylation agents and/or HDAC inhibitors might serve as a possible treatment modality for classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Abstract B69: Development of a screening strategy for detecting rituximab-resistant mutations and characterization of mechanisms for acquiring resistant phenotype.

Abstract Background: Previously we reported that gene mutations of CD20 were found in patients with B-cell non-Hodgkin's lymphoma, and we proposed that C-terminal deletion mutations of CD20 might be related to relapse/resistance after rituximab therapy. Most of the cases that had these mutations were diagnosed as CD20 negative by immunohistochemical analysis using L26 monoclonal antibody. L26 recognizes the cytoplasmic region of CD20 molecules, but no more detailed information about its epitope had been reported. To make this clear, we determined the binding site of L26 antibody on CD20 protein. In addition, we developed novel antibodies that recognize wide variety of CD20 molecular subtypes including those having mutations. Methods: To determine the binding site of L26 antibody on CD20, we established six sub-lines that expressed a various length of C-terminal truncated CD20 using an originally CD20 negative myeloma cell line. These cell lines were used for epitope-mapping for L26 antibody. To detect comprehensive CD20 molecules including CD20 having mutation in C-terminal region, we developed antibodies that recognize near the amino terminal of CD20 molecules. Using these antibodies, we re-screened the specimens of CD20 negative cases determined by L26-based immunohistochemistry. Results: The epitope analysis revealed that L26 recognizes near the C-terminus of CD20 cytoplasmic region. This result showed that most of CD20 molecules with the C-terminal deletion mutation and frame-shift mutation could not be recognized by L26. So we screened previously diagnosed specimens and found several cases that included the cells stained by the N-terminal antibody but not by L26. Genetic analysis revealed that these cells had a mutation in the coding region of CD20. One of these cases, we successfully analyzed the phenotype of lymphoma cells having mutated CD20 in detail using cryopreserves living specimens. In this case, a frame shift mutation occurred due to one base nucleotide deletion, resulting in the translation of peptide of another reading frame of 41 amino acids after the amino acid position 250 with a slight early termination. Interestingly, although mutant CD20 molecule expressed adjacent to the plasma membrane, rituximab could not bind to the cells. DNA sequence analysis about genome and mRNA of CD20 gene indicated that the lymphoma cells of this patient had one mutated allele among two CD20 alleles. Discussions: The C-terminal region of CD20 undertakes a critical role in presentation of the large loop where the rituximab binding site locates. Thus, deletion or frame-shift mutation of C-terminal region impairs the antigenicity against rituximab and it may cause resistance to rituximab therapy. We propose here that finding a mutation by screening using antibodies of two kinds of epitope is useful in detection of irreversible resistant mutation for molecular targeted drug including rituximab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B69.

Secretion of Tumor-Specific Antigen by Myeloma Cells Is Required for Cancer Immunosurveillance by CD4+ T Cells

Tumor-specific CD4(+) T cells orchestrate the adaptive immune responses against cancer. We have previously shown that CD4(+) T cells recognize MHC class II-negative myeloma cells indirectly by collaborating with tumor-infiltrating macrophages. We, here, hypothesize that this critical step may be dependent on secretion of tumor-specific antigens by cancer cells. This was investigated using T-cell receptor-transgenic mice, in which CD4(+) T cells mediate rejection of syngeneic MOPC315 myeloma cells. We analyzed the immune response against myeloma cell variants, which either secrete or retain intracellularly a tumor-specific idiotypic (Id) antigen. Our results reveal that CD4(+) T cells helped by macrophages are capable of detecting nonsecreted tumor antigens from MHC class II-negative cancer cells. However, Id secretion was required for successful myeloma immunosurveillance. Antigen secretion resulted in stronger priming of naive myeloma-specific CD4(+) T cells in tumor-draining lymph nodes. Secretion of antigen by at least some cancer cells within a tumor was shown to facilitate immunosurveillance. Treatment by local injection of purified tumor-specific antigen successfully enhanced immunity against nonsecreting myeloma cells. Collectively, the data indicate that antigen concentration within the tumor extracellular matrix must reach a certain threshold to allow successful cancer immunosurveillance by CD4(+) T cells.

UCH-L1 Provides An Essential Survival Signal in Malignant B-Cells by Enhancing NF-κB Activation.

Using an activity-based approach, we previously described the over-expression of the deubiquitinase ubiquitin carboxy terminal hydroxylase 1 (UCH-L1) in several B-cell malignancies including multiple myeloma, Burkitt lymphoma, non-Hodgkin lymphoma and chronic lymphocytic leukemia. UCH-L1 over expression also correlates with a poor prognosis in non-lymphoid tumors including non-small cell lung, esophageal, colorectal and pancreatic cancers. Whether UCH-L1 expression contributes to the malignant phenotype in these conditions, however, has not been established. We therefore sought to determine the role of UCH-L1 in lymphoid malignancies through the use of lentivirusencoded short hairpin RNA (shRNA) targeting UCH-L1. When compared with a control non-silencing shRNA, reduced UCH-L1 levels are associated with a profound block in proliferation and loss of viability (>90%) in the UCH-L1 expressing myeloma cell line KMS11. The introduction of these shRNAs into the UCH-L1 negative myeloma cell line KMS12 does not affect proliferation or viability. The association of cell death with reduced UCH-L1 levels was further reinforced using a doxycycline inducible shRNA construct. KMS11 and KMS12 cells grow equally well following transduction and selection of stable viral integrants. However, upon induction of shRNA production with doxycycline, greater than 90% of KMS11 cells die within 48-hours timed with the depletion of UCH-L1 while KMS12 cells continue to proliferate. These results strongly suggest that UCH-L1 is essential for survival in UCH-L1 expressing myeloma cells. Given the critical role of NF-kB signaling in many lymphoid malignancies, we examined the impact of UCH-L1 depletion on NF-kB activity. Using a secreted luciferase reporter assay, we determined NF-kB activity in control and UCH-L1 depleted KMS11 cells. We observed a 5-fold reduction of NF-kB activity in UCH-L1 knockdown cells compared to control. In order to establish a correlation between loss of UCH-L1 expression and NF-kB down-regulation, cells transduced with an inducible shRNA were treated with doxycycline and monitored daily for UCH-L1 and NF-kB activation. We observed a direct relationship between the level of UCH-L1 and NF-kB activity in these cells with a 5-fold reduction in NF-kB activity by 48-hours accompanied by reduced viability. These effects are entirely reversible with the removal of doxycycline from the growth medium. Doxycycline induction had no effect on the NF-kB activity of the UCH-L1 negative cell line KMS12. Knockdown of UCH-L1 does not affect TNF-alpha-induced NF-kB activity in these cells suggesting that UCH-L1 modulates the non-canonical NF-kB pathway. A relationship between UCH-L1 and NF-kB is further suggested by observing a 3.8-fold increase in baseline NF-kB activity in HeLa cells over-expressing UCH-L1. These data strongly indicate that UCH-L1 enhances NF-kB through the non-canonical pathway. In order to determine whether UCH-L1 over-expression can by itself promote tumorigenesis in vivo, we have engineered a UCH-L1 transgenic mouse. These mice will provide us the opportunity to study the effect of UCH-L1 expression on spontaneous as well as induced (carcinogen or oncogene) tumorigenesis. Further studies are examining the effect of forced UCH-L1 expression on B-cell development. This study will provide a better understanding of the role of UCH-L1 in hematological malignancies and may validate UCH-L1 as an important drug target for B-cell malignancies.

Recognition of Myeloma by MAGE-A3 Specific Cytotoxic T Lymphocytes Induced by Treatment with Azacitidine and MGCD0103

Demethylating agents and histone deacetylase inhibitors (HDACi) are epigenetic modulators that can induce re-expression of tumor suppressor and cell cycle proteins that have been silenced through aberrant hypermethylation associated with tumoral transformation. Azacitidine (Aza) is a cytosine analogue that primarily affects RNA during transcription. However, DNA is also a target for demethylation during replication thereby increasing gene re-expression. Treatment with HDACi, such as MGCD0103 (MGC), can synergize with demethylating agents to boost the epigenetic effects of either drug used alone. Our gene expression profiling data shows that the cancer-testis antigen MAGE-A3 is expressed in 31% of myeloma patients at diagnosis and the frequency of expression is increased at relapse to 49% (n=51 paired samples, p<0.001, unpublished data). However, expression of MAGE-A3 is often heterogeneous. We hypothesized that the combination of Aza and MGC could induce MAGE-A3 expression, thus facilitating killing of myeloma cells by MAGE-A3 specific CTLs isolated from a HLA-A68 positive patient post MAGE-A3 protein vaccination (J Immunother 2007; 30:847). The MAGE-A3 negative myeloma cell line LP1 was first transfected with HLA-A68. MAGE-A3 protein production was optimized by dose finding and time course experiments using Aza alone or Aza and MGC sequentially. Induction of MAGE-A3 RNA expression was assessed by real time PCR and protein expression by Western blotting. 51Cr-release assays were used to measure killing of Aza/MGC treated cell lines by MAGE-A3 specific CTLs. MAGE-A3 RNA expression was detected in LP1-A68 treated with 500nM Aza for 3 days and expression was enhanced by sequential treatment with 1mM MGC for 1 day when compared to Aza treatment alone. However, protein expression was low. In an effort to optimize protein production, we increased the time of treatment with 500nM Aza to 5 days and with 500nM MGC to 2 days. After this sequential treatment, protein was clearly expressed (Figure 1) and LP1-A68 cells were killed by MAGE-A3 specific CTLs (specific lysis: 70% ± 9% at E:T ratio of 5:1), whilst untreated controls only showed background killing (specific lysis: 12% ± 5%) (Figure 2). Repeat experiments are in progress to verify these results. 500nM Aza in vitro is comparable to a clinically achievable in vivo dose of 12.5mg/m2 (Leukemia 2008; 22:965). 500nM MGC is comparable to a 280mg/m2in vivo dose (Blood 2006; 108: 1954). Additional titration experiments with MGC will be tested to achieve clinically relevant concentrations in vivo that can induce MAGE-A3 expression. In conclusion, epigenetic modulation by Aza and MGC can enhance MAGE-A3 expression and result in increased killing by MAGE-A3 specific CTLs. Hypomethylating agents and HDACi may be useful to sensitize tumor cells to immune effectors. [Display omitted] [Display omitted]

P90RSK2 as a Therapeutic Target in Treatment of FGFR3-Expressing t(4;14) Multiple Myeloma.

p90 Ribosomal S6 Kinase 2 (p90RSK2) is a substrate of ERK and plays an active role in anti-apoptosis signaling and cell cycle regulation. Our proteomics studies revealed that p90RSK2 is not only activated but also tyrosine-phosphorylated in hematopoietic cells expressing constitutively activated leukemogenic FGFR3 mutants. FGFR3 is a receptor-tyrosine kinase that responds to fibroblast growth factor (FGF). Recent studies have suggested that FGFR3 may play a significant role in the pathogenesis and disease progression of some hematopoietic malignancies including t(4;14)(p16.3;q32.3) multiple myeloma, which is associated with dysregulation of FGFR3. We found that the MEK1 inhibitor U0126 effectively inhibited RSK2 activation induced by FGFR3, suggesting that FGFR3 activates RSK2 through the MEK/ERK pathway. Interestingly, FGFR3-dependent tyrosine phosphorylation at Y529 was required for phosphorylation and activation of the Ser/Thr kinase RSK2 by ERK. By using a specific phospho-RSK2 (Y529) antibody, we identified FGFR3 as the direct tyrosine kinase that phosphorylates RSK2 at Y529 in an in vitro kinase assay, which only contains purified recombinant active FGFR3 and RSK2 proteins. Substitution of Y529 in RSK2 attenuates inactive ERK binding to RSK2, which is prior to and required for ERK-dependent phosphorylation and activation of RSK2. In addition, reconstitution of RSK2 phosphorylation at Y529 consequently enhanced inactive ERK binding by a GST pull down, suggesting that phosphorylation at Y529 by FGFR3 facilitates ERK binding to RSK2, which is a sequential event and precedes inactive ERK binding. These observations support a novel two-step model that FGFR3 activates RSK2 by both assisting inactive ERK binding via tyrosine phosphorylation of RSK2 at Y529 and activating the MEK/ERK pathway. Moreover, we observed that RSK2 is specifically phosphorylated at Y529 and activated in FGFR3 expressing various human t(4;14) positive MM cell lines. Targeted down-regulation of RSK2 by siRNA induced significant apoptosis in FGFR3-expressing human myeloma cell lines including OPM1 and KMS11. In addition, a specific RSK inhibitor fmk induced significant apoptosis in t(4;14) positive human myeloma cells including KMS11, OPM1, LP1 and KMS18, but not in control t(4;14) negative myeloma cell lines including ANBL6 and U266. Similar results were obtained by using another RSK specific and potent inhibitor BI-D1870. Furthermore, we observed that fmk induced significant apoptosis in primary CD138-positive, FGFR3-expressing myeloma cells from a t(4;14) positive multiple myeloma patient, but not in the control CD138-negative cells from the same patient, nor primary samples from a t(4;14)-negative patient as a control. Together, these data not only support our model that RSK2 is a critical signaling effector of FGFR3 in hematopoietic transformation, but also provide “proof of principle” for the therapeutic potential of RSK2 inhibitors such as fmk in treatment of t(4;14) positive, FGFR3-expressing multiple myeloma with minimal non-specific cytotoxicity.

PU.1 Is Downregulated in a Subset of Multiple Myeloma by the Methylation of Its -17 kb Upstream Regulatory Region and the Promoter.

It has been reported that disruption of transcription factors critical for hematopoiesis, such as C/EBPa and AML1, is involved in leukemogenesis. PU.1 is a transcription factor important for both myeloid and lymphoid development. We reported that mice in which the levels of PU.1 were 20% of that of wild-type developed acute myeloid leukemia, T cell lymphoma, and a CLL-like disease. These findings strongly suggest that PU.1 has tumor suppressive activity in multiple hematopoietic lineages. Last year, we reported that PU.1 is downregulated in a majority of multiple myeloma cell lines and and freshly isolated CD138 positive myeloma cells from certain number of myeloma patients, and that tet-off inducible exogenous expression of PU.1 in PU.1 negative myeloma cell lines induced cell growth arrest and apoptosis. Based on their PU.1 expression levels, we divided the myeloma patients into two groups, namely PU.1 high and PU.1 low-to-negative, (cutoff index of 25th percentile of the PU.1 expression level distribution among all patients). The PU.1 low-to-negative patients had a significantly poorer prognosis than the PU.1 high patients. To elucidate the mechanisms of downregulation of PU.1, we performed sequence and epigenetic analysis of the promoter region and the -17 kb upstream region that is conserved among mammalians and important for proper expression of PU.1. There are no mutations in these regions of all five myeloma cell lines. In contrast, the -17 kb upstream region was highly methylated in 3 of 4 PU.1 negative myeloma cell lines, while the promoter region was also methylated to various levels in all five myeloma cell lines including one PU.1 positive cell line. These data suggested that the downregulation of PU.1 in myeloma cell lines might be dependent on the methylation of both regulatory regions of PU.1 gene, especially the -17 kb upstream region. We also evaluated the mechanisms of cell growth arrest and apoptosis of myeloma cell lines induced by PU.1. Among apoptosis-related genes, we identified that TRAIL was upregulated after PU.1 induction. To evaluate the effect of upregulation of TRAIL, we stably introduced siRNA for TRAIL into myeloma cell lines expressing PU.1, and we found that apoptosis of these cells was partially suppressed by siRNA for TRAIL, suggesting that apoptosis of myeloma cells induced by PU.1 might be at least partially due to TRAIL upregulation. We are currently performing DNA microarray analysis to compare the expression levels of genes between before and after PU.1 induction, in order to further elucidate the mechanisms of cell growth arrest and apoptosis.

Primary Antitumor Immune Response Mediated by CD4 + T Cells

Gene-targeted mice have recently revealed a role for lymphocytes and interferon-gamma (IFNgamma) in conferring protection against cancer, but the mechanisms remain unclear. Here, we have characterized a successful primary antitumor immune response initiated by naive CD4+ T cells. Major histocompatibility complex class II (MHC-II)-negative myeloma cells injected subcutaneously into syngeneic mice were surrounded within 3 days by macrophages that captured tumor antigens. Within 6 days, naive myeloma-specific CD4+ T cells became activated in draining lymph nodes and subsequently migrated to the incipient tumor site. Upon recognition of tumor-derived antigenic peptides presented on MHC-II by macrophages, the myeloma-specific CD4+ T cells were reactivated and started to secrete cytokines. T cell-derived IFNgamma activated macrophages in close proximity to the tumor cells. Tumor cell growth was completely inhibited by such locally activated macrophages. These data indicate a mechanism for immunosurveillance of MHC-II-negative cancer cells by tumor-specific CD4+ T cells through collaboration with macrophages.

A cellular model for myeloma cell growth and maturation based on an intraclonal CD45 hierarchy.

Multiple myeloma (MM) is a plasma cell malignancy mainly characterized by the accumulation of malignant plasma cells within the bone marrow. This review shows that the biology of CD45 illuminates that of MM and, more specifically, provides a better delineation of a tumor cell 'hierarchy' of clinical interest. We show that in MM, as in normal plasma cell differentiation, there is an intraclonal CD45 hierarchy that is a gradient of CD45 expression on myeloma cells directly related to their proliferation rate and differentiation status. This CD45 hierarchy allows for the design of a cellular model for MM-cell growth and maturation in which CD45 bright myeloma cells represent the proliferating compartment and CD45 low myeloma cells the quiescent compartment. This model includes an aberrant phenotype that is annihilation rather than decline of CD45, annihilation reflecting the terminal phase of the disease and/or an aggressive presentation of MM. Data from the literature suggest that CD45 bright myeloma cells are targeted by interleukin (IL)-6, whereas CD45 negative myeloma cells with a high clonogenic capacity are targeted by insulin/insulin-like growth factor 1 (IGF-1). This model will be useful for both a better understanding of the basic biology of MM and a better stratification of and therapeutic approach to the patients. Finally, this model presents MM as a self-renewing plasma cell disease, although the first oncogenic events such as 14q32 translocations clearly occur earlier in a B cell.

Antitumor monoclonal antibodies enhance cross-presentation ofcCellular antigens and the generation of myeloma-specific killer T cells by dendritic cells.

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti–syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti–syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell–loaded mature DCs induced a strong CD8+ T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8+ T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti–syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1–specific response. Cross-presentation was inhibited by pretreatment of DCs with Fcγ receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.

Establishment and Characterization of a CD95 (Fas/Apo-1)-Negative Myeloma Cell Line

Although expression of CD95 (Fas/Apo-1) on myeloma cells has been reported, its significance is not clearly understood. We established a myeloma cell line, KHM-11ad (11ad), from a parental cell line, KHM-11, by collecting cells adhered to a plastic dish. KHM-11 cells have been reported to be positive for CD45 and CD95 (Fas/Apo1), and negative for a myelomonocytic antigen, CD13. Interestingly, CD95 was not detected in 11ad. Expression of CD45 was also significantly decreased in 11ad cells while expression of CD13 was detected in these cells. The growth rate of 11ad cells was 1.7 times lower than that of KHM-11 cells. Analysis of adhesion molecules showed that expression of VLA4 and CD44 was significantly suppressed in 11ad. The IC₅₀ of melphalan (L-PAM) for 11ad cells was 50 times higher than that for KHM-11, indicating that 11ad is significantly refractory to L-PAM than KHM-11 cells. Induction of apoptosis by doxorubicin and cycloheximide was suppressed in 11ad cells compared with those in KHM-11 cells. Western blot analysis for Bcl-2 family of proteins showed that Bax was expressed at a 2.2 times lower level in 11ad cells than in KHM-11 cells while there was no difference in expression of Bcl-2, Bcl-Xs nor Bcl-X_L. These results suggest that CD95-negative myeloma cells may have characteristics as follows: (1) slow proliferation; (2) low sensitivity to apoptosis; (3) low expression of VLA4, CD44 and Bax. Although these intraclonal variations were based on the findings of cell lines, these may reflect similar variations in vivo. The 11ad line may be a suitable model for analyzing intraclonal variation of myeloma cells.